WO2001059113A2 - G-protein coupled receptor proteins and nucleic acids encoding same - Google Patents

G-protein coupled receptor proteins and nucleic acids encoding same Download PDF

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WO2001059113A2
WO2001059113A2 PCT/US2001/004404 US0104404W WO0159113A2 WO 2001059113 A2 WO2001059113 A2 WO 2001059113A2 US 0104404 W US0104404 W US 0104404W WO 0159113 A2 WO0159113 A2 WO 0159113A2
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nucleic acid
amino acid
polypeptide
ofthe
gpcrx
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PCT/US2001/004404
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French (fr)
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WO2001059113A3 (en
Inventor
Raymond J. Taupier, Jr.
Catherine E. Burgess
Muralidhara Padigaru
Velizar T. Tchernev
Vishnu S. Mishra
Stacie Casman
Robert Ballinger
Corine A. M. Vernet
Li Li
Kimberly A. Spytek
David P. Andrew
Peter S. Mezes
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Curagen Corporation
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Priority to AU2001239756A priority Critical patent/AU2001239756A1/en
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Publication of WO2001059113A3 publication Critical patent/WO2001059113A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the mvention generally relates to novel GPCR1/GPCR2, GPCR3, GPCR4, GPCR5, GPCR6 and GPCR7 nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleic acids encoding novel polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
  • the invention is based in part upon the discovery of novel nucleic acid sequences encoding novel polypeptides.
  • the disclosed GPCR1, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6 and GPCR7 nucleic acids and polypeptides encoded therefrom, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "GPCRX” nucleic acid or polypeptide sequences.
  • the invention provides an isolated GPCRX nucleic acid molecule encoding a GPCRX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21.
  • the GPCRX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein- coding sequence of a GPCRX nucleic acid sequence.
  • the invention also includes an isolated nucleic acid that encodes a GPCRX polypeptide, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21.
  • oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous .nucleotides of a GPCRX nucleic acid (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21) or a complement of said oligonucleotide.
  • GPCRX nucleic acid e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21
  • complement of said oligonucleotide e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21
  • GPCRX polypeptides SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22.
  • the GPCRX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human GPCRX polypeptide.
  • the invention also features antibodies that immunoselectively-binds to GPCRX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier.
  • the therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or an antibody specific for a GPCRX polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes a GPCRX nucleic acid, under conditions allowing for expression ofthe GPCRX polypeptide encoded by the DNA. If desired, the GPCRX polypeptide can then be recovered.
  • the invention mcludes a method of detecting the presence of a
  • GPCRX polypeptide in a sample in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the GPCRX polypeptide within the sample.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a GPCRX.
  • Also included in the invention is a method of detecting the presence of a GPCRX nucleic acid molecule in a sample by contacting the sample with a GPCRX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a GPCRX nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of a GPCRX polypeptide by contacting a cell sample that includes the GPCRX polypeptide with a , compound that binds to the GPCRX polypeptide in an amount sufficient to modulate the activity of said polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a Therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, or other disorders related to cell signal processing and metabolic pathway modulation.
  • the Therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or a GPCRX-specific antibody, or biologically-active derivatives or fragments thereof.
  • the invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation.
  • the method includes contacting a test compound with a GPCRX polypeptide and determining if the test compound binds to said GPCRX polypeptide. Binding ofthe test compound to the GPCRX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • Also within the scope ofthe invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a GPCRX nucleic acid.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a GPCRX polypeptide, a GPCRX nucleic acid, or both, in a subject (e.g., a human subject).
  • the method includes measuring the amount ofthe GPCRX polypeptide in a test sample from the subject and comparing the amount ofthe polypeptide in the test sample to the amount ofthe GPCRX polypeptide present in a control sample.
  • An alteration in the level ofthe GPCRX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
  • the expression levels ofthe new polypeptides ofthe invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
  • the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a GPCRX polypeptide, a GPCRX nucleic acid, or a GPCRX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition.
  • the disorder includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
  • the invention can be used in a method to identity the cellular receptors and downstream effectors ofthe invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
  • the invention is based, in part, upon the discovery of novel nucleic acid sequences that encode novel polypeptides.
  • novel nucleic acids and their encoded polypeptides are referred to individually as GPCR1, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, and GPCR7.
  • the nucleic acids, and their encoded polypeptides are collectively designated herein as "GPCRX”.
  • the novel GPCRX nucleic acids ofthe invention include the nucleic acids whose sequences are provided in Tables 1 A, 2A, 2C, 3 A, 4A, 4C, 5 A, 5C, 5E, 6A, and 7A inclusive ("Tables 1 A - 7 A”), or a fragment, derivative, analog or homolog thereof.
  • the novel GPCRX proteins ofthe invention include the protein fragments whose sequences are provided in Tables IB, IC, 2B, 2D, 3B, 4B, 4D, 5B, 5D, 5F, 6B, and 7B inclusive ("Tables IB - 7B").
  • Tables IB - 7B The individual GPCRX nucleic acids and proteins are described below. Within the scope of this invention is a method of using these nucleic acids and peptides in the treatment or prevention of a disorder related to cell signaling or metabolic pathway modulation.
  • GPCR1 Novel GPCR1 is a G-protein coupled receptor ("GPCR") protein related to the cysteinyl leukotriene receptor.
  • GPCR1 maps to human chromosome 13.
  • the GPC 1 nucleic acid of 1260 nucleotides is shown in Table 1 A.
  • the GPCR1 open reading frame (“ORF”) begins at one of two alternative ATG initiation codons, shown in bold in Table 1 A.
  • the GPCR1 ORF begins with an initiation codon at nucleotides 105-107, and the encoded polypeptide is alternatively referred to herein as GPCRla or as AL137118A.
  • the GPCR1 ORF begins with an ATG initiation codon at nucleotides 120-122, and the encoded polypeptide is alternatively referred to herein as GPCRlb or as CG54236-02. In either embodiment, the GPCR1 ORF terminates at a TAA codon at nucleotides 1143-1145. As shown in Table 1 A, putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters. Table 1A. GPCR1 nucleotide sequence (SEQ ID NO:l).
  • the encoded GPCR1 protein is translated from nucleotides 105 through 1145 and has 346 amino acid residues, referred to as the GPCRla protein.
  • the GPCRla protein was analyzed for signal peptide prediction and cellular localization. SignalP results predict that GPCRla is cleaved between position 59 and 60 of SEQ ID NO:2, i.e, at the dash in the amino acid sequence GLS-IYN. Psort and Hydropathy profiles also predict that GPCR1 contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000).
  • the GPCRla polypeptide sequence is presented in Table IB using the one-letter amino acid code.
  • an encoded GPCR1 protein referred to alternatively as the GPCRlb or CG54236-02 polypeptide is translated from nucleotides 120 through 1145 and has a polypeptide sequence of 341 amino acid residues.
  • the predicted GPCRlb polypeptide sequence includes amino acids 5 through 346 of SEQ ID NO:2 and is presented in Table IC using the one-letter code.
  • the identical predicted signal cleavage site in GPCRla occurs in GPCRlb between position 54 and 55 ofthe sequence shown in Table IC.
  • GPCRla or GPCRlb any reference to a GPCR1 polypeptide or nucleic acid is assumed to encompass all variants.
  • GPCRl was initially identified with a TblastN analysis of a proprietary sequence file for a G-protein coupled receptor probe or homolog which was run against the Genomic Daily Files made available by GenBank.
  • a proprietary software program (GenScanTM) was used to further predict the nucleic acid sequence and the selection of exons.
  • the resulting sequences were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • the GPCRl nucleic acid sequence has 269 of 422 bases (63%) identical to a Gallus gallus activated T cell-specific G protein-coupled receptor mRNA (GenBank Ace. No. L06109) (SEQ ID NO:23) shown in Table ID.
  • the "E-value” or "Expect” value is a numeric indication ofthe probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • the probability that the subject (“Sbjct”) retrieved from the GPCRl BLAST analysis, in this case the Gallus gallus activated T cell- specific G protein-coupled receptor mRNA, matched the Query GPCRl sequence purely by chance is 1.3xl0 "13 .
  • the GPCRl nucleic acid sequence has a 100% homology across 1260 nucleotides to the Homo sapiens cystemyl leukotriene CysLT2 receptor (SEQ ID NO:24), as shown in Table IE.
  • GenBank XM_007164 sequence was directly deposited to National Center for Biotechnology Information, NIH, Bethesda, MD 20894, USA, and provided to the public on November 16, 2000.
  • the GPCRla protein has 113 of 313 amino acid residues (36 %) identical to, and 177 of 313 residues (56 %) positive with, the 367 amino acid residue P2Y-like G-protein coupled receptor from Homo sapiens ( ⁇ tnr:TREMBLNEW-CAA73144) (SEQ ID NO:25).
  • the GPCRla protein was also found to have 346 of 346 amino acid residues (100%) identical to, and 346 of 346 residues (100%) positive with, the 346 amino acid sequence of Homo sapiens cysteinyl leukotriene CysLT2 receptor
  • cysteinyl leukotriene CysLT2 receptor (ptnr:XP_007164) (SEQ ID NO:26).
  • GenBank XM_007164 sequence (SEQ LD NO:24), above, and was also directly deposited to National Center for Biotechnology Information, NTH, and made public on November 16, 2000. Table 1G.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • the "strong" group of conserved amino acid residues maybe any one ofthe following groups of amino acids: STA, NEOK. NHOK. NDEO. OHRK. MILN. MILF. HY. FYW.
  • Table II lists the statistics and domain description from DOMAIN analysis results against GPCRl .
  • the region from amino acid residue 63 through 247 (numbered with respect to SEO ID NO:2) most probably (E 3xlQ- 30 ) contains a "seven transmembrane receptor (rhodopsin family fragment" domain; aligned here with residues 1-177 ofthe 7tm 1 entry (SEQ ID NO:29) ofthe Pfam database. This indicates that the GPCRl sequence has properties similar to those of other proteins known to contain this domain as well as to the 7tm 1 domain itself.
  • GPCRX RNA was derived using tissue sources including, but not limited to. proprietary database sources, public EST sources, literature sources, and/or RACE sources, as described in the Examples. GPCRl is expressed in at least the following tissues: adrenal gland/suprarenal gland, heart, placenta, spleen, and peripheral blood leukocytes.
  • the nucleic acids and proteins of GPCRl are useful in potential therapeutic applications implicated in various GPCR- or OR-related pathologies and/or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding GPCRl protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from: cardiomyopathv. atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASP), atrioventricular (A-N) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions ofthe like.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma. lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophv. Additional GPCR-related diseases and disorders are mentioned throughout the Specification. Further, the protein similarity information, expression pattern, and map location for
  • GPCRl suggests that GPCRl may have important structural and/or physiological functions characteristic ofthe GPCR family. Therefore, the nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration ⁇ n v jf ro and ⁇ n v j vo (vi) biological defense weapon.
  • a contemplated GPCRl epitope is from aa 30 to 60. In another embodiment, a GPCRl epitope is from aa 80 to 95. In additional embodiments. GPCRl epitopes are from aa 110 to 170. from aa 180 to 240: from aa 250 to 270. and from aa 280 to 305.
  • GPCR2 is an Olfactory Receptor ("OR")-like protein.
  • OR Olfactory Receptor
  • Two alternative novel GPCR2 nucleic acids and encoded polypeptides are disclosed.
  • a GPCR2a variant (alternatively referred to herein as
  • AC022289 A includes the 1039 nucleotide sequence (SEQ ID NO:3) shown in Table 2A.
  • a GPCR2a ORF begins with an ATG initiation codon at nucleotides 54-56 and ends with a TGA codon at nucleotides 996-998. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 2A, and the start and stop codons are in bold letters.
  • the GPCR2a polypeptide (SEO ID NO:4) encoded by SEO ID NO:3 is 314 aa and is presented using the one-letter amino acid code in Table 2B.
  • the Psort profile for GPCR2 predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a GPCR2a peptide is between amino acids 43 and 44, e ., at the dash in the amino acid sequence NNG-NLG, based on the SignalP result.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or. in the case ofthe reverse primer, until the stop codon was reached. Such suitable sequences were then used as the forward and reverse primers in a PCR amplification based on a wide range of cDNA libraries. The resulting amplicon was gel purified, cloned and sequenced to high redundancy, as described in the Examples. The cloned sequence is disclosed as an alternative embodiment of GPCR2 (SEO ID
  • GPCR2b is referred to herein as the GPCR2b and reported in Tables 2C and 2D.
  • GPCR2b is alternatively referred to herein as AC022289 Al.
  • the GPCR2b ORF begins with an ATG initiation codon at nucleotides 54-56 and ends with a TGA codon at nucleotides 996-998. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 2C. and the start and stop codons are in bold letters.
  • the GPCR2b protein (SEO ID NO:6) encoded bv SEO ID NO:5 is 314 amino acid in length, has a molecular weight of 35806.5 Daltons, and is presented using the one-letter code in Table 2D.
  • the most likely cleavage site for a GPCR2b peptide is between amino acids 43 and 44, / esammlung at the dash in the amino acid sequence VNG-NLG. based on the SignalP result.
  • any reference to GPCR2 is assumed to encompass all variants. Residue differences between any GPCRX variant sequences herein are written to show the residue in the "a” variant, the residue position with respect to the "a” variant, and the residue in the "b” variant. GPCRX residues in all following sequence alignments that differ between the individual GPCRX variants are highlighted in black and marked with the (o) symbol above the variant residue in all alignments herein.
  • the GPCR2 nucleic acid sequences differ at the following two positions: C648T and C922T.
  • the GPCR2 polypeptides differ only at one residue, namely P290L.
  • GPCR2a nucleic acid sequence has 471 of 648 bases (72%) identical to R a t f S norvegicus taste bud receptor protein (SEO ID NO: 30). as shown in Table 2E.
  • SEO ID NO: 30 R a t f S norvegicus taste bud receptor protein
  • Table 2E BLASTN of GPCR2 against rat taste bud receptor protein.
  • GPCR2 nucleic acid sequence has homology to two regions ofthe ff m sapiens olfactory receptor ("OR5D3") gene, as shown in Table 2F.
  • OR5D3 residues 437-644 (SEO ID NO:31) has 168 of 208 bases (80%) identical to GPCR2. with an E value of 4x10-”.
  • QR5D3 residues 121-219 (SEO ID NO:32) has 82 of 99 bases (82%) identical to GPCR2. with an E value of 6xlQ- 7 .
  • the full GPCR2 amino acid sequence has 159 of306 amino acid residues (51 %) identical to, and 214 of306 residues (69 %) positive with, the 314 amino acid residue proteins & m_ omo sapiens Olfactory Receptor-like protein OLF1 (pt ⁇ r: SPTREMBL-ACC: O13606) (SEO ID NO:33) (Table 2G).
  • the residue that differs between GPCR2a and GPCR2b is highlighted in black and marked with the (o) symbol.
  • the full amino acid sequence ofthe GPCR2 protein ofthe invention has 152 of 301 amino acid residues (50%) identical to, and 207 of 301 residues (68%) positive with, the 312 amino acid residue proteins from Gallus gallus olfactory receptor 4 (ptnr: SPTREMBL-ACC: CAA64370.1) (SEO ID NO:34) (Table 2H).
  • the residue that differs between GPCR2a and GPCR2b is highlighted in black and marked with the (o) symbol.
  • Novel_Human_OLF i.e. GPCR2, SEQ ID NO: 4
  • Rattus norvegicus OLF Ace # Q63395, SEQ ID NO: 36
  • GPCR2 gg ⁇ F ⁇ itdA ⁇ ' ii- ⁇ AlCTiloigLBaLiTOa ⁇ g-WLWB BGPSELLCYSiRBl-Jlgs GPlitf-HMitatij HUMANJDLF CHICK_OLF4 ⁇ K ⁇ PSQrKAVg R[jQKgL ⁇ 3sE3AFLNg ⁇ gGL ⁇ 3K ⁇ Sg vJ30S3 RAT_OLF ⁇ 3 ⁇ ER[3353N33 ⁇ Es
  • DOMAIN results for GPCR2 were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 2J with the statistics and domain description.
  • the 7tm 1. a seven transmembrane receptor (rhodopsin family), was shown to have two segments with significant homology to GPCR2.
  • An alignment of GPCR2 with residues 1-170 (SEO ID NO:29) and residues 310-377 (SEO ID NO:37) of 7tm 1 are shown in Table 2J.
  • nucleic acids and proteins of GPCR2 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further below.
  • a cDNA encoding the 0 if ac tory rece pt or -like protein may be useful in gene therapy, and the 0 if ac tory receptor" u ⁇ e protein may be useful when administered to a subject in need thereof.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy.
  • Other GPCR-1 diseases and disorders are contemplated.
  • novel nucleic acid encoding GPCR2, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • This novel protein also has immense value in development of powerful assay system for functional analysis.
  • GPCR3 An additional GPCR-like protein ofthe invention, referred to herein as GPCR3, is an Olfactory Receptor ("OR")-like protein.
  • the GPCR3 nucleic acid of 1001 nucleotides (also designated APOOl 112 A) is shown in Table 3 A.
  • An ORF was identified beginning with an ATG initiation codon at nucleotides 12-14 and ending with a TAA codon at nucleotides 945- 47.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 3 A. and the start and stop codons are in bold letters.
  • Table 3A GPCR3 Nucleotide Sequence (SEQ ID NO:7)
  • the GPCR3 nucleic acid sequence has 609 of 923 bases (65%) identical to a and 609/923 bases (65%) positive with p an troglodytes species Olfactory Receptor OR93 gene (SEO ID NO:38), as shown in Table 3C.
  • the BLASTN alignment shown in Table 3D indicates that two fragments of GPCR3 have homology to fragments of Mus musculus olfactory receptor 4 cluster, gene 3 ("Qlfr4-3") (GENBANK-ID: NM 013728.1). Residues 827-907 (SEO ID NO:39) and residues 163-210 (SEQ ID NO-.40) ofthe O1&4-3 gene are shown below.
  • the full GPCR3 amino acid sequence has 166 of 305 amino acid residues (54%) identical to, and 214 of 305 residues (70%) positive with, the 314 amino acid residue OR93CH protein from Pan troglodytes f ⁇ tnr: SPTREMBL-ACC: 077756) (SEO ID NO:41) (Table 3E).
  • the full amino acid sequence ofthe GPCR3 protein was also found to have 166 of 311 amino acid residues (53%) identical to, and 215 of 311 residues (68%) positive with, the 311 40 amino acid residue proteins from j,f l ⁇ S m s us olfactory receptor 4 cluster, gene 3 (SEO ID NO:42), shown in Table 3F.
  • olfactory receptor 4 cluster gene 3 [Mus musculus], derived from gb
  • Hyloba tes lar (Common Gibbon) OLF, SPTREMBL -Ace # 077758 , SEQ ID NO : 43
  • GPCR3 ⁇ aS ⁇ CTPiitdit-WGSRlBig ⁇ Sv5sFffllTVtgMY ⁇ LTGLMElHlMWTYNll ⁇ Krirf ilEiiSt3g ⁇ a
  • nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in various in various GPCR-related pathological disorders and/or OR- related pathological disorders, described further below.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma. prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy.
  • Other GPCR-related diseases and disorders are contemplated.
  • novel nucleic acid encodin g olfactory receptor 'l ke protein, and the olfactory receptor -u ke protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • This novel protein also has immense value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders
  • GPCR4 is an Olfactory Receptor ("OR")-like protein, wherein two alternative novel GPCR4 nucleic acids and encoded polypeptides are disclosed.
  • the novel GPCR4a nucleic acid of 980 nucleotides (also referred to as AP001112 B) is shown in Table 4A.
  • An ORF begins with an ATG initiation codon at nucleotides 19-21 and ends with a TAA codon at nucleotides 940-42.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 4A, and the start and stop codons are in bold letters.
  • the GPCR4a protein encoded by SEO ID NO: 9 has 307 amino acid residues and is presented using the one-letter code in Table 4B.
  • the Psort profile for GPCR4 predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a peptide is between amino acids 39 and 40, / e .. at the dash in the amino acid sequence MNG-NIG, based on the SignalP result.
  • the target GPCR4a sequence was subjected an the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy.
  • GPCR4b also referred to as AC020597A
  • the 980 nucleotides of GPCR4b are provided in Table 4C.
  • the resulting GPCR4b nucleotide sequence differs from that of GPCR4a at nine positions, namely A75G, A100G. C102T. C264T. T270A. C582T. A610C, T627C and T759C.
  • the GPCR4b protein encoded by SEO ID NO:l 1 has 314 amino acid residues and a molecular weight of 35155.8 Daltons, as presented using the one-letter code in Table 4D.
  • GPCR4a differs from GPCR4b at four residues, namely I28N. N45L. F84L and I198L.
  • the signal peptide and Psort analyses for both GPCR4 variants are the same.
  • GPCR4a Unless specifically addressing GPCR4a or 4b, assume any reference to GPCR4 to encompass all variants. hi a search of sequence databases, it was found, for example, that the nucleic acid sequence for GPCR4ahas 591 of 940 bases (62%) identical to and 591 of 940 bases (62%) positive with Rgttus norvegicus species taste bud Receptor clone (GENBANK-ID: U50948) 25 (SEO ID NO:44) (Table 4E). The residues that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
  • the full GPCR4a amino acid sequence has 155 of 304 amino acid residues (50 %) identical to, and 215 of 304 residues (70%) positive with, the 314 amino acid residue proteins from p an troglodytes OR93CH (ptnr: SPTREMBL-ACC: 077756) (SEO ID NO:41) (Table 41).
  • the residue that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
  • the GPCR4a amino acid sequence has 139 of 303 amino acid residues (45%) identical to. and 192 of 303 residues (62%) positive with the 303 amino acid OR93Gib protein from Hylobates lar (GENBANK-ID:AAC63971.1) (SEO ID NO:43) (Table 4J).
  • the residue that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
  • the GPCR4a protein has 137 of 304 amino acid residues (45%) identical to. and 191 of 304 residues (62%) positive with, the 308 amino acid odorant receptor K42 from Mu musculus (GENBANK-ID:AAG39876.1) (SEO ID NO:49) (Table 4K). The residue that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
  • the GPCR4b protein has 141 of 301 amino acid residues (46%) identical to, and 189 of 301 residues (61%) positive with, the 314 amino acid OR 511 from Mii musculus (GENBANK-FD:AAG39876.1) (SEO ID NO:50) (Table 4L). The residue that-differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
  • the full amino acid sequence ofthe GPCR4b protein has 140 of 303 amino acid residues (46%) identical to, and 193 of 303 residues (63 %) positive with, the 312 amino acid OR4 protein from Mu musculus (GENBANK-ID:AAG39876.1) (SEO ID NO:34) (Table 4J).
  • the residue that differs between GPCR4a and GPCR4b are highlighted in black and marked 15 with the (o) symbol.
  • DOMAIN results for GPCR4a were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 4L with the statistics and domain description. Residues 1-163 (SEO ID NO:29) and residues 313-377 (SEO ID NO:37) of 7tm_l are aligned with GPCR4 in Table 4O. The residue that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
  • the nucleic acids and proteins of GPCR4 are useful in potential therapeutic applications.
  • compositions ofthe present invention will have efficacy for treatment of patients
  • neoplasm adenocarcinoma. lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy.
  • Other GPCR-related diseases and disorders are contemplated.
  • novel GPCR4 nucleic acid and protein, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein
  • GPCR5 is an Olfactory Receptor ("OR")-like protein, wherein three alternative novel GPCR5 nucleic acids and encoded polypeptides are disclosed.
  • the novel GPCR5a nucleic acid of 980 nucleotides (also referred to as APOOl 112 C) 35 is shown in Table 5A.
  • An ORF begins with an ATG initiation codon at nucleotides 26-28 and ends with a TGA codon at nucleotides 941-43.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5A, and the start and stop codons are in bold letters.
  • Table 5A GPCR5a Nucleotide Sequence (SEQ ID NO:13)
  • the SignalP. Psort and/or Hydropathy profile for GPCR5a predict that GPCR5a has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the signalP shows a signal sequence is coded for in the first 44 amino acids, esammlung with a cleavage site at the dash in the sequence NLG-MIM, between amino acids 44 and 45. This is typical of this type of membrane protein.
  • the resulting GPCR5b sequence (also referred to herein as AC0170103B1) is reported below in Table 5C .
  • the GPCR5b protein encoded by SEO ID NO: 15 has 305 amino acid residues and is presented using the one-letter code in Table 5D.
  • the SignalP, Psort and/or Hydropathy profiles for GPCR5b are the same as for GPCR5a.
  • the GPCR5c protein encoded by SEQ ID NO: 17 has 305 amino acid residues and is presented using the one-letter code in Table 5F.
  • the SignalP, Psort and/or Hydropathy profiles for GPCR5c are the same as for GPCR5a and GPCR5b.
  • GPCR5 variants differ at four nucleotide residues, namely GPCR5a and GPCR5b differ from GPCR5c at T29C.
  • GPCR5a and GPCR5c differ from GPCR5b at A537G.
  • GPCR5 variants differ at four amino acid residues, namely GPCR5a and GPCR5b differ from GPCR5c at S2P, A230V and A299G, while GPCR5a and GPCR5c differ from GPCR5b at N171S. All numbering is in reference to GPCR5a. Unless specifically addressing GPCR5a or GPCR5b or GPCR5c, assume any reference to GPCR5 to encompass all variants.
  • GPCR5a has 633 of 959 bases (66 %) identical to and 633 of 959 bases (66%) positive with a G a u lls galh ⁇ s species Olfactory Receptor clone (GENBANK-ID: X94742) (SEO ID NO:51) (Table 5G). The residue that differs between GPCR5a. GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol. Table 5G-BLASTN of GPCR5a against OR 2 (SEQ ID NO:51)
  • the GPCR5a amino acid has 154 of 306 amino acid residues (55%) identical to, and
  • the GPCR5a amino acid has 148 of 301 amino acid residues (49%) identical to, and 198 of 301 residues (65%) positive with, the 308 amino acid K42 from M S musC ulus 25 (GENBANK-ID:AAG39876.1) (SEO ID NO:53) (Table 51).
  • the residue that differs between GPCR5a, GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol.
  • the GPCR5b amino acid sequence has 153 of 306 amino acid residues (50%) identical to. and 198 of 306 residues (64%) positive with, the 309 amino acid M71 from M US musculus
  • GPCR5a 60 GPCR5a. GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol. Table 5K. BLASTX of GPCR5b against M71
  • Hyloba tes lar (Common Gibbon) OLF, SPTREMBL-Acc # 077758, SEQ ID NO: 43
  • GPCR5a lOTlTJSGSAl ⁇ -taUMd TJgjCld-lMs LtWgvi-raivvHl.ylii ⁇ iLSSlLiaOTiMBMfdi-ltMdit.W ⁇
  • GPCR5 a feliWeMilMMBroil ia elnlBgcMH ⁇ iaaPT DKTVEE sj ⁇ IA-gSiiilFfflsjgv ⁇ rcidftifeMdMgDBJg
  • RATJDLF The presence of identifiable domains in GPCR5 was determined by searches using algorithms such as PROSITE. Blocks, Pfam. ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The results indicate that this protein contains the following protein domains (as defined by Interpro) at the indicated positions: domain name 7tm 1 (InterPro) 7 transmembrane receptor (rhodopsin family) at amino acid positions 41 to 289. This indicates that the sequence of GPCR5 has properties similar to those of other proteins known to contain this/these domain(s) and similar to the properties of these domains.
  • DOMAIN results for GPCR5a were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 5M with the statistics and domain description. Residues 1-180 (SEO ID NO:29) and residues 313-377 (SEO ID NO:37) of 7tm 1 are aligned with GPCR4 in Table 40. The residue that differs between GPCR5a, GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol.
  • GPCR5 is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neuro genie cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue.
  • SNPs single nucleotide polymorphisms
  • Table 5N “Depth” represents the number of clones covering the region ofthe SNP.
  • the Putative Allele Frequency (Putative AUele Freq.) is the fraction of all the clones containing the SNP.
  • a dash (“-"). when shown, means that a base is not present. The sign ">” means "is changed to”.
  • nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody). (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • compositions ofthe present invention are useful in potential diagnostic and therapeutic applications implicated in various GPCR- or OR-related diseases and disorders described below and/or other pathologies.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from: developmental diseases. MHC ⁇ and UI diseases (immune diseases). Taste and scent detectability Disorders. Burkitt's lymphoma. Corticoneuro enie disease. Signal Transduction pathway disorders. Retinal diseases including those involving photoreception. Cell Growth rate disorders: Cell Shape disorders.
  • Feeding disorders;control of feeding potential obesity due to over-eating: potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (MDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2). pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma: prostate cancer: uterus cancer), anorexia, bulimia, asthma. Parkinson's disease. acute heart failure, hypotension, hypertension, urinary retention, osteoporosis. Crohn's disease: multiple sclerosis: Albright Hereditary Ostoeodystrophy, angina pectoris.
  • DRLA Dentatorubro-pallidoluysian atrophy
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cD ⁇ A encoding GPCR5 may be useful in gene therapy, and GPCR5 may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2). pain, cancer (including but not limited to Neoplasm: adenocarcinoma: lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis. Crohn's disease; multiple sclerosis: and Treatment of Albright Hereditary Ostoeodystrophy.
  • GPCR5 angina pectoris. myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • the novel nucleic acid encoding GPCR5, and the GPCR5 protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods and other diseases, disorders and conditions ofthe like. Other GPCR-related diseases and disorders are contemplated.
  • a contemplated epitope of GPCR5c would be from amino acid 10 to 50. In another embodiment, a contemplated epitope of GPCR5c would be from amino acid 35 to 45. In yet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 80 to 120. In vet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 135 to 160.
  • a contemplated epitope of GPCR5c would be from amino acid 205 to 235. In yet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 245 to 260. In yet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 275 to 290.
  • the novel nucleic acid of 1050 nucleotides GPCR6 (also designated APOOl 112 D) encoding a novel Olfactory Receptor-like protein is shown in Table 6A.
  • An ORF was identified beginning with an ATG initiation codon at nucleotides 53-55 and ending with a TAA codon at nucleotides 1007-1009.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
  • the GPCR6 protein encoded by SEO ID NO: 19 has 318 amino acid residues and is presented using the one-letter code in Table 6B.
  • the GPCR6 nucleic acid sequence has 621 of 939 bases (66 %) identical to a and 621 of 939 bases (66%) positive with GaUus ga u us species Olfactory Receptor 2 (GENBANK-ID: X94742) (Table 6C).
  • Gallus gallus (SEQ ID NO: 51) 996 bp.
  • the GPCR6 amino acid has 165 of 307 amino acid residues (53 %) identical to, and 30 226 of 307 residues (73%) positive with, the 312 amino acid OR4 from Gallus gallus (ptnr: SPTREMBL-ACC: 077756) (SEO ID NO:56) (Table 6D).
  • the GPCR6 amino acid has 153 of 313 amino acid residues (48%) identical to, and 199 of 313 residues (62%) positive with, the 314 amino acid OR93Ch from p f] troglodytes OR93Ch (GENBANK-ID:AAC63969.1) (SEO ID NO:57) (Table 6E).
  • the GPCR6 amino acid has 150 of 312 amino acid residues (48%) identical to. and 5 198 of 312 residues (63%) positive with, the 313 amino acid OR93Gib from Hylobates lar (GENBANK-ID:AAC63971.1) (SEO ID NO:58) (Table 6F).
  • the GPCR6 amino acid has 143 of 307 amino acid residues (46%) identical to, and 0 193 of 307 residues (62%) positive with, the 332 amino acid OR2 from Gallus gallus (embCAA64368.1) (SEO ID NO:59) (Table 6G).
  • the GPCR6 amino acid has 150 of 311 amino acid residues (48%) identical to, and 193 of 311 residues (61%) positive with, the 311 amino acid K30 from jt ⁇ fj . musculus (GENBANK-ID:AAG39871.1) (SEO ID NO:60) (Table 6H). 5 Table 6H.-BLASTX of GPCR6 against K30
  • the GPCR6 amino acid has 149 of 311 amino acid residues (47%) identical to, and 192 of 311 residues (60%) positive with, the 314 amino acid Kl 1 from Mi l s musculus (GENBANK-ID: AAG39856.1) (SEO ID NO:61) (Table 61).
  • Hyloba tes lar (Common Gibbon) OLF, SPTREMBL-Acc # 077758, SEQ ID NO: 43 -. 3. Homo sapiens OLF, SWISSPROT-Acc # Q13606, SEQ ID NO: 33
  • GPCR6 MT.vp ⁇ l.foraGlHsliiil.gaaahM ⁇
  • DOMAIN results for GPCR6 were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 6K with the statistics and domain description. Residues 1-158 (SEO ID NO:29) and residues 313-377 (SEO ID NO:37) of 7tm 1 are aligned with GPCR4 in Table 6K.
  • nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in various in various GPCR-related pathological disorders and/or OR- related pathological disorders, described further below.
  • a cDNA encoding the olfactory receptor -like protein may be useful in gene therapy, and the olfactory receptor -like protein may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from Neoplasm: adenocarcinoma; lymphoma: prostate cancer: uterus cancer; Immune response; AIDS; asthma; Crohn's disease; multiple sclerosis; and Albright Hereditary Ostoeodystrophy.
  • GPCR6 nucleic acid and protein . or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • This novel protein also has immense value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the novel nucleic acid of 981 nucleotides GPCR7 (also designated. AP001112 dal) encoding a novel OR-like protein is shown in Table 7A.
  • An ORF begins with an ATG initiation codon at nucleotides 27-29 and ends with a TGA codon at nucleotides 942-944. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon.
  • the GPCR7 protein encoded by SEO ID NO:20 has 305 amino acid residues and is 10 presented using the one-letter code in Table 7B.
  • the SignalP, Psort and/orHydropathyprofile for GPCR7 predict that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of0.6000.
  • the SignalP shows a cleavage site between amino acids 44 and 45, / g protagonist at the dash in the sequence amino acid NLG-MIM. This is typical ofa membrane protein.
  • nucleic acid sequence of GPCR7 has 633 of 959 bases (66%) identical to a gb:GENBANK- ID:GGCOR2GEN lacc:X94742.1 mRNA from Gallus gallus OR2 (SEO ID NO:51) (Table.
  • the GPCR7 amino acid has 164 of 305 amino acid residues (53%) identical to, and 214 of 305 amino acid residues (70%) similar to, the 309 amino acid OR M72 ( ptnr:TREMBLNE -Acc No.:AAG09780 ) protein from Mu musculus QR M72 - f SEQ DP NO:52) (Table 7D).
  • Table 7D BLASTP alignments of GPCR7 against OR M72, (SEQ ID NO:52)
  • GPCR7 The presence of identifiable domains in GPCR7 was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, and Prints followed by determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The results indicate that this protein contains the following protein domains (as defined by Interpro) at the indicated positions: domain name 7tm 1 (InterPro) 7 transmembrane receptor (rhodopsin family) at amino acid positions 41 to 289. Tins indicates that the sequence of GPCR7 has properties similar to those of other proteins known to contain this domain. GPCR7 maps to chromosome 11. This information was assigned using the Online
  • OMIM Mendelian Inheritance in Man
  • GPCR7 is expressed in at least the following tissues: Apical micro villi ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue.
  • GPCR7 may have important structural and/or physiological functions characteristic ofthe Olfactory Receptor family. Therefore. GPCR7 are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • GPCR7 is useful in potential diagnostic and therapeutic applications implicated in various GPCR- or OR-related diseases and disorders described below and/or other pathologies.
  • the compositions of GPCR7 will have efficacy for treatment of patients suffering from: : Familial Mediterranian Fever, developmental diseases, MHCII and III diseases (immune diseases).
  • Taste and scent detectability Disorders Burkitt's lymphoma, Corticoneuro enie disease.
  • Signal Transduction pathway disorders Retinal diseases including those involving photoreception.
  • Cell Growth rate disorders Cell Shape disorders. Feeding disordersxontrol of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of apetite).
  • noninsulin-dependent diabetes mellitus NfDDMl
  • bacterial, fungal, protozoal and viral infections particularly infections caused by HIN-1 or HIN-2
  • pain cancer
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma: prostate cancer; uterus cancer
  • anorexia bulimia
  • asthma Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis. Crohn's disease; multiple sclerosis; and
  • DRL Hereditary Ostoeodystrophy
  • angina pectoris myocardial infarction
  • ulcers ulcers
  • asthma allergies
  • benign prostatic hypertrophy and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation.
  • Dentatorubro-pallidoluysian atrophy(DRPLA) Hypophosphatemic rickets, autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders ofthe like.
  • the polypeptides can be used as immunogens to produce antibodies specific for GPCR7. and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR7-like protein may be useful in gene therapy, and the GPCR7-like protein may be useful when administered to a subject in need thereof.
  • the compositions of GPCR7 will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal arid viral infections (particularly infections caused by HIN-1 or HIN-2).
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer
  • anorexia, bulimia asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis
  • the GPCR7 nucleic acid and protein , or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to GPCR7 for use in therapeutic or diagnostic methods. Other GPCR-related diseases and disorders are contemplated.
  • a contemplated GPCR7 epitope is from aa 15 to 70. In another embodiment, a GPCR7 epitope is from aa 85 to 125. In additional embodiments, GPCR7 epitopes are from aa 140 to 175. from aa 210 to 235, from aa 240 to 260, and from aa 275 to 290.
  • Table 8A A summary of homologous sequences identified in searches of available sequence databases is provided in Table 8B.
  • nucleic acid molecules that encode GPCRX polypeptides or biologically-active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify GPCRX- encoding nucleic acids (e .g- GPCRX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of GPCRX nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e g-., cDNA or genomic DNA), RNA molecules (e g., mRNA), analogs ofthe DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.
  • GPCRX nucleic acid can encode a mature GPCRX polypeptide.
  • a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
  • Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an ORF. or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N. where residue 1 is the N-terminal methionine. would have residues 2 through N remaining after removal ofthe N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, g ycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • probes refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, eêt , 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • isolated nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid ( g though sequences located at the 5'- and 3'-termini ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated GPCRX nucleic acid molecules can contain less than about 5 kb.4 kb, 3 kb. 2 kb.
  • nucleic acid molecule ofthe invention e g.. a nucleic acid molecule having the nucleotide sequence of SEO ID NOS:l. 3, 5, 7. 9, 11. 13. 15. 17. 19 and 21.
  • GPCRX molecules can be isolated using standard hybridization and cloning techniques (e.
  • a nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to GPCRX nucleotide sequences can be prepared by standard synthetic techniques, eschreib., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEO ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17. 19 and 21, or a complement thereof. Oligonucleotides maybe chemically synthesized and may also be used as probes.
  • an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NOS:l. 3. 5, 7. 9. 11. 13. 15. 17. 19 and 21, or a portion of this nucleotide sequence (e . g . - a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an GPCRX polypeptide).
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEO ID NOS:l, 3, 5. 7. 9. 11. 13. 15. 17. 19 and 21. is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:l. 3, 5. 7. 9. 11, 13. 15, 17. 19 and 21. that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains.
  • Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
  • Derivatives and analogs maybe full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs ofthe nucleic acids or proteins ofthe invention include, but are not limited to.
  • nucleic acids or proteins ofthe invention comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e cilantro Ausubel, e ⁇ /u-CuRRENT
  • homologous nucleic acid sequence or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of GPCRX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for an GPCRX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include.
  • Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human GPCRX protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEO ID NOS:2. 4. 6. 8. 10. 12, 14. 16. 18, 20 and 22, as well as a polypeptide possessing GPCRX biological activity. Various biological activities ofthe GPCRX proteins are described below.
  • An GPCRX polypeptide is encoded by the open reading frame ("ORF") of an GPCRX nucleic acid.
  • An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide.
  • a stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.
  • An ORF that represents the coding sequence for a full protein begins with an ATG "start” codon and terminates with one ofthe three “stop” codons, namely, TAA, TAG, or TGA.
  • an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both.
  • a minimum size requirement is often set, e ⁇ a stretch of DNA that would encode a protein of 50 amino acids or more.
  • the nucleotide sequences determined from the cloning ofthe human GPCRX genes allows for the generation of probes and primers designed for use in identifying and/or cloning GPCRX homologues in other cell types, e g. from other tissues, as well as GPCRX homologues from other vertebrates.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25. 50, 100, 150, 200. 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEO ID NOS:l, 3. 5. 7. 9, 11, 13. 15. 17.
  • Probes based on the human GPCRX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto. eargent.
  • the label group can be a radioisotope. a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an GPCRX protein, such as by measuring a level of an GPCRX-encoding nucleic acid in a sample of cells from a subject e g bank detecting GPCRX mRNA levels or determining whether a genomic GPCRX gene has been mutated or deleted.
  • a polypeptide having a biologically-active portion of an GPCRX polypeptide refers to polypeptides exhibiting activity similar, but not necessarily identical to. an activity of a polypeptide ofthe mvention. including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically- active portion of GPCRX” can be prepared by isolating a portion of SEO ID NOS:l, 3, 5, 7, 9, 11, 13. 15. 17. 19 and 21, that encodes a polypeptide having an GPCRX biological activity (the biological activities ofthe GPCRX proteins are described below), expressing the encoded portion of GPCRX protein ( ⁇ . g- by recombinant expression m v # r ) and assessing the activity ofthe encoded portion of GPCRX.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. due to degeneracy ofthe genetic code and thus encode the same GPCRX proteins as that encoded by the nucleotide sequences shown in SEO ID NOS.T. 3, 5, 7, 9, 11, 13, 15. 17, 19 and 21.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEO ID NOS:2, 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22.
  • GPCRX nucleotide sequences shown in SEO ID NOS:l. 3. 5. 7. 9, 11. 13. 15. 17. 19 and 21, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences ofthe GPCRX polypeptides may exist within a population ⁇ . - the human population). Such genetic polymorphism in the GPCRX genes may exist among individuals within a population due to natural allelic variation.
  • gene and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an GPCRX protein, preferably a vertebrate GPCRX protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe GPCRX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the GPCRX polypeptides. which are the result of natural allelic variation and that do not alter the functional activity ofthe GPCRX polypeptides, are intended to be within the scope ofthe invention.
  • nucleic acid molecules encoding GPCRX proteins from other species and thus that have a nucleotide sequence that differs from the human sequence of SEO ID NOS:l, 3. 5, 7, 9. 11. 13. 15. 17. 19 and 21. are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe GPCRX cDNAs ofthe invention can be isolated based on their homology to the human GPCRX nucleic acids disclosed herein using the human cDNAs. or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEO ID NOS: 1. 3. 5. 7. 9. 11. 13, 15. 17. 19 and 21.
  • the nucleic acid is at least 10, 25. 50. 100. 250, 500, 750. 1000, 1500, or 2000 or more nucleotides in length.
  • an isolated nucleic acid molecule ofthe invention hybridizes to the coding region.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs /_ g nucleic acids encoding GPCRX proteins derived from species other than human
  • other related sequences eenne paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50%) ofthe probes are occupied at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e . g . - 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in Ausubel, et ah- ( eds -). CUR ENT PROTOCOLS IN MOLECULAR BIO OGY. Joh Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%. 75%. 85%. 90%. 95%. 98%. or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5). 1 mM EDTA.
  • nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences of SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e. g- encodes a natural protein).
  • a nucleic acid sequence that is hvbridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well-known within the art. See, e-g .
  • a nucleic acid that is hvbridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS:l, 3. 5. 7. 9. 11. 13. 15, 17. 19 and 21. or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • a non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide. 5X SSC.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequences ofthe GPCRX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
  • amino acid residues that are conserved among the GPCRX proteins ofthe invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
  • nucleic acid molecules encoding GPCRX proteins that contain changes in amino acid residues that are not essential for activity. Such GPCRX proteins differ in amino acid sequence from SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. vet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEO ID NOS.-2.4. 6. 8. 10. 12, 14, 16, 18, 20 and 22.
  • the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEO ID NOS:2, 4, 6. 8. 10. 12. 14. 16. 18.
  • An isolated nucleic acid molecule encoding an GPCRX protein homologous to the protein ofSEO ID NOS :2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein- Mutations can be introduced into SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16, 18. 20 and 22. by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e . g- lysine. arginine, histidine), acidic side chains ( e. g- aspartic acid, glutamic acid), uncharged polar side chains (e. g- glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e . g- alanine. valine, leucine. isoleucine. proline. phenylalanine. methionine.
  • a predicted non-essential amino acid residue in the GPCRX protein is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an GPCRX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GPCRX biological activity to identify mutants that retain activity. Following mutagenesis of SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22.
  • the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
  • the relatedness of amino acid families may also be determined based on side chain interactions.
  • Substituted amino acids may be fully conserved "strong” residues or fully conserved “weak” residues.
  • the "strong” group of conserved amino acid residues maybe any one ofthe following groups: STA. NEOK. NHOK. NDEQ. OHRK. MILV. MILF. HY. FYW. wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other.
  • the "weak" group of conserved residues may be any one ofthe following: CSA. ATV. SAG. STNK. STPA. SGND.
  • a mutant GPCRX protein can be assayed for (;) the ability to form proteimprotein interactions with other GPCRX proteins, other cell-surface proteins, or biologically-active portions thereof, (//) complex formation between a mutant GPCRX protein and an GPCRX ligand; or ( ) the ability of a mutant GPCRX protein to bind to an intracellular target protein or biolo ically-active portion thereof; ( e perhaps. avidin proteins).
  • a mutant GPCRX protein can be assayed for the ability to regulate a specific biological function (e . g., regulation of insulin release).
  • Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEO ID NOS:l. 3, 5. 7. 9. 11. 13. 15. 17. 19 and 21. or fragments. analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e. g- complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence).
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire GPCRX coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding an GPCRX protein.
  • coding region refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region” ofthe coding strand of a nucleotide sequence encoding the GPCRX protein.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids ( ? - g bank also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or H ⁇ ogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of GPCRX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of GPCRX mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GPCRX mRNA.
  • An antisense oligonucleotide can be. for example, about 5, 10, 15, 20. 25. 30, 35. 40.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e . g- an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e. - phosphorothioate derivatives and acridine substituted nucleotides can be used).
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhvdroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine. 1-methylguanine, 1-methylinosine.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation ( g ., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated j n s j u such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an GPCRX protein to thereby inhibit expression ofthe protein ( e. g- by inhibiting transcription and/or translation).
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or. for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site. Alternatively.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e . g- bv linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in wliich the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other. See, g-g-> Gaultier, et fl /., 1987. jf uc ⁇ Acids Res. ⁇ S 6625-6641.
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (see, g y Inoue, ef a j. 1987. Nucl . Acids Res.
  • Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA. to which they have a complementary region.
  • ribozymes e. g- hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591
  • a ribozyme having specificity for an GPCRX-encoding nucleic acid can be designed based upon the nucleotide sequence of an GPCRX cDNA disclosed herein ( 7 - g Stamm SEO ID NOS: 1. 3, 5. 7. 9, 11, 13, 15. 17. 19 and 21).
  • a derivative of a Tetrahymena L ⁇ i9 INS R A can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an GPCRX-encoding mRNA. See, e o-., U.S. Patent 4,987,071 to Cech, e a U and U.S.
  • Patent 5,116,742 to Cech, e f a f GPCRX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, g o-., Bartel et ah, (1993) Science 261:1411-1418.
  • GPCRX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe GPCRX nucleic acid (e.g- th e GPCRX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe GPCRX gene in target cells, gee, e.g., Helene, 1991. Anticancer Drug Des- 6: 569-84; Helene, et a 1992. Ann ⁇ -N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
  • nucleotide sequences complementary to the regulatory region ofthe GPCRX nucleic acid e.g- th e GPCRX promoter and/or enhancers
  • the GPCRX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e perhaps., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids, ⁇ e , e.g., Hyrup. g ⁇ j., 1996. BioorgMed Chem 4: 5-23.
  • the terms "peptide nucleic acids” or "PNAs" refer to nucleic acid mimics (e .
  • PNA oligomers in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, e t fl /., 1996. Sl ⁇ ra- Perry-O'Keefe, et ⁇ /., 1996. Proc _ NatL Acad S ci. USA 93: 14670-14675.
  • PNAs of GPCRX can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, esammlung., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of GPCRX can also be used, for example, in the analysis of single base pair mutations in a gene ( e. g- PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes. g protagonist., S ⁇ nucleases ( see> Hyrup, e f a i t 1996.
  • PNAs of GPCRX can be modified, g p-., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of GPCRX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e .
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation ( see Hyrup, etal., 1996. suprc - The synthesis of PNA-DNA chimeras can be performed as described in Hyrup ⁇ e t a ⁇ 1996. SM pra and Finn, et a 1996. ⁇ uc ⁇ Acids Res 2 : 3357-3363.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs.
  • e p-., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite. can be used between the PNA and the 5' end of DNA.
  • $ ⁇ ee e.g., Mag, e f a 1989.
  • oligonucleotide may include other appended groups such as peptides ( e.
  • oligonucleotides can be modified with hybridization triggered cleavage agents ( se e , e.g, Krol, et q , 1988. BioTechniques 6:958-976) or intercalating agents ( see> e g ., Zon, 1988. Pharm. Res- $' ⁇ 539-549).
  • the oligonucleotide may be conjugated to another molecule, g g chord a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
  • a polypeptide according to the invention includes a polypeptide including the amino acid sequence of GPCRX polypeptides whose sequences are provided in SEO ID NOS:2. 4. 6. 8. 10. 12, 14, 16, 18. 20 and 22.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. while still encoding a protein that maintains its GPCRX activities and physiological functions, or a functional fragment thereof.

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Abstract

Disclosed herein are novel human nucleic acid sequences which encode polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies which immunospecifically-bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The invention further discloses therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involding any one of these novel human nucleic acids and proteins.

Description

NOVEL PROTEINS AND NUCLEIC ACIDS ENCODING SAME
FIELD OF THE INVENTION
The mvention generally relates to novel GPCR1/GPCR2, GPCR3, GPCR4, GPCR5, GPCR6 and GPCR7 nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleic acids encoding novel polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of novel nucleic acid sequences encoding novel polypeptides. The disclosed GPCR1, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6 and GPCR7 nucleic acids and polypeptides encoded therefrom, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "GPCRX" nucleic acid or polypeptide sequences.
In one aspect, the invention provides an isolated GPCRX nucleic acid molecule encoding a GPCRX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21. In some embodiments, the GPCRX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein- coding sequence of a GPCRX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a GPCRX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21. ■ "' Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous .nucleotides of a GPCRX nucleic acid (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21) or a complement of said oligonucleotide.
Also included in the invention are substantially purified GPCRX polypeptides (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). In certain embodiments, the GPCRX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human GPCRX polypeptide.
• The invention also features antibodies that immunoselectively-binds to GPCRX polypeptides, or fragments, homologs, analogs or derivatives thereof. In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier. The therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or an antibody specific for a GPCRX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a GPCRX nucleic acid, under conditions allowing for expression ofthe GPCRX polypeptide encoded by the DNA. If desired, the GPCRX polypeptide can then be recovered. In another aspect, the invention mcludes a method of detecting the presence of a
GPCRX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the GPCRX polypeptide within the sample. The invention also includes methods to identify specific cell or tissue types based on their expression of a GPCRX.
Also included in the invention is a method of detecting the presence of a GPCRX nucleic acid molecule in a sample by contacting the sample with a GPCRX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a GPCRX nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the activity of a GPCRX polypeptide by contacting a cell sample that includes the GPCRX polypeptide with a , compound that binds to the GPCRX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
Also within the scope ofthe invention is the use of a Therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, or other disorders related to cell signal processing and metabolic pathway modulation. The Therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or a GPCRX-specific antibody, or biologically-active derivatives or fragments thereof.
The invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation. The method includes contacting a test compound with a GPCRX polypeptide and determining if the test compound binds to said GPCRX polypeptide. Binding ofthe test compound to the GPCRX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
Also within the scope ofthe invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a GPCRX nucleic acid. Expression or activity of GPCRX polypeptide is then measured in the test animal, as is expression or activity ofthe protein in a control animal which recombinantly- expresses GPCRX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of GPCRX polypeptide in both the test animal and the control animal is compared. A change in the activity of GPCRX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of he disorder or syndrome. In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a GPCRX polypeptide, a GPCRX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount ofthe GPCRX polypeptide in a test sample from the subject and comparing the amount ofthe polypeptide in the test sample to the amount ofthe GPCRX polypeptide present in a control sample. An alteration in the level ofthe GPCRX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders. Also, the expression levels ofthe new polypeptides ofthe invention can be used in a method to screen for various cancers as well as to determine the stage of cancers. In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a GPCRX polypeptide, a GPCRX nucleic acid, or a GPCRX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors ofthe invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages ofthe invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION
The invention is based, in part, upon the discovery of novel nucleic acid sequences that encode novel polypeptides. The novel nucleic acids and their encoded polypeptides are referred to individually as GPCR1, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, and GPCR7. The nucleic acids, and their encoded polypeptides, are collectively designated herein as "GPCRX".
The novel GPCRX nucleic acids ofthe invention include the nucleic acids whose sequences are provided in Tables 1 A, 2A, 2C, 3 A, 4A, 4C, 5 A, 5C, 5E, 6A, and 7A inclusive ("Tables 1 A - 7 A"), or a fragment, derivative, analog or homolog thereof. The novel GPCRX proteins ofthe invention include the protein fragments whose sequences are provided in Tables IB, IC, 2B, 2D, 3B, 4B, 4D, 5B, 5D, 5F, 6B, and 7B inclusive ("Tables IB - 7B"). The individual GPCRX nucleic acids and proteins are described below. Within the scope of this invention is a method of using these nucleic acids and peptides in the treatment or prevention of a disorder related to cell signaling or metabolic pathway modulation.
GPCR1 Novel GPCR1 is a G-protein coupled receptor ("GPCR") protein related to the cysteinyl leukotriene receptor. GPCR1 maps to human chromosome 13. The GPC 1 nucleic acid of 1260 nucleotides is shown in Table 1 A. The GPCR1 open reading frame ("ORF") begins at one of two alternative ATG initiation codons, shown in bold in Table 1 A. In one embodiment, the GPCR1 ORF begins with an initiation codon at nucleotides 105-107, and the encoded polypeptide is alternatively referred to herein as GPCRla or as AL137118A. In another embodiment, the GPCR1 ORF begins with an ATG initiation codon at nucleotides 120-122, and the encoded polypeptide is alternatively referred to herein as GPCRlb or as CG54236-02. In either embodiment, the GPCR1 ORF terminates at a TAA codon at nucleotides 1143-1145. As shown in Table 1 A, putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters. Table 1A. GPCR1 nucleotide sequence (SEQ ID NO:l).
TGCTCCCTGTTTCATTAAAACCTAGAGAGATGTAATCAGTAAGCAAGAAGGAAAAAGGGAAATTCACAAAGTAACTTTTTGTGT CTGTTTCTTTTTAACCCAGCATGGAGAGAAAATTTATGTCCTTGCAACCATCCATCTCCGTATCAGAAATGGAACCAAATGGCA CCTTCAGCAATAACAACAGCAGGAACTGCACAATTGAAAACTTCAAGAGAGAATTTTTCCCAATTGTATATCTGATAATATTTT TCTGGGGAGTCTTGGGAAATGGGTTGTCCATATATGTTTTCCTGCAGCCTTATAAGAAGTCCACATCTGTGAACGTTTTCATGC TAAATCTGGCCATTTCAGATCTCCTGTTCATAAGCACGCTTCCCTTCAGGGCTGACTATTATCTTAGAGGCTCCAATTGGATAT TTGGAGACCTGGCCTGCAGGATTATGTCTTATTCCTTGTATGTCAACATGTACAGCAGTATTTATTTCCTGACCGTGCTGAGTG TTGTGCGTTTCCTGGCAATGGTTCACCCCTTTCGGCTTCTGCATGTCACCAGCATCAGGAGTGCCTGGATCCTCTGTGGGATCA TATGGATCCTTATCATGGCTTCCTCAATAATGCTCCTGGACAGTGGCTCTGAGCAGAACGGCAGTGTCACATCATGCTTAGAGC TGAATCTCTATAAAATTGCTAAGCTGCAGACCATGAACTATATTGCCTTGGTGGTGGGCTGCCTGCTGCCATTTTTCACACTCA GCATCTGTTATCTGCTGATCATTCGGGTTCTGTTAAAAGTGGAGGTCCCAGAATCGGGGCTGCGGGTTTCTCACAGGAAGGCAC TGACCACCATCATCATCACCTTGATCATCTTCTTCTTGTGTTTCCTGCCCTATCACACACTGAGGACCGTCCACTTGACGACAT GGAAAGTGGGTTTATGCAAAGACAGACTGCATAAAGCTTTGGTTATCACACTGGCCTTGGCAGCAGCCAATGCCTGCTTCAATC CTCTGCTCTATTACTTTGCTGGGGAGAATTTTAAGGACAGACTAAAGTCTGCACTCAGAAAAGGCCATCCACAGAAGGCAAAGA CAAAGTGTGTTTTCCCTGTTAGTGTGTGGTTGAGAAAGGAAACAAGAGTATAAGGAGCTCTTAGATGAGACCTGTTCTTGTATC CTTGTGTCCATCTTCATTCACTCATAGTCTCCAAATGACTTTGTATTTACATCACTCCCAACAAATGTTGATTCTTAATATTTA
In one embodiment, the encoded GPCR1 protein is translated from nucleotides 105 through 1145 and has 346 amino acid residues, referred to as the GPCRla protein. The GPCRla protein was analyzed for signal peptide prediction and cellular localization. SignalP results predict that GPCRla is cleaved between position 59 and 60 of SEQ ID NO:2, i.e, at the dash in the amino acid sequence GLS-IYN. Psort and Hydropathy profiles also predict that GPCR1 contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000). The GPCRla polypeptide sequence is presented in Table IB using the one-letter amino acid code.
Table IB. Encoded GPCRla protein sequence (SEQ ID ΝO:2).
MERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFPIVYLIIFF GVLGNG SIYVFLQPYKKSTSVNVFM NLAISD LLFISTLPFRADYYLRGSN IFGD ACRIMSYSLYVNMYSSIYFLTVLSVVRF AMVHPFRLLHVTSIRSA ILCGIIWILIMA SSIML DSGSEQNGSVTSCLE NLYKIAKLQTMNYIALVVGC LPFFTLSICY LIIRVL KVEVPESGLRVSHR A TTIIIT LIIFFLCFLPYHT RTVH TTWKVGLCKDR HKA VITLALAAANACFNPLLYYFAGENFKDRLKSALRKGHPQKAKTKCVFPV SV LRKETRV
In an alternative embodiment, an encoded GPCR1 protein referred to alternatively as the GPCRlb or CG54236-02 polypeptide is translated from nucleotides 120 through 1145 and has a polypeptide sequence of 341 amino acid residues. The predicted GPCRlb polypeptide sequence includes amino acids 5 through 346 of SEQ ID NO:2 and is presented in Table IC using the one-letter code. The identical predicted signal cleavage site in GPCRla occurs in GPCRlb between position 54 and 55 ofthe sequence shown in Table IC.
Table IC. Encoded GPCRlb protein sequence. MSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFPIVYLIIFFWGVLGNGLSIYVFLQPYKKSTSVNVF LN AISDLLFIS
TLPFRADYYLRGSNWIFGDLACRIMSYSLYvT^MYSSIYFLTVLSVVRFLA VHPFRLLHVTSIRSA ILCGII ILIMASSI L LDSGSEQNGSVTSCLELNLYKIAKLQTMNYIA WGC LPFFTLSICYL IIRVLLKVEVPESG RVSHRKALTTIIIT IIFF LCFLPYHTLRTVH TT KVG CKDRLHKALVIT ALAAANACFNP LYYFAGENFKDRLKSALRKGHPQKAKTKCVFPVSV LR KETRV
Unless specifically addressed as GPCRla or GPCRlb, any reference to a GPCR1 polypeptide or nucleic acid is assumed to encompass all variants. GPCRl was initially identified with a TblastN analysis of a proprietary sequence file for a G-protein coupled receptor probe or homolog which was run against the Genomic Daily Files made available by GenBank. A proprietary software program (GenScan™) was used to further predict the nucleic acid sequence and the selection of exons. The resulting sequences were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
In an analysis of sequence databases, it was found, for example, that the GPCRl nucleic acid sequence has 269 of 422 bases (63%) identical to a Gallus gallus activated T cell- specific G protein-coupled receptor mRNA (GenBank Ace. No. L06109) (SEQ ID NO:23) shown in Table ID. In all BLAST alignments herein, the "E-value" or "Expect" value is a numeric indication ofthe probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, as shown in Table IE, the probability that the subject ("Sbjct") retrieved from the GPCRl BLAST analysis, in this case the Gallus gallus activated T cell- specific G protein-coupled receptor mRNA, matched the Query GPCRl sequence purely by chance is 1.3xl0"13.
Table ID. BLASTN of GPCRl against activated T cell-specific GPCR
GENBANK-ID:CHKGPCR|acc:L06109 Gallus gallus activated T cell-specific G protein- coupled receptor mRNA (SEQ ID Nθ:23); Length = 1438 Score = 509 (76.4 bits), Expect = 1.3e-13 Identities = 269/422 (63%); Strand = Plus / Plus
Query: 186 AGCAGGAACTGCACAATTGAAAACTTCAAGA-G-AGAATTTTTCCCAAT-TGTATATCTG 242 IM I I II I I I I II I IM I I I I I I I I I I I II I II I
Sbjct: 91 AGCTCTAACTGCTCCACTGAGGACTCCTTTAAGTACACTTTGTATGGCTGTGTCT-TCAG 149
Query: 243 -ATAATATTTTTCTGGGGAGTCTTGGGAAA-TGGGTTGTCCATATATGTTTTC-CTGCAG 299
II lllll ll II ll l l II II I 111 I I I 11 llll II Sbjct: 150 CATGGTATTTGTCCTCGGCCTCATAGCCAACTGCGTTG-CTATCTACATTTTTACTTTTA 208
Query: 300 CCTTATAAGAAGTCCACATCTGTGA—ACGTTTT-CATGCTAAATCTGGCCATTTCAGAT 356
I II II lll l l l l ll III I III llll II II II Sbjct: 209 CATTG-AA--AGTGCGGAAC-GAGACCACGACGTACATGCTGAATTTGGCGATATCGGAC 264
Query: 357 CTCCTGTTCATAAGCACGCTTCCCTTCAGGGCTGACTATTATCTTAGAGGCTCC-AATTG 415 ll lllll I I I I I I II I llll II II I I I I ll ll Sbjct: 265 CTGCTGTTTGTGTTTACGTTGCCCTTCAGGA-T--CTATTA-CTTCGTGGTGAGGAACTG 320 Query: 416 GATATTTGGAGACCTGGCCTGCAGGAT-TATGTCTTATTCCTTGTATGTCAACATGTACA 474
I II II I I I I I I II I I I I I III I II I I Ml I I lllll Sbjct: 321 GCCCTTCGGAGACGTTCTGTGCAAGATCTCCGTCACGCTG-TTCTACACCAACATGTACG 379
Query: 475 GCAGTATT-TATTTCCTGACCGTGC-TGAGTGTTGTGCGTTTCCTGGCAATGGTTCACCC 532 I I I I I I I I II I 1 I I 1 II I II I I I I I I I I I 1 I I I I I I I I 1 I I II II
Sbjct: 380 GGAGCATTCTATT-CCTGACC-TGCATCAGCGTGGATCGCTTCCTGGCCATAGTGCACCC 437
Query: 533 CTTTCGGCT-TCTGCATGTCACCAGCATCAGGAGTGCCTGGATCCTCTGTGGGATCATAT 591
II I I II 11 I I I I ll ll MM III II 111 1 I I I I I I Sbjct: 438 CTTTCG-CTCTAAGACTCTTCGCACCAAAAGGAACGCCAGGATCGTGTGCGTGGCGGTGT 496 Query : 592 GGATCCTTATCATGGC 607 l l l l l I l l l l
Sbj ct : 497 GGATCACCGTGCTGGC 512
In addition, the GPCRl nucleic acid sequence has a 100% homology across 1260 nucleotides to the Homo sapiens cystemyl leukotriene CysLT2 receptor (SEQ ID NO:24), as shown in Table IE. The GenBank XM_007164 sequence (SEQ ID NO:24) was directly deposited to National Center for Biotechnology Information, NIH, Bethesda, MD 20894, USA, and provided to the public on November 16, 2000.
Table IE. BLASTN of GPCRl against CysLT2 receptor ref |X _007164 Homo sapiens cysteinyl leukotriene CysLT2 receptor; cDNA: PSEC0146 from clone PLACE1006979 (LOC57105) , mRNA (SEQ ID Nθ:24)
Score = 2498 bits (1260), Expect = 0.0
Identities = 1260/1260 (100%); Strand = Plus / Plus
Query: 1 tgctccctgtttcattaaaacctagagagatgtaatcagtaagcaagaaggaaaaaggga 60 III I 1 M I II I I II I I I II II M Mll ill II M II II II II M M Ml I MM
Sbjct: 160 tgctccctgtttcattaaaacctagagagatgtaatcagtaagcaagaaggaaaaaggga 219
Query: 61 aattcacaaagtaactttttgtgtctgtttctttttaacccagcatggagagaaaattta 120 llll IMMMMIMIMMMMMMMMIMMMMMMMMMMMMM
Sbjct: 220 aattcacaaagtaactttttgtgtctgtttctttttaacccagcatggagagaaaattta 279
Query: 121 tgtccttgcaaccatccatctccgtatcagaaatggaaccaaatggcaccttcagcaata 180
III II II II I I II II I I II II II M II I I II II II I MMMl I I I II II II I I II MM Sbjct: 280 tgtccttgcaaccatccatctccgtatcagaaatggaaccaaatggcaccttcagcaata 339
Query: 181 acaacagcaggaactgcacaattgaaaacttcaagagagaatttttcccaattgtatatc 240
IM I I III I I I lllll I II 1 II lllll II MMMl II II II II I I I III III I Sbjct: 340 acaacagcaggaactgcacaattgaaaacttcaagagagaatttttcccaattgtatatc 399
Query: 241 tgataatatttttctggggagtcttgggaaatgggttgtccatatatgttttcctgcagc 300
III I I II II II III llll II II MM II III llll 11 I I II I lllll I Sbjct: 400 tgataatatttttctggggagtcttgggaaatgggttgtccatatatgttttcctgcagc 459
Query: 301 cttataagaagtccacatctgtgaacgttttcatgctaaatctggccatttcagatctcc 360
I I I I M I M M M I M M M M M M M M M M I M M M M M M M M M l l l l l l l Sbjct : 460 cttataagaagtccacatctgtgaacgttttcatgctaaatctggccatttcagatctcc 519
Query: 361 tgttcataagcacgcttcccttcagggctgactattatcttagaggctccaattggatat 420 II I I II I II I I I II I I I III II Mill I MM II II llll II I I II III I I I I I I MM I Sbjct: 520 tgttcataagcacgcttcccttcagggctgactattatcttagaggctccaattggatat 579
Query: 421 ttggagacctggcctgcaggattatgtcttattccttgtatgtcaacatgtacagcagta 480 II I I llll I I lllll I I II II llll II II I Mill I llll II 111 II lllll I I Sbjct: 580 ttggagacctggcctgcaggattatgtcttattccttgtatgtcaacatgtacagcagta 639
Query: 481 tttatttcctgaccgtgctgagtgttgtgcgtttcctggcaatggttcacccctttcggc 540
II I I I II II I I llll I II III lllll llll III II II II II I I I lllll II I I I lllll I Sbjct: 640 tttatttcctgaccgtgctgagtgttgtgcgtttcctggcaatggttcacccctttcggc 699
Query: 541 ttctgcatgtcaccagcatcaggagtgcctggatcctctgtgggatcatatggatcctta 600
II I I llll I I III II I I II II llll II III III III II I II II I I llll II I I MUM I Sbjct: 700 ttctgcatgtcaccagcatcaggagtgcctggatcctctgtgggatcatatggatcctta 759
Query: 601 tcatggcttcctcaataatgctcctggacagtggctctgagcagaacggcagtgtcacat 660
II I I II II I I lllll II II II III II II I I III II II I I I II I I II I I lllll I Sbjct: 760 tcatggcttcctcaataatgctcctggacagtggctctgagcagaacggcagtgtcacat 819
Query: 661 catgcttagagctgaatctctataaaattgctaagctgcagaccatgaactatattgcct 720 MIIMMMMMMMMMMMMMMMMMMMMMMIMMIMMM Sbjct: 820 catgcttagagctgaatctctataaaattgctaagctgcagaccatgaactatattgcct 879
Query: 721 tggtggtgggctgcctgctgccatttttcacactcagcatctgttatctgctgatcattc 780
II II II I MM I I MM I I MM II III II II MMM I II II II I I I II II II II llll Sbjct: 880 tggtggtgggctgcctgctgccatttttcacactcagcatctgttatctgctgatcattc 939
Query: 781 gggttctgttaaaagtggaggtcccagaatcggggctgcgggtttctcacaggaaggcac 840 llll I I I II I II I MMIMMMMM II III II II 111 llll I I I I I I I llll II III Sbjct: 940 gggttctgttaaaagtggaggtcccagaatcggggctgcgggtttctcacaggaaggcac 999
Query: 841 tgaccaccatcatcatcaccttgatcatcttcttcttgtgtttcctgccctatcacacac 900
IMMIIIMI MMMMMMMMMMMMMIMMMMMMMM HUM Sbjct: 1000 tgaccaccatcatcatcaccttgatcatcttcttcttgtgtttcctgccctatcacacac 1059 Query: 901 tgaggaccgtccacttgacgacatggaaagtgggtttatgcaaagacagactgcataaag 960 II II I I I II I I I I II II I III I Mill II I I II I MM I I I I I I I II I I I llll Sbjct: 1060 tgaggaccgtccacttgacgacatggaaagtgggtttatgcaaagacagactgcataaag 1119
Query: 961 ctttggttatcacactggccttggcagcagccaatgcctgcttcaatcctctgctctatt 1020 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Sbjct: 1120 ctttggttatcacactggccttggcagcagccaatgcctgcttcaatcctctgctctatt 1179
Query: 1021 actttgctggggagaattttaaggacagactaaagtctgcactcagaaaaggccatccac 1080 MMIMIMMMMMMMMMMMMMMMIMMIMM I lllll Sbjct: 1180 actttgctggggagaattttaaggacagactaaagtctgcactcagaaaaggccatccac 1239
Query: 1081 agaaggcaaagacaaagtgtgttttccctgttagtgtgtggttgagaaaggaaacaagag 1140
Mill I I II I I I Mill I llll II III I I II III III II I llll I I I I I I IMI I I I II I Sbjct: 1240 agaaggcaaagacaaagtgtgttttccctgttagtgtgtggttgagaaaggaaacaagag 1299
Query: 1141 tataaggagctcttagatgagacctgttcttgtatccttgtgtccatcttcattcactca 1200 llll I I II II I I III II III III II I II II II II III I 11 I I II II I I lllll Sbjct: 1300 tataaggagctcttagatgagacctgttcttgtatccttgtgtccatcttcattcactca 1359 Query: 1201 tagtctccaaatgactttgtatttacatcactcccaacaaatgttgattcttaatattta 1260 M I 11 I I 1 I 1 I I M I 11 I I I I I M I I 1 I 1 I I 1 I I I I I M 11 I 1 I I I I I I I I I I 1 I I 111 I
Sbjct: 1360 tagtctccaaatgactttgtatttacatcactcccaacaaatgttgattcttaatattta 1419
A BLASTX search was performed against public protein databases. As shown in Table IF, the GPCRla protein has 113 of 313 amino acid residues (36 %) identical to, and 177 of 313 residues (56 %) positive with, the 367 amino acid residue P2Y-like G-protein coupled receptor from Homo sapiens (ρtnr:TREMBLNEW-CAA73144) (SEQ ID NO:25).
Table IF. BLASTX of GPCRla against P2Y-Like GPCR
>ptnr:TREMBLNE -ACC:CAA7314 P2Y-Like G-Protein Coupled Receptor-Homo sapiens (Human), 367 aa (SEQ ID Nθ:25)
Score = 477 (167.9 bits), Expect = 1.3e-44, P = 1.3e-44 Identities = 113/313 (36%), Positives = 177/313 (56%), Frame = +3.
Query: 135 SISVSEMEPNG TFSNNNSRNCTIEN-FKREFFPIVYLIIFFWGVLGNGLSIYVFLQP 302
I++ 1+ 1 I II + 1 1 + I 11+ I ++11 I++++I++
Sbjct 28 SMNGLEVAPPGLITNFSLATAEQCGQETPLEN LFASFYLLDFILALVGNTLALWLFIRD 87
Query 303 YKKSTSVNVFMLNLAISDLLFISTLPFRADYYLRGSN IFGDLACRIMSYSLYVNMYSSI 482 +1 I III+++II++II + 11 1 1+ I++I II++III+ + l+ MI+ ll
Sbjct 88 HKSGTPANVFLMHLAVADLSCVLVLPTRLVYHFSGNH PFGEIACRLTGFLFYLNMYASI 147
Query 483 YFLTVLSVVRFLA VHPFRLLHVTSIRSA ILCGIIWILI- ASSIMLLDSGSEQNGSVT 659
111 I +1 I I ll+lll + I + I + I +I+++ +1 + +1+ + 1
Sbjct 148 YFLTCISADRFLAIVHPVKSLKLRRPLYAHLACAFL VWAVAMAPLLVSPQTVQTNHTV 207 Query: 660 SCLELNLYKIAKLQT NYIALWGCLLPFFTLSICYLLIIRVLLKVEVPESGLRVSHR— 833
I I + 1 I + I ++ 1 I M l I I II 1 I I I IMI I
Sbjct: 208 VCLQL — YR-EKASHHALVSLAVAFTFPFITTVTCYLLIIRSL RQGLRVEKRLK 258 Query: 834 -KALTTIIITLIIFFLCFLPYHTLRTVHLTT KV-GL-CKDRLHKALV — ITLALAAANA 998
11+ I I I II +11 + 11 I I + I++ ++ 1 I + II II I + I Sbjct: '259 TKAVRMIAIVLAIFLVCFVPYHVNRSVYVLHYRSHGASCATQRILALANRITSCLTSLNG 318
Query: 999 CFNPLLYYFAGENFKDRLKSAL 1064 +I++I+I I 1+ I + I
Sbjct: 319 ALDPIMYFFVAEKFRHALCNLL 340
As shown in Table 1G, the GPCRla protein was also found to have 346 of 346 amino acid residues (100%) identical to, and 346 of 346 residues (100%) positive with, the 346 amino acid sequence of Homo sapiens cysteinyl leukotriene CysLT2 receptor
(ptnr:XP_007164) (SEQ ID NO:26). The cysteinyl leukotriene CysLT2 receptor (SEQ ED NO:26) is the protein encoded by GenBank XM_007164 sequence (SEQ LD NO:24), above, and was also directly deposited to National Center for Biotechnology Information, NTH, and made public on November 16, 2000. Table 1G. BLASTX of GPCRla against CysLT2 receptor ptnr:XP__007164 cysteinyl leukotriene CysLT2 receptor; cDNA: PSEC0146 from clone PLACE1006979 [Homo sapiens] (SEQ ID NO:26); Length = 346 Score = 657 bits (1696), Expect = 0.0
Identities = 346/346 (100%), Positives = 346/346 (100%)
Query: 1 ERKFMSLQPSISVSE EPNGTFSNNNSRNCTIENFKREFFPIVY IIFFWGV GNGLSI 60
I I I I 11 II 1 II I II I II I II II I II II M M II I M I II I II II I 11 I II I I II II II I I Sbjct: 1 MERKFMS QPSISVSEMEPNGTFSNNNSRNCTIENFKREFFPIVYLIIFFWGVLGNG SI 60 Query: 61 YVFLQPYKKSTSVNVFMNLAISDLLFIST PFRADYYLRGSN IFGDLACRIMSYS YV 120
II I I II I II I III Ml Ml 111111111111 II II I II I I I II I I I I II I I III I Mill Sbjct: 61 YVF QPYKKSTSVNVFMLNLAISDL FISTLPFRADYY RGSN IFGDLACRIMSYSLYV 120
Query: 121 N YSSIYFLTVLSVVRF AMVHPFRLLHVTSIRSA ILCGIIWILI ASSI LLDSGSEQ 180 I I I I I I I I I I II I I II II I II I II I I 11 II I I II I I II I I I II I I I II I I II I I I I I I I I
Sbjct: 121 N YSSIYFLTVLSWRF AMVHPFRLLHVTSIRSAWILCGIIWILI ASSIMLLDSGSEQ 180
Query: 181 NGSVTSCLE NLYKIAKLQTMNYIA WGCLLPFFTLSICYLLIIRVL KVEVPESGLRV 240 II I I II II I I I II III I llll II II II II I III II III II I II I I I I Ml I II I 1 lllll Sbjct: 181 NGSVTSC ELN YKIA LQTMNYIALWGCLLPFFT SICY IIRVL KVEVPESGLRV 240
Query: 241 SHRKALTTIIITLIIFFLCFLPYHT RTVH TT KVG CKDRLHKALVITLALAAANACF 300
I I I I I I I I I II I llll II II II II III I II II II I II I I I I I II I III I II I I I I I I III Sbjct: 241 SHRKA TTIIITLIIFFLCFLPYHTLRTVHLTT KVGLCKDR HKALVITLAAAANACF 300
Query: 301 NP YYFAGENFKDRL SA RKGHPQKAKTKCVFPVSVWLRKETRV 346
II 1 I) I I II I I I lllll MM II II MM II II I Mill I III I I I Sbjct: 301 NPLLYYFAGENFKDR KSALRKGHPQKA TKCVFPVSV LRKETRV 346 A ClustalW analysis comparing the protein of the invention with related protein sequences is given in Table IH, with GPCRl shown on line 2. In the ClustalW alignment of the GPCRl protein, as well as all other ClustalW analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
Table IH. ClustalW Analysis of GPCRl
1) patp_W75799_Human, SEQ ID NO: 27
2) (AL137118A) Novel GPCRla, SEQ ID NO: 2
3) (sptr-ACC:P34996) P2Y Purinoceptor 1 (ATP Receptor) (P2Y1) (Purinergic Receptor) Gallus gallus (Chicken), 362 aa, SEQ ID NO: 28
4) (STREMB -ACC:CAA73144) P2Y-like G-Protein Coupled Receptor Homo sapiens (Human), 367 aa., SEQ ID NO:25 patp__W75799_Human
GPCRla
ACC_P34996_P2YR_CHICK
ACC_CAA73144_Human patp__W75799_Human GPCRla
ACC_P34996_P2YR_CHICK ACC_CAA73144_Human patp_W75799_Human GPCRla
ACC_P34996_P2YR_CHICK ACC_CAA73144_Human
Figure imgf000013_0001
patp_W75799_Human |VG[BF|ILT|S|F|M-AKPQKD|KNN|K|F|PPQ|NQT|NHVL|LHU FJ|IJ| GPCRla |GlBHlM|s|lMΪD-|SEQN|-Sv|s|H- LYlI^~~L^TMNlI !4-----iC LH
ACC_P34996_P2YR_CHICK SSLV|Ag^|VlBl|FYBJTG|RR|κ|lT|YDTTADEYLR-SYF|YSMCTT|FM|Cj| ACC_CAA731 4_Hu an ||AFLJVV AVAMHJV-1PQT|QT|H|W|Q|Y E| SH HA JHA |TTB| patp_W75799_Human VI|IVJ|TMJ TBJ[SMKKN — LSS|KHIGBMVI IΆH--S|MBBIQBIB|HFL
GPCRla F|LSIBBJ|M ---BVEVP.BG RVSB---ILTT.----ITII.--IF C.-M-ITL.MB TW
ACC_P34996_P2YR_CHICK |V|LG|G|VKA|IY|DLDN| — PLR JS|YL v J[ v|a|s Y JFBMK|LN|RA
ACC_CAA731 4_Human H'T τ H-H-isiRQGLRVlκ — K B ΓRH HHHICIVHB I1SIYV:LHY patp_W75799_Human HNE— TKp|DSV |MQKSV'MBS---laslC--iMLMBslGB-l B~lI'FB~---lLSl GPCRla K VG |KD- -|HBLBBAHAAlA-_fr^lLHYBB-BIKDH IH---lGlpQKA
ACC_P34996_P2YR_CHICK LDFQ PQMJ|F DKVYATYQV|RG|SHS|VHIHL|D BRHSR|TB-SHR ACC_CAA73144_Human RSH— G | ORI| L|NRΪSC| HGALB|^^IHVA|KBH |CNL|CG— K| K patp_W75799_Human VTY|PR|K A|PLE|G|EICKV
GPCRla KTKCV|P V|VWLRK|--|RV
ACC_P34996_P2YR_CHICK EPN|QS|SEEMTLNILT|Y|QNGD|SL
ACC CAA73144 Human GPPPS|EG KTN|SSLSAKSEL
The presence of identifiable domains in GPCRl, as well as all other GPCRX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks,
Pfam, ProDomain, and Prints, and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/ interpro).
DOMAIN results, e g., for GPCRl as disclosed in Table II, were collected from the
Conserved Domain Database (CDD) with Reverse Position Specific BLAST analyses. This
BLAST analysis software samples domains found in the Smart and Pfam collections. For
Table II and all successive DOMAIN sequence alignments, fully conserved single residues are indicated by f *) and "strong" semi-conserved residues are indicated by (:). The "strong" group of conserved amino acid residues maybe any one ofthe following groups of amino acids: STA, NEOK. NHOK. NDEO. OHRK. MILN. MILF. HY. FYW.
Table II lists the statistics and domain description from DOMAIN analysis results against GPCRl . The region from amino acid residue 63 through 247 (numbered with respect to SEO ID NO:2) most probably (E = 3xlQ-30) contains a "seven transmembrane receptor (rhodopsin family fragment" domain; aligned here with residues 1-177 ofthe 7tm 1 entry (SEQ ID NO:29) ofthe Pfam database. This indicates that the GPCRl sequence has properties similar to those of other proteins known to contain this domain as well as to the 7tm 1 domain itself.
Table II. DOMAIN results for GPCRl
Sbjct: 7 transmembrane receptor (rhodopsin family) fragment (SEQ ID NO: 29) gnllPfa lpfamOOOOl; Length = 377 Score = 125 bits (315), Expect = 3e-30
Query : 63 GNGLSIYVFLQPYKKSTSVNVFMLNLAISDLLFISTLPFRADYYLRGSNWIFGDLACRIM 122
Sbj ct : 1 GNVLVCMAVSREI aLQTTTNYLIVSLAVADLLVATLVMP VVYLEVVGEWKFSRIHCDIF 60
Query: 123 SYSLYVNMYSSIYFLTVLSVVRFLAMVHPFR-LLHVTSIRSA ILCGII ILIMASSI L 181
Sbj ct : 61 VTLDVMMCTASILNLCAISIDRYTAVA PMLYNTRYSSKRRVTVMIAIVWVLSFTISCPM 120
Query : 182 LDSGSEQNGSVTSCLELNLYKIAKLQTMNYIALWGCLLPFFTLSICYLLIIRVLLKVEV 241
Sbj ct : 121 LFGLNNTDQN— ECIIA NPAFWYSSIVSFYVPFIVTLLVYIKIYIVLRRRRK 171
* : : : * : : : * : ** : * : * ** :
Query: 242 PESGLR 247
Sbjct: 172 RVNTKR 177
Expression information for GPCRX RNA was derived using tissue sources including, but not limited to. proprietary database sources, public EST sources, literature sources, and/or RACE sources, as described in the Examples. GPCRl is expressed in at least the following tissues: adrenal gland/suprarenal gland, heart, placenta, spleen, and peripheral blood leukocytes.
The nucleic acids and proteins of GPCRl are useful in potential therapeutic applications implicated in various GPCR- or OR-related pathologies and/or disorders. For example, a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof. The novel nucleic acid encoding GPCRl protein, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. The GPCRX nucleic acids and proteins are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from: cardiomyopathv. atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASP), atrioventricular (A-N) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions ofthe like. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma. lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophv. Additional GPCR-related diseases and disorders are mentioned throughout the Specification. Further, the protein similarity information, expression pattern, and map location for
GPCRl suggests that GPCRl may have important structural and/or physiological functions characteristic ofthe GPCR family. Therefore, the nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration \n vjfro and \n vjvo (vi) biological defense weapon.
These materials are further useful in the generation of antibodies that bind immunospecifically to the novel GPCRl substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. In one embodiment, a contemplated GPCRl epitope is from aa 30 to 60. In another embodiment, a GPCRl epitope is from aa 80 to 95. In additional embodiments. GPCRl epitopes are from aa 110 to 170. from aa 180 to 240: from aa 250 to 270. and from aa 280 to 305. GPCR2
A second GPCR-like protein ofthe invention, referred to herein as GPCR2, is an Olfactory Receptor ("OR")-like protein. Two alternative novel GPCR2 nucleic acids and encoded polypeptides are disclosed. In one embodiment, a GPCR2a variant (alternatively referred to herein as
AC022289 A) includes the 1039 nucleotide sequence (SEQ ID NO:3) shown in Table 2A. A GPCR2a ORF begins with an ATG initiation codon at nucleotides 54-56 and ends with a TGA codon at nucleotides 996-998. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 2A, and the start and stop codons are in bold letters.
Table 2A. GPCR2a Nucleotide Sequence (SEQ ID NO:3).
ATATTTTGCTTTGGCAGGAACAATTCTCTTCAACCCTTCCATTAAAAGGAATTATGATGATGGTTTTAAGGAATCTGAGCATGG AGCCCACCTTTGCCCTTTTAGGTTTCACAGATTACCCAAAGCTTCAGATTCCTCTCTTCCTTGTGTTTCTGCTCATGTATGTTA TCACAGTGGTAGGAAACCTTGGGATGATCATAATAATCAAGATTAACCCCAAATTTCACACTCCTATGTACTTTTTCCTTAGTC ACCTCTCTTTTGTTGATTTTTGTTACTCTTCCATTGTCACTCCCAAGCTGCTTGAGAACTTGGTAATGGCAGATAAAAGCATCT TCTACTTTAGCTGCATGATGCAGTACTTCCTGTCCTGCACTGCTGTGGTGACAGAGTCTTTCTTGCTGGCAGTGATGGCCTATG ACCGCTTTGTGGCCATCTGCAATCCTCTGCTTTATACAGTGGCCATGTCACAGAGGCTCTGTGCCCTGCTGGTGGCTGGGTCAT ATCTCTGGGGCATGTTTGGCCCCTTGGTACTCCTTTGTTATGCTCTCCGGTTAAACTTCTCTGGACCTAATGTAATCAACCACT TCTTTTGTGAGTATACTGCTCTCATCTCTGTGTCTGGCTCTGATATACTCATCCCCCACCTGCTGCTTTTCAGCTTCGCCACCT TCAATGAGATGTGTACACTACTGATCATCCTCACTTCCTATGTTTTCATTTTTGTGACTGTACTAAAAATCCGTTCTGTTAGTG GGCGCCACAAAGCCTTCTCCACCTGGGCCTCCCACCTGACTGCTATCACCATCTTCCATGGGACCATCCTTTTCCTTTACTGTG TACCCAACTCCAAAAACTCTCGGCAAACAGTCAAAGTGGCCTCTGTATTTTACACAGTTGTCAACCCCATGCTGAACCCTCCGA TCTACAGCCTAAGGAATAAAGACGTGAAGGATGCTTTCTGGAAGTTAATACATACACAAGTTCCATTTCACTGAACCAGTCTCA AAAGTTGTTTTCAATCCAAATGAACAACCCA
The GPCR2a polypeptide (SEO ID NO:4) encoded by SEO ID NO:3 is 314 aa and is presented using the one-letter amino acid code in Table 2B. The Psort profile for GPCR2 predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The most likely cleavage site for a GPCR2a peptide is between amino acids 43 and 44, e., at the dash in the amino acid sequence NNG-NLG, based on the SignalP result.
Table 2B. GPCR2a protein sequence (SEQ ID NO:4)
M MVLRNLSMEPTFALLGFTDYPKLQIPLFLVFLLMYVITVVGNLGMIIIIKINPKFHTPMYFFLSHLSFVDFCYSSIVTP LL ENLV ADKSIFYFSCM QYFLSCTAVVTESFLLAVMAYDRFVAICNPLLYTVA SQRLCALLVAGSYLWGMFGPLVLLCYALRL NFSGPNVINHFFCEYTALISVSGSDILIPHLLLFSFATFNE CTLLIILTSYVFIFVTVLKIRSVSGRHKAFSTWASHLTAITI FHGTILFLYCVPNSKNSRQTVKVASVFYTWNPMLNPPIYSLRNKDVKDAF KLIHTQVPFH
The predicted GPCR2a sequence, above, was subjected an the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or. in the case ofthe reverse primer, until the stop codon was reached. Such suitable sequences were then used as the forward and reverse primers in a PCR amplification based on a wide range of cDNA libraries. The resulting amplicon was gel purified, cloned and sequenced to high redundancy, as described in the Examples. The cloned sequence is disclosed as an alternative embodiment of GPCR2 (SEO ID
NO:5). referred to herein as the GPCR2b and reported in Tables 2C and 2D. GPCR2b is alternatively referred to herein as AC022289 Al. The GPCR2b ORF begins with an ATG initiation codon at nucleotides 54-56 and ends with a TGA codon at nucleotides 996-998. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 2C. and the start and stop codons are in bold letters.
Table 2C. GPCR2b Nucleotide Sequence
ATATTTTGCTTTGGCAGGAACAATTCTCTTCAΆCCCTTCCATTAΆΆAGGAATTATGATGATGGTTTTAAGGAATCTGAGCATGG AGCCCACCTTTGCCCTTTTAGGTTTCACAGATTACCCAAAGCTTCAGATTCCTCTCTTCCTTGTGTTTCTGCTCATGTATGTTA TCACAGTGGTAGGAAACCTTGGGATGATCATAATAATCAAGATTAACCCCAAATTTCACACTCCTATGTACTTTTTCCTTAGTC ACCTCTCTTTTGTTGATTTTTGTTACTCTTCCATTGTCACTCCCAAGCTGCTTGAGAACTTGGTAATGGCAGATAAAAGCATCT TCTACTTTAGCTGCATGATGCAGTACTTCCTGTCCTGCACTGCTGTGGTGACAGAGTCTTTCTTGCTGGCAGTGATGGCCTATG ACCGCTTTGTGGCCATCTGCAATCCTCTGCTTTATACAGTGGCCATGTCACAGAGGCTCTGTGCCCTGCTGGTGGCTGGGTCAT ATCTCTGGGGCATGTTTGGCCCCTTGGTACTCCTTTGTTATGCTCTCCGGTTAAACTTCTCTGGACCTAATGTAATCAACCACT TCTTTTGTGAGTATACTGCTCTCATCTCTGTGTCTGGCTCTGATATACTCATCCCCCACTTGCTGCTTTTCAGCTTCGCCACCT TCAATGAGATGTGTACACTACTGATCATCCTCACTTCCTATGTTTTCATTTTTGTGACTGTACTAAAAATCCGTTCTGTTAGTG GGCGCCACAAAGCCTTCTCCACCTGGGCCTCCCACCTGACTGCTATCACCATCTTCCATGGGACCATCCTTTTCCTTTACTGTG TACCCAACTCCAAAAACTCTCGGCAAACAGTCAAAGTGGCCTCTGTATTTTACACAGTTGTCAACCCCATGCTGAACCCTCTGA TCTACAGCCTAAGGAATAAAGACGTGAAGGATGCTTTCTGGAAGTTAATACATACACAAGTTCCATTTCACTGAACCAGTCTCA ^
The GPCR2b protein (SEO ID NO:6) encoded bv SEO ID NO:5 is 314 amino acid in length, has a molecular weight of 35806.5 Daltons, and is presented using the one-letter code in Table 2D. As with GPCR2a, the most likely cleavage site for a GPCR2b peptide is between amino acids 43 and 44, / e„ at the dash in the amino acid sequence VNG-NLG. based on the SignalP result.
Table 2D-GPCR2b protein sequence
M MVLRNLSMEPTFALLGFTDYPKLQIPLFLVFLLMYVITWGNLGMIIIIKINPKFHTPMYFFLSHLSFVDFCYSSIVTPKLL ENLV ADKSIFYFSC MQYFLSCTAVVTESFLLAVMAYDRFVAICNPLLYTVAMSQRLCALLVAGSYLWGMFGPLVLLCYALRL NFSGPNVINHFFCEYTALISVSGSDILIPHLLLFSFATFNEMCTLLIILTSYVFIFVTVLKIRSVSGRHKAFSTWASHLTAITI FHGTILFLYCVPNSKNSRQTVKVASVFYTWNPMLNPLIYSLRNKDVKDAF KLIHTQVPFH
Unless specifically addressed as GPCR2a or GPCR2b, any reference to GPCR2 is assumed to encompass all variants. Residue differences between any GPCRX variant sequences herein are written to show the residue in the "a" variant, the residue position with respect to the "a" variant, and the residue in the "b" variant. GPCRX residues in all following sequence alignments that differ between the individual GPCRX variants are highlighted in black and marked with the (o) symbol above the variant residue in all alignments herein. For example, the GPCR2 nucleic acid sequences differ at the following two positions: C648T and C922T. The GPCR2 polypeptides differ only at one residue, namely P290L.
In a BLASTN search of sequence databases, it was found, for example, that the GPCR2a nucleic acid sequence has 471 of 648 bases (72%) identical to Ratf S norvegicus taste bud receptor protein (SEO ID NO: 30). as shown in Table 2E. The BLASTN alignment shown in Table 2E result from a search utilizing the nucleotide sequence for GPCR2a. The residue that differs between GPCR2a and GPCR2b is highlighted in black and marked with the (o) symbol.
Table 2E. BLASTN of GPCR2 against rat taste bud receptor protein.
10 >gb:GENBAN -ID:RNU50948 |acc:U50948 Ra ttus norvegicus taste bud receptor protein TB
567 (TB 567) gene, complete eds - Ra ttus norvegicus, 1299 bp. (SEQ ID NO: 30) Score = 1221 (183.2 bits), Expect = 3.3e-49, P = 3.3e-49 Identities = 591/940 (62%), Positives = 591/940 (62%), Strand = Plus / Plus
15. Query: 523 TTTGTTGATTTTTGTTACTCTTCCATTGTCACTCCCAAGCTGCTTGAGAACTTGGTAATG 582 I llll I I I II I l I I II III I II II II III II III I I II I I I I II Sbjct: 2 TTTGTTGATTTCTGTTATTCCACCACAATTACACCAAAACTGCTGGAGAACTTGGTTGTG 61
Query: 583 GCAGATAAAAGCATCTTCTACTTTAGCTGCATGATGCAGTACTTCCTGTCCTGCACTGCT 642
20 I II I I II II III I I I I I MM III II I III I I llll l I
Sbjct: 62 GAAGACAGAATCATCTCCTTCACAGGATGCATCATGCAATTCTTCTTTGCCTGTATATTT 121 Query: 643 GTGGTGACAGAGTCTTTCTTGCTGGCAGTGATGGCCTATGACCGCTTTGTGGCCATCTGC 702.
25 llll I I I III I I 111 II I llll II MUM I I I II I II I I M
Sbjct: 122 GTGGTGACAGAAACATTCATGCTGGCAGCGATGGCTTATGACAGATTTGTGGCAGTGTGT 181
Query: 703 AATCCTCTGCTTTATACAGTGGCCATGTCACAGAGGCTCTGTGCCCTGCTGGTGGCTGGG 762
II II I II I I I III lllll II II III llll III I II II II I Sbjct: 182 AACCCTCTGCTTTACACAGTTGCAATGTCCCAGAGGCTTTGCTCCTTGTTAGTGGCTGCA 241
30
Query: 763 TCATATCTCTGGG-GCAT-GTTTGGCCCCTTGG-TACTCCTTTGTTATGCTCTCCGGTTA 819
M M I I I I I I I I l l l l l l I I I l l l l l I I I I I I I I I
Sbjct: 242 TCATA-CTCTTGGAGTTTAGTTTGTTCCTTAACATACACATACTTTCTGTTGACT—TTA 298
35 Query: 820 AACTTCTCT-GGACCTAATGTAATCAACCACTTCTTTTGTGAGTATACTGCTCTCATCTC 878
I I I I M l I I I I l l l l l l l l l I I I I I I I I l l l l I I I
Sbjct: 299 TCTTTTTGTAGGAC-TAACTTCATTAATAACTTTGTCTGTGAGCACGCTGCCATTGTTGC 357 o Query: 879 TGTGTCTGGCTCTGATATACT-CATCCCCCACgTGCTGCTTTTCAGCTTC-GCCACCTTC 936
40 II I II 111 I I llll II I II I I II II I Sbjct: 358 TGTGTCCTGCTCTGACCC-CTACATGAGCCAGAAGGTCATTTT-AGTTTCTGCAACATTC 415
Query: 937 AATGAGATGTGTACACTACTGATCATCCTCACTTCCTATGTTTTCATTTTTGTGACTGTA 996 , - lllll ll I I II I I II II I II II II II I I II I II I I II I I I I II I I I
- . Sbjct: 416 AATGAAATAAGCAGCCTGGTAATCATTCTCACTTCCTATGCTTTCATTTTTATCACTGTC 475
Query: 997 CTAAAAATCCGTTCTGTTAGTGGGCGCCACAAAGCCTTCTCCACCTGGGCCTCCCACCTG 1056 I II II 1 III I I I lllll II _n Sbjct: 476 ATGAAGATGCCTTCCACTGGGGGGCGCAAGAAAGCGTTCTCCACGTGTGCCTCCCACCTG 535
Query: 1057 ACTGCTATCACCATCTTCCATGGGACCATCCTTTTCCTTTACTGTGTACCCAACTCCAAA 1116
II II II I I III I I I III II III I I II II I I I II III I Sbjct: 536 ACCGCCATTACCATTTTCCATGGGACTATCCTTTTTCTCTACTGTGTTCCTAACTCCAAA 595
55. Query: 1117 AACTCTCGGCAAACAGTCAAAGTGGCCTCTGTATTTTACACAGTTGTCAACCCC 1170 I II III I II II I I lllll lllll III II II III Sbjct: 596 AGTTCATGGCTCATGGTCAAGGTGGCCTCTGTCTTTTACACAGTGGTCATTCCC 649 The GPCR2 nucleic acid sequence has homology to two regions ofthe ff m sapiens olfactory receptor ("OR5D3") gene, as shown in Table 2F. OR5D3 residues 437-644 (SEO ID NO:31) has 168 of 208 bases (80%) identical to GPCR2. with an E value of 4x10-". QR5D3 residues 121-219 (SEO ID NO:32) has 82 of 99 bases (82%) identical to GPCR2. with an E value of 6xlQ-7.
Table 2F. BLASTN of GPCR2 against the OR5D3 gene
>gb|AF065860.1 IAF065860 Homo sapiens olfactory receptor (OR5D3) gene, partial eds Length = 649; Score = 95.6 bits (48), Expect = 4e-17; Identities = 168/208 (80%); Strand = Plus / Plus
Query: 696 atcatcctcacttcctatgttttcatttttgtgactgtactaaaaatccgttctgttagt 755
II I I I I I II III MM II II II II II I I I I II II I II II I III I I Sbjct: 437 atcattctcacttcctatgctttcatttttatcactgtcatgaagatgccttccactggg 496
Query: 756 gggcgccacaaagccttctccacctgggcctcccacctgactgctatcaccatcttccat 815
MUM I lllll II I 111111111 I II I II II lllll Sbjct: 497 gggcgcaagaaagcgttctccacgtgtgcctcccacctgaccgccattaccattttccat 556
Query: 816 gggaccatccttttcctttactgtgtacccaactccaaaaactctcggcaaacagtcaaa 875
II I I I I I II I I I I II II I 111 II II llll Ml II I II 111 I Mill Sbjct: 557 gggactatcctttttctctactgtgttcctaactccaaaagttcatggctcatggtcaag 616
Query: 876 gtggcctctgtattttacacagttgtca 903
Ml Mill III lllll MM Sbjct: 617 gtggcctctgtcttttacacagtggtca 644 (SEQ ID NO: 31)
Score = 61.9 bits (31), Expect = 6e-07 Identities = 82/99 (82%) Strand = Plus / Plus
Query: 380 tgtggtgacagagtctttcttgctggcagtgatggcctatgaccgctttgtggccatctg 439
- II I I III II II I I III II II llll I MUM 1 I II Sbjct: 121 tgtggtgacagaaacattcatgctggcagcgatggcttatgacagatttgtggcagtgtg 180
Query: 440 caatcctctgctttatacagtggccatgtcacagaggct 478
II I I I I I II I I I I I III I II II III Sbjct: 181 taaccctctgctttacacagttgcaatgtcccagaggct 219 (SEQ ID NO: 32)
The full GPCR2 amino acid sequence has 159 of306 amino acid residues (51 %) identical to, and 214 of306 residues (69 %) positive with, the 314 amino acid residue proteins & m_ omo sapiens Olfactory Receptor-like protein OLF1 (ptαr: SPTREMBL-ACC: O13606) (SEO ID NO:33) (Table 2G). The residue that differs between GPCR2a and GPCR2b is highlighted in black and marked with the (o) symbol.
Table 2G. BLASTX of GPCR2 against OR-like Protein OLF1
>ptnr:SWISSPROT-ACC:Q13606 OLFACTORY RECEPTOR-LIKE PROTEIN 0LF1 - Homo sapiens
(Human), 314 aa. (SEQ ID NO:33)
Score = 814 (286.5 bits). Expect = 1.6e-80, P = 1.6e-80
Identities = 159/306 (51%), Positives = 214/306 (69%), Frame = +1
Query: 6 RNLSMEPTFALLGFTDYPKLQIPLFLVFLLMYVITWGNLGMIIIIKINPKFHTPMYFFL 65
II ++ l llll 1 + 1 II lll + l.l +1 I ++II + I++++I + I + I Sbjct: 6 RNYTLVTEFILLGFPTRPELQIVLFLMFLTLYAIILIGNIGLMLLIRIDPHLQTP YFFL 65
Query: 66 SHLSFVDFCYSSIVTPKLLENLVMADKSIFYFSCMMQYFLSCTAWTESFLLAVMAYDRF 125 l + l 111 I II I + I l + l I + +111 1+ I +I++ II III l + ll I II 11 + Sbjct: 66 SNLSFVDLCYFSDIVPKMLVNFLSENKSISYYGCALQFYFFCTFADTESFILAAAYDRY 125
Query: 126 VAICNPLLYTVAMSQRLCALLVAGSYLWGMFGPLVLLCYALRLNFSGPNVINHFFCEYTA 185
I II II I I I I I I 11+ +1 1+ lll l II +1 I + II I I II 11 + Sbjct: 126 VAICNPLLYTWMSRGICMRLIVLSYLGGNMSSLVHTSFAFILKYCDKNVINHFFCDLPP 185
Query: 186 LISVSGSDILIPHLLLFSFATFNEMCTLLIILTSYVFIFVTVLKIRSVSGRHKAFST AS 245
1+ +1 +1 I I I ++ + 1+ +11+ II II ++I1I I II 111 I II I II Sbjct: 186 LLKLSCTDTTINE LLSTYGSSVEIICFIIIIISYFFILLSVLKIRSFSGRK TFSTCAS 245 o Query: 246 HLTAITIFHGTILFLYCVPNSKNSRQTVKVASVFYTWNPMLNP@IYSLRNKDVKDAF K 305
II I++I 1+ I l + M + l 1+ I I 1+ llll 1+ l + ll I I I III I I 111 I I 1 Sbjct: 246 HLTSVTIYQGTLLFIYSRPSYLYSPNTDKIISVFYTIFIPVLNPLIYSLRNKDVKDAAE 305
Query: 306 LIHTQV 311
++ ++| Sbjct: 306 VLRS V 311
The full amino acid sequence ofthe GPCR2 protein ofthe invention has 152 of 301 amino acid residues (50%) identical to, and 207 of 301 residues (68%) positive with, the 312 amino acid residue proteins from Gallus gallus olfactory receptor 4 (ptnr: SPTREMBL-ACC: CAA64370.1) (SEO ID NO:34) (Table 2H). The residue that differs between GPCR2a and GPCR2b is highlighted in black and marked with the (o) symbol.
Table 2H. BLASTX of GPCR2 against OR-4
>em I CAA64370.il (X94744) olfactory receptor 4 [ Gallus gallus] ; (SEQ ID NO:34), 312 aa
Statistics for GPCR2a: Score = 304 bits (779), Expect = 8e-82
Identities = 152/301 (50%), Positives = 207/301 (68%) Statistics for GPCR2b: Score = 320 bits (821), Expect = le-86
Identities = 160/306 (52%), Positives = 215/306 (69%)
Query: 7 NLSMEPTFALLGFTDYPKLQIPLFLVFLLMYVITWGNLGMIIIIKINPKFHTPMYFFLS 66
I ++ I l + l +1 + 1 I++ M + IIM + M II III I + II + I+ +1+ II llllll Sbjct: 5 NHTLASEFILVGLSDHPKMKAALFWFLLIYVITFQGNLGIIILIQGDPRLHTS YFFLS 64
Query: 67 HLSFVDFCYSSIVTPKLLENLVMADKSIFYFSCMMQYFLSCTAWTESFLLAV AYDRFV 126
I I I I l + l I ++ 1 + I I + ++ I + I I I I I I I I I I I I I I I l + l
Sbj ct : 65 SLSWDICFSSVIAPRTLVNFLSERRTISFTGCTGQTFFYIVFVTTECFLLAVMAYDRYV 124
Query: 127 AICNPLLYTVAMSQRLCALLVAGSYL G FGPLVLLCYALRLNFSGPNVINHFFCEYTAL 186 llll I I I 1+ I++I I II I 11+ 1+ ++ + +11 I I l + l I I 111+ 1 Sbjct: 125 AICNPLLYSTI TRRQC QLVVGSYIGGILNAIIQTTFIIRLPFCGSNIINHFFCDVPPL 184
Query: 187 ISVSGSDILIPHLLLFSFATFNEMCTLLIILTSYVFIFVTVLKIRSVSGRHKAFSTWASH 246
+++I + I ++I II I 1+ 1+ II I l + ll +I + M I I I II I I III
Sbj ct : 185 LALSLASTYISEMILFSLAGIIELSTVTSILVSYIFISCAILRIRSAEGRQKALSTCASH 244 o Query : 247 LTAITIFHGTILFLYCVPNSKNSRQTVKVASVFYTWNPMLNPglYSLRNKDVKDAFWKL 306
II 1 + 1+ +11 +1 I l + l I I II lllll I I III I++I I I ++
Sbjct: 245 LTAVTLLYGTTIFTYLRPSSSYSLNTDKVVSVFYTWIPMLNPLIYSLRNQEVKGALSRV 304
Query: 307 I 307
+ Sbjct: 305 V 305
A multiple sequence alignment is given in Table 21, with the GPCR2 protein ofthe invention being shown on line 1. in a ClustalW analysis comparing GPCR2 with related protein sequences. Table 2I.-Information for the ClustalW proteins:
1. Novel_Human_OLF, i.e. GPCR2, SEQ ID NO: 4
2. Homo sapiens OLF, ptnr-SWISSPROT Ace # Q13606, SEQ ID NO: 33
3. Gallus gallus OLF, ptnr-SWISSPROT Ace # P37070, SEQ ID NO: 35
4. Rattus norvegicus OLF, Ace # Q63395, SEQ ID NO: 36
Figure imgf000021_0001
GPCR2 gg^F^^^itdA^' ii-^AlCTiloigLBaLiTOa^g-WLWB BGPSELLCYSiRBl-Jlgs GPlitf-HMitatij HUMANJDLF CHICK_OLF4 ^^^^^ K^^PSQrKAVg R[jQKgL{3sE3AFLNg^^gGL{3K^^Sg vJ30S3 RAT_OLF ^3^^ER[3353N33ϊEs|33gS J33L A SgEL[^AVgJΪEAMMN[ESEκgYI0SgjYS
Figure imgf000021_0002
DOMAIN results for GPCR2 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 2J with the statistics and domain description. The 7tm 1. a seven transmembrane receptor (rhodopsin family), was shown to have two segments with significant homology to GPCR2. An alignment of GPCR2 with residues 1-170 (SEO ID NO:29) and residues 310-377 (SEO ID NO:37) of 7tm 1 are shown in Table 2J.
Table 2 J.-DOMAIN results for GPCR2 gnl I Pfa lpfamOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family) (SEQ ID Nθ:29)
Length = 377; Score = 83.2 bits (204), Expect = 2e-17 Query: 43 GNLGMIIIIKINP FHTPMYFFLSHLSFVDFCYSSIVTPKLLΞNLVMADKSIFYFSCMMQ 102 Sbjct: 1 GNVLVC AVSREKALQTTTNYLIVSLAVADLLVATLVMP WYLEWGEWKFSRIHCDIF 60 **. . . . * . . * . * ...* * .. *. . * .
Query : 103 YFLSCTAWTESFLLAVMAYDRFVAICNPLLYTVA SQRLCALLVAGSYLWG FGPLVLL 162 Sbj ct : 61 VTLDVMMCTASILNLCAISIDRYTAVAMP LYNTRYSSKRRVTVM IAI 108
Query : 163 CYALRLNFSGPNVINHFFCEYTALISVSGSDILIPHLLLFSFATFNEMCTLLIILTSYVF 222 Sbj ct : 109 V VLSFTISCP LF GLNNTDQNECIIANPAFWYSSIVSF-YVPFIVTLLVYI 161 Query: 223 IFVTVLKIR 231 Sbjct: 162 IYIVLRRRR 170 *: : : : * gnl I PfamlpfamOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family) (SEQ ID
NO:37)
Length = 377
Score = 35.8 bits (81), Expect = 0.003 Query: 226 TVLKIRSVSGRHKAFST ASHLTAITIFHGT-ILFLYCVPNSKNSRQTVKVASVFYTWN 284 Sbjct: 310 SRRKLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYSAFTWLGYVN 369
: *: : * : * : : * . * . ** o Query: 285 PMLNPJJlY 292 Sbjct: 370 SAVNPIIY 377
The nucleic acids and proteins of GPCR2 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further below. For example, a cDNA encoding the 0ifactory receptor-like protein may be useful in gene therapy, and the 0ifactory receptor"u^e protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Other GPCR-1 diseases and disorders are contemplated.
The novel nucleic acid encoding GPCR2, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. This novel protein also has immense value in development of powerful assay system for functional analysis.
GPCR3
An additional GPCR-like protein ofthe invention, referred to herein as GPCR3, is an Olfactory Receptor ("OR")-like protein. The GPCR3 nucleic acid of 1001 nucleotides (also designated APOOl 112 A) is shown in Table 3 A. An ORF was identified beginning with an ATG initiation codon at nucleotides 12-14 and ending with a TAA codon at nucleotides 945- 47. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 3 A. and the start and stop codons are in bold letters. Table 3A. GPCR3 Nucleotide Sequence (SEQ ID NO:7)
CGAAGGAAATTATGAGAAGAAACTGCACGTTGGTGACTGAGTTCATTCTCCTGGGACTGACCAGTCGCCGGGAATTACAAATTC TCCTCTTCACGCTGTTTCTGGCCATTTACATGGTCACGGTGGCAGGGAACCTTGGCATGATTGTCCTCATCCAGGCCAACGCCT GGCTCCACATGCCCATGTACTTTTTCCTGAGCCACTTATCCTTCGTGGATCTGTGCTTCTCTTCCAATGTGACTCCAAAGATGC TGGAGATTTTCCTTTCAGAGAAGAAAAGCATTTCCTATCCTGCCTGTCTTGTGCAGTGTTACCTTTTTATCGCCTTGGTCCATG TTGAGATCTACATCCTGGCTGTGATGGCCTTTGACCGGTACATGGCCATCTGCAACCCTCTGCTTTATGGCAGCAGAATGTCCA AGAGTGTGTGCTCCTTCCTCATCACGGTGCCTTATGTGTATGGAGCGCTCACTGGCCTGATGGAGACCATGTGGACCTACAACC TAGCCTTCTGTGGCCCCAATGAAATTAATCACTTCTACTGTGCGGACCCACCACTGATTAAGCTGGCTTGTTCTGACACCTACA ACAAGGAGTTGTCAATGTTTATTGTGGCTGGCTGGAACCTTTCTTTTTCTCTCTTCATCATATGTATTTCCTACCTTTACATTT TCCCTGCTATTTTAAAGATTCGCTCTACAGAGGGCAGGCAAAAAGCTTTTTCTACCTGTGGCTCCCATCTGACAGCTGTCACTA TATTCTATGCAACCCTTTTCTTCATGTATCTCAGACCCCCCTCAAAGGAATCTGTTGAACAGGGTAAAATGGTAGCTGTATTTT ATACCACAGTAATCCCTATGCTGAACCTTATAATTTATAGCCTTAGAAATAAAAATGTAAAAGAAGCATTAATCAAAGAGCTGT CAATGAAGATATACTTTTCTTAAAAATCAGTATTCTTTTGGTTTCTAAAGCCCTTCCTAGACTTTTTTCTTTAGCTG The GPCR3 polypeptide (SEO ID NO:8) encoded by SEO ID NO:7 is 311 amino acid residues and is presented using the one-letter code in Table 3B.
Table 3B. Encoded GPCR3 protein sequence (SEQ ID NO:8).
MRRNCTLVTEFILLGLTSRRELQILLFTLFLAIYMVTVAGNLGMIVLIQANAWLHMPMYFFLSHLSFVDLCFSSNVTPKMLEIF LSEK SISYPACLVQCYLFIALVHVEIYILAVMAFDRY AICNPLLYGSRMSKSVCSFLITVPYVYGALTGLMETM TYNLAFC GPNEINHFYCADPPLIKLACSDTYNKELSMFIVAGWNLSFSLFIICISYLYIFPAILKIRSTEGRQKAFSTCGSHLTAVTIFYA TLFFMYLRPPSKESVEQGKMVAVFYTTVIP LNLIIYSLRNKNVKEALIKELSMKIYFS
In a search of sequence databases, it was found, for example, that the GPCR3 nucleic acid sequence has 609 of 923 bases (65%) identical to a and 609/923 bases (65%) positive with pan troglodytes species Olfactory Receptor OR93 gene (SEO ID NO:38), as shown in Table 3C.
Table 3C. BLASTN of GPCR3 against Chimpanzee OR93 gene
>gb:GENBANK-ID:AF045577 |acc:AF045577 Pan troglodytes olfactory receptor OR93Ch
(0R93) gene, complete eds - Pan troglodytes, (SEQ ID Nθ:38), 989 bp Score = 1405 (210.8 bits), Expect = 2.1e-57, P = 2. le-57 Identities = 609/923 (65%), Positives = 609/923 (65%), Strand = Plus / Plus
Query: 20 AAACTGCACGTTGGTGACTGAGTTCATTCTCCTGGGACTGACC-AGTCGCCGGGAATTAC 78
I I II I I II II I II I I I II I II II II III I II l ll l Sbjct: 12 AAACTACACAAAGGTCACCGAATTCATTTTCACAGGCTTGAATTACAATCCTC-AGTTGC 70
Query: 79 AAATTCTCCTCTTCACGCTGTTTCTGGCCATTTAC-ATGGTCA-CGGTGGCAG-GGAACC 135
I I I I I M i l l I I I I I I M I I I I I I I I I I I I I I I l l l l l
Sbjct: 71 AGGTCTTCCTCTTCCTACTCTTTCTGACAACTTTCTATG-TCATCAATGTAACTGGAAAC 129
Query: 136 TTGGCA-TGATTGTCCTCATCCAGGCCAACGCCTGGCTCCACATGCCCATGTACTTTTTC 194
I I I I I I II II I M I I II II I I I I I II I M I II I 111 I I II II Ml Sbjct: 130 TTGGGAATGATTGTCCTTATCCGAATCGATTCCCGCCTTCACACACCCATGTACTTTTTC 189
Query: 195 CTGAGCCACTTATCCTTCGTGGATCTGTGCTTCTCTTCCAATGTGACTCCAAAGATGCTG 254
I I I I I I II I II I II II I II I llll I I I I I I lllll II I I Sbjct: 190 CTCAGCCACCTGTCCTTTGTGGACATCTGCTTCTCCTCAGTTGTGAGCCCCAAGATGCTC 249
Query: 255 GA-GATTTTCCTTTCAGAGAAGAAAAGC-ATTTCCTATCCT-GCCTGT-CTT-GT-GCAG 308
I I II Ml I II II I I I I I I II I I I II I I I IM I II I I I I II Sbjct: 250 ACTGACTT—CTTTGTGAAGAGGAAAGCCATTTCTT-TCCTTGGCTGTGCTTTGCAGCAG 306
Query: 309 TGTTACCTTTTTATCGCCTTGGTCCATGTTGAGATCTACATCCTGGCTGTG-ATGGCCTT 367 ll l l lll I l lll II I III I I I I III I II Sbjct: 307 TGGTTC-TTTGGGTT-CTTTGTGGCA-GCAGAGTGTTTCCTCTTGGC-GTCCATGGCCTA 362
Query: 368 TGACCGGTACATGGCCATCTGCAACCCTCTGCTTTATGGCAGCAGA-ATGTCCAAGAGTG 426 I II M I M II II III ll l ll III I I Ml I I III I Sbjct: 363 TGACCGCTATGTGGCCATCTGTAACCCATTGTTATACT-CAGTTGCTATGTCCCAGAGGC 421
Query: 427 TGTGC-TCCTTCCTCATCACGGTGCCTTATGTGTATGGAGCGC-TCACTGGCCTGATGGA 484 l lll lll I I I II ll lllll I III I I I I I I I II I Sbjct: 422 TCTGCATCCAGC-TAGTGGTGGGT'CCCTATGTCATTGGA-CTCATGAATACCATGACTCA 479
Query: 485 GAC—CATGTGGACCTACAACCTAGCCTTCTGTGGCCCCAATGAAATTAATCACTTCTAC 542
II II II I I I I I I II II I II I II II I I I I I II I I I I II I Sbjct: 480 CACAACAAATGCATTT-CGTC-TCCCTTTTTGTGGCCCTAATGTCATCAATCATTTCTTC 537
Query: 543 TGTGCGGACCCACCACTGA-TTAAGCTGGCTTGTTCTGACACCTACAACAAGGAGTTGTC 601
MM I II II I II II I III I II I I II III I II II I Sbjct: 538 TGTGATATGTCCCC-CTTACTTTCCCTTGTATGTGCTGATACCAGGCTCAATAAGTTGGC 596
Query: 602 AATGTTTATTGTGGCTGG—CTG-GAACCT-TTCTTTT-TCTCTCTTCATCATATGTATT 656
I I II II lll l I III I III II lll l l lll
Sbjct: 597 AGTTTTCATCGTGGCTGGAGCTGCGGGAGTCTTCAGTGGTCTGACT—ATCCT—G-ATT 651
Query: 657 TCCTACCTTTACATTTTCCCTGCTATTTTAAAGATTCGCTCTACAGAGGGCAGGCAAAAA 716
III I II II II II 1 II ll ll I I I I I II II II I II I I II I III Sbjct: 652 TCCTACATTTACATCCTCATGGCCATCCTGAGGATCCGCTCTGCTGATGGGAGGTGCAAA 711
Query: 717 GCTTTTTCTACCTGTGGCTCCCATCTGACAGCTGTCACTATATTCTATGCAACCCTTTTC 776
I II II II MIMMIMI II II MM IMMllll Sbjct: 712 ACCTTTTCTACTTGCTCTTCTCACCTGACAGCTGTTTTCATCTTGTATGGTACCCTTTTC 771
Query: 777 TTCATGTATCTCAGACCCCCCTCAAAGGAATCTGTTGAACAGGGTAAAATGGTAGCTGTA 836 ll ll lll l I II 111 II l ll l llll l ll llll Sbjct: 772 TTTATTTATGTACGTCCTAGTGCAAGCTTCTCCCTGGATCTCAATAAATTAGTGTCTGTG 831
Query: 837 TTTTATACCACAGTAATCCCTATGCTGAACCTTATAATTTATAGCCTTAGAAATAAAAAT 896 lllll ll llll II llll jl I II II III I II III II I Sbjct: 832 TTTTACACAGCAGTGATTCCTATGTTGAACCCACTTATCTACAGCTTGAGAAACAAGGAA 891
Query: 897 GTAAAAGAAGC-ATTAATCAAAGAGCTGTCAATGAAGATATACTTTT 942
I I I I III I I II I II II I MM I I I I I l llll Sbjct: 892 GTCAAAGATGCCATCCA-CAG-GA-CTGTCACTCA-GAGGAAGTTTT 934
The BLASTN alignment shown in Table 3D indicates that two fragments of GPCR3 have homology to fragments of Mus musculus olfactory receptor 4 cluster, gene 3 ("Qlfr4-3") (GENBANK-ID: NM 013728.1). Residues 827-907 (SEO ID NO:39) and residues 163-210 (SEQ ID NO-.40) ofthe O1&4-3 gene are shown below.
Table 3DJBLASTN of GPCR3 against Olfr4-3
>ref |NM_013728.1 | Mus musculus olfactory receptor 4 cluster, gene 3 (01fr4-3), mRNA Length = 957 (SEQ ID NO: 39) Score = 58.0 bits (29), Expect = 9e-06 Identities = 68/81 (83%) Strand = Plus / Plus
Query: 835 tattttataccacagtaatccctatgctgaaccttataatttatagccttagaaataaaa 894
I II II I I II II II II I I II II I II I I I11 II II I I I Sbjct: 827 tattttataccactgttatccctatgttgaacccattcatttatagcctgagaaataagg 886 Query: 895 atgtaaaagaagcattaatca 915 I II I II I I II 1 I II llll
Sbjct: 887 aagtcaaagatgcattaatca 907
>ref |NM_013728.11 Mus musculus olfactory receptor 4 cluster, gene 3 (01fr4-3) , mRNA Length = 957 (SEQ ID NO: 40) Score = 56.0 bits (28), Expect = 4e-05 Identities = 43/48 (89%) Strand = Plus / Plus Query: 171 ctccacatgcccatgtactttttcctgagccacttatccttcgtggat 218 llllllll Ml II II I Ml II I II llllllll lllll llllll Sbjct: 163 ctccacatccccatgtacttttttctcagccacttgtcctttgtggat 210
The full GPCR3 amino acid sequence has 166 of 305 amino acid residues (54%) identical to, and 214 of 305 residues (70%) positive with, the 314 amino acid residue OR93CH protein from Pan troglodytes føtnr: SPTREMBL-ACC: 077756) (SEO ID NO:41) (Table 3E).
Table 3E. BLASTX of GPCR3 against OR93CH - (SEQ ID NO:41)
>SPTRE BL-ACC:077756 OLFACTORY RECEPTOR 0R93CH - Pan' troglodytes (Chimpanzee), 314
10 aa.
Score = 832 (292.9 bits), Expect = 3.6e-82, P = 3.6e-82
Identities = 166/305 (54%), Positives = 214/305 (70%), Frame = +3
Query: 4 NCTLVTEFILLGLTSRRELQILLFTLFLA-IYMVTVAGNLGMIVLIQANA LHMP YFFL 62
15 l l lllll II +11+ II 1 II I++ I I II II II I 1+ ++ II I lllll Sbjct: 5 NYTKVTEFIFTGLNYNPQLQVFLFLLFLTTFYVINVTGNLGMIVLIRIDSRLHTPMYFFL 64
Query: 63 SHLSFVDLCFSSNVTPKMLEIFLSEKKSISYPACLVQCYLFIALVHVEIYILAVMAFDRY 122
20 III I I I l + l I I I l + ll I I I ++I + I 1+ I +1 + I I I ++I I I l + lll
Sbjct: 65 SHLSFVDICFSSVVSPKMLTDFFVKRKAISFLGCALQQ FFGFFVAAECFLLASMAYDRY 124
Query: 123 MAICNPLLYGSRMSKSVCSFLITVPYVYGALTGLMETMWTYNLAFCGPNEINHFYCADPP 182
+ 111 I I I II 11+ +1 1+ III I + + I + I III II II I l + l I Sbjct: 125 VAICNPLLYSVA SQRLCIQLWGPYVIGL NTMTHTTNAFRLPFCGPNVINHFFCD SP 184
25
Query: 183 LIKLACSDTYNKELS FIVAG NLSFSLFIICISYLYIFPAILKIRSTEGRQKAFSTCGS 242
1+ I l + ll +I++I Ml I II I I 11 + 11 I ll + l II +11 1 II II 1 Sbjct: 185 LLSLVCADTRLNKLAVFIVAGAAGVFSGLTILISYIYIL AILRIRSADGRCKTFSTCSS 244
30 Query: 243 HLTAVTIFYATLFFMYLRPPSKESVEQGKMVAVFYTTVIPMLNLIIYSLRNKNVKEALIK 302 lllll I I II ll + l + ll + I++ l + l + ll II +11 II I II 11 + 1+ + Sbjct: 245 HLTAVFILYGTLFFIYVRPSASFSLDLNKLVSVFYTAVIPMLNPLIYSLRNKEVKDAIHR 304
Query: 303 ELSMK 307
35 ++ + Sbjct: 305 TVTQR 309
The full amino acid sequence ofthe GPCR3 protein was also found to have 166 of 311 amino acid residues (53%) identical to, and 215 of 311 residues (68%) positive with, the 311 40 amino acid residue proteins from j,fl{S m s us olfactory receptor 4 cluster, gene 3 (SEO ID NO:42), shown in Table 3F.
Table 3F.-BLASTX of GPCR3 against mouse OR4 cluster, gene 3
>ref ]NP__038756.11 olfactory receptor 4 cluster, gene 3 [Mus musculus], derived from gb|AAF20365.1|AF146372_l (AF146372) olfactory receptor 0R912-93 [Mus musculus
45 domesticus] (SEQ ID Nθ:42). Length = 318; Score = 321 bits (823), Expect = 6e-87 Identities = 166/311 (53%), Positives = 215/311 (68%)
Query: 1 MRRNCTLVTEFILLGLTSRRELQILLFTLFLAIY VTVAGNLG IVLIQANA LHMPMYF 60
50 I I I l + ll I I llll lll + ll II +1+ 1+ II II I l + ll + 11 + 1111 Sbjct: 2 MHRNQTWTEFFFTGLTSSFHLQIVLFLTFLCVYLATLLGNLG IILIHLDTRLHIPMYF 61
Query: 61 FLSHLSFVDLCFSSNVTPKMLEIFLSEKKSISYPACLVQCYLFIALVHVEIYILAVMAFD 120
« 111 II llll I M ++MI I +1 I I 1+ l +l II I I ++I I ll + l
^2. Sbjct: 62 FLSHLSFVDACSSSVISPKMLSDMFVDKKVISFLGCAIQLCLFSQFWTECFLLASMAYD 121
Query: 121 RYMAICNPLLYGSRMS SVCSFLITVPYVYGALTGLMETMWTYNLAFCGPNEINHFYCAD 180 ll + l I I I I I I 11+ II 1+ II 1 ++ ++ + + I +11 II II ll + l Sbjct: 122 RYVAICKPLLYTLIMSQRVCVQLVIGPYSIGFISTMVHIISAFVLPYCGPNLINHFFCDL 181
Query: 181 PPLIKLACSDTYNKELS FIVAGWNLSFSLFIICISYLYIFPAILKIRSTEGRQKAFSTC 240
I++ II I++I + +1 llll II II +11 + 11 llll I +1 l + l I 1 III Sbjct: 182 LPVLSLACANTQ NKRLLFIVAGILGVFSGIIILVSYVYIAITILKISSADGRRKAFSTC 241
Query: 241 GSHLTAVTIFYATLFFMYLRPPSKESVEQGKMVAVFYTTVIPMLNLIIYSLRNKNVKEAL 300
Mill l + l I II ll + l + ll I I++ I + I++II I I I II IM I l + ll Sbjct: 242 SSHLTAVSILYGTLFFIYVRPSSSFSLDINKWSLFYTTVIPMLNPFIYSLRNKEVKDAL 301
Query: 301 IKELSMKIYFS 311
1+ + +1 Sbjct: 302 IRTFEKQFCYS 312
A multiple sequence alignment is given in Table 3G, with GPCR3 being shown on line 4, in a ClustalW analysis comparing GPCR3 with related protein sequences.
Table 3G. Information for the ClustalW proteins:
1 . Hyloba tes lar (Common Gibbon) OLF, SPTREMBL -Ace # 077758 , SEQ ID NO : 43
2 . Pan troglodytes (Chimpanzee ) OLF, SPTREMBL-Acc # 077756, SEQ ID NO : 41
3 . Mus musculus OLF, GENBANK-Acc # AAF20365 , SEQ ID NO : 42
4 . Novel HumanjDLF, GPCR3 , SEQ ID NO : 8
Figure imgf000026_0001
GIBBON_OLF ftrø HWJW^^^
CHIMPANZEE_0LF BωaMMiMdbtf.ιMd.^M^^^
MOUSE_OLF røfataijg^s gAgsi^iigaOTsiBMi^^
GPCR3 tf -fet.tfctmaLHg>ϊlN-aτ[3 iEiraLSEκigsiigγp 5lLvicYLi3iALfflHVl-liYii-^ira
GIEBON_OLF JJi.MIWt.hhttMl...^^
CHIMPANZEE_0LF MMM«MM g^^)^^
MOUSE_OLF ^ i v . ^KB-ππTLiiπas^vHvEϊgEii-ϊπs^Fis^i iiis^ gE ^^L^i^i^
GPCR3 πaS^CTPiitdit-WGSRlBigκSv5sFffllTVtgMY^LTGLMElHlMWTYNllΑKrirf ilEiiSt3gγa
Figure imgf000026_0002
DOMAIN results for GPCR3 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 3H with the statistics and domain description.
Table 3H.-DOMAIN results for GPCR3. gnl I Pfaml famOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family) (SEQ ID Nθ:29) Length = 377
Score = 92.8 bits (229), Expect = 2e-20
Query: 40 GNLGMIVLIQANA LHMP YFFLSHLSFVDLCFSSNVTPKMLEIFLSEKKSISYPACLVQ 99 Sbjct: 1 GNVLVCMAVSRE ALQTTTNYLIVSLAVADLLVATLVMPWWYLEWGE KFSRIHCDIF 60
Query: 100 CYLFIALVHVEIYILAVMAFDRYMAICNPLLYGSRM-SKSVCSFLITVPYVYGALTGLME 158
Sbjct: 61 VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPM 120
* . . * * .. *** *. *.** :* ** : :* : :*
Query: 159 T WTYNLAFCGPNEINHFYCADPPLIKLACSDTYNKELSMFIVAGWNLSFSLFIICISYL 218
Sbjct: 121 LFGLNNTDQNE CIIANPAFWYSSIVS—FYVPFIVTLLVYI 160
* * : : : * :
Query: 219 YIFPAILKIRSTEGRQK 235 Sbjct: 161 KIYIVLRRRRKRVNTKR 177 * . . . * The nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in various in various GPCR-related pathological disorders and/or OR- related pathological disorders, described further below. For example, a cDNA encoding the olfactory receptor -like protein may be useful in gene therapy, and the olfactory receptor =Hke protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma. prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Other GPCR-related diseases and disorders are contemplated.
The novel nucleic acid encoding olfactory receptor 'l ke protein, and the olfactory receptor -uke protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. This novel protein also has immense value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders
GPCR4
GPCR4 is an Olfactory Receptor ("OR")-like protein, wherein two alternative novel GPCR4 nucleic acids and encoded polypeptides are disclosed.
The novel GPCR4a nucleic acid of 980 nucleotides (also referred to as AP001112 B) is shown in Table 4A. An ORF begins with an ATG initiation codon at nucleotides 19-21 and ends with a TAA codon at nucleotides 940-42. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 4A, and the start and stop codons are in bold letters.
Table 4A. GPCR4a Nucleotide Sequence (SEQ ID NO:9)
TGACTTAGAATTCAGAAAATGCTCAATTTCACCGATGTGACAGAGTTCATTCTTTTGGGGCTAACGAGCCGTCGAGAATGGCAA GTTCTCTTCTTCATCATCTTTCTTGTGGTCTACATCATCACCATGGTGGGCAATATCGGCATGATGGTGTTAATCAAGGTCAGT CCTCAGCTTAACAACCCCATGTACTTTTTCCTCAGTCACTTGTCATTTGTTGATGTGTGGTTTTCTTCCAATGTCACCCCTAAA ATGTTGGAAAACCTGTTTTCAGATAAAAAAACAATTACTTATGCTGGTTGTTTAGTACAGTGTTTCTTCTTCATTGCTCTTGTC CATGTGGAAATTTTTATTCTTGCTGCGATGGCCTTTGATAGATACATGGCAATTGGGAATCCTCTGCTTTATGGCAGTAAAATG TCAAGGGTTGTCTGTATTCGACTGATTACTTTCCCTTACATTTATGGTTTTCTGACGAGTCTGGCAGCAACATTATGGACTTAC GGCTTGTACTTCTGTGGAAAAATTGAGATCAACCATTTCTACTGTGCAGATCCACCTCTCATCAAAATGGCCTGTGCCGGGACC TTTGTAAAAGAATATACAATGATCATACTTGCCGGCATTAACTTCACATATTCCCTGACTGTAATTATCATCTCTTACTTATTC ATCCTCATTGCCATTCTGCGAATGCGCTCAGCAGAAGGAAGGCAGAAGGCCTTTTCCACATGTGGGTCCCATCTGACAGCTGTC ATTATATTCTATGGTACTCTGATCTTCATGTATCTCAGACGTCCCACAGAGGAGTCTGTGGAGCAGGGGAAGATGGTGGCTGTG TTCTATACCACAGTGATCCCCATGTTGAATCCCATGATCTACAGTCTGAGGAACAAGGATGTGAAAAAGGCCATGATGAAAGTG ATCAGCAGATCATGTTAAACAAAATAAAATCAAATTTGATTTAATTTTATCTTCTA
The GPCR4a protein encoded by SEO ID NO: 9 has 307 amino acid residues and is presented using the one-letter code in Table 4B. The Psort profile for GPCR4 predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The most likely cleavage site for a peptide is between amino acids 39 and 40, / e.. at the dash in the amino acid sequence MNG-NIG, based on the SignalP result.
Table 4B. Encoded GPCR4a protein sequence (SEQ ID NO:10).
MLNFTDVTEFILLGLTSRREWQVLFFIIFLWYIIT VGNIGMMVLIKVSPQLNNPMYFFLSHLSFVDVWFSSNVTPK LENLF SDKKTITYAGCLVQCFFFIALVHVEIFILAAMAFDRYMAIGNPLLYGSKMSRWCIRLITFPYIYGFLTSLAATL TYGLYFCG KIEINHFYCADPPLIK ACAGTFVKEYTMIILAGINFTYSLTVIIISYLFILIAILRMRSAEGRQ AFSTCGSHLTAVIIFYGT LIFMYLRRPTEESVEQGK VAVFYTTVIPMLNPMIYSLRNKDVKKA MKVISRSC
The target GPCR4a sequence was subjected an the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy. The 980 nucleotides of GPCR4b (also referred to as AC020597A) are provided in Table 4C. The resulting GPCR4b nucleotide sequence differs from that of GPCR4a at nine positions, namely A75G, A100G. C102T. C264T. T270A. C582T. A610C, T627C and T759C.
Table 4C.-GPCR4b Nucleotide Sequence (SEQ ID NO:ll)
TGACTTAGAATTCAGAAAATGCTCAATTTCACCGATGTGACAGAGTTCATTCTTTTGGGGCTAACGAGCCGTCGGGAATGGCAA GTTCTCTTCTTCATCGTTTTTCTTGTGGTCTACATCATCACCATGGTGGGCAATATCGGCATGATGTTGTTAATCAAGGTCAGT CCTCAGCTTAACAACCCCATGTACTTTTTCCTCAGTCACTTGTCATTTGTTGATGTGTGGTTTTCTTCCAATGTCACCCCTAAA ATGTTGGAAAATCTGTTATCAGATAAAAAAACAATTACTTATGCTGGTTGTTTAGTACAGTGTTTCTTCTTCATTGCTCTTGTC CATGTGGAAATTTTTATTCTTGCTGCGATGGCCTTTGATAGATACATGGCAATTGGGAATCCTCTGCTTTATGGCAGTAAAATG TCAAGGGTTGTCTGTATTCGACTGATTACTTTCCCTTACATTTATGGTTTTCTGACGAGTCTGGCAGCAACATTATGGACTTAC GGCTTGTACTTCTGTGGAAAAATTGAGATCAACCATTTCTACTGTGCAGATCCACCTCTCATCAAAATGGCCTGTGCTGGGACC TTTGTAAAAGAATATACAATGCTCATACTTGCCGGCATCAACTTCACATATTCCCTGACTGTAATTATCATCTCTTACTTATTC ATCCTCATTGCCATTCTGCGAATGCGCTCAGCAGAAGGAAGGCAGAAGGCCTTTTCCACATGTGGGTCCCATCTGACAGCTGTC ATCATATTCTATGGTACTCTGATCTTCATGTATCTCAGACGTCCCACAGAGGAGTCTGTGGAGCAGGGGAAGATGGTGGCTGTG TTCTATACCACAGTGATCCCCATGTTGAATCCCATGATCTACAGTCTGAGGAACAAGGATGTGAAAAAGGCCATGATGAAAGTG ATCAGCAGATCATGTTAAACAAAATAAAATCAAATTTGATTTAATTTTATCTTCTA
10 The GPCR4b protein encoded by SEO ID NO:l 1 has 314 amino acid residues and a molecular weight of 35155.8 Daltons, as presented using the one-letter code in Table 4D. GPCR4a differs from GPCR4b at four residues, namely I28N. N45L. F84L and I198L. The signal peptide and Psort analyses for both GPCR4 variants are the same.
Table 4D.-Encoded GPCR4b protein sequence (SEQ ID ΝO:12)
15 MLNFTDVTEFILLGLTSRRE QVLFFIVFLVVYIIT VGNIGMMLLIKVSPQLNNPMYFFLSHLSFVDV FSSNVTPKiylLENLL SDKKTITYAGCLVQCFFFIALVHVEIFILAAMAFDRYMAIGNPLLYGSKMSRVVCIRLITFPYIYGFLTSLAATLWTYGLYFCG KIEINHFYCADPPLIKMACAGTFVKEYTMLILAGINFTYSLTVIIISYLFILIAILRMRSAEGRQKAFSTCGSHLTAVIIFYGT LIF YLRRPTEESVEQGKMVAVFYTTVIP LNPMIYSLRNKDV KAMMKVISRSC
20 Unless specifically addressing GPCR4a or 4b, assume any reference to GPCR4 to encompass all variants. hi a search of sequence databases, it was found, for example, that the nucleic acid sequence for GPCR4ahas 591 of 940 bases (62%) identical to and 591 of 940 bases (62%) positive with Rgttus norvegicus species taste bud Receptor clone (GENBANK-ID: U50948) 25 (SEO ID NO:44) (Table 4E). The residues that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
Table 4E. BLASTN of GPCR4a against rat TB567 gene
>gb:GENBANK-ID:RNU50948|acc:O50948 Rattus norvegicus taste bud receptor protein TB 567 (TB 567) gene, complete eds - Rattus norvegicus, 1299 bp. (SEQ ID NO: 44)
30 Score = 1221 (183.2 bits), Expect = 3.3e-49, P = 3.3e-49
Identities = 591/940 (62%), Positives = 591/940 (62%), Strand = Plus / Plus o
Query: 25 AATTTCACCGATGTGACAGAGTTCATTCTTTTGGGGCTAACGAG-CCGTCGgGAATGGCA 83
35 III I I I I I I II I I I I I II llll llll I lll ll l l lll
Sbj ct : 46 AATGCCACCGAAGTCACTGACTTCTATCTTCTGGGATTTG-GAGTCCAGCAA-AATACTC 103 o o
Query: 84 AGT-TCTCTTCTTCATCgτgτTTCTTGTGGTCTACATCATCA-CCATGGTGGGCAATATC 141 III I II I III lllll llll III II llll I I II I I I I I I
Sbjct: 104 AGTGTGTCCTCTTCATTGTATTTTTTGTGATCTATGTCA-CATCCATGGTGGGCAACACT 162
40 o
Query: 142 GGCATGATGgTGTTAATCAAGG-TCAGTCCTCAGCTTAACAACCCCATGTACTTTTTCCT 200 ll lllll I l lllll l l lll l lll l l II I I I I I II I I III I
Sbjct: 163 GGGATGATCCTCCTCATCAACACTAACTCCAGA-CTTCAGACTCCCATGTACTTCTTCTT 221
45 Query: 201 -CAGTCACTTGTCATTTGTTGATGTGTGGTTTTCTTCCAATGTCACCCCTAAAATGTTGG 259
II I I I I I I II II II I I II I 1 11 I II I I I I I I II I I Sbjct: 222 ACAAA-ACCTGGCTTTTGTGGATATCTGTTACACGTCTGCCATCACTCCCAAGATGCTGC 280 o o _n Query: 260 AAAAgCTGTTJJTCAGATAAAAAAACAATTACTTATGCTGGTTGTTTAGTACAGTGTTTCT 319 ^ l l l l l lll l I II I I I I I I II I I I I I I I I II
Sbjct: 281 AGAGCTTCATGGTAGAAGACTGTTCCATATCATACACAGGATGTGTAATACAATTGTTGG 340
Query: 320 TCTTCATTGCTCTTGTCCATGTGGAAATTTTTAT-T-CTTGCTGCGATGGCCTTTGATAG 377 Sbjct: 341 TAT---ATGCCACATTTGCAACCAGTGACTGTTACCTACTCGCTGTTATGGCAGTGGACCG 398
Query: 378 ATACATGGCAATTGGGAATCCTCTGCTTTATGGCAGTAAA-ATGTCAAGGGTTGTCTGTA 436
II lllllll l ll ll ll l II l lll lllll I lllll Sbjct: 399 GTATGTGGCAATCTGTAAGCCCCTCCGGTACCCGA-TAATCATGTCTCGACAGGTCTGCT 457
Query: 437 TTCGACTGATTACTTTCCCTTACATTTATGGTTTTCTGACGAGTC-TG-GCAGCA-ACAT 493
I I II I II I llll 1 llll 1 I l ll l l l ll ll) Sbjct: 458 TACTGTTGGTCGCTCTTTCTTATCTC-ATGGGATCAATAA-ATTCCTCTGTA-CACACAG 514
Query: 494 TATGGACTTACGGCTTGTACTTCTGTGGAAAA—ATTGAGATCAACCATTTCTACTGTGC 551
II ll l l I II I II I I II I I 1 I I II II I I II I lllll Sbjct: 515 GATTTACATTCTCATTGT-CTTAT-TGTAACTCCAAAAATATCAATCACTTTTTCTGTGA 572 o Query: 552 AGATCCACCTCTCATCAAAATGGCCTGTGCgGGGACCTTTGTAAAAGAAT-ATACAA-TG 609
I I II 1 I II I I II I II I ll ll l l l Sbjct: 573 TGTTGTCCCAATCATCAGTCTTTCATGCTC-GA-ACACTGATATTAATATCATGCTACTT 630 o o
Query: 610 gjTCATACTTGCCGGCATJjAACTTCACATATTCCCTGACTGTAATTATCATCTCTTACTTA 669
II I III II I II II I I II I I II II I I I II I 1 II I I III II Sbjct: 631 ATTGTTTTTGTTGGATTTAACCTGACATTCACTGTGTTGGTCATTATCTTCTCTTACATA 690
Query: 670 TTCATCCTCATTGCCATTCTGCGAATGCGCTCAGCAGAAGGAAGGCAGAAGGCCTTTTCC 729 l llll l lllll ll III 111 I I I I I II I I I I I llll lll
Sbjct: 691 TACATCATGGCCGCCATCCTAAAGATGTCCTCTACTGCAGGGAGGAAGAAAACCTTCTCC 750 o
Query: 730 ACATGTGGGTCCCATCTGACAGCTGTCATJjATATTCTATGGTACTCTGATCTTC-ATGTA 788 II llll lllll llll II II II llll lllll
Sbjct: 751 ACGTGTGCCTCCCACCTGACAGCAGTCACCATTTTCTATGGAACCCTT-TCTTATATGTA 809
Query: 789 TCT-CAGACGTCCCACAGAGGAGTCTGTGGAGCAGGGGAAGATGGTGGCTGTGTTCTATA 847 l lll l ll I II I I II I I III I III III I I I I I I I II I Sbjct: 810 CTTACAGCC-TCATTCGGACAATTCTGAGGAGAATATGAAAGTGGCCTCTGTGTTTTATG 868
Query: 848 CCACAGTGATCCCCATGTTGAATCCCATGATCTACAGTCTGAGGAACAAGGATGTGAAA- 906
II lllll llll II I llll I I II I III II III Sbjct: 869 GCATTGTGATTCCCATGCTGAACCCTCTCATCTACAGCTTGAGAAACAAGGAAGTCAAAG 928
Query: 907 AAGGCCATGATGAAAGTGATCAGCAGATCATGTTAAACAAAATAAAATCAAAT—TTGAT 964 l l l l I I I I I I I I I I I I I I l l l l l 1 1 1 i i m m i l l l l l
Sbjct: 929 AAGGTTTTAAAGCAA-TGAGCAGAAGGT—TCTTAAG AATGAAATCAAATCCTTGAT 982
BLASTN alignments also found homology between two fragments of GPCR4 and M S musculus odorant receptor M72 (GENBANK-ID:AF247656) shown if Table 4F. M72 residues 821-890 (SEO ID NO:45) and residues 160-201 (SEO ID NO:46). are aligned with GPCR4 in Table 4F.
Table 4F. BLASTN of GPCR4a against M72
>gb|AF247656.1 |AF247656 Mus musculus odorant receptor M72 (M72) gene, complete eds Length = 930 Score = 83.8 bits (42), Expect = 2e-13 Identities = 63/70 (90%), Strand = Plus / Plus
Query: 836 ctgtgttctataccacagtgatccccatgttgaatcccatgatctacagtctgaggaaca 895
II I I I I 111 I I II II I II II II II III I II II III I I II I I I I I I 11)11 III) Sbjct: 821 ctgtgttctacaccacagtgatccccatgttcaaccccctgatctacagcctgagaaaca 880
Query: 896 aggatgtgaa 905 llll lllll Sbjct: 881 aggaggtgaa 890 (SEQ ID NO: 45)
Score = 44.1 bits (22), Expect = 0.13 Identities = 37/42 (88% ) , Strand = Plus / Plus
Query: 172 cagcttaacaaccccatgtactttttcctcagtcacttgtca 213 l l l l l l I I I I I I I I I I I I I I I l l l l l l l l l I I l l l l l Sbj ct : 160 cagcttcacacccccatgtacttcttcctcagtaacctgtca 201 (SEQ ID NO : 46)
BLASTN alignments found homology between fragments of GPCR4 and jfus musculus odorant receptor K42 (GENBANK-ID: AF282291)(SEO ID NO:47) shown if Table 4G.
Table 4G.-BLASTN of GPCR4b against OR K42
>gb|AF282291.1 IAF282291 Mus musculus odorant receptor K42 gene, complete eds (SEQ ID NO: 7) Length = 927 Score = 77.8 bits (39), Expect = le-11 Identities = 60/67 (89%); Strand = Plus / Plus
Query: 836 ctgtgttctataccacagtgatccccatgttgaatcccatgatctacagtctgaggaaca 895
I II I II I I I I I I II II I I IMI II III I I II I II I I I I I I II I I III Sbjct: 815 ctgtgttctacaccacagtgatcccaatgctaaatcccctcatatacagtctgaggaaca 874
Query: 896 aggatgt 902 Sbjct: 875 aggatgt 881
BLASTN alignments found homology between GPCR4 and tøus musculus odorant receptor K40 (GENBANK-ID :AF282289 ) (SEO ID NO:48) shown in Table 4G.
Table 4H.-BLASTN of GPCR4 against OR K40
>gb|AF282289.1 IAF282289 Mus musculus odorant receptor K40 gene, complete eds (SEQ ID NO: 48) Length = 927 Score = 75.8 bits (38), Expect = 4e-ll Identities = 62/70 (88%); Strand = Plus / Plus
Query: 836 ctgtgttctataccacagtgatccccatgttgaatcccatgatctacagtctgaggaaca 895
I II I I I I II I I I I I II I I I III I I II II I I I II II I II lllllllll Sbjct: 824 ctgtgttctataccacagtgatccccatgctgaacccattaatatacagtttgaggaaca 883
Query: 896 aggatgtgaa 905
I llllllll Sbjct: 884 aagatgtgaa 893
The full GPCR4a amino acid sequence has 155 of 304 amino acid residues (50 %) identical to, and 215 of 304 residues (70%) positive with, the 314 amino acid residue proteins from pan troglodytes OR93CH (ptnr: SPTREMBL-ACC: 077756) (SEO ID NO:41) (Table 41). The residue that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
Table 41. BLASTX of GPCR4a against OR93CH
>ptnr: SPTREMBL-ACC: 077756 OLFACTORY RECEPTOR 0R93CH - Pan troglodytes (Chimpanzee),
314 aa. (SEQ ID Nθ:41)
Score = 806 (283.7 bits), Expect = 2.0e-79, P = 2.0e-79
Identities = 155/304 (50%), Positives = 215/304 (70%), Frame = +1 o o
Query: 25 NFTDVTEFILLGLTSRRE QVLFFlBFLW-YIITMVGNIGMMgLIKVSPQLNNPMYFFL 201 l + l I I I I I II + 11 I++I.I l + l + I l + l l + ll I++ +1+ I III I I
Sbj ct : 5 NYTKVTEFIFTGLNYNPQLQVFLFLLFLTTFYVINVTGNLGMIVLIRIDSRLHTPMYFFL 64 o Query : 202 SHLSFVDV FSSNVTPKMLENLgSDKKTITYAGCLVQCFFFIALVHVEIFILAAMAFDRY 381
I I II I I 1+ I I I l + l II I + I +1 I++ I I +1 +11 I I l + ll + ll + lll Sbjct: 65 SHLSFVDICFSSWSPKMLTDFFVKRKAISFLGCALQQ FFGFFVAAECFLLASMAYDRY 124
Query: 382 MAIGNPLLYGSKMSRWCIRLITFPYIYGFLTSLAATLWTYGLYFCGKIEINHFYCADPP 561 + 11 lllll 11+ +11 + 1+ 11+ I + ++ I + I I I I III l + l I - Sbjct: 125 VAICNPLLYSVAMSQRLCIQLWGPYVIGLMNTMTHTTNAFRLPFCGPNVINHFFCDMSP 184 Query: 562 LIKMACAGTFVKEYTM0ILAGINFTYSLTVIIISYLFILIAILRMRSAEGRQKAFSTCGS 741 1+ + I I I + + + l + ll +1 l + l I1++II + I I I l + l ll + l I I llll I Sbjct: 185 LLSLVCADTRLNKLAVFIVAGAAGVFSGLTILISYIYILMAILRIRSADGRCKTFSTCSS 244
Query : 742 HLTAVIIFYGTLIFMYLRRPTEESVEQGKMVAVFYTTVIPMLNPMIYSLRNKDVKKAMMK 921 l l l l l I l l l l l + l + l I ++ l + l + I M I l l l l l l l + l l l l l l l + l l 1 + +
Sbj ct : 245 HLTAVFILYGTLFFIYVRPSASFSLDLNKLVSVFYTAVIPMLNPLIYSLRNKEVKDAIHR 304
Query: 922 VISR 933 +++
Sbjct: 305 TVTQ 308
The GPCR4a amino acid sequence has 139 of 303 amino acid residues (45%) identical to. and 192 of 303 residues (62%) positive with the 303 amino acid OR93Gib protein from Hylobates lar (GENBANK-ID:AAC63971.1) (SEO ID NO:43) (Table 4J). The residue that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
Table 4J.-BLASTX of GPCR4a against OR93Gib
>gb|AAC63971.1| (AF045580) olfactory receptor 0R93Gib [Hylobates lar] (SEQ ID NO: 43) Length = 313
Score = 272 bits (695), Expect = 4e-72
Identities = 155/303 (51%), Positives = 208/303 (69%) o o
Query: 3 NFTDVTEFILLGLTSRREWQVLFFI0FLVVYIITMVGNIG.4MJSLIKVSPQLNNPMYFFLS 62 l + l I I I I I II + 1 1 II I I++ II ll + l I I++ +1+ I II llll
Sbjct: 5 YTKVTEFIFTGLNYNPQLQVFLFLLFLTFYVISVTGNFGMIVLIRMDSRLHTPMYFFLS 64 o Query: 63 HLSFVDVWFSSNVTPKMLENL@SDKKTITYAGCLVQCFFFIALVHVEIFILAAMAFDRYM 122
I II I I 1+ I I I l + l III + I +1 I++ II +1 +11 I I l + l l + l l + l 11 + Sbjct: 65 HLSFVDICFSSWSPKMLTDFFVKRKAISFLGCALQQWFFGFFVAAECFLLAS AYDRYV 124
Query: 123 AIGNPLLYGSKMSRVVCIRLITFPYIYGFLTSLAATL TYGLYFCGKIEINHFYCADPPL 182
Sbjct: 125 AICNPLLYSVF SQRLCIQLWGPYVIGLMNTMTHTTNAFRLPFCGLNVINHFFCDMSPL 184 o Query: 183 IKMACAGTFVKEYTM0ILAGINFTYSLTVIIISYLFILIAILRMRSAEGRQKAFSTCGSH 242 + + I I I + + + l + ll +1 l lll I I I I I l + l 1 l + ll I I I I I II Sbjct: 185 LSLVCADTRLNKLAVFIMAGAVGVFSGLTILISYIYILMAILRIRSADGRCKTFSTCSSH 244
Query: 243 LTAVIIFYGTLIFMYLRRPTEESVEQGKMVAVFYTTVIPMLNPMIYSLRNKDVKKAMMKV 302 llll I llll l+l+l ++ l+l+ll II 1+ + Sbjct: 245 LTAVFILYGTLFFIYVRPSASFPLDLNKWSVFYTAVIPMLNPLIYSLRNKEVKDAIHRT 304
Query: 303 ISR 305
+++ Sbjct: 305 VTQ 307
The GPCR4a protein has 137 of 304 amino acid residues (45%) identical to. and 191 of 304 residues (62%) positive with, the 308 amino acid odorant receptor K42 from Mu musculus (GENBANK-ID:AAG39876.1) (SEO ID NO:49) (Table 4K). The residue that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
Table 4K.-BLASTX of GPCR4a against K42
>gb|AAG39876.1| (AF282291) odorant receptor K42 [Λfus iϊiusculus] (SEQ ID Nθ:49), 30E aa
Statistics for GPCR4a: Score = 271 bits (694), Expect = 6e-72
Identities = 149/304 (49%), Positives = 203/304 (67%) Statistics for GPCR4b: Score = 273 bits (699), Expect = 2e-72
Identities = 138/304 (45%), Positives = 192/304 (62%)
10
Query: 2 LNFTDVTEFILLGLTSRREWQVLFFlgFLWYIITMVGNIGMMgLIKVSPQLNNPMYFFL 61
+ 1 + I l + ll I I I I I I I I I 11 + 11 I + II++I I +1 I I++I I l + ll Sbjct: 1 MNHSSVTDFILEGLTKRPELQLPLFLLFLGIHVITWGNLGMILLINISSQLHSPMYYFL 60
15 Query: 62 SHLSFVDV FSSNVTP-CMLENLgSDKKTITYAGCLVQCFFFIALVHVEIFILAAMAFDRY 121
I IIII + I+ +1 I +11 I I I I I I I++ 1+ I +11+ I I ++I I I l + lll
Sbjct: 61 SHLSFIDLCYSSVITPK LVNFVCAKNTISFKECMTQLYFFLLLAISEGYLLTAMAYDRY 120
Query: 122 MAIGNPLLYGSKMSRWCIRLITFPYIYGFLTSLAATLWTYGLYFCGKIEINHFYCADPP 181
20 + II + 1 I I I + I I II ++ I II + I l lll I I ++ 1 I
Sbjct: 121 VAICSPLLYNTVMSHKVCSIMMAVVYSLGFFGATVHTTRMTMLSFCGSHIIRHYFCDILP 180
Query: 182 LIKMACAGTFVKEYTMglLAGINFTYSLTVIIISYLFILIAILRMRSAEGRQKAFSTCGS 241
|+ ++|+ l + l + |4- l + l llll lll I I l + l I I I I II I I I I
25 Sbjct: 181 LLTLSCSSTHINEVLLFIIGGVNTLAPTLAVIISYAFILTSILRIRSNEGRSKAFGTCSS 240
Query: 242 HLTAVIIFYGTLIFMYLRRPTEESVEQGK VAVFYTTVIPMLNPMIYSLRNKDVKKAMMK 301 l+ ll l I + I++ I II + 1+ ++II 1+ +1 III III II II l + l I I II I I II I l+ l Sbjct: 241 HIMAVGIFFGSITFMYFKPPSSNNMEQEKVSSVFYTTVIPMLNPLIYSLRNKDVKTALKK 300
30
Query: 302 VISR 305
++ I Sbjct: 301 MVGR 304
35 The GPCR4b protein has 141 of 301 amino acid residues (46%) identical to, and 189 of 301 residues (61%) positive with, the 314 amino acid OR 511 from Mii musculus (GENBANK-FD:AAG39876.1) (SEO ID NO:50) (Table 4L). The residue that-differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
Table 4L.-BLASTX of GPCR4b against OR 511
40 ref |NP_006628.1 I olfactory receptor, family 5, subfamily I, member 1 [Homo sapiens] splQ13606|O5Il_HUMAN OLFACTORY RECEPTOR 511 (OLFACTORY RECEPTOR-LIKE PROTEIN OLF1) gb I AAB01214.il (U56420) HsOLFl [Homo sapiens] (SEQ ID NO:50); Length = 314
Score = 274 bits (700), Expect = le-72
Identities = 141/301 (46%), Positives = 189/301 (61%)
45 o o
Query: 3 NFTDVTEFILLGLTSRREWQVLFFigFLWYIIT VGNIGMMgLIKVSPQLNNPMYFFLS 62 l + l I I I I II I I +1 I II I II I I ++I I ll + l II I++ I I lllllll Sbjct: 7 NYTLVTEFILLGFPTRPELQIVLFLMFLTLYAIILIGNIGLMLLIRIDPHLQTPMYFFLS 66
^Hn Query: 63 HLSFVDV FSSNVTPK LENLJ °3SDKKTITYAGCLVQCFFFIALVHVEIFILAAMAFDRYM 122
+ 11 I 11+ + I++ llll I 11+ l + l + l I I +1 +1 I I I II II I I + III +
Sbjct: 67 NLSFVDLCYFSDIVPKMLVNFLSENKSISYYGCALQFYFFCTFADTESFILAAMAYDRYV 126
Query: 123 AIGNPLLYGSKMSRWCIRLITFPYIYGFLTSLAATLWTYGLYFCGKIEINHFYCADPPL 182
55 II ll lll III +I + III 1+ I ++I I I + + I +1 I I II l + l III
Sbjct: 127 AICNPLLYTVVMSRGICMRLIVLSYLGGN SSLVHTSFAFILKYCDKNVINHFFCDLPPL 186 o
Query: 183 IKMACAGTFVKEYTMglLAGINFTYSLTVIIISYLFILIAILRMRSAEGRQKAFSTCGSH 242 + I++I I + 1+ + +11 II I I I I I++I I I l + l I I II II Sbjct: 187 L LSCTDTTINE LLSTYGSSVEIICFIIIIISYFFILLSVLKIRSFSGRKKTFSTCASH 246
Query: 243 LTAVIIFYGTLIFMYLRRPTEESVEQGKMVAVFYTTVIPMLNPMIYSLRNKDVKKAMMKV 302 ll + l 1+ II l + l + l I I I+++I1II I l + l ll + l 111 II I I II I II Sbjct: 247 LTSVTIYQGTLLFIYSRPSYLYSPNTDKIISVFYTIFIPVLNPLIYSLRNKDVKDAAEKV -306
Query: 303 I 303"
+ Sbjct: 307 L 307
10
The full amino acid sequence ofthe GPCR4b protein has 140 of 303 amino acid residues (46%) identical to, and 193 of 303 residues (63 %) positive with, the 312 amino acid OR4 protein from Mu musculus (GENBANK-ID:AAG39876.1) (SEO ID NO:34) (Table 4J). The residue that differs between GPCR4a and GPCR4b are highlighted in black and marked 15 with the (o) symbol.
Table 4M.-BLASTX of GPCR4b and OR 4 emb|CAA64370.1| (X94744) olfactory receptor 4 [Gallus gallus] ; (SEQ ID NO:34) Length = 312; Score = 271 bits (693), Expect = 7e-72 -n Identities = 140/303 (46%), Positives = 193/303 (63%) ^- o o
Query: 3 NFTDVTEFILLGLTSRREWQVLFFlrøFLWYIITMVGNIGMMJJLIKVSPQLNNPMYFFLS 62
I I +1111 + 11+ + + l lll 1 I I I I + I+++I 1+ 1 + 1+ Sbjct: 5 NHTLASEFILVGLSDHPKMKAALFWFLLIYVITFQGNLGIIILIQGDPRLHTSMYFFLS 64
9<; ° ώ- Query : 63 HLSFVDVWFSSNVTPKMLENL0SDKKTITYAGCLVQCFFFIALVHVEIFILAAMAFDRYM 122
I I I I + I I I + I + I 1 I I +++ 1 I ++ 1 I I I I + 1 I I I + 1 I I I + 1 I I + Sbj ct : 65 SLSWDICFSSVIAPRTLVNFLSERRTISFTGCTGQTFFYIVFVTTECFLLAVMAYDRYV 124
Query : 123 AIGNPLLYGSKMSRWCIRLITFPYIYGFLTSLAATLWTYGLYFCGKIEINHFYCADPPL 182
30 I I l l l l l + l + l I ++ I + I I I I ++ I + l l l l I I l l + l I I I Sbj ct : 125 AICNPLLYSTIMTRRQCMQLWGSYIGGILNAIIQTTFIIRLPFCGSNIINHFFCDVPPL 184 o Query: 183 I MACAGTFVKEYTMgJlLAGINFTYSLTVIIISYLFILIAILRMRSAEGRQKAFSTCGSH 242
35 + ++ I I++ I + llll ++I I I I I I III l + l I III I I I I I II II
Sbj ct : 185 LALSLASTYISEMILFSLAGIIELSTVTSILVSYIFIS.CAILRIRSAEGRQKALSTCASH 244
Query : 243 LTAVIIFYGTLIFMYLRRPTEESVEQGKMVAVFYTTVIPMLNPMIYSLRNKDVKKAMMKV 302
II I I + I II I I I I I + 1+ l + l + l III I I I I II l + l I II II++II 1+ +1 Sbjct: 245 LTAVTLLYGTTIFTYLRPSSSYSLNTDKVVSVFYTWIPMLNPLIYSLRNQEVKGALSRV 304
40
Query: 303 ISR 305
+ I Sbjct: 305 VER 307
45 A multiple sequence alignment is given in Table 4K, with GPCR4a being shown on line 1, in a ClustalW analysis comparing GPCR4a with related protein sequences.
Table 4N.-Information for the ClustalW proteins:
1. Novel HumanJDLF, GPCR4a, SEQ ID NO: 10
2. Hylobates lar (Common Gibbon) OLF, SPTREMBL-Acc # 077758, SEQ ID NO: 43
50 3. Pan troglodytes (Chimpanzee) OLF, SPTREMBL-Acc # 077756, SEQ ID NO: 41
4. Mus musculus OLF, GENBANK-Acc # AAF20365, SEQ ID NO: 42
5. Homo sapiens OLF, SWISSPROT-Acc # Q13606, SEQ ID NO: 33
Figure imgf000035_0001
GPCR4 a
GIBBON_OLF
CHIMPANZEE DLF MOUSEJDLF
HOMAN_OLF
GPCR4a
GIBBONJDLF
CHIMPANZEEjOLF
MOUSE_OLF
HUMAN_OLF
Figure imgf000035_0002
Figure imgf000035_0003
GPCR4a
GIBBON_OLF
CHIMPANZEEJDLF
MOUSE_OLF
HUMANJDLF
Figure imgf000035_0004
DOMAIN results for GPCR4a were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 4L with the statistics and domain description. Residues 1-163 (SEO ID NO:29) and residues 313-377 (SEO ID NO:37) of 7tm_l are aligned with GPCR4 in Table 4O. The residue that differs between GPCR4a and GPCR4b are highlighted in black and marked with the (o) symbol.
Table 4OJDOMATN results for GPCR4a. gnl I PfamlpfamOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family)
(SEQ ID NO: 29) Length = 377
Statistics for GPCR4a: Score = 89.7 bits (221), Expect = 2e-19 Statistics for GPCR4b: Score = 90.5 bits (223), Expect = le-19 o o
Query: 39 GNIGMMSLIKVSPQLNNPMYFFLSHLSFVDVWFSSNVTPKMLENLgSDK TITYAGCLVQ 98
Sbjct: 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMP VVYLEVVGE KFSRIHCDIF 60
Query: 99 CFFFIALVHVEIFILAAMAFDRYMAIGNPLLYGSKMSRV-VCIRLITFPYIYGFLTSLAA 157 Sbjct: 61 VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIV VLSFTISCPM 120
Query : 158 TLWTYGLYFCGKIEINHFYCADPPLIKMACAGTFVKEYTM0ILΑGINFTYSLTV 211 Sbj ct : 121 LFGLNNTDQNE CHAN PAFWYSSIVSFYVPFIVTL LVYIKIY 163 Sbjct: 7 transmembrane receptor (rhodopsin family) fragment (SEQ ID NO: 37) gnl I PfamlpfamOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family) 377 aa Statistics for GPCR4a: Score = 40.4 bits (93), Expect = le-04 Statistics for GPCR4b: Score = 40.4 bits (93), Expect = le-04
Query: 225 RMRSAEGRQKAFSTCGSHLTAVIIFYGTLIFMYLRRPTEESVEQG-KMVAVFYTTVIPML 283 Sbjct: 313 KLSQQKEKKATQMLAIVLGVFIIC LPFFITHILNIHCDCNIPPVLYSAFTWLGYVNSAV 372 .. . .. : * : * * : : : : * : ifi Query: 284 NPMIY 288 Sbjct: 373 NPIIY 377 ** . **
The nucleic acids and proteins of GPCR4 are useful in potential therapeutic
15 applications implicated in various in various GPCR-related pathological disorders and/or OR- related pathological disorders, described further below. For example, a cDNA encoding the olfactory receptor -like protein may be useful in gene therapy, and the olfactory receptor ^ protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients
20 suffering from neoplasm, adenocarcinoma. lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Other GPCR-related diseases and disorders are contemplated.
The novel GPCR4 nucleic acid and protein, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein
25 are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. This novel protein also has immense value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various
30 disorders
GPCR5
GPCR5 is an Olfactory Receptor ("OR")-like protein, wherein three alternative novel GPCR5 nucleic acids and encoded polypeptides are disclosed.
The novel GPCR5a nucleic acid of 980 nucleotides (also referred to as APOOl 112 C) 35 is shown in Table 5A. An ORF begins with an ATG initiation codon at nucleotides 26-28 and ends with a TGA codon at nucleotides 941-43. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5A, and the start and stop codons are in bold letters. Table 5A. GPCR5a Nucleotide Sequence (SEQ ID NO:13)
AGCTTGAAGAGCAAACTGTCAGGAAATGTCCAACACAAATGGCAGTGCAATCACAGAATTCATTTTACTTGGGCTCACAGATTG CCCGGAACTCCAGTCTCTGCTTTTTGTGCTGTTTCTGGTTGTTTACCTCGTCACCCTGCTAGGCAACCTGGGCATGATAATGTT AATGAGACTGGACTCTCGCCTTCACACGCCCATGTACTTCTTCCTCACTAACTTAGCCTTTGTGGATTTGTGCTATACATCAAA TGCAACCCCGCAGATGTCGACTAATATCGTATCTGAGAAGACCATTTCCTTTGCTGGTTGCTTTACACAGTGCTACATTTTCAT TGCCCTTCTACTCACTGAGTTTTACATGCTGGCAGCAATGGCCTATGACCGCTATGTGGCCATATATGACCCTCTGCGCTACAG TGTGAAAACGTCCAGGAGAGTTTGCATCTGCTTGGCCACATTTCCCTATGTCTATGGCTTCTCAGATGGACTCTTCCAGGCCAT CCTGACCTTCCGCCTGACCTTCTGTAGATCCAATGTCATCAACCACTTCTACTGTGCTGACCCGCCGCTCATTAAGCTTTCTTG TTCTGATACTTATGTCAAAGAGCATGCCATGTTCATATCTGCTGGCTTCAACCTCTCCAGCTCCCTCACCATCGTCTTGGTGTC CTATGCCTTCATTCTTGCTGCCATCCTCCGGATCAAATCAGCAGAGGGAAGGCACAAGGCATTCTCCACCTGTGGTTCCCATAT GATGGCTGTCACCCTGTTTTATGGGACTCTCTTTTGCATGTATATAAGACCACCAACAGATAAGACTGTTGAGGAATCTAAAAT AATAGCTGTCTTTTACACCTTTGTGAGTCCGGTACTTAATCCATTGATCTACAGTCTGAGGAATAAAGATGTGAAGCAGGCCTT GAAGAATGTCCTGAGATGAAATATTGTCATGACCATGGTGATGCCTTTGTTTCCTA The GPCR5a protein encoded by SEO ID NO:13 has 305 amino acid residues, and is presented using the one-letter code in Table 5B. The SignalP. Psort and/or Hydropathy profile for GPCR5a predict that GPCR5a has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The signalP shows a signal sequence is coded for in the first 44 amino acids, e„ with a cleavage site at the dash in the sequence NLG-MIM, between amino acids 44 and 45. This is typical of this type of membrane protein.
Table 5B. Encoded GPCR5a protein sequence (SEQ ID NO:14).
MSNTNGSAITEFILLGLTDCPELQSLLFVLFLWYLVTLLGNLGMIMLMRLDSRLHTPMYFFLTNLAFVDLCYTSNATPQMSTN IVSEKTISFAGCFTQCYIFIALLLTEFYMLAAMAYDRYVAIYDPLRYSVKTSRRVCICLATFPYVYGFSDGLFQAILTFRLTFC RSNVINHFYCADPPLIKLSCSDTYVKEHAMFISAGFNLSSSLTIVLVSYAFILAAILRIKSAEGRHKAFSTCGSHMMAVTLFYG TLFCMYIRPPTDKTVEESKIIAVFYTFVSPVLNPLIYSLRNKDVKQALKNVLR
The target GPCR5a sequence, above, was subjected an the exon linking process to confirm the sequence, as reported for GPCR2 and GPCR4, above. The resulting GPCR5b sequence (also referred to herein as AC0170103B1) is reported below in Table 5C .
Table 5C.-GPCR5 Nucleotide Sequence (SEQ ID NO.15)
AGCTTGAAGAGCAAACTGTCAGGAAATGTCCAACACAAATGGCAGTGCAATCACAGAATTCATTTTACTTGGGCTCACAGATTG
CCCGGAACTCCAGTCTCTGCTTTTTGTGCTGTTTCTGGTTGTTTACCTCGTCACCCTGCTAGGCAACCTGGGCATGATAATGTT AATGAGACTGGACTCTCGCCTTCACACGCCCATGTACTTCTTCCTCACTAACTTAGCCTTTGTGGATTTGTGCTATACATCAAA TGCAACCCCGCAGATGTCGACTAATATCGTATCTGAGAAGACCATTTCCTTTGCTGGTTGCTTTACACAGTGCTACATTTTCAT TGCCCTTCTACTCACTGAGTTTTACATGCTGGCAGCAATGGCCTATGACCGCTATGTGGCCATATATGACCCTCTGCGCTACAG TGTGAAAACGTCCAGGAGAGTTTGCATCTGCTTGGCCACATTTCCCTATGTCTATGGCTTCTCAGATGGACTCTTCCAGGCCAT CCTGACCTTCCGCCTGACCTTCTGTAGATCCAGTGTCATCAACCACTTCTACTGTGCTGACCCGCCGCTCATTAAGCTTTCTTG TTCTGATACTTATGTCAAAGAGCATGCCATGTTCATATCTGCTGGCTTCAACCTCTCCAGCTCCCTCACCATCGTCTTGGTGTC CTATGCCTTCATTCTTGCTGCCATCCTCCGGATCAAATCAGCAGAGGGAAGGCACAAGGCATTCTCCACCTGTGGTTCCCATAT GATGGCTGTCACCCTGTTTTATGGGACTCTCTTTTGCATGTATATAAGACCACCAACAGATAAGACTGTTGAGGAATCTAAAAT AATAGCTGTCTTTTACACCTTTGTGAGTCCGGTACTTAATCCATTGATCTACAGTCTGAGGAATAAAGATGTGAAGCAGGCCTT GAAGAATGTCCTGAGATGAAATATTGTCATGACCATGGTGATGCCTTTGTTTCCTA
The GPCR5b protein encoded by SEO ID NO: 15 has 305 amino acid residues and is presented using the one-letter code in Table 5D. The SignalP, Psort and/or Hydropathy profiles for GPCR5b are the same as for GPCR5a.
Table 5D.-Encoded GPCR5 protein (SEQ ID NO:16).
MSNTNGSAITEFILLGLTDCPELQSLLFVLFLWYLVTLLGNLGMIMLMRLDSRLHTPMYFFLTNLAFVDLCYTSNATPQMSTN IVSEKTISFAGCFTQCYIFIALLLTEFYMLAAMAYDRYVAIYDPLRYSVKTSRRVCICLATFPYVYGFSDGLFQAILTFRLTFC RSSVINHFYCADPPLIKLSCSDTYVKEHAMFISAGFNLSSSLTIVLVSYAFILAAILRIKSAEGRHKAFSTCGSHMMAVTLFYG TLFCMYIRPPTDKTVEΞSKIIAVFYTFVSPVLNPLIYSLRNKDVKQAL NVLR In an alternative embodiment, a novel GPCR5c nucleic acid of 1006 nucleotides (also referred to herein as CG50173-01) is shown in Table 5E. An ORF was identified beginning with an ATG initiation codon at nucleotides 83-85 and ending with a TGA codon at nucleotides 998-1000. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon.
Table 5E.-GPCR5c Nucleotide Sequence (SEQ ID NO:17)
AATTCAAATGAACATTAACATGGTATGTGCATTTGTTTATATTGGCTTTATTTCCATAGCTTGAAGAGCAAACTGTCAGGAAAT GCCCAACACAAATGGCAGTGCAATCACAGAATTCATTTTACTTGGGCTCACAGATTGCCCGGAACTCCAGTCTCTGCTTTTTGT GCTGTTTCTGGTTGTTTACCTCGTCACCCTGCTAGGCAACCTGGGCATGATAATGTTAATGAGACTGGACTCTCGCCTTCACAC GCCCATGTACTTCTTCCTCACTAACTTAGCCTTTGTGGATTTGTGCTATACATCAAATGCAACCCCGCAGATGTCGACTAATAT CGTATCTGAGAAGACCATTTCCTTTGCTGGTTGCTTTACACAGTGCTACATTTTCATTGCCCTTCTACTCACTGAGTTTTACAT GCTGGCAGCAATGGCCTATGACCGCTATGTGGCCATATATGACCCTCTGCGCTACAGTGTGAAAACGTCCAGGAGAGTTTGCAT CTGCTTGGCCACATTTCCCTATGTCTATGGCTTCTCAGATGGACTCTTCCAGGCCATCCTGACCTTCCGCCTGACCTTCTGTAG ATCCAATGTCATCAACCACTTCTACTGTGCTGACCCGCCGCTCATTAAGCTTTCTTGTTCTGATACTTATGTCAAAGAGCATGC CATGTTCATATCTGCTGGCTTCAACCTCTCCAGCTCCCTCACCATCGTCTTGGTGTCCTATGCCTTCATTCTTGCTGCCATCCT CCGGATCAAATCAGTAGAGGGAAGGCACAAGGCATTCTCCACCTGTGGTTCCCATATGATGGCTGTCACCCTGTTTTATGGGAC TCTCTTTTGCATGTATATAAGACCACCAACAGATAAGACTGTTGAGGAATCTAAAATAATAGCTGTCTTTTACACCTTTGTGAG TCCGGTACTTAATCCATTGATCTACAGTCTGAGGAATAAAGATGTGAAGCAGGGCTTGAAGAATGTCCTGAGATGAAATATT
The GPCR5c protein encoded by SEQ ID NO: 17 has 305 amino acid residues and is presented using the one-letter code in Table 5F. The SignalP, Psort and/or Hydropathy profiles for GPCR5c are the same as for GPCR5a and GPCR5b.
Table 5F.-Encoded GPCR5c protein sequence (SEQ ID NO:18).
MPNTNGSAITEFILLGLTDCPELQSLLFVLFLWYLVTLLGNLGMIMLMRLDSRLHTPMYFFLTNLAFVDLCYTSNATPQMSTN IVSEKTISFAGCFTQCYIFIALLLTEFYMLAAMAYDRYVAIYDPLRYSVKTSRRVCICLATFPYVYGFSDGLFQAILTFRLTFC RSNVINHFYCADPPLIKLSCSDTYVKEHAMFISAGFNLSSSLTIVLVSYAFILAAILRIKSVEGRHKAFSTCGSHMMAVTLFYG TLFCMYIRPPTDKTVEESKIIAVFYTFVSPVLNPLIYSLRNKDVKQGLKNVLR GPCR5 variants differ at four nucleotide residues, namely GPCR5a and GPCR5b differ from GPCR5c at T29C. C714T and C921G. while GPCR5a and GPCR5c differ from GPCR5b at A537G. GPCR5 variants differ at four amino acid residues, namely GPCR5a and GPCR5b differ from GPCR5c at S2P, A230V and A299G, while GPCR5a and GPCR5c differ from GPCR5b at N171S. All numbering is in reference to GPCR5a. Unless specifically addressing GPCR5a or GPCR5b or GPCR5c, assume any reference to GPCR5 to encompass all variants. ha a search of sequence databases, it was found, for example, that the nucleic acid sequence GPCR5a has 633 of 959 bases (66 %) identical to and 633 of 959 bases (66%) positive with a Gaulls galhιs species Olfactory Receptor clone (GENBANK-ID: X94742) (SEO ID NO:51) (Table 5G). The residue that differs between GPCR5a. GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol. Table 5G-BLASTN of GPCR5a against OR 2 (SEQ ID NO:51)
>gb:GENBANK-ID:GGCOR2GEN|acc:X94742 G. gallus cor2 DNA for olfactory receptor 2
Gallus gallus, 996 bp. (SEQ ID NO: 51)
Statistics for GPCR5a: Score = 1409 (211.4 bits), Expect = 1.4e-57, P = 1.4e-57
Identities = 633/959 (66%), Positives = 633/959 (66%), Statistics for GPCR5c: Score = 1379 (206.9 bits), Expect = 2.5e-56, P = 2.5e-56
Identities = 623/944 (65%), Positives = 623/944 (65%) Strand = Plus / Plus o Query: 7 AAGAGCAAACTGTCA-G-GAA-ATGJJCCAACACAAATGGCAGTGCAATCACAGAATTCAT 63
II llll llll I II lll llll llll III I II I I I I I I II I Sbjct: 37 AACTGCAA-CTGTGTTGTGATGATGGCCAAGGGAAATCACAGCTCCATCACTGAATTTGT 95
Query 64 TTTACTTGGGCT-CACAGATTGCCCGGAACTCCAGTCTCTGCTTTTTGTGCTGTTTCTGG 122 I I I I II II lllll II 1 II I II II l llll l Sbjct 96 GCT-CTTGGGATTCTCTGAAAAGAGGGCCATCCAGGCTGTTCTCTTTATGG-GCTTCTTG 153 Query 123 TTGTT-TACCTCGTCACCCTGCTAGGCAACCTGGGCATGATAATGTTAATGAGACTGGAC 181
II I lllll llll I II I II I II I I I I II II II II I II II II Sbjct 154 CTGATCTACCTGATCACTCTGCTAGGCAATGTGGGCATGATCACATTGATCAGGCTGGAC 213 Query 182 TCTCGCCTTCACACGCCCATGTACTTCTTCCTCACTAACTTAGCCTTTGTGGATTTGTGC 241
II I I II 111 I II I I II I I I III II llll I l lll llll l lll l lll Sbjct 214 TCCCGGCTTCACACCCCTATGTACTTCTTCCTGAGCAGCTTGTCCTTCCTCGATATCTGC 273 Query 242 TATACATC-A-AATGCAACCC-CGCA-GATGTC-GACTAATATC-GTATCTGAGAAGACC 295 ii i i 11 i m i i n 11 i i u in i i : i ; mi i Sbjct 274 TATTCCTCCACAAT-CACTCCTCGAGTGCTCTCAGACC—TCCCAGCATCACAGAAAGTC 330 Query 296 ATTTCCTTTGCTGGTTGCTTTACACAGTGCTACATTTTCATTGCCCTT-CTACTCACTGA 354 III lll l II I I I I I I I I I I I I I I I I Sbjct 331 ATTTCCCACTCTGCATGCCTGGCACAGTTTTATTTCTACGCTGTCTTTGCCAC-CACAGA 389 Query 355 GTTTTACATGCTGGCAGCAATGGCCTATGACCGCTATGTGGCCATATATGACCCTCTGCG 414
II II I I III 1 I II I I I I II llll lllll I llll II I I Sbjct 390 GTGCTATCTTTTGGCCGCAATGGCATATGACCGCTACGTGGCCATCTGCAGCCCTCTGCT 449 Query 415 CTACAGTGTGAAA-ACGTCCAGGAGAGTTTGCAT-CTGCTTG—GCCACATT-TCCCTAT 469
III II I I I I II I I lllll III l lllll l II I l lll l Sbjct 450 CTAT-GTCTTCTCCATGTCCAGCAGAGTTTGTGTGCTGCTGGTTGCTGGCTCATACCT-T 507 Query 470 GTCTATGGCTTCTCAGATGG-ACTCTTCCAGGCCATCCTGAC-CTTCCGCCTGACCTTCT 527
111 ll ll l l lll II lll l I II I I I I II I I lllll I Sbjct 508 GTCGG-GG-TTGTGA-ATGCCACCATTC-ACACAGGGCTTGCACTGCAGC-TGTCCTTCT 562 o Query 528 GTAGATCCASTGTCATCAACCACTTCTACTGTG-CTGACCCGCCGCTCATTAAGCTTTCT 586
I I I I I I I I I I I I I I iinniiin I I l i m I I I I I I i i I I in Sbjct 563 GTGGTCCCAACATCATCAATCACTTCTACTGTGACGGTCCC-CCGCTC-T-ACGCCATCT 619 Query 587 TGTTCTGATACTT-ATGTCAΆA-GAGCATGCCATGTTCATATCTGCT-GGCTTCAACCT- 642
I I I I I I I l l l l l l l l l l l l l I I I I I I I I I I I I 11 I Sbjct 620 CGTGCACAGACCCCACCACCAACGAGATTGCGATATTTCT-TGTGGTTGGCTTCAACATG 678 Query 643 CTCCAGCTCCCTCACCATCGTCTTGGTGTCCTATGCCTTCATTCTTGCT-GCCATCCTCC 701 lll l l ll I I I I II I l lllll III II I II I I I I lllll Sbjct 679 CTC-ATCACCAGCGTGACCATCTTCATCTCCTACACCTACATCCT-GTTCGCTGTCCTCA 736 o Query 702 GGAT-CAAATCAGgAG-AGGGAAGGCACAAGGCATTCTCCACCTGTGGTTCCCATATGAT 759
I I I I I I I I I II I II I II l lll I III III II I I II lllll II Sbjct 737 GGATGCACA-CAGCTGCAGGCAAA-CGCAAAACCTTCTCCACGTGTGCGTCCCACCTGGC 794 Query 760 GGCTGTCACCCTGTTTTATGGGACTCT-CTT-TTGCATGTATATAAGACC-ACCAACAGA 816
I lllll I I I II I I II llll ll ll l l ll l l III Sbjct 795 CACCGTCACCCTATTCTATGC—CTCTGCTGGTTCCATGTACTCACGGCCCAGCTCCAGG 852 Query 817 TAAGACT-GTTGAGGAATCTAAAATAATAGCTGTCTTTTACACCTTTGTGAGTCCGGTAC 875
I I I I I I II I II II II I I I I I I I I I I I II I I Sbjct 853 CACTCCCAGGACCTGGA-C-AAGGTGGCCTCTGTGTTCTACACCATGGTGACCCCCATGC 910 o Query: 876 TTAATCCATTGATCTACAGTCTGAGGAATAAAGATGTGAAGCAGGgCTTGAAGAATGTCC 935 l ll ll 1 I II I I III III 111 II I II II I II I I II lll ll Sbjct: 911 TGAACCCCCTCATCTACAGCCTGAGGAACCAGGAGGTAAAGGATGTTTTAGGGAAAGTGA 970
Query: 936 TGAGATGAAATATTGTCA-TGACCA 959
II I l ll l llll llll l Sbjct: 971 TGGGGAGGAAGAGTGTCTCTGACAA 995
10 The full amino acid sequence ofthe protein of GPCR5a has 160 of 301 amino acid residues (53 %) identical to. and 215 of 301 residues (71%) positive with, the 313 amino acid OR93GIB from HyIobates lar (ptnr: SPTREMBL-ACC: 077758) (SEO ID NO:43) (Table
5H). The residue that differs between GPCR5a. GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol.
15 Table 5H.-BLASTX of GPCR5a against OR93GIB
>ptnr: SPTREMBL-ACC: 077758 OLFACTORY RECEPTOR OR93GIB - Hylobates lar (Common gibbon), 313 aa. (SEQ ID NO: 43)
Score = 803 (282.7 bits), Expect = 4.2e-79, P = 4.2e-79 Identities = 160/301 (53%), Positives = 215/301 (71%), Frame = +2 -=. o
Query: 26 M@NTNGSAITEFILLGLTDCPELQSLLFVLFLWYLVTLLGNLGMIMLMRLDSRLHTPMY 205 l +l I + +1 III II l+ll ll +lll I++++ I I lll +l +l+l I
Sbjct: 1 MANENYTKVTEFIFTGLNYNPQLQVFLFLLFLTFYVISVTGNFGMIVLIRMDSRLHTPMY 60 . Query: 206 FFLTNLAFVDLCYTSNATPQMSTNI-VSEKTISFAGCFTQCYIFIALLLTEFYMLAAMAY 382
II I++I+I I I+I++I +1 +1 1+ I l lll ll l + l + I ++1I+I I I Sbjct: 61 FFLSHLSFVDICFSSVVSPKMLTDFFVKRKAISFLGCALQQWFFGFFVAAECFLLASMAY 120 o Query: 383 DRYVAIYDPLRYSVKTSRRVCICLATFPYVYGFSDGLFQAILTFRLTFCRS|J]VINHFYCA 562 ^ III I I I +1 I II I l+ l+ll I lll l + + lll ll I I I II l+l
Sbjct: 121 DRYVAICNPLLYSVFMSQRLCIQLWGPYVIGLMNTMTHTTNAFRLPFCGLNVINHFFCD 180 o Query: 563 DPPLIKLSCSDTYVKEHAMFISAG-FNLSSSLTIVLVSYAFILAAILRIKSgEGRHKAFS 739
35 ll+ l l +l I ++ l+ll II + 1 III l+ll +1 I I I I I l +ll + l I I II Sbjct: 181 MSPLLSLVCADTRLNKLAVFIMAGAVGVFSGLTI-LISYIYILMAILRIRSADGRCKTFS 239
Query: 740 TCGSHMMAVTLFYGTLFCMYIRPPTDKTVEESKIIAVFYTFVSPVLNPLIYSLRNKDVKQ 919
II 11+ II + lllll +1+11 ++ +1+++I III I l +l I III 111 I ll+l I
Sbjct: 240 TCSSHLTAVFILYGTLFFIYVRPSASFPLDLNKWSVFYTAVIPMLNPLIYSLRNKEVKD 299
40 0 Query: 920 gL 925
1 + Sbjct: 300 Al 301
45 The GPCR5a amino acid has 154 of 306 amino acid residues (55%) identical to, and
199 of 306 residues (64%) positive with, the 309 amino acid M72 from Mus musculus (GENBANK-ID: AAG09870.1) (SEO ID NO:53) (Table 51). The residue that differs between GPCR5a, GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol.
Table 5L-BLASTX of GPCR5a against M72
50 >gb | AAG09780. 1 | AF247656_l (AF247656 ) odorant receptor M72 [Mus musculus]
( SEQ ID NO : 53 ) Length = 309
Score = 294 bits (752), Expect = le-78
Identities = 161/306 (53%), Positives = 206/306 (68%), Gaps = 2/306 (0%) _ o 2- Query: 1 MgJNTNGSAITEFILLGLTDCPELQSLLFVLFLWYLVTLLGNLGMIMLMRLDSRLHTPMY 60
1+ I I +1 II II I 11+ llll I III 1 II III I II 1+ l+l +l lllll Sbjct: 1 MAAENQSTVTEFILRGLTNRPELQLPLLLLFLGIYIVTMVGNLGMITLIGLNSQLHTPMY 60
Query: 61 FFLTNLAFVDLCYTSNATPQMSTNIVSEKT-ISFAGCFTQCYIFIALLLTEFYMLAAMAY 119 lll + l 1+ I I I I l + l ll + l I II++ 11+ II +1 I 1+ ++ I II I III Sbjct: 61 FFLSNLSLVDLCYSSVITPKMLINFVSQRNLISYVGCMSQLYFFLVFVIAECYMLTVMAY 120 o Query: 120 DRYVAIYDPLRYSVKTSRRVCICLATFPYvYGFSDGLFQAILTFRLTFCRS|J]VINHFYCA 179
111 I I I I I I++ I +1 I I I 1 + I +1 +1 ++I + I++I Sbjct: 121 DRYVAICQPLLYNIIMSPALCSLLWFVYAMGLIGSTIETSLMLKLNYCE-DLISHYFCD 179
10 o Query: 180 DPPLIKLSCSDTYVKEHAMFISAGFNLSSSLTIVLVSYAFILAAILRIKSgEGRHKAFST 239
I l + ll II I I I l l + l III 1+ + ll + l llll I++I III I III lllll Sbjct: 180 ILPLMKLSCSSTYDIEMAVFFLAGFNIIVTSLTVLISYAFILSSILRISSNEGRSKAFST 239 o
15 Query: 240 CGSHMMAVTLFYGTLFCMYIRPPTDKTVEESKIIAVFYTFVSPVLNPLIYSLRNKDVKQS 299
I II II I 111+ I I++I I ++ + + +1 I I I 1 1+ I II llll I I l + ll I Sbjct: 240 CSSHFAAVGLFYGSTAFMYLKPSTASSLAQENVASVFYTTVIPMFNPLIYSLRNKEVKTA 299
Query: 300 LKNVLR 305
20 I I I Sbjct: 300 LDKTLR 305
The GPCR5a amino acid has 148 of 301 amino acid residues (49%) identical to, and 198 of 301 residues (65%) positive with, the 308 amino acid K42 from M S musCulus 25 (GENBANK-ID:AAG39876.1) (SEO ID NO:53) (Table 51). The residue that differs between GPCR5a, GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol.
Table 5J.-BLASTX of GPCR5a against K42
>gb|AAG39876.1|AF282291_l (AF282291) odorant receptor K42 [Mus musculus] (SEQ ID NO: 53) Length = 308 2U Score = 293 bits (751), Expect = lβ-78
Identities = 153/301 (51%), Positives = 203/301 (67%), Gaps = 1/301 (0%)
Query: 5 NGSAITEFILLGLTDCPELQSLLFVLFLWYLVTLLGNLGMIMLMRLDΞRLHTPMYFFLT 64
,, I I++I+I ll lll llll ll lll lllll 1+1+ + l+l l+l 11+11+
22. Sbjct: 2 NHSSVTDFILEGLTKRPELQLPLFLLFLGIHVITVVGNLGMILLINISSQLHSPMYYFLS 61
Query: 65 NLAFVDLCYTSNATPQMSTNIVSEK-TISFAGCFTQCYIFIALLLTEFYMLAAMAYDRYV 123
+ 1 + 1 + 1 l l l + l l l + l I I l l l l l I I I I 1 + I ++ I l + l I I I I I I I I
■π Sbjct: 62 HLSFIDLCYSSVITPKMLVNFVCAKNTISFKECMTQLYFFLLLAISEGYLLTAMAYDRYV 121
Query: 124 AIYDPLRYSVKTSRRVCICLATFPYVYGFSDGLFQAILTFRLTFCRSUJVINHFYCADPPL 183
II 11 1+ l +ll + I II I + II I ++ I I ++ I II Sbjct: 122 AICSPLLYNTVMSHKVCSIMMAVVYSLGFFGATVHTTRMTMLSFCGSHIIRHYFCDILPL 181
. - o
H Query: 184 IKLSCSDTYVKEHAMFISAGFNLSSSLTIVLVSYAFILAAILRIKSgEGRHKAFSTCGSH 243 + I I II I++ I +11 I I + I++II llll +1 I I l + l III III II II Sbjct: 182 LTLSCSSTHINEVLLFIIGGVNTLAPTLAVIISYAFILTSILRIRSNEGRSKAFGTCSSH 241 o Query: 244 MMAVTLFYGTLFCMYIRPPTDKTVEESKIIAVFYTFVSPVLNPLIYSLRNKDVKQgLKNV 303
50 + 1 I I +I + I++ 11 +11+ +1+ 1+ +1111 I l + l I I II I II I II I I I III + Sbjct: 242 IMAVGIFFGSITFMYFKPPSSNNMEQEKVSSVFYTTVIPMLNPLIYSLRNKDVKTALKM 301
Query: 304 L 304
+
55 Sbjct: 302 V 302
The GPCR5b amino acid sequence has 153 of 306 amino acid residues (50%) identical to. and 198 of 306 residues (64%) positive with, the 309 amino acid M71 from MUS musculus
(GENBANK-ID:AAG29379.1) (SEO ID NO:54) (Table 5K). The residue that differs between
60 GPCR5a. GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol. Table 5K. BLASTX of GPCR5b against M71
>gb|AAG29379.1|AF281061_l (AF281061) odorant receptor M71 [Mus musculus] (SEQ ID NO: 54) Length = 309 Score = 290 bits (743), Expect = le-77
Identities = 161/306 (53%), Positives = 206/306 (67%), Gaps = 2/306 (0%) o Query: 1 MgNTNGSAITEFILLGLTDCPELQSLLFVLFLWYLVTLLGNLGMIMLMRLDSRLHTPMY 60
1+ I I +1 I I II II 1+ II II ll lll I II 111111 1+ l + l + ll llll Sbjct: 1 MTAENQSTVTEFILGGLTNRPELQLPLFLLFLGIYWTMVGNLGMITLIGLNSQLHTPMY 60
10
Query: 61 FFLTNLAFVDLCYTSNATPQMSTNIVSEKT-ISFAGCFTQCYIFIALLLTEFYMLAAMAY 119
II l + l 1+ II I I l + l ll + l I I I++ I 1+ II +1 I 1+ ++ I I II III Sbjct: 61 FFLSNLSLVDLCYSSVITPKMLINFVSQRNLISYVGCMSQLYFFLVFVIAECYMLTVMAY 120 Query: 120 DRYVAIYDPLRYSVKTSRRVCICLATFPYVYGFSDGLFQAILTFRLTFCRS@°VINHFYCA 179 II I ++ I +1 I I I I + I +1 +1 + I + I ++ I Sbjct: 121 DRYVAICQPLLYNIIMSPALCSLLVAFVYAVGLIGSAIETGLMLKLNYCED-LISHYFCD 179
0 Query: 180 DPPLIKLSCSDTYVKEHAMFISAGFNLSSSLTIVLVSYAFILAAILRIKSgEGRHKAFST 239
20 ll + l I I I I II l l + l I I I++ + ll + l II I I1++1111 I III lllll Sbjct: 180 ILPLMKLSCSSTYDVEMAVFFLAGFDIIVTSLTVLISYAFILSSILRISSNEGRSKAFST 239 o Query: 240 CGSHMMAVTLFYGTLFCMYIRPPTDKTVEESKIIAVFYTFVSPVLNPLIYSLRNKDVKQgJ 299
25 I II I I II I 1+ I I++I I ++ + + +1 II I 1 1+ I III I I II 11 + 11 I Sbjct: 240 CSSHFAAVGLFYGSTAFMYLKPSTASSLAQENVASVFYTTVIPMFNPLIYSLRNKEVKTA 299
Query: 300 LKNVLR 305
I II Sbjct: 300 LDKTLR 305
30
A multiple sequence alignment is given in Table 5L. with GPCR5a being shown on line 1. in a ClustalW analysis comparing GPCR5a with related protein sequences.
Table 5L-Information for the ClustalW proteins:
1 Novel HumanjDLF, GPCR5a, SEQ ID NO: 14
35 2. Homo sapiens OLF, S ISSPROT-Acc # Q13606, SEQ ID NO: 33
3. Hyloba tes lar (Common Gibbon) OLF, SPTREMBL-Acc # 077758, SEQ ID NO: 43
4. .Rattus norvegicus OLF, SPTREMBL-Acc # Q63394, SEQ ID NO: 55 o
GPCR5a — lOTlTJSGSAl^-taUMd TJgjCld-lMs LtWgvi-raivvHl.yliiπiLSSlLiaOTiMBMfdi-ltMdit.W^
40 HUMANJDLF MEFTD l^TLgggroiiiBFP iag^IVl^LMtSiTL^piHIl^lE f-lΪHgiiglPHBQBg GIBBONJDLF - -l8lAiSlElSHτκ^-iιtalali<FT@LNYNi3Q^VF3 LWaW FHvpsvτS5lF^lgiivBπS|M|^tj5M-l RATJDLF - -ggV JJJES I SRJSgHEFSig&gE PJEvvgjVsiilLE.I F@S]MMigjVSi23JgKJEBB
45
Figure imgf000042_0001
GPCR5a W d: WΛ<YD^RE -^κτ|5R^5iicBEITFtg^γl3^SD HlF0AiLτt5aiτiiBRSlitf- ^)i>jY
50 HUMANJDLF >Λ' i^ W. <»tttd*^ πV!^RGlBMRBlΞ SB G@HMS^VHiSSFAi lBKYBDK[^i ^iJ3 GIBBONJDLF ^E3g^E33jSH^Fi^QlSLfflQBvQGi^ii ]S MTHiEN SJ2 [a^ |jS232333 RATjDLF A JjSFLgERSJHB^l HQi g H[EAAC Isg|SJJgVLQSgWTLQM@ ggHKEVD[ g@
GPCR5a gADf331ιiaW-Sls[SSYVK|gHg iBs^FNB|S Sg gB fflv|^AahMAHll-bHκigEli5BigHia l3
55 HUMANJDLF ^Lg33Jgj^TJ^l|J@ L[JSTYGSS0EI0CFlEHl^gF[3HLSv|κS^FSg3 iτ GIBBONJDLF g^S[^Sgvg J^R JSK ^JΪ^ggAgGVFgGLT^^^IYSjMgl3R|3^Dg3ciτ|3 RAT OLF BEviaΑi-nraΪRgvlBiffiiΑfelAEWaFi svfflFLLilpyliilLWbfc VilaHvoEllvi-iKHtWAalcigRiSAra
GPCR5 a feliWeMilMMBroil ia elnlBgcMHτiaaPT DKTVEE sj^IA-gSiiilFfflsjgvπrcidftifeMdMgDBJg
60 HUMAN_0LF fSBBJAia^S lYOE^LfilHSiagSY YiaPNTDlffll^^
GIBBONJDLF l^sMitft^FILHSiai lE vfgaSASFP^
RATJDLF lllfrf^HllV^Lfaπ^IYM^LO^P PS[-|K^RG[ Wf^L|^GpiT|gM>*i>dftCτ[BπSlEE-Sg
Figure imgf000042_0002
HUMANJDLF
GIBBONJDLF
RATJDLF
Figure imgf000043_0001
The presence of identifiable domains in GPCR5 was determined by searches using algorithms such as PROSITE. Blocks, Pfam. ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The results indicate that this protein contains the following protein domains (as defined by Interpro) at the indicated positions: domain name 7tm 1 (InterPro) 7 transmembrane receptor (rhodopsin family) at amino acid positions 41 to 289. This indicates that the sequence of GPCR5 has properties similar to those of other proteins known to contain this/these domain(s) and similar to the properties of these domains.
DOMAIN results for GPCR5a were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 5M with the statistics and domain description. Residues 1-180 (SEO ID NO:29) and residues 313-377 (SEO ID NO:37) of 7tm 1 are aligned with GPCR4 in Table 40. The residue that differs between GPCR5a, GPCR5b and GPCR5c are highlighted in black and marked with the (o) symbol.
Table 5M.-DOMAIN results for GPCR5a.
Sbjct: 7 transmembrane receptor (rhodopsin family) fragment (SEQ ID NO: 29) gnl 1 PfamlpfamOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family), 377 aa Statistics for GPCR5a: Score = 92.8 bits (229), Expect = 2e-20 Statistics for GPCR5b: Score = 92.8 bits (229), Expect = 2e-20
Query: 41 GNLGMIMLMRLDSRLHTPMYFFLTNLAFVDLCYTSNATPQMSTNIVS-EKTISFAGCFTQ 99
Sbjct: 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYLEWGE KFSRIHCDIF 60 **. . * . . * * . • .** ** . * . * * * *
Query: 100 CYIFIALLLTEFYMLAAMAYDRYVAIYDPLRYS-VKTSRRVCICLATFPYVYGFSDGLFQ 158
Sbjct: 61 VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPM 120
Query : 159 AILTFRLTFCRSJSVINHFYCADPPLIKLSCSDTYVKEHAMFISAGFNLSSSLTIVLVSYA 218 Sbj ct : 121 LFGLNNTDQN- -ECIIANPAFWYSSIVSFYV — PFIVTLLVYI 160
*
Query: 219 FIL-AAILRIKSgjEGRHKAF 237 Sbjct: 161 KIYIVLRRRRKRVNTKRSSR 180
Sbjct: 7 transmembrane receptor (rhodopsin family) fragment (SEQ ID NO: 37) gnl I PfamlpfamOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family), 377 aa Statistics for GPCR5a: Score = 35.8 bits (81), Expect = 0.003 Statistics for GPCR5b: Score = 35.8 bits (81), Expect = 0.003 o Query: 226 RIKSgEGRHKAFSTCGSHMMAVTLFYGTLFCMYIRP-PTDKTVEESKIIAVFYTFVSPVL 284 Sbjct: 313 KLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYSAFT LGYVNSAV 372
Query: 285 NPLIY 289 Sbjct: 373 NPIIY 377 GPCR5 is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neuro genie cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue. Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to Public EST sources. Literature sources, and/or RACE sources.
In the following positions, one or more consensus positions of GPCR5 have been identified as single nucleotide polymorphisms ("SNPs"). As shown in Table 5N. "Depth" represents the number of clones covering the region ofthe SNP. The Putative Allele Frequency (Putative AUele Freq.) is the fraction of all the clones containing the SNP. A dash ("-"). when shown, means that a base is not present. The sign ">" means "is changed to".
Table 5N: GPCR5 Single Nucleotide Polymorphisms
Figure imgf000044_0001
The protein similarity information, expression pattern, and map location for the GPCR5 protein and nucleic acid disclosed herein suggest that GPCR5 may have important structural and/or physiological functions characteristic ofthe Olfactory Receptor family and the GPCR family. Therefore, the nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody). (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
The nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various GPCR- or OR-related diseases and disorders described below and/or other pathologies. For example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from: developmental diseases. MHCπ and UI diseases (immune diseases). Taste and scent detectability Disorders. Burkitt's lymphoma. Corticoneuro enie disease. Signal Transduction pathway disorders. Retinal diseases including those involving photoreception. Cell Growth rate disorders: Cell Shape disorders. Feeding disorders;control of feeding: potential obesity due to over-eating: potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (MDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2). pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma: prostate cancer: uterus cancer), anorexia, bulimia, asthma. Parkinson's disease. acute heart failure, hypotension, hypertension, urinary retention, osteoporosis. Crohn's disease: multiple sclerosis: Albright Hereditary Ostoeodystrophy, angina pectoris. myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation. Dentatorubro-pallidoluysian atrophy (DRPLA) Hvpophosphatemic rickets, autosomal dominant (2) Acrocallosal syndrome and dvskinesias. such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders ofthe like. The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDΝA encoding GPCR5 may be useful in gene therapy, and GPCR5 may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2). pain, cancer (including but not limited to Neoplasm: adenocarcinoma: lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis. Crohn's disease; multiple sclerosis: and Treatment of Albright Hereditary Ostoeodystrophy. angina pectoris. myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. The novel nucleic acid encoding GPCR5, and the GPCR5 protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods and other diseases, disorders and conditions ofthe like. Other GPCR-related diseases and disorders are contemplated.
These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. In one embodiment, a contemplated epitope of GPCR5c would be from amino acid 10 to 50. In another embodiment, a contemplated epitope of GPCR5c would be from amino acid 35 to 45. In yet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 80 to 120. In vet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 135 to 160. In vet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 205 to 235. In yet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 245 to 260. In yet another embodiment, a contemplated epitope of GPCR5c would be from amino acid 275 to 290.
GPCR6
The novel nucleic acid of 1050 nucleotides GPCR6 (also designated APOOl 112 D) encoding a novel Olfactory Receptor-like protein is shown in Table 6A. An ORF was identified beginning with an ATG initiation codon at nucleotides 53-55 and ending with a TAA codon at nucleotides 1007-1009. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
Table 6A GPCR6 Nucleotide Sequence (SEQ ID NO:19)
TGTAATGGACTTCTCATTCACCTTGATTTATTTTCATCCATTTAAGTGAAAAATGTTGGTACCTAAGAAAATGGTTAGAGGAAA TTCTACTTTGGTGACGGAATTTATTCTCTTGGGATTAAAGGATCTTCCAGAGCTTCAGCCCATCCTCTTTGTACTGTTCCTGCT AATCTACCTGATCACTGTCGGGGGGAACCTTGGGATGTTGGTGTTGATCAGGATAGATTCACGCCTCCACACCCCCATGTATTT CTTTCTTGCTAGTTTGTCCTGCTTGGATTTGTATTACTCCACTAATGTGACTCCCAAGATGTTGGTGAACTTCTTCTCAGACAA GAAAGCCATTTCCTATGCTGCTTGTTTAGTCCAGTGCTATTTTTTCATTGCTGTGGTGATTACTGAATATTATATGCTAGCTGT AATGGCCTATGATAGGTATGTGGCCATCTGTAACCCTTTGCTTTACAGCAGCAAGATGTCCAAAGGGCTCTGTATTCGCCTGAT TGCTGGTCCATATGTCTATGGGTTTCTTAGTGGACTGATGGAAACCATGTGGACATACCACTTGACCTTCTGTGGCTCCAATAT CATTAATCACTTCTACTGTGCTGACCCACCCCTCATCCGACTTTCCTGCTCTGACACTTTCATTAAGGAAACATCCATGTTTGT GGTAGCATGOTTTAACCTCTCCAGCTCCCTCATCATAATCCTCATCTCCTACATCTTCATTCTCATTGCCATCCTGAGGATGCG TTCTGCTGAAAGTAGGCGCAAAGCGTTCTCCACCTGCGGGTCCCACCTGGTGGCAGTGACTGTGTTTTATGGAACCCTGTTCTG CATGTACGTTAGACCTCCCACGGACAGGTCAGTGGAACAGTCCAAAGTCATTGCTGTTTTCTACACTTTTGTAAGCCCTATGTT GAACCCCATCATCTATAGTTTGAGGAACAAGGATGTGAAACAAGCTTTTTGGAAACTGATCAGAAGAAACGTGCTTTTGAAGTA AAATCAGTGTATCTTTATTAGTCAAATAAAAAAATCTTTCTA
T - sequence change from A to T to correct a stop codon.
The GPCR6 protein encoded by SEO ID NO: 19 has 318 amino acid residues and is presented using the one-letter code in Table 6B.
Table 6B. Encoded GPCR6 protein sequence (SEQ ID NO:20).
MLVPKKMVRGNSTLVTEFILLGLKDLPELQPILFVLFLLIYLITVGGNLGMLVLIRIDSRLHTPMYFFLASLSCLDLYYSTNVT PKMLVNFFSDKKAISYAACLVQCYFFIAVVITEYYMLAVMAYDRYVAICNPLLYSSKMSKGLCIRLIAGPYVYGFLSGLMETM TYHLTFCGSNIINHFYCADPPLIRLSCSDTFIKETSMFVVACFNLSSSLIIILISYIFILIAILRMRSAESRRKAFSTCGSHLV AVTVFYGTLFCMYVRPPTDRSVEQSKVIAVFYTFVSPMLNPIIYSLRNKDVKQAFWKLIRRNVLLK
In a search of sequence databases, it was found, for example, that the GPCR6 nucleic acid sequence has 621 of 939 bases (66 %) identical to a and 621 of 939 bases (66%) positive with GaUus gauus species Olfactory Receptor 2 (GENBANK-ID: X94742) (Table 6C).
Table 6C. BLASTN of GPCR6 against OR2 (SEQ ID NO:51)
>gb:GENBANK-ID:GGC0R2GEN|acc:X94742 G. gallus cor2 DNA for olfactory receptor 2
Gallus gallus, (SEQ ID NO: 51) 996 bp.
Score = 1539 (230.9 bits), Expect = 1.8e-63, P = 1.8e-63
Identities = 621/939 (66%), Positives = 621/939 (66%), Strand = Plus / Plus
Query: 67 GAAAATGGTTAGAGGAAATTCTACTTTGGTGACGGAATTTATTCTCTTGGGATTAAAGGA 126
II I II I I I I 1 I I 111 II I I I III 1 I II II I II Sbjct: 54 GATGATGGCCAAGGGAAATCACAGCTCCATCACTGAATTTGTGCTCTTGGGATTCTCTGA 113
Query: 127 TCTTCCAGAGCTTCAGCCCATCCTCTTTGTACTGTTCCTGCTAATCTACCTGATCACTGT 186
I l lll l l l I I I I II I III I I I I I I I 1 I III I Sbjct: 114 AAAGAGGGCCATCCAGGCTGTTCTCTTTATGGGCTTCTTGCTGATCTACCTGATCACTCT 173
Query: 187 CGGGGGGAACCTTGGGATGTTGGTGTTGATCAGGATAGATTCACGCCTCCACACCCCCAT 246
II I I l ll lll l II I II II I I II II I I I III I I Sbjct: 174 GCTAGGCAATGTGGGCATGATCACATTGATCAGGCTGGACTCCCGGCTTCACACCCCTAT 233
Query: 247 GTATTTCTTTCTT-GCTAGTTTGTCCTGCTTGGAT-T-TG-TATTACTCCACTAATGTGA 302
III I I I II II II II I I III I II llll 111 1
Sbjct: 234 GTACTTCTTCCTGAGC-AGCTTGTCCTTCCTCGATATCTGCTATTCCTCCAC-AATC—A 289
Query: 303 CTCC-CAAGATGT,TGGTGA-ACTTCTTCTCAGACA-AGAAAGCCATTTCCTATGCTGCTT 359
I II I 1 I I II I l l ll ll II II II III I II II II I I llll l Sbjct: 290 CTCCTCGAG-TGCTC-TCAGACCTCCCAGCAT-CACAGAAAGTCATTTCCCACTCTGCAT 346
Query: 360 GTTTAGTCCAGTGCTATTTTTTCATTG-CTGTGGTGATTACTGAATATTATATGCTAGCT 418
1 I I llll lllll l l II II I I I ll ll l lll l I II Sbjct: 347 GCCTGGCACAGTTTTATTTCTACGCTGTCTTTGCC-ACCACAGAGTGCTATCTTTTGGCC 405
Query: 419 GTAATGGCCTATGATAGGTATGTGGCCATCTGTAACCCTTTGCTTTACAGCAGCAAGATG 478
I I I II I I I llll I II II llll lllll I 111 I II I I I I I I 111 Sbjct: 406 GCAATGGCATATGACCGCTACGTGGCCATCTGCAGCCCTCTGCTCTATGTCTTCTCCATG 465
Query: 479 TCCAA-AGGGCTCTGTATTCGCCTGATTGCTGGTCCATATGTCTAT-GGGTTTCTTAGTG 536 llll ll l l lll l l II I I llll II llll I I I II I I I I I I I Sbjct: 466 TCCAGCAGAGTT-TGTGTGCTGCTGGTTGCTGGCTCATACCT-TGTCGGGGTTGTGAATG 523
Query: 537 G-ACTGATGGAAACCATG-TGGACATACCACTTGACCTTCTGTGGCTCCAATATCATTAA 594
II II I II I I I I I II II llll I I I III I II I I llll I I ' Sbjct: 524 CCACC-ATTCACACAGGGCTTG-CACTGCAGCTGTCCTTCTGTGGTCCCAACATCATCAA 581 Query: 595 TCACTTCTACTGTG-CTGACCCACCCCTCATCCGACTT-TCCTGCTCTGACACTTTCATT 652
II I II II I llll II I I III II 111 I II I I II III 1 III I II Sbjct: 582 TCACTTCTACTGTGACGGTCCC-CCGCTC-TACGCCATCTCGTGCACAGACCCCACCACC 639
Query: 653 AAGGAAACATCCATGTTTGTGGTAGCATGATTTAACCT-CTCCAGCTCCCTCATCATAAT 711
I I I I I I I I I I I I I I I I II III l lll l l II I I I II Sbjct: 640 AACGAGATTGCGATATTTCTTGTGGTTGGCTTCAACATGCTC-ATCACCAGCGTGACCAT 698
Query: 712 CCTCATCTCCTACATCTTCATTCTCATTGCCATCCTGAGGATGCGTTCTGCTGAAAGTAG 771
10 I I II I I I I I I II I II II I II I II II I I l llll l l l Sbjct: 699 CTTCATCTCCTACACCTACATCCTGTTCGCTGTCCTCAGGATGCACACAGCTGCAGGCAA 758
Query: 772 GCGCAAAGCGTTCTCCACCTGCGGGTCCCACCTGGTGGCAGTGACTGTGTTTTATGGAAC 831
15 llllll I II I l ll ll I II I Ml I Sbjct: 759 ACGCAAAACCTTCTCCACGTGTGCGTCCCACCTGGCCACCGTCACCCTATTCTATGCCTC 818
Query: 832 C-CTGTTCTGCATGTACGTTA—GACCTCCCACGGACAGGTCAGTGGAACAGTCCAAAGT 888
I II I I I I I II I l l ll II lll l l lll ll Sbjct: 819 TGCTGGT-TCCATGTAC-TCACGGCCCAGCTCCAGGCAC-TCCCAGGACCTGGACAAGGT 875
20
Query: 889 CATTGCTGTTTTCTACACTTTTGTAAGCCCTATGTTGAACCCCATCATCTATAGTTTGAG 948
II I I I II I III III llll 111 I II llll Sbjct: 876 GGCCTCTGTGTTCTACACCATGGTGACCCCCATGCTGAACCCCCTCATCTACAGCCTGAG 935
25 Query: 949 GAACAAGGATGTGAAACAAGCTTTTTGGAAACTGATCAGAAGAAAC-GTGCTTTTGA 1004
II I I I I II II II l l lll lllll llll l ll ll III l lll Sbjct: 936 GAACCAGGAGGTAAAGGATGTTTTAGGGAAAGTGATGGGGAGGAAGAGTGTCTCTGA 992
The GPCR6 amino acid has 165 of 307 amino acid residues (53 %) identical to, and 30 226 of 307 residues (73%) positive with, the 312 amino acid OR4 from Gallus gallus (ptnr: SPTREMBL-ACC: 077756) (SEO ID NO:56) (Table 6D).
Table 6D. BLASTX of GPCR6 against OR4
>ptnr:SPTREMBL-ACC:Q90808 OLFACTORY RECEPTOR 4 - Gallus gallus (Chicken), 312 aa (fragment). (SEQ ID NO:56) 22. Score = 867 (305.2 bits). Expect = 7.0e-86, P = 7.0e-86
Identities = 165/307 (53%), Positives = 226/307 (73%), Frame = +2
Query: 71 MVRGNSTLVTEFILLGLKDLPELQPILFVLFLLIYLITVGGNLGMLVLIRIDSRLHTPMY 250
40 I II I I +1 II l + ll I I+++ I ll + l II I l + ll II II+++II+ I llll II Sbjct: 1 MAEGNHTLASEFILVGLSDHPKMKAALFVVFLLIYVITFQGNLGIIILIQGDPRLHTSMY 60 Query: 251 FFLASLSCLDLYYSTNVTPKMLVNFFSDKKAISYAACLVQCYFFIAWITEYYMLAVMAY 430 lll+lll +1+ +1+ + |+ llll 1+++ 11+ I I +1+1 I II Sbjct: 61 FFLSSLSVVDICFSSVIAPRTLVNFLSERRTISFTGCTGQTFFYIVFVTTECFLLAVMAY 120
45 Query: 431 DRYVAICNPLLYSSKMSKGLCIRLIAGPYVYGFLSGLMETMWTYHLTFCGSNIINHFYCA 610
1111111111111+ 1++ 1++I+ 1 1+ 1 1+ +++I + I lllll I llll+l Sbjct: 121 DRYVAICNPLLYSTIMTRRQCMQLVVGSYIGGILNAIIQTTFIIRLPFCGSNIINHFFCD 180
50 Query: 611 DPPLIRLSCSDTFIKETSMFVVA*FNLSSSLIIILISYIFILIAILRMRSAESRRKAFST 790
I I 1+ I I + l + l I +1 +1 I++ ll + lll I I I I I l + l II I l + ll I I Sbjct: 181 VPPLLALSLAΞTYISEMILFSLAGIIELSTVTSILVSYIFISCAILRIRSAEGRQKALST 240 Query: 791 CGSHLVAVTVFYGTLFCMYVRPPTDRSVEQSKVIAVFYTFVSPMLNPIIYSLRNKDVKQA 970
55 I II I I 11+ I I I l + l I + 1+ I I++I III I II II l + l II I I I++II I Sbjct: 241 CASHLTAVTLLYGTTIFTYLRPSSSYSLNTDKVVSVFYTWIPMLNPLIYSLRNQEVKGA 300 Query: 971 F KLIRR 991
+++ I
60 Sbj ct : 301 LSRWER 307 The GPCR6 amino acid has 153 of 313 amino acid residues (48%) identical to, and 199 of 313 residues (62%) positive with, the 314 amino acid OR93Ch from p f] troglodytes OR93Ch (GENBANK-ID:AAC63969.1) (SEO ID NO:57) (Table 6E).
Table 6E.-BLASTX of GPCR6 against OR93Ch
>gb|AAC63969.1| (AF045577) olfactory receptor OR93Ch [Pan troglodytes] (SEQ ID NO: 57) Length = 314
Score = 293 bits (749), Expect = 2e-78
Identities = 169/313 (54%), Positives = 215/313 (69%), Gaps = 1/313 (0%)
10 Query: 7 MVRGNSTLVTEFILLGLKDLPELQPILFVLFLLIYLI-TVGGNLGMLVLIRIDSRLHTPM 65
I l l lllll II l + l |. ll lll I I II I l + ll I I I I II I I I II Sbjct: 1 MANENYTKVTEFIFTGLNYNPQLQVFLFLLFLTTFYVINVTGNLGMIVLIRIDSRLHTPM 60
Query: 66 YFFLASLSCLDLYYSTNVTPKMLVNFFSDKKAISYAACLVQCYFFIAWITEYYMLAVMA 125
15 I 111+ II +1+ +1+ l + l II 1 +11 +1111+ I +1 +11 I I ++I I II Sbjct: 61 YFFLSHLSFVDICFSSWSPKMLTDFFVKRKAISFLGCALQQWFFGFFVAAECFLLASMA 120
Query: 126 YDRYVAICNPLLYSSKMSKGLCIRLIAGPYVYGFLSGLMETM TYHLTFCGSNIINHFYC 185- 0 I lllll I II I I I I I 11+ II 1 + 1+ llll 1 ++ + I + I I I I l + l II l + l Sbjct: 121 YDRYVAICNPLLYSVAMSQRLCIQLVVGPYVIGLMNTMTHTTNAFRLPFCGPNVINHFFC 180
Query: 186 ADPPLIRLSCSDTFIKETSMFWACFNLSSSLIIILISYIFILIAILRMRSAESRRKAFS 245 ll+ l l + ll + + ++I + U I llllll II I I ll + l I 1+ I I II Sbjct: 181 DMSPLLSLVCADTRLNKLAVFIVAGAAGVFSGLTILISYIYILMAILRIRSADGRCKTFS 240 5
Query: 246 TCGSHLVAVTVFYGTLFCMYVRPPTDRSVEQSKVIAVFYTFVSPMLNPIIYSLRNKDVKQ 305
II 111 II + I III I +1111 I++ +I+++III I I I I I ll + l I 111 I l + l 1 Sbjct: 241 TCSSHLTAVFILYGTLFFIYVRPSASFSLDLNKLVSVFYTAVIPMLNPLIYSLRNKEVKD 300 0 Query: 306 AFWKLIRRNVLL 318
I + + + I Sbjct: 301 AIHRTVTQRKFC 313
The GPCR6 amino acid has 150 of 312 amino acid residues (48%) identical to. and 5 198 of 312 residues (63%) positive with, the 313 amino acid OR93Gib from Hylobates lar (GENBANK-ID:AAC63971.1) (SEO ID NO:58) (Table 6F).
Table 6F.-BLASTX of GPCR6 against OR93Gib
>gb|AAC63971.1| (AF045580) olfactory receptor 0R93Gib [Hyloba tes lar] (SEQ ID NO: 58) Length = 313 0 Score = 291 bits (745), Expect = 7e-78
Identities = 168/312 (54%), Positives = 216/312 (69%)
Query: 7 MVRGNSTLVTEFILLGLKDLPELQPILFVLFLLIYLITVGGNLGMLVLIRIDSRLHTPMY 66 ,<- I l l lllll II l + ll ll lll I l + l II I l + lll l + lll I I I III Sbjct: 1 MANENYTKVTEFIFTGLNYNPQLQVFLFLLFLTFYVISVTGNFGMIVLIRMDSRLHTPMY 60
Query: 67 FFLASLSCLDLYYSTNVTPKMLVNFFSDKKAISYAACLVQCYFFIAWITEYYMLAVMAY 126
111+ II +1+ +1+ l + lll I +11 +1111+ I +1 +1 I I I ++I1 III Sbjct: 61 FFLSHLSFVDICFSΞVVSPKMLTDFFVKRKAISFLGCALQQ FFGFFVAAECFLLASMAY 120 0
Query: 127 DRYVAICNPLLYSSKMSKGLCIRLIAGPYVYGFLSGLMETM TYHLTFCGSNIINHFYCA 186 lllll I III I I I I ll+ l 11 + 1+ llll I ++ + I + I II I l + ll ll + l Sbjct: 121 DRYVAICNPLLYSVFMSQRLCIQLVVGPYVIGLMNTMTHTTNAFRLPFCGLNVINHFFCD 180 5 Query: 187 DPPLIRLSCSDTFIKETSMFWACFNLSSSLIIILISYIFILIAILRMRSAESRRKAFST 246 ll+ l l + l I + + ++I++I I lllll II I I I l + ll 1+ I I III Sbjct: 181 MSPLLSLVCADTRLNKLAVFIMAGAVGVFSGLTILISYIYILMAILRIRSADGRCKTFST 240 Query: 247 CGSHLVAVTVFYGTLFCMYVRPPTDRSVEQSKVIAVFYTFVSPMLNPIIYSLRNKDVKQA 306
I II I I I + I I I I I +1 III ++ +II++III I I II I I l + ll I I I ll + l I I Sbjct: 241 CSSHLTAVFILYGTLFFIYVRPSASFPLDLNKWSVFYTAVIPMLNPLIYSLRNKEVKDA 300 . Query: 307 FWKLIRRNVLLK 318
+ + + I Sbjct: 301 IHRTVTQRKFCK 312
The GPCR6 amino acid has 143 of 307 amino acid residues (46%) identical to, and 0 193 of 307 residues (62%) positive with, the 332 amino acid OR2 from Gallus gallus (embCAA64368.1) (SEO ID NO:59) (Table 6G).
Table 6G.-BLASTX of GPCR6 against OR2
>emb I CAA64368.il (X94742) olfactory receptor 2 [ Gallus gallus] (SEQ ID O:59), 332 aa 5 Score = 290 bits (743), Expect = le-77
Identities = 160/307 (52%), Positives = 210/307 (68%)
Query: 7 MVRGNSTLVTEFILLGLKDLPELQPILFVLFLLIYLITVGGNLGMLVLIRIDSRLHTPMY 66 0 I +11 + +11 l + lll + +1 II llll 1111+ ll + l 1+ II l + l II 1 lllll Sbjct: 20 MAKGNHSSITEFVLLGFSEKRAIQAVLFMGFLLIYLITLLGNVGMITLIRLDSRLHTPMY 79
Query: 67 FFLASLSCLDLYYSTNVTP MLVNFFSDKKAISYAACLVQCYFFIAWITEYYMLAVMAY 126 lll + l I I 11+ I 1+ +1 I++I + + +1 II++I I I I 11+ I I l + l I III
Sbjct: 80 FFLSSLSFLDICYSSTITPRVLSDLPASQKVISHSACLAQFYFYAVFATTECYLLAAMAY 139 5 Query: 127 DRYVAICNPLLYSSKMSKGLCIRLIAGPYVYGFLSGLMETM TYHLTFCGSNIINHFYCA 186 llllll l + llll II +1+ l + ll 1+ I ++ + I l + ll I III lllll
Sbjct: 140 DRYVAICSPLLYVFSMSSRVCVLLVAGSYLVGWNATIHTGLALQLSFCGPNIINHFYCD 199 0 Query: 187 DPPLIRLSCSDTFIKETSMFWACFNLSSSLIIILISYIFILIAILRMRSAESRRKAFST 246
III +1 l + l I ++I + I II I I I II II I I II +1 +1 I I II Sbjct: 200 GPPLYAISCTDPTTNEIAIFLWGFNMLITSVTIFISYTYILFAVLRMHTAAGKRKTFST 259
Query: 247 CGSHLVAVTVFYGTLFCMYVRPPTDRSVEQSKVIAVFYTFVSPMLNPIIYSLRNKDVKQA 306 5 l lll 11 + 11 + I I II + I + II +1 I II l + ll I ll + l I III I++II Sbjct: 260 CASHLATVTLFYASAGSMYSRPSSRHSQDLDKVASVFYTMVTPMLNPLIYSLRNQEVKDV 319
Query: 307 F KLIRR 313 0 I++ I Sbjct: 320 LGKVMGR 326
The GPCR6 amino acid has 150 of 311 amino acid residues (48%) identical to, and 193 of 311 residues (61%) positive with, the 311 amino acid K30 from jtøfj. musculus (GENBANK-ID:AAG39871.1) (SEO ID NO:60) (Table 6H). 5 Table 6H.-BLASTX of GPCR6 against K30
>gb|AAG39871.1|AF282286_l (AF282286) odorant receptor K30 [Mus musculus] (SEQ ID NO: 60) Length = 311 Score = 290 bits (743), Expect = le-77 Identities = 166/311 (53%), Positives = 206/311 (66%) 0
Query : 7 MVRGNSTLVTEFILLGLKDLPELQPILFVLFLLIYLITVGGNLGMLVLIRIDSRLHTPMY 66
I ++ I I + I I I I I I I I + I I I I l l l l l l l l l l l l l I ++ I I + I l l l l l l Sbj ct : 1 MLKGNLSEVTEFILAGLTNKPELQLPLFLLFLAIYWTWGNLGMIILILLSSHLHTPMY 60 5 Query : 67 FFLASLSCLDLYYSTNVTPKMLVNFFSDKKAISYAACLVQCYFFIAWITEYYMLAVMAY 126
+ 1 l + l I I +1 I I I + II I III I + I III 1+ I II 1+ I I +1 I lllll Sbjct: 61 YFLSSLSFIDLCQSTVIIPKMLVNFVTVKNIISYPECMTQLYFFVTFAIAECHMLAVMAY 120
Query: 127 DRYVAICNPLLYSSKMSKGLCIRLIAGPYVYGFLSGLMETMWTYHLTFCGSNIINHFYCA 186 I I I I I I I I I I I I++ I I +1 +1 1 1 + 1+ + 11 +1 + 1 I I++I Sbjct: 121 DRYVAICNPLLYNAVMSFQVCSSMIFGVYSIALIGATTHTVCMLRVNFCKANVINHYFCD 180
Query: 187 DPPLIRLSCSDTFIKETSMFWACFNLSSSLIIILISYIFILIAILRMRSAESRR AFST 246
II + I II II I I I + + 11 ll lllll I I +++ 1 I I I II I I Sbjct: 181 LFPLLELPCSDTFINEVWLCFSVFNIFIPTLTILTSYIFIIASILQIKSTEGRSKAFST 240
Query: 247 CGSHLVAVTVFYGTLFCMYVRPPTDRSVEQSKVIAVFYTFVSPMLNPIIYSLRNKDVKQA 306
10 I 11+ II +1 + 1 + 1 I I++I + I++I II +111 I I I I II l + l III I II I II I Sbjct: 241 CSSHISAVAIFFGSLAFMYLQPSSVSSMDQGKVSSVFYTIWPMLNPLIYSLRNKDVKVA 300
Query: 307 FWKLIRRNVLL 317
I I I Sbjct: 301 LNKFFERKFFL 311
15
The GPCR6 amino acid has 149 of 311 amino acid residues (47%) identical to, and 192 of 311 residues (60%) positive with, the 314 amino acid Kl 1 from Mils musculus (GENBANK-ID: AAG39856.1) (SEO ID NO:61) (Table 61).
Table 6I.-BLASTX of GPCR6 against Kll (SEQ ID NO:61)
20 >gb|AAG39856.1|AF282271_l (AF282271) odorant receptor Kll [Mus musculus] (SEQ ID NO: 61) , 314 aa
Score = 289 bits (739), Expect = 4e-77 Identities = 164/311 (53%), Positives = 207/311 (66%)
25 Query: 7 MVRGNSTLVTEFILLGLKDLPELQPILFVLFLLIYLITVGGNLGMLVLIRIDSRLHTPMY 66
I II I I I I 1 I I + 1 II I ll ll I I II I I I 1 I I ++ 1 I + I I II I I I Sbjct: 4 MTSGNYCTVTEFFLAGLSEKPELQLPLFFLFIGIYMITVAGNLGMIILIGLSSHLHTPMY 63
Query: 67 FFLASLSCLDLYYSTNVTPKMLVNFFSDKKAISYAACLVQCYFFIAVVITEYYMLAVMAY 126
30 + 11 + 1 I I +1 II I I llll II I ++I III 1+ I I I 1+ I I l + l I III Sbjct: 64 YFLSSLSFIDFCQSTVVTPKMLVNFVTEKNIISYPGCMTQLYFFLIFAIAECYILAAMAY 123
Query: 127 DRYVAICNPLLYSSKMSKGLCIRLIAGPYVYGFLSGLMETM TYHLTFCGSNIINHFYCA 186
35 111111111111+ II + I ll+l 1+ I + I + + 11 ++I I I++I Sbjct: 124 DRYVAICNPLLYNVTMSYQIYIFLISGVYIIGVICASAHTGFMVRIRFCKLDVINHYFCD 183
Query: 187 DPPLIRLSCSDTFIKETSMFVVACFNLSSSLIIILISYIFILIAILRMRSAESRRKAFST 246
1I++I+I l+l+l I + I I lllll I ll+l I I I lllll Sbjct: 184 LLPLLKLACSNTYINEMLILFFGTLNIFVPILTIITSYIFIIASILRIRSTEGRSKAFST 243
40
Query: 247 CGSHLVAVTVFYGTLFCMYVRPPTDRSVEQSKVIAVFYTFVSPMLNPIIYSLRNKDVKQA 306
I II++I I I l + l + l I I++I + I++I II +1 I II I II I ll + l I lllll II I Sbjct: 244 CSSHILAVAVFFGSLAFMYLQPSSVSSMDQGKVSSVFYTIVVPMLNPLIYSLRNKDVAVA 303
45 Query: 307 FWKLIRRNVLL 317 l + l I + Sbjct: 304 LKKIIERKTFM 314
A multiple sequence alignment is given in Table 6J, with the GPCR6 protein being 50 shown on line 4. in a ClustalW analysis comparing GPCR6 with related protein sequences.
Table 6J. Information for the ClustalW proteins:
1. Gallus gallus fCHICKEN) OLF, SPTREMBL-Acc # Q90808, SEQ ID NO: 56
2. Hyloba tes lar (Common Gibbon) OLF, SPTREMBL-Acc # 077758, SEQ ID NO: 43 -. 3. Homo sapiens OLF, SWISSPROT-Acc # Q13606, SEQ ID NO: 33
22. 4. Novel HumanjDLF, GPCR6, SEQ ID NO: 20
5. Rattus norvegicus OR, Ace # G264617, SEQ ID NO: 62 CHI CKENJDLF ^GlSHJ^SJgBJV^SJBHBK -AAWa-^BiLI^TOFOE^LBlilliiilOGlilPig
GIBBON_01 F — . g[NE§YBκgBiiaiMFTEπilYNBθHI8IVFBiaι EaiT F0V0S VTgJjFfMgViSgMggg
HϋMAN_OLF MEF nR^YlntA^Jatalirt-BlFPTRfEilglVggl JgTLgAgiLlgSl LMLlBgljgPH
GPCR6 MT.vpκκl.foraGlHsliiil.gaaahM^
RAT_OLF ^SVA§ESISRgji5EFSSR| gE P[3J0vS3Vs lL[ilFgJ]MMlB VS[gL^K
Figure imgf000052_0001
CHICKENjDLF Ol-ffiKlGl5lLSi3vVEllTVRV-
GIBBONJDLF i5-SiSD)3τHpjτVTORKFCKA
HUMANJDLF [gDggDgAEKVLRSKVDSS-
GPCR6 DggQgFWKLIRJ SlVLLK-
RATJDLF Eg5@GgF g Mκ|lILIGK
DOMAIN results for GPCR6 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 6K with the statistics and domain description. Residues 1-158 (SEO ID NO:29) and residues 313-377 (SEO ID NO:37) of 7tm 1 are aligned with GPCR4 in Table 6K.
Table 6K. DOMAIN results for GPCR6.
Sbjct: 7 transmembrane receptor (rhodopsin family) fragment (SEQ ID NO: 29) gnl I PfamlpfamOOOOl, 7tm_l, 7 transmembrane receptor (.rhodopsin family)
Length = 377
Score = 95.1 bits -(235), Expect = 5e-21
Query: 47 GNLGMLVLIRIDSRLHTPMYFFLASLSCLDLYYSTNVTPKMLVNFFSDKKAISYAACLVQ 106 Sbjct: 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMP WYLEWGEWKFSRIHCDIF 60
Query: 107 CYFFIAWITEYYMLAVMAYDRYVAICNPLLYSSKM-SKGLCIRLIAGPYVYGFLSGLME 165 Sbjct: 61 VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPM 120 . . * .. *** *. *.**... ** .** . * *
Query: 166 -TMWTYHLTF-CGSNIINHFYCADPPLIRLSCSDTFIKETSMFWA 209 Sbjct: 121 LFGLNNTDQNECIIANPAFWY SSIVSFYVPFIVTLLV 158
Sbjct: 7 transmembrane receptor (rhodopsin family) fragment (SEQ ID NO: 37) gnl I Pfaml famOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family) Length = 377 Score = 38.5 bits (88), Expect = 5e-04
Query: 233 RMRSAESRRKAFSTCGSHLVAVTVFYGTLFCMYVRP-PTDRSVEQSKVIAVFYTFVSPML 291 Sbjct: 313 KLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYSAFTWLGYVNSAV 372
Query: 292 NPIIY 296 Sbjct: 373 NPIIY 377 *****
The nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in various in various GPCR-related pathological disorders and/or OR- related pathological disorders, described further below. For example, a cDNA encoding the olfactory receptor -like protein may be useful in gene therapy, and the olfactory receptor -like protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from Neoplasm: adenocarcinoma; lymphoma: prostate cancer: uterus cancer; Immune response; AIDS; asthma; Crohn's disease; multiple sclerosis; and Albright Hereditary Ostoeodystrophy. Other GPCR-related diseases and disorders are contemplated. The GPCR6 nucleic acid and protein . or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. This novel protein also has immense value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
GPCR7
The novel nucleic acid of 981 nucleotides GPCR7 (also designated. AP001112 dal) encoding a novel OR-like protein is shown in Table 7A. An ORF begins with an ATG initiation codon at nucleotides 27-29 and ends with a TGA codon at nucleotides 942-944. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon.
Table 7A. GPCR7 Nucleotide Sequence (SEQ ID NO:21)
AGCTTGAAGAGCAAACTGTCAGGAATATGTCCAACACAAATGGCAGTGCAATCACAGAATTCATTTTACTTGGGCT CACAGATTGCCCGG ACTCCAGTCTCTGCTTTTTGTGCTGTTTCTGGTTGTTTACCTCGTCACCCTGCTAGGCAAC CTGGGCATGATAATGTTAATGAGACTGGACTCTCGCCTTCACACGCCCATGTACTTCTTCCTCACTAACTTAGCCT TTGTGGATTTGTGCTATACATCAAΆTGCAACCCCGCAGATGTCGACTAATATCGTATCTGAGAAGACCATTTCCTT TGCTGGTTGCTTTACACAGTGCTACATTTTCATTGCCCTTCTACTCACTGAGTTTTACATGCTGGCAGCAATGGCC TATGACCGCTATGTGGCCATATATGACCCTCTGCGCTACAGTGTGAAAACGTCCAGGAGAGTTTGCATCTGCTTGG CCACATTTCCCTATGTCTATGGCTTCTCAGATGGACTCTTCCAGGCCATCCTGACCTTCCGCCTGACCTTCTGTAG ATCCAATGTCATCAΆCCACTTCTACTGTGCTGACCCGCCGCTCATTAAGCTTTCTTGTTCTGATACTTATGTCAAA GAGCATGCCATGTTCATATCTGCTGGCTTCAACCTCTCCAGCTCCCTCACCATCGTCTTGGTGTCCTATGCCTTCA TTCTTGCTGCCATCCTCCGGATCAAATCAGCAGAGGGAAGGCACAAGGCATTCTCCACCTGTGGTTCCCATATGAT 2. GGCTGTCACCCTGTTTTATGGGACTCTCTTTTGCATGTATATAAGACCACCAACAGATAΆGACTGTTGAGGAATCT AAAATAATAGCTGTCTTTTACACCTTTGTGAGTCCGGTACTTAATCCATTGATCTACAGTCTGAGGAATAAAGATG TGAAGCAGGCCTTGAAGAATGTCCTGAGATGAAATATTGTCATGACCATGGTGATGCCTTTGTTTCCTA
The GPCR7 protein encoded by SEO ID NO:20 has 305 amino acid residues and is 10 presented using the one-letter code in Table 7B. The SignalP, Psort and/orHydropathyprofile for GPCR7 predict that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of0.6000. The SignalP shows a cleavage site between amino acids 44 and 45, / g„ at the dash in the sequence amino acid NLG-MIM. This is typical ofa membrane protein.
I5- Table 7B. Encoded GPCR7 protein sequence (SEQ ID NO:22).
MSNTNGSAITEFILLGLTDCPELQSL FVLFLVVYLVTLLGNLGMIMLMRLDSRLHTPMYFFLTNLAFVD LCYTSNATPQMSTNIVSEKTISFAGCFTQCYIFIALLLTEFY LAAMAYDRYVAIYDPLRYSVKTSRRVC ICLATFPYVYGFSDGLFQAILTFRLTFCRSNVINHFYCADPPLIKLSCSDTYVKEHAMFISAGFNLSSSL TIVLVSYAFILAAILRIKSAEGRHKAFSTCGSHMMAVTLFYGTLFCMYIRPPTDKTVEESKIIAVFYTFV
20 SPVLNPLIYSLRNKDVKQALKNVLR
In a search of sequence databases, it was found, for example, that the nucleic acid sequence of GPCR7 has 633 of 959 bases (66%) identical to a gb:GENBANK- ID:GGCOR2GEN lacc:X94742.1 mRNA from Gallus gallus OR2 (SEO ID NO:51) (Table.
25 7Q,
Table 7C. BLASTN of GPCR7 against COR2
>gb:GENBANK-ID:GGCOR2GEN|acc:X94742.1 G. gallus cor2 DNA for olfactory receptor 2 -
(SEQ ID NO:51) Gallus gallus, 996 bp.
Score = 1415 (212.3 bits), Expect = 5.9e-58, P = 5.9e-58
30 Identities = 633/959 (66%), Positives = 633/959 (66%), Strand = Plus / Plus
Query: 7 AAGAGCAAACTGTCAGGA-AT-ATGTCCAACACAAATGGCAGTGCAATCACAGAATTCAT 64
II llll llll I ll lll llll llll 111 I 111 II I llll I Sbjct: 37 AACTGCAA-CTGTGTTGTGATGATGGCCAAGGGAAATCACAGCTCCATCACTGAATTTGT 95
35
Query: 65 TTTACTTGGGCT-CACAGATTGCCCGGAACTCCAGTCTCTGCTTTTTGTGCTGTTTCTGG 123
I I I 1 I I II II II I II I I I III II l llll l Sbjct: 96 GCT-CTTGGGATTCTCTGAAAAGAGGGCCATCCAGGCTGTTCTCTTTATGG-GCTTCTTG 153
40 Query: 124 TTGTT-TACCTCGTCACCCTGCTAGGCAACCTGGGCATGATAATGTTAATGAGACTGGAC 182 ll l lllll llll I II III I I III 1 I I I I I 1 III 111 Sbjct: 154 CTGATCTACCTGATCACTCTGCTAGGCAATGTGGGCATGATCACATTGATCAGGCTGGAC 213
Query: 183 TCTCGCCTTCACACGCCCATGTACTTCTTCCTCACTAACTTAGCCTTTGTGGATTTGTGC 242
45 I I II I III llll II I 1 I II I II II l lll l lll Sbjct: 214 TCCCGGCTTCACACCCCTATGTACTTCTTCCTGAGCAGCTTGTCCTTCCTCGATATCTGC 273
Query: 243 TATACATC-A-AATGCAACCC-CGCA-GATGTC-GACTAATATC-GTATCTGAGAAGACC 296
50 I I I I II I II I I I ll ll l l ll lll I I I II I llll I Sbjct: 274 TATTCCTCCACAAT-CACTCCTCGAGTGCTCTCAGACC—TCCCAGCATCACAGAAAGTC 330
Query: 297 ATTTCCTTTGCTGGTTGCTTTACACAGTGCTACATTTTCATTGCCCTT-CTACTCACTGA 355 III I II I I I I I I I • I I I I I I I I I I I I I III I I Sbjct: 331 ATTTCCCACTCTGCATGCCTGGCACAGTTTTATTTCTACGCTGTCTTTGCCAC-CACAGA 389 Query: 356 GTTTTACATGCTGGCAGCAATGGCCTATGACCGCTATGTGGCCATATATGACCCTCTGCG 415
II II I II II I llll I II I II II I I II II II I II III I Sbjct: 390 GTGCTATCTTTTGGCCGCAATGGCATATGACCGCTACGTGGCCATCTGCAGCCCTCTGCT 449
Query: 416 CTACAGTGTGAAA-ACGTCCAGGAGAGTTTGCAT-CTGCTTG—GCCACATT-TCCCTAT 470
III II I I I I llll II I II II I I I I I I I I II I I I II I Sbjct: 450 CTAT-GTCTTCTCCATGTCCAGCAGAGTTTGTGTGCTGCTGGTTGCTGGCTCATACCT-T 507
Query: 471 GTCTATGGCTTCTCAGATGG-ACTCTTCCAGGCCATCCTGAC-CTTCCGCCTGACCTTCT 528
III ll ll l l lll II lll l I II I I I I I I II I I III I Sbjct: 508 GTCGG-GG-TTGTGA-ATGCCACCATTC-ACACAGGGCTTGCACTGCAGC-TGTCCTTCT 562
Query: 529 GTAGATCCAATGTCATCAACCACTTCTACTGTG-CTGACCCGCCGCTCATTAAGCTTTCT 587
II I III 1 II I I I II lllll I I I III lllll 1 I I I I III Sbjct: 563 GTGGTCCCAACATCATCAATCACTTCTACTGTGACGGTCCC-CCGCTC-T-ACGCCATCT 619
Query: 588 TGTTCTGATACTT-ATGTCAAA-GAGCATGCCATGTTCATATCTGCT-GGCTTCAACCT- 643
11 1 I II I l ll lll lll ll ll I I II 1 III I Sbjct: 620 CGTGCACAGACCCCACCACCAACGAGATTGCGATATTTCT-TGTGGTTGGCTTCAACATG 678
Query: 644 CTCCAGCTCCCTCACCATCGTCTTGGTGTCCTATGCCTTCATTCTTGCT-GCCATCCTCC 702 lll l l ll I l l llll l lllll 111 III II I I II lllll Sbjct: 679 CTC-ATCACCAGCGTGACCATCTTCATCTCCTACACCTACATCCT-GTTCGCTGTCCTCA 736
Query: 703 GGAT-CAAATCAGCAG-AGGGAAGGCACAAGGCATTCTCCACCTGTGGTTCCCATATGAT 760
I I I I II I I 111 I III II l lll I I I I I II lllll II Sbjct: 737 GGATGCACA-CAGCTGCAGGCAAA-CGCAAAACCTTCTCCACGTGTGCGTCCCACCTGGC 794
Query: 761 GGCTGTCACCCTGTTTTATGGGACTCT-CTT-TTGCATGTATATAAGACC-ACCAACAGA 817
I llll I I I I II III I llll ll ll l l ll l l III Sbjct: 795 CACCGTCACCCTATTCTATGC--CTCTGCTGGTTCCATGTACTCACGGCCCAGCTCCAGG 852
Query: 818 TAAGACT-GTTGAGGAATCTAAAATAATAGCTGTCTTTTACACCTTTGTGAGTCCGGTAC 876
I I I l l l ll I III I II llll II I I I II I 1 I I Sbjct: 853 CACTCCCAGGACCTGGA-C-AAGGTGGCCTCTGTGTTCTACACCATGGTGACCCCCATGC 910
Query: 877 TTAATCCATTGATCTACAGTCTGAGGAATAAAGATGTGAAGCAGGCCTTGAAGAATGTCC 936 l ll ll I III II III lllll III I II II II I I I II lll ll Sbjct: 911 TGAACCCCCTCATCTACAGCCTGAGGAACCAGGAGGTAAAGGATGTTTTAGGGAAAGTGA 970
Query: 937 TGAGATGAAATATTGTCA-TGACCA 960
I I I I I I I I I I I n n i
Sbj ct : 971 TGGGGAGGAAGAGTGTCTCTGACAA 995
The GPCR7 amino acid has 164 of 305 amino acid residues (53%) identical to, and 214 of 305 amino acid residues (70%) similar to, the 309 amino acid OR M72 (ptnr:TREMBLNE -Acc No.:AAG09780) protein from Mu musculus QR M72- f SEQ DP NO:52) (Table 7D). Table 7D. BLASTP alignments of GPCR7 against OR M72, (SEQ ID NO:52)
>ptnr:TREMBLNEW-ACC:AAG09780 ODORANT RECEPTOR M72 - Mus musculus (Mouse), 309 aa. Score = 811 (285.5 bits), Expect = 1.2e-80, P = 1.2e-80 Identities = 164/305 (53%), Positives = 214/305 (70%) Query: 1 MSNTNGSAITEFILLGLTDCPELQSLLFVLFLWYLVTLLGNLGMIMLMRLDSRLHTPMY 60
1+ I I +1 II II I 11+ I II I l +lll +1 + 1 I++I lllll 1+ l + l + lllll I Sbjct: 1 MAAENQSTVTEFILRGLTNRPELQLPLLLLFLGIYIVTMVGNLGMITLIGLNSQLHTPMY 60
Query: 61 FFLTNLAFVDLCYTSNATPQMSTNIVSEKT-ISFAGCFTQCYIFIALLLTEFYMLAAMAY 119
I I l + l 1+ I I II l + l ll + l I II++ 11+ I I +1 I 1+ ++ I I I I III Sbjct: 61 FFLSNLSLVDLCYSSVITPKMLINFVSQRNLISYVGCMSQLYFFLVFVIAECYMLTVMAY 120 Query: 120 DRYVAIYDPLRYSVKTSRRVCICLATFPYVYGFSDGLFQAILTFRLTFCRSNVINHFYCA 179 llll I I I I |++ I +1 I I 1 I + I +1 +1 ++1 + I++I Sbjct: 121 DRYVAICQPLLYNIIMSPALCSLLWFVYAMGLIGSTIETSLMLKLNYCE-DLISHYFCD 179
Query: 180 DPPLIKLSCSDTYVKEHAMFISAGFNLS-SSLTIVLVSYAFILAAILRIKSAEGRHKAFS 238 ll + l I I I I II I l + l I II 1+ +111 I l + ll 111 I++II 11 I III llll Sbjct: 180 ILPLMKLSCSSTYDIEMAVFFLAGFNIIVTSLT-VLISYAFILSSILRISSNEGRSKAFS 238
Query: 239 TCGSHMMAVTLFYGTLFCMYIRPPTDKTVEESKIIAVFYTFVSPVLNPLIYSLRNKDVKQ 298
M il I I I I I I + I I ++ 1 I ++ + + + II II I I + I I I I II I I II + 1 I Sbjct: 239 TCSSHFAAVGLFYGSTAFMYLKPSTASSLAQENVASVFYTTVIPMFNPLIYSLRNKEVKT 298
Query: 299 ALKNVLR 305
II II Sbjct: 299 ALDKTLR 305
The presence of identifiable domains in GPCR7 was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, and Prints followed by determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The results indicate that this protein contains the following protein domains (as defined by Interpro) at the indicated positions: domain name 7tm 1 (InterPro) 7 transmembrane receptor (rhodopsin family) at amino acid positions 41 to 289. Tins indicates that the sequence of GPCR7 has properties similar to those of other proteins known to contain this domain. GPCR7 maps to chromosome 11. This information was assigned using the Online
Mendelian Inheritance in Man (OMIM) database, the electronic northern bioinformatic tool implemented by CuraGen Corporation, public ESTs, public literature references and/or genomic clone homologies. This was executed to derive the chromosomal mapping ofthe Genomic clones, literature references and/or EST sequences that were included in the invention.
GPCR7 is expressed in at least the following tissues: Apical micro villi ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue. Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to Public EST sources. Genomic Clone sources. Literature sources, and/or RACE sources.
The protein similarity information, expression pattern, and map location for the GPCR7 protein and nucleic acid suggest that GPCR7 may have important structural and/or physiological functions characteristic ofthe Olfactory Receptor family. Therefore. GPCR7 are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
GPCR7 is useful in potential diagnostic and therapeutic applications implicated in various GPCR- or OR-related diseases and disorders described below and/or other pathologies. For example, the compositions of GPCR7 will have efficacy for treatment of patients suffering from: : Familial Mediterranian Fever, developmental diseases, MHCII and III diseases (immune diseases). Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneuro enie disease. Signal Transduction pathway disorders. Retinal diseases including those involving photoreception. Cell Growth rate disorders: Cell Shape disorders. Feeding disordersxontrol of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of apetite). noninsulin-dependent diabetes mellitus (NfDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma: prostate cancer; uterus cancer), anorexia, bulimia, asthma. Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis. Crohn's disease; multiple sclerosis; and
Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation. Dentatorubro-pallidoluysian atrophy(DRPLA) Hypophosphatemic rickets, autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders ofthe like. The polypeptides can be used as immunogens to produce antibodies specific for GPCR7. and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding the GPCR7-like protein may be useful in gene therapy, and the GPCR7-like protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions of GPCR7 will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal arid viral infections (particularly infections caused by HIN-1 or HIN-2). pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety. schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. The GPCR7 nucleic acid and protein , or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to GPCR7 for use in therapeutic or diagnostic methods. Other GPCR-related diseases and disorders are contemplated.
These materials are further useful in the generation of antibodies that bind immunospecifically to the novel GPCR7 substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. In one embodiment, a contemplated GPCR7 epitope is from aa 15 to 70. In another embodiment, a GPCR7 epitope is from aa 85 to 125. In additional embodiments, GPCR7 epitopes are from aa 140 to 175. from aa 210 to 235, from aa 240 to 260, and from aa 275 to 290. A summary ofthe GPCRX nucleic acids and proteins ofthe invention is provided in
Table 8A. A summary of homologous sequences identified in searches of available sequence databases is provided in Table 8B.
TABLE 8 : Summary Of Nucleic Acids And Proteins Of The Invention
Figure imgf000058_0001
Figure imgf000059_0001
TABLE 8B: Summary of Query Sequences Disclosed
Figure imgf000059_0002
Figure imgf000060_0001
GPCRX Nucleic Acids and Polypeptides
One aspect ofthe invention pertains to isolated nucleic acid molecules that encode GPCRX polypeptides or biologically-active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify GPCRX- encoding nucleic acids (e.g- GPCRX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of GPCRX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e g-., cDNA or genomic DNA), RNA molecules (e g., mRNA), analogs ofthe DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.
An GPCRX nucleic acid can encode a mature GPCRX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an ORF. or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N. where residue 1 is the N-terminal methionine. would have residues 2 through N remaining after removal ofthe N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, g ycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e „ , 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid ( g„ sequences located at the 5'- and 3'-termini ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, in various embodiments, the isolated GPCRX nucleic acid molecules can contain less than about 5 kb.4 kb, 3 kb. 2 kb. 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell/tissue from which the nucleic acid is derived (e g.. brain, heart. liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized. A nucleic acid molecule ofthe invention, e g.. a nucleic acid molecule having the nucleotide sequence of SEO ID NOS:l. 3, 5, 7. 9, 11. 13. 15. 17. 19 and 21. or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion ofthe nucleic acid sequence ofSEO ID NOS: 1. 3. 5, 7. 9. 11. 13. 15. 17. 19 and 21 as a hybridization probe, GPCRX molecules can be isolated using standard hybridization and cloning techniques (e.g- as described in Sambrook, et α/., (eds.), M0LECULAR CLONING A LABORATORY MANUAL-2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, ef a , (eds.), CUR ENT PROTOCOLS IN MOLECULAR BIO OGY. John Wiley & Sons, New York, NY, 1993.)
A nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to GPCRX nucleotide sequences can be prepared by standard synthetic techniques, e „., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment ofthe invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEO ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17. 19 and 21, or a complement thereof. Oligonucleotides maybe chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NOS:l. 3. 5, 7. 9. 11. 13. 15. 17. 19 and 21, or a portion of this nucleotide sequence (e.g.- a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an GPCRX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEO ID NOS:l, 3, 5. 7. 9. 11. 13. 15. 17. 19 and 21. is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:l. 3, 5. 7. 9. 11, 13. 15, 17. 19 and 21. that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species. Derivatives and analogs maybe full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs ofthe nucleic acids or proteins ofthe invention include, but are not limited to. molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e „ Ausubel, e α/u-CuRRENT
PROTOCOLS IN MOLECULAR BIOLOGY- John wilev & Sons- New York- NY- 1993- md below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of GPCRX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an GPCRX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include. e g- , frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human GPCRX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEO ID NOS:2. 4. 6. 8. 10. 12, 14. 16. 18, 20 and 22, as well as a polypeptide possessing GPCRX biological activity. Various biological activities ofthe GPCRX proteins are described below.
An GPCRX polypeptide is encoded by the open reading frame ("ORF") of an GPCRX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one ofthe three "stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a fcona fide cellular protein, a minimum size requirement is often set, e σ a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning ofthe human GPCRX genes allows for the generation of probes and primers designed for use in identifying and/or cloning GPCRX homologues in other cell types, e g. from other tissues, as well as GPCRX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25. 50, 100, 150, 200. 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEO ID NOS:l, 3. 5. 7. 9, 11, 13. 15. 17. 19 and 21: or an anti-sense strand nucleotide sequence of SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21: or of a naturally occurring mutant of SEO ID NOS:!. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21.
Probes based on the human GPCRX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto. e „. the label group can be a radioisotope. a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an GPCRX protein, such as by measuring a level of an GPCRX-encoding nucleic acid in a sample of cells from a subject e g„ detecting GPCRX mRNA levels or determining whether a genomic GPCRX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of an GPCRX polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to. an activity of a polypeptide ofthe mvention. including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically- active portion of GPCRX" can be prepared by isolating a portion of SEO ID NOS:l, 3, 5, 7, 9, 11, 13. 15. 17. 19 and 21, that encodes a polypeptide having an GPCRX biological activity (the biological activities ofthe GPCRX proteins are described below), expressing the encoded portion of GPCRX protein (β.g- by recombinant expression m v#r ) and assessing the activity ofthe encoded portion of GPCRX.
GPCRX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. due to degeneracy ofthe genetic code and thus encode the same GPCRX proteins as that encoded by the nucleotide sequences shown in SEO ID NO NOS.T. 3, 5, 7, 9, 11, 13, 15. 17, 19 and 21. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEO ID NOS:2, 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22.
In addition to the human GPCRX nucleotide sequences shown in SEO ID NOS:l. 3. 5. 7. 9, 11. 13. 15. 17. 19 and 21, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences ofthe GPCRX polypeptides may exist within a population β. - the human population). Such genetic polymorphism in the GPCRX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an GPCRX protein, preferably a vertebrate GPCRX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe GPCRX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the GPCRX polypeptides. which are the result of natural allelic variation and that do not alter the functional activity ofthe GPCRX polypeptides, are intended to be within the scope ofthe invention.
Moreover, nucleic acid molecules encoding GPCRX proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence of SEO ID NOS:l, 3. 5, 7, 9. 11. 13. 15. 17. 19 and 21. are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe GPCRX cDNAs ofthe invention can be isolated based on their homology to the human GPCRX nucleic acids disclosed herein using the human cDNAs. or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEO ID NOS: 1. 3. 5. 7. 9. 11. 13, 15. 17. 19 and 21. In another embodiment, the nucleic acid is at least 10, 25. 50. 100. 250, 500, 750. 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule ofthe invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Homologs (/_g nucleic acids encoding GPCRX proteins derived from species other than human) or other related sequences (e „ paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion ofthe particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50%) ofthe probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g.- 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et ah- (eds-). CUR ENT PROTOCOLS IN MOLECULAR BIO OGY. Joh Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%. 75%. 85%. 90%. 95%. 98%. or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5). 1 mM EDTA. 0.02% PVP, 0.02% Ficoll, 0.02% BSA. and 500 mg/ml denatured salmon sperm DNA at 65°C. followed by one or more washes in 0.2X SSC. 0.01% BSA at 50°C. An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences of SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g- encodes a natural protein). h a second embodiment, a nucleic acid sequence that is hvbridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well-known within the art. See, e-g. , Ausubel, et fl/_ (eds.), 1993, CU RENT PROTOCOLS IN MOLECULAR BIOLOGY. ohn Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND
Figure imgf000067_0001
LABORATORY MANUAL. Stockton Press, NY. In a third embodiment, a nucleic acid that is hvbridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS:l, 3. 5. 7. 9. 11. 13. 15, 17. 19 and 21. or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide. 5X SSC. 50 mM Tris-HCl (pH 7.5). 5 mM EDTA. 0.02% PVP. 0.02% FicoU. 0.2% BSA. 100 mg/ml denatured salmon sperm DNA. 10% (wt/vol) dextran sulfate at 40°C. followed by one or more washes in 2X SSC. 25 mM Tris-HCl (pH 7.4). 5 mM EDTA. and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g.- as employed for cross-species hybridizations). See. g g Ausubel. ef α (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. Joh Wiley & Sons, NY, and Kriegler, 1990, IENE ∑RANSFER AND
Figure imgf000068_0001
MANUAL. Stockton Press, NY; Shilo and Weinberg, 1981. proc Natl Acad Sci USA IS- 6789-6792.
Conservative Mutations hi addition to naturally-occurring allelic variants of GPCRX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEO ID NO NOS:l, 3. 5. 7, 9. 11. 13. 15. 17, 19 and 21, thereby leading to changes in the amino acid sequences ofthe encoded GPCRX proteins, without altering the functional ability of said GPCRX proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. A
"non-essential" amino acid residue is a residue that can be altered from the wild-type sequences ofthe GPCRX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the GPCRX proteins ofthe invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect ofthe invention pertains to nucleic acid molecules encoding GPCRX proteins that contain changes in amino acid residues that are not essential for activity. Such GPCRX proteins differ in amino acid sequence from SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. vet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEO ID NOS.-2.4. 6. 8. 10. 12, 14, 16, 18, 20 and 22. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEO ID NOS:2, 4, 6. 8. 10. 12. 14. 16. 18. 20 and 22; more preferably at least about 70% homologous to SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22: still more preferably at least about 80% homologous to SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22: even more preferably at least about 90% homologous to SEQ ID NOS:2. 4. 6. 8. 10, 12. 14. 16. 18. 20 and 22: and most preferably at least about 95% homologous to SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22.
An isolated nucleic acid molecule encoding an GPCRX protein homologous to the protein ofSEO ID NOS :2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEO ID NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein- Mutations can be introduced into SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16, 18. 20 and 22. by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g- lysine. arginine, histidine), acidic side chains (e.g- aspartic acid, glutamic acid), uncharged polar side chains (e.g- glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g- alanine. valine, leucine. isoleucine. proline. phenylalanine. methionine. tryptophan), beta-branched side chains (e.g- threonine, valine, isoleucine) and aromatic side chains (e.g- tyrosine, phenylalanine, tryptophan. histidine). Thus, a predicted non-essential amino acid residue in the GPCRX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an GPCRX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GPCRX biological activity to identify mutants that retain activity. Following mutagenesis of SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined. The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues maybe any one ofthe following groups: STA. NEOK. NHOK. NDEQ. OHRK. MILV. MILF. HY. FYW. wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one ofthe following: CSA. ATV. SAG. STNK. STPA. SGND. SNDEOK. NDEOHK. NEOHRK. VLIM, HFY, wherein the letters within each group represent the single letter amino acid code- In one embodiment, a mutant GPCRX protein can be assayed for (;) the ability to form proteimprotein interactions with other GPCRX proteins, other cell-surface proteins, or biologically-active portions thereof, (//) complex formation between a mutant GPCRX protein and an GPCRX ligand; or ( ) the ability of a mutant GPCRX protein to bind to an intracellular target protein or biolo ically-active portion thereof; (e „. avidin proteins).
In vet another embodiment, a mutant GPCRX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids
Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEO ID NOS:l. 3, 5. 7. 9. 11. 13. 15. 17. 19 and 21. or fragments. analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g- complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire GPCRX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs. derivatives and analogs of an GPCRX protein of SEQ ID NOS:2. 4, 6, 8. 10. 12. 14. 16. 18. 20 and 22; or antisense nucleic acids complementary to an GPCRX nucleic acid sequence of SEO ID NOS:l. 3. 5. 7. 9, 11, 13, 15, 17, 19 and 21. are additionally provided. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding an GPCRX protein. The term "coding region" refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding the GPCRX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (?- g„ also referred to as 5' and 3' untranslated regions). Given the coding strand sequences encoding the GPCRX protein disclosed herein, antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hόogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of GPCRX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of GPCRX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GPCRX mRNA. An antisense oligonucleotide can be. for example, about 5, 10, 15, 20. 25. 30, 35. 40. 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g- an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e. - phosphorothioate derivatives and acridine substituted nucleotides can be used). Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhvdroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine. 1-methylguanine, 1-methylinosine. 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcvtosine, 5-methylcvtosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil. 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v). wybutoxosine. pseudouracil. queosine. 2-thiocytosine, 5-methyl-2-thiouracil. 2-thiouracil. 4-thiouracil. 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w. and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation ( g., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated jn sj u such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an GPCRX protein to thereby inhibit expression ofthe protein (e.g- by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or. for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site. Alternatively. antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g- bv linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in wliich the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In vet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other. See, g-g-> Gaultier, et fl/., 1987. jfucι Acids Res.ΛS 6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (see, g y Inoue, ef aj. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (see. e „f Inoue, ef aj., 1987. FEBSLett. 215: 327-330. Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid ofthe invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA. to which they have a complementary region. Thus, ribozymes (_e.g- hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave GPCRX mRNA transcripts to thereby inhibit translation of GPCRX mRNA. A ribozyme having specificity for an GPCRX-encoding nucleic acid can be designed based upon the nucleotide sequence of an GPCRX cDNA disclosed herein (7- g„ SEO ID NOS: 1. 3, 5. 7. 9, 11, 13, 15. 17. 19 and 21). For example, a derivative of a Tetrahymena L~i9 INS R A can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an GPCRX-encoding mRNA. See, e o-., U.S. Patent 4,987,071 to Cech, e aU and U.S. Patent 5,116,742 to Cech, ef af GPCRX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, g o-., Bartel et ah, (1993) Science 261:1411-1418.
Alternatively, GPCRX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe GPCRX nucleic acid (e.g- the GPCRX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe GPCRX gene in target cells, gee, e.g., Helene, 1991. Anticancer Drug Des- 6: 569-84; Helene, et a 1992. Ann^-N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the GPCRX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e „., the stability, hybridization, or solubility ofthe molecule. For example, the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids, ^ e, e.g., Hyrup. g Ωj., 1996. BioorgMed Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics (e.g- DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et fl/., 1996. Slφra- Perry-O'Keefe, et α/., 1996. Proc_ NatL Acad Sci. USA 93: 14670-14675.
PNAs of GPCRX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e „., inducing transcription or translation arrest or inhibiting replication. PNAs of GPCRX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g- PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes. g „., Sχ nucleases (see> Hyrup, ef ait 1996.S pra) or as probes or primers for DNA sequence and hybridization (see> Hyrup, et fl/., 1996, supra- Perry-O'Keefe, et al > 1996. supraX In another embodiment. PNAs of GPCRX can be modified, g p-., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of GPCRX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g- RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see Hyrup, etal., 1996. suprc - The synthesis of PNA-DNA chimeras can be performed as described in Hyrup^ et a\ 1996. SMpra and Finn, et a 1996. χucι Acids Res 2 : 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs. e p-., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite. can be used between the PNA and the 5' end of DNA. $ < ee, e.g., Mag, ef a 1989. yy?/g/ Acid Res ^: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment, ^ee, e.g., Finn, ef #/., 1996. supra- Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. $ee, e.g., Petersen, et g/., 1975. Bioorg. Med. Chem. Lett. >i 1119-11124. In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g- for targeting host cell receptors m
Figure imgf000074_0001
the cell membrane (see,.e.g-- Letsinger, et a/., 1989. prQC, Natl. Acad. Sci. U.S.A. l 6553-6556; Lemaitre, ef Ω/„ 1987. pr c_ Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g- p τ Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g, Krol, et q , 1988. BioTechniques 6:958-976) or intercalating agents (see> e g., Zon, 1988. Pharm. Res- $'■ 539-549). To this end, the oligonucleotide may be conjugated to another molecule, g g„ a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
GPCRX Polypeptides
A polypeptide according to the invention includes a polypeptide including the amino acid sequence of GPCRX polypeptides whose sequences are provided in SEO ID NOS:2. 4. 6. 8. 10. 12, 14, 16, 18. 20 and 22. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. while still encoding a protein that maintains its GPCRX activities and physiological functions, or a functional fragment thereof.
In general, an GPCRX variant that preserves GPCRX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect ofthe invention pertains to isolated GPCRX proteins, and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-GPCRX antibodies. In one embodiment, native GPCRX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment. GPCRX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, an GPCRX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the GPCRX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of GPCRX proteins in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly- produced, hi one embodiment, the language "substantially free of cellular material" includes preparations of GPCRX proteins having less than about 30% (by dry weight) of non-GPCRX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-GPCRX proteins, still more preferably less than about 10% of non-GPCRX proteins, and most preferably less than about 5% of non-GPCRX proteins. When the GPCRX protein or biologically-active portion thereof is recombinantiy-produced, it is also preferably substantially free of culture medium, ; g„ culture medium represents less than about 20%>, more preferably less than about 10%, and most preferably less than about 5% ofthe volume of the GPCRX protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of GPCRX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of GPCRX proteins having less than about 30% (by dry weight) of chemical precursors or non-GPCRX chemicals, more preferably less than about 20% chemical precursors or non-GPCRX chemicals, still more preferably less than about 10% chemical precursors or non-GPCRX chemicals, and most preferably less than about 5%> chemical precursors or non-GPCRX chemicals.
Biologically-active portions of GPCRX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences ofthe GPCRX proteins (e g.. the amino acid sequence shown in SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16, 18. 20 and 22) that include fewer amino acids than the full-length GPCRX proteins, and exhibit at least one activity of an GPCRX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity ofthe GPCRX protein. A biologically- active portion of an GPCRX protein can be a polypeptide which is. for example. 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native GPCRX protein. In an embodiment, the GPCRX protein has an amino acid sequence shown in SEO ID
NOS:2. 4. 6. 8. 10. 12, 14. 16, 18, 20 and 22. hi other embodiments, the GPCRX protein is substantially homologous to SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. and retains the functional activity ofthe protein of SEO ID NOS:2, 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the GPCRX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEO 3D NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. and retains the functional activity of the GPCRX proteins of SEQ ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22.
Determining Homology Between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g- gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position ( g„ as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. gg Needleman and Wunsch, 1970. JMol Biol 8: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region ofthe analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%. 75%. 80%, 85%. 90%. 95%. 98%, or 99%. with the CDS (encoding) part ofthe DNA sequence shown in SEO 3D NOS:l. 3. 5. 7. 9, 11. 13. 15. 17. 19 and 21. The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A. T. C. G. U. or I. in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (; e the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
Chimeric and Fusion Proteins
The invention also provides GPCRX chimeric or fusion proteins. As used herein, an GPCRX "chimeric protein" or "fusion protein" comprises an GPCRX polypeptide operatively- linked to a non-GPCRX polypeptide. An "GPCRX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an GPCRX protein (SEQ ID NOS:2, 4, 6, 8, 10. 12. 14, 16. 18. 20 and 22). whereas a "non-GPCRX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the GPCRX protein, e g„ a protein that is different from the GPCRX protein and that is derived from the same or a different organism. Within an GPCRX fusion protein the GPCRX polypeptide can correspond to all or a portion of an GPCRX protein. In one embodiment, an GPCRX fusion protein comprises at least one biologically-active portion of an GPCRX protein. In another embodiment, an GPCRX fusion protein comprises at least two biologically-active portions of an GPCRX protein. In vet another embodiment, an GPCRX fusion protein comprises at least three biologically-active portions of an GPCRX protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the GPCRX polypeptide and the non-GPCRX polypeptide are fused in-frame with one another. The non-GPCRX polypeptide can be fused to the N-terminus or C-terminus ofthe GPCRX polypeptide.
In one embodiment, the fusion protein is a GST-GPCRX fusion protein in which the GPCRX sequences are fused to the C-terminus ofthe GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant GPCRX polypeptides. hi another embodiment, the fusion protein is an GPCRX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g- mammalian host cells), expression and/or secretion of GPCRX can be increased through use of a heterologous signal sequence. In yet another embodiment, the fusion protein is an GPCRX-immuno globulin fusion protein in which the GPCRX sequences are fused to sequences derived from a member ofthe immuno globulin protein family. The GPCRX-immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an GPCRX ligand and an GPCRX protein on the surface of a cell, to thereby suppress GPCRX-mediated signal transduction fn vjvo. The GPCRX- immuno globulin fusion proteins can be used to affect the bioavailability of an GPCRX cognate ligand. Inhibition ofthe GPCRX ligand/GPCRX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e g. promoting or inl ibiting) cell survival. Moreover, the GPCRX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-GPCRX antibodies in a subject, to purify GPCRX ligands, and in screening assays to identify molecules that inhibit the interaction of GPCRX with an GPCRX ligand. An GPCRX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, βg . by employing blunt-ended or stagger-ended termini for ligation, restriction enzvme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see> &g ι Ausubel, et fl/. (eds.) CURRENT PROTOCOLS IN MOLECULAR Bjr)τ_QGγ, John Wiley & Sons. 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g- a GST polypeptide). An GPCRX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GPCRX protein.
GPCRX Agonists and Antagonists The invention also pertains to variants ofthe GPCRX proteins that function as either
GPCRX agonists (j_e mimetics) or as GPCRX antagonists. Variants ofthe GPCRX protein can be generated by mutagenesis (e.g- discrete point mutation or truncation ofthe GPCRX protein). An agonist ofthe GPCRX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe GPCRX protein. An antagonist ofthe GPCRX protein can inhibit one or more ofthe activities ofthe naturally occurring fonn ofthe GPCRX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the GPCRX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occumng form ofthe GPCRX proteins- Variants ofthe GPCRX proteins that function as either GPCRX agonists ( e mimetics) or as GPCRX antagonists can be identified by screening combinatorial libraries of mutants (e.g- truncation mutants) ofthe GPCRX proteins for GPCRX protein agonist or antagonist activity. In one embodiment, a variegated library of GPCRX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of GPCPvX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential GPCRX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g- f°r phage display) containing the set of GPCPvX sequences therein. There are a variety of methods which can be used to produce libraries of potential GPCRX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential GPCRX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art, gee, e.g., Narang. 1983. Tetrahedron 39: 3: Itakura, et α/., 1984. Annu. Rev. Biochem. 53 : 323; Itakura, et α/., 1984. Science 198: 1°56; M^et al- 9S3. Nucl. Acids Res. 11: 477. Polypeptide Libraries
In addition, libraries of fragments ofthe GPCPvX protein coding sequences can be used to generate a variegated population of GPCPvX fragments for screening and subsequent selection of variants of an GPCPvX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an GPCRX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA. renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Sχ nuclease. and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes ofthe GPCRX proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of GPCRX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation ofthe vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM). a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify GPCRX variants. See, e.g., Arkin and Yourvan, 1992. Proc_ jVαt/. Acad. Sci. USA 89: 7811-7815; Delgrave, et al-ι 1993. Protein Engineering 6:327-331.
Anti-GPCRX Antibodies
The invention encompasses antibodies and antibody fragments, such as Fab or Fab)2 that bind immunospecifically to any ofthe GPCPvX polypeptides of said invention. An isolated GPCRX protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind to GPCRX polypeptides using standard techniques for polyclonal and monoclonal antibody preparation. The full-length GPCRX proteins can be used or, alternatively, the invention provides antigenic peptide fragments of GPCRX proteins for use as immunogens. The antigenic GPCRX peptides comprises at least 4 amino acid residues ofthe amino acid sequence shown in SEO 3D NO NOS:2, 4, 6, 8, 10, 12, 14, 16. 18, 20 and 22, and encompasses an epitope of GPCPvX such that an antibody raised against the peptide forms a specific immune complex with GPCPvX. Preferably, the antigenic peptide comprises at least 6, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on use and according to methods well known to someone skilled in the art.
In certain embodiments ofthe invention, at least one epitope encompassed by the antigenic peptide is a region of GPCPvX that is located on the surface ofthe protein (e.g- a hydrophihc region). As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well lαiown in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation (see> g.g-. Hopp and Woods, 1981. proc_ χat. Acad. Sci. USA 78: 3824-3828: Kvte and Doolittle. 1982. j Mol. Biol- 157: 105-142. each incoφorated herein by reference in their entirety). As disclosed herein, GPCPvX protein sequences of SEO 3D NOS:2, 4. 6, 8, 10, 12, 14,
16, 18, 20 and 22, or derivatives, fragments, analogs or homologs thereof, maybe utilized as immunogens in the generation of antibodies that immunospecifically-bind these protein components. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically-active portions of immuno globulin molecules. g.. molecules that contain an antigen binding site that specifically-binds (immunoreacts with) an antigen, such as GPCRX. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F fc and F, L„2 fragments, and an F , expression library. In a specific embodiment, antibodies to human GPCPvX proteins are disclosed. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to an GPCRX protein sequence of SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20 and 22. or a derivative, fragment, analog or homolog thereof. Some of these proteins are discussed below.
For the production of polyclonal antibodies, various suitable host animals (e.g- rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative ofthe foregoing. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed GPCRX protein or a chemically-synthesized GPCRX polypeptide. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to. Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lvsolecithin. pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille-Calmette-Guerin mά Corynebacterium pan m- or similar immunostimulatory agents. If desired, the antibody molecules directed against GPCPvX can be isolated from the mammal (e.g- from the blood) and further purified by well known techniques, such as protein A chromato raphy to obtain the IgG fraction. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of GPCRX. A monoclonal antibody composition thus typically displays a single binding affinity for a particular GPCRX protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular GPCRX protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., ohler & Milstein, 1975. Nature 2 6: 495-497); the trioma technique; the human B-cell hybridoma technique (see> e.g., Kozbor, et al., 9 .Immunol. Today 4'- 72) and the EBV hybridoma technique to produce human monoclonal antibodies (See, e.g., Cole, e al >
1985. hi: M0N0CLONAL ANTIBODIES AND CANCER IHERAPY. Alan R- Liss. k10-. PP- 77-96). Human monoclonal antibodies may be utilized in the practice ofthe invention and may be produced by using human hybridomas (see> e.g., Cote, et ait 1983. proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus jn vitro see, e.g., Cole, et al, g . hi: MONOCLONAL ANTIBODIES AND GANGER IHERAPY. Alan R. Liss, Inc., pp. 77-96). Each ofthe above citations is incorporated herein by reference in their entirety.
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an GPCPvX protein ( gg e „ U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fdb expression libraries ( gg e „ Huse, et al, 1989. Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fal fragments with the desired specificity for an GPCPvX protein or derivatives, fragments, analogs or homologs thereof. Non-human antibodies can be "humanized" by techniques well known in the art. See, e.g., U-S- Patent No. 5,225,539. Antibody fragments that contain the idiotypes to an GPCPvX protein may be produced by techniques known in the art including, but not limited to: ( ) an F(dl>,)2 fragment produced by pepsin digestion of an antibody molecule; (?γ) an Fa fragment generated by reducing the disulfide bridges of an F,ύV}2 fragment; ( /) an Fdb fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent; and (?v) F fragments. - Additionally, recombinant anti-GPCRX antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope ofthe invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Application No. PCT/US86/02269: European Patent Application No. 184.187; European Patent
Application No. 171.496; European Patent Application No. 173.494; PCT International Publication No. WO 86/01533; U.S. Patent No. 4.816.567; U.S. Pat. No. 5.225.539; European Patent Application No. 125.023; Better. et aj.. 1988. Science 0: 1041-1043: Liu. t j.. 1987. Proc. Natl Acad. Sci. USA 84: 3439-3443; Liu, et al- 987- J. Immunol- I39: 3521-3526; Sun, et g I987- Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et a 1987. Cancer Res. 999-1005; Wood, et α/„ 1985. Nature 314 :446-449; Shaw, et a I988- J. Natl. Cancer Inst. 80: 1553-1559); Morrison(1985) Science 229:1202-1207; Oi, et al- 0986) BioTechniques 4:214; Jones, et al- - Nature 321: 552-525; Verhoeyan, et α/., 1988. & gnce 239: 1534; and Beidler. g/ fl/„ 1988. j Immunol. 41 : 4053-4060. Each ofthe above citations are incorporated herein by reference in their entirety.
In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an GPCRX protein is facilitated by generation of hybridomas that bind to the fragment of an GPCRX protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an GPCRX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-GPCRX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an GPCRX protein e.g., f°r use m measuring levels ofthe GPCRX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for GPCRX proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
An anti-GPCRX antibody (e.g- monoclonal antibody) can be used to isolate an GPCRX polypeptide by standard techniques, such as affinity chromato raphy or immunoprecipitation. An anti-GPCRX antibody can facilitate the purification of natural GPCRX polypeptide from cells and of recombinantly-produced GPCRX polypeptide expressed in host cells. Moreover, an anti-GPCRX antibody can be used to detect GPCRX protein (e.g- in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe GPCRX protein. Anti-GPCRX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, β.g- o. for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling ( g„ physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, ftuorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes lummol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1 51. 1311. 35S or 3H.
GPCRX Recombinant Expression Vectors and Host Cells
Another aspect ofthe invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an GPCPvX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid". which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g- bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g- non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g- replication defective retroviruses. adenoviruses and adeno-associated viruses), which serve equivalent functions. The recombinant expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g- m n in vjtro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g- polvadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION XECHNOLOGY METHODS IN E ZYMOLOQY 1 5. Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g- tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g- GPCRX proteins, mutant forms of GPCRX proteins, fusion proteins, etc.).
The recombinant expression vectors ofthe invention can be designed for expression of GPCRX proteins in prokaryotic or eukaryotic cells. For example, GPCRX proteins can be expressed in bacterial cells such as Escherichia coli- insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel. GENE EXPRESSION T-ECHNOLOGY METHODS IN NZYMOLQGY 185. Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated m v?γr , for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli w m vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein. Such fusion vectors typically serve three purposes: ( ) to increase expression of recombinant protein; ( ) to increase the solubility ofthe recombinant protein; and ( ) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa. thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech fric; Smith and Johnson, 1988. Gene 7: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia. Piscatawav, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion pr cojj expression vectors include pTrc (Anrrann et α ., (1988) Gene 69:301-315) and pET 1 Id (Studier et al S i E EXPRESSION IECHNOLOGYIMETHODS IN ENZYMOLOGY I85. Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in g c / is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION IECHNOLOGY METHODS IN ENZYMQLOGY 185. Academic Press. San Diego. Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in p; coli-Lsee, e.g., Wada. et al- 1992. Nucl Acids Res- 20-' 2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques. In another embodiment, the GPCRX expression vector is a yeast expression vector-
Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari. et a 1987. ∑MBO J. 6 229-234). pMFa (Kurian and Herskowitz, 1982. Cell 3Q: 933-943), pJRY88 (Schultz et a 1987. Gene 54: 113-123). pYES2 (Invitrogen Corporation. San Diego, Calif), and picZ (InVitrogen Corp. San Diego, Calif.). Alternatively, GPCRX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g- SF9 cells) include the pAc series (Smith, et al- I 83- o/. Cell. Biol. : 2156-2165) and the pVL series (Lucklow and Summers, 1989. Vir l gy 170: 31-39). In vet another embodiment, a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include ρCDM8 (Seed, 1987. Nature 329'- 840> and pMT2PC (Kaufman. et d LjEMBO j 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2. cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, β.g. - Chapters 16 and 17 of
Figure imgf000087_0001
CLONINGIALABORATORY MANUAL- 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.. 1989. hi another embodiment, the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g- tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al- 1987. Genes Dev. ± 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 3: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immuno globulins (Banerii. et a 19 . Cell 3 : 729-740; Queen and Baltimore, 1983. Cell : 741-748), neuron-specific promoters (e g., the neurofilament promoter; Byrne and Ruddle, 1989. pro Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al- 1 85. Science 230: 912-916). and mammary gland-specific promoters (e.g- milk whey promoter; U.S. Pat. No. 4.873.316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed. e.g. - e murine hox promoters (Kessel and Gruss. 1990. Science 249: 374-379) and the κ-fetoprotem promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription ofthe DNA molecule) of an RNA molecule that is antisense to GPCPvX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression ofthe antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion ofthe regulation of gene expression using antisense genes See, e.g., Weintraub, et al- "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics^ Vo1- 1(1) I986-
Another aspect ofthe invention pertains to host cells into which a recombinant expression vector ofthe invention has been introduced. The terms "host cell" and
"recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example. GPCRX protein can be expressed in bacterial cells such as E. coli- insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g- DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et «/__(MOLECULAR CLQNING: A LABORATORY MANUAL- 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. 3h order to identify and select these integrants, a gene that encodes a selectable marker (e.g- resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromvcin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding GPCRX or can be introduced on a separate vector. Cells stablv transfected with the introduced nucleic acid can be identified by drug selection (e.g- cells that have incorporated the selectable marker gene will survive, while the other cells die). A host cell ofthe invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce ( e- express) GPCRX protein. Accordingly, the invention further provides methods for producing GPCRX protein using the host cells ofthe invention, hi one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding GPCPvX protein has been introduced) in a suitable medium such that GPCPvX protein is produced. In another embodiment, the method further comprises isolating GPCRX protein from the medium or the host cell.
Transgenic GPCRX Animals
The host cells ofthe invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which GPCRX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous GPCRX sequences have been introduced into their genome or homologous recombinant animals in which endogenous GPCRX sequences have been altered. Such animals are useful for studying the function, and/or activity of GPCRX protein and for identifying and/or evaluating modulators of GPCRX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous GPCRX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g- an embryonic cell ofthe animal, prior to development ofthe animal.
A transgenic animal ofthe invention can be created by introducing GPCRX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g- bv microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human GPCRX cDNA sequences of SEO 3D NOS:l. 3. 5. 7. 9. 11. 13. 15. 17. 19 and 21. can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non- human homologue ofthe human GPCPvX gene, such as a mouse GPCPvX gene, can be isolated based on hybridization to the human GPCPvX cDNA (described further Suprc an(l use(l as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the GPCPvX transgene to direct expression of GPCPvX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4.870.009: and 4,873.191; and Hogan, 1986. In: MANIPL11ATING THE
Figure imgf000090_0001
Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence ofthe GPCPvX transgene in its genome and/or expression of GPCPvX mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding GPCRX protein can further be bred to other transgenic animals carrying other transgenes. To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an GPCRX gene into which a deletion, addition or substitution has been introduced to thereby alter. e „., functionally disrupt, the GPCRX gene. The GPCRX gene can be a human gene (e g.. the cDNA of SEO 3D NOS:l. 3, 5, 7. 9, 11. 13. 15. 17. 19 and 21), but more preferably, is a non-human homologue of a human GPCPvX gene. For example, a mouse homologue of human GPCRX gene of SEO 3D NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, can be used to construct a homologous recombination vector suitable for altering an endogenous GPCPvX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous GPCPvX gene is functionally disrupted ( e- o longer encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous GPCPvX gene is mutated or otherwise altered but still encodes functional protein (e.g- the upstream regulatory region can be altered to thereby alter the expression of the endogenous GPCPvX protein). In the homologous recombination vector, the altered portion ofthe GPCPvX gene is flanked at its 5'- and 3'-termini by additional nucleic acid ofthe GPCRX gene to allow for homologous recombination to occur between the exogenous GPCRX gene carried by the vector and an endogenous GPCRX gene in an embryonic stem cell. The additional flanking GPCRX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3 '-termini) are included in the vector. See, e.g., Thomas, et aj.. 1987. Cell 51 : 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (β.g- by electroporation) and cells in which the introduced GPCRX gene has homolo ously-recombined with the endogenous GPCRX gene are selected. See, e.g- Li. et a/--. 1992. ce// 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g- a mouse) to form aggregation chimeras. See, e.g- Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM £ELLS: A PRACTICAL APPROACH. Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously-recombined DNA by germline transmission ofthe transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140: WO 92/0968: and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description ofthe cre/loxP recombinase system, See, e.g- Lakso, et a 1"2- Proc. Natl. Acad. Sci. USA ® 6232-6236. Another example of a recombinase system is the FLP recombinase system of
Saccharomyces cerevisiae.-See, O'Gorman, et a 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression ofthe transgene. animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals. g p-., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al- 1997. Nature 38 : 810-813. In brief, a cell (e.g- a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter GQ phase. The quiescent cell can then be fused. e.g- through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone ofthe animal from which the cell (e.g- the somatic cell) is isolated.
Pharmaceutical Compositions
The GPCRX nucleic acid molecules. GPCRX proteins, and anti-GPCRX antibodies (also referred to herein as "active compounds") ofthe invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to. water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e g., intravenous, intradermal, subcutaneous, oral (e.g- inhalation), transdermal (?- g topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens: antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous 5 solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water. Cremophor EL™ (BASF. Parsippany. N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under
10 the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by
15 the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants. Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the
20 composition. Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin- Sterile injectable solutions can be prepared by incorporating the active compound (e.g- an GPCPvX protein or anti-GPCRX antibody) in the required amount in an appropriate solvent
25 with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above, i the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of
3.0 the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin: an excipient such as starch or lactose, a disintegrating agent such as alginic acid. Primogel. or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin: or a flavoring agent such as peppermint. methyl salicylate. or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant. g o-.. a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g- w h conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polvglvcolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4.522.811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by. for example, intravenous injection, local administration (see, e.g., US. Patent No. 5.328,470) or by stereotactic injection (see> .e g., Chen, et aj., 1994. pro Nat Acad. Sci. USA 9i : 3054-3057). The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g- retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules ofthe invention can be used to express GPCRX protein (e.g- v a recombinant expression vector in a host cell in gene therapy applications), to detect GPCRX mRNA (e.g- i a biological sample) or a genetic lesion in an GPCRX gene, and to modulate GPCRX activity, as described further, below. In addition, the GPCRX proteins can be used to screen drugs or compounds that modulate the GPCRX protein activity or expression as well as to treat disorders characterizedby insufficient or excessive production of GPCRX protein or production of GPCRX protein forms that have decreased or aberrant activity compared to GPCRX wild-type protein (e.g- diabetes (regulates insulin release): obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dvslipidemias. In addition, the anti-GPCPvX antibodies ofthe invention can be used to detect and isolate GPCRX proteins and modulate GPCRX activity, hi yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra-
Screening Assays
The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, e- candidate or test compounds or agents (e g-., peptides, peptidomimetics, small molecules or other drugs) that bind to GPCRX proteins or have a stimulatory or inhibitory effect on, e „ GPCRX protein expression or GPCRX protein activity. The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity ofthe membrane-bound form of an GPCRX protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromato raphy selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design l2'- U5-
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be. e.g- nucleic acids, peptides, polypeptides, peptidomimetics. carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any ofthe assays ofthe invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et a/., 1993. Proc. Natl Acad. Sci. U.S.A. 90: 6909; Erb, et a 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et fl/., 1994. j_ Med. Chem. 3 : 2678; Cho, et al- l"3- Science 1^- I30 ; Carrell, et a ^^- Angew. Chem. Int. Ed. Engl. l 2059; Carell, et a 1994. Angew. Chem. Int. Ed. Engl. 33 : 2061; and Gallop, et a 1994. j Med. Chem. 3 : 1233. Libraries of compounds maybe presented in solution (e.g- Houghten, 1992. Bjntech ique* 13 : 412-421). or on beads (Lam. 1991. Nature 54: 82-84). on chips (Fodor. 1993. Nature 364: 555-556). bacteria (Ladner. U.S. Patent No. 5.223.409). spores (Ladner. U.S. Patent 5,233,409), plasmids (Cull, et a 1"2. proc. Natl. Acad. Sci. USA &L 1865-1869) or on phage (Scott and Smith. 1990. Science 249: 386-390: Deylin. 1990. Science 249: 404-406; Cwirla, et a ^^- Proc. Natl Acad. Sci. U.S.A. ^- 6378-6382; Felici, 1991. J. Mol. Biol 222: 301-310; Ladner. U.S. Patent No. 5.233.409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of GPCPvX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to an GPCPvX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability ofthe test compound to bind to the GPCRX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the GPCRX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 1 51, 35S, 14C, or 3H. either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product, hi one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of GPCPvX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds GPCRX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an GPCRX protein, wherein determining the ability ofthe test compound to interact with an GPCRX protein comprises determining the ability ofthe test compound to preferentially bind to GPCRX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of GPCPvX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability ofthe test compound to modulate (e.g- stimulate or inhibit) the activity ofthe GPCRX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of GPCRX or a biologically-active portion thereof can be accomplished, for example, by determining the ability ofthe GPCRX protein to bind to or interact with an GPCRX target molecule. As used herein, a "target molecule" is a molecule with which an GPCRX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an GPCRX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An GPCRX target molecule can be a non-GPCRX molecule or an GPCRX protein or polypeptide ofthe invention. In one embodiment, an GPCRX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound GPCRX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with GPCRX.
Determining the ability ofthe GPCRX protein to bind to or interact with an GPCRX target molecule can be accomplished by one ofthe methods described above for determining direct binding. In one embodiment, determining the ability ofthe GPCPvX protein to bind to or interact with an GPCPvX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger ofthe target (/ g. intracellular Ca2+, diacylglycerol, 3P3, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of a reporter gene (comprising an GPCRX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g- luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In vet another embodiment, an assay ofthe invention is a cell-free assay comprising contacting an GPCPvX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to bind to the GPCPvX protein or biologically- active portion thereof. Binding ofthe test compound to the GPCPvX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the GPCPvX protein or biologically-active portion thereof with a known compound which binds GPCPvX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an GPCPvX protein, wherein determimng the ability ofthe test compound to interact with an GPCPvX protein comprises determining the ability ofthe test compound to preferentially bind to GPCPvX or biologically-active portion thereof as compared to the known compound. In still another embodiment, an assay is a cell-free assay comprising contacting GPCRX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to modulate (e g. stimulate or inhibit) the activity ofthe GPCRX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of GPCRX can be accomplished, for example, by determining the ability ofthe GPCRX protein to bind to an GPCRX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability ofthe test compound to modulate the activity of GPCPvX protein can be accomplished by determining the ability ofthe GPCPvX protein further modulate an GPCPvX target molecule. For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as described, supra- hi yet another embodiment, the cell-free assay comprises contacting the GPCPvX protein or biologically-active portion thereof with a known compound which binds GPCPvX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an GPCPvX protein, wherein determining the ability ofthe test compound to interact with an GPCPvX protein comprises determining the ability ofthe GPCPvX protein to preferentially bind to or modulate the activity of an GPCPvX target molecule.
The cell-free assays ofthe invention are amenable to use of both the soluble form or the membrane-bound form of GPCPvX protein. In the case of cell-free assays comprising the membrane-bound form of GPCPvX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of GPCPvX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside. n-dodecylglucoside. n-dodecylmaltoside. octanoyl-N-methylglucamide. decanoyl-N-methylglucamide. Triton® X-100. Triton® X-114. Thesit®.
Isotridecypolv(ethylene glycol ether)^, N-dodecyl— N.N-dimethyl-3 -ammonio- 1 -propane sulfonate. 3-(3-cholamidoproρyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroχy-l-propane sulfonate (CHAPSO).
In more than one embodiment ofthe above assay methods ofthe invention, it may be desirable to immobilize either GPCPvX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both ofthe proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to GPCRX protein, or interaction of GPCRX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example. GST-GPCRX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or GPCRX protein, and the mixture is incubated under conditions conducive to complex formation (e.g- at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra-
Alternatively, the complexes can be dissociated from the matrix, and the level of GPCRX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays ofthe invention. For example, either the GPCRX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotmylated GPCRX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g- biotinylation kit. Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with GPCRX protein or target molecules, but which do not interfere with binding ofthe GPCRX protein to its target molecule, can be derivatized to the wells ofthe plate, and unbound target or GPCRX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the GPCRX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the GPCRX protein or target molecule.
In another embodiment, modulators of GPCPvX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of GPCPvX mPvNA or protein in the cell is determined. The level of expression of GPCPvX mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of GPCRX mRNA or protein in the absence ofthe candidate compound. The candidate compound can then be identified as a modulator of GPCRX mRNA or protein expression based upon this comparison. For example, when expression of GPCRX mRNA or protein is greater (/_e statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of GPCRX mRNA or protein expression. Alternatively, when expression of GPCRX mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of GPCRX mRNA or protein expression. 5 The level of GPCRX mRNA or protein expression in the cells can be determined by methods described herein for detecting GPCRX mRNA or protein.
In vet another aspect ofthe invention, the GPCPvX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see> e.g- U-S- Patent No. 5,283,317; Zervos, et /., 1993. Cell 72: 223-232; Madura, et al- 1993, J Biol Chem. 268: 12046-12054; 0 Bartel, et al- 1993. Biotechniques u'- 920-924; Iwabuchi, et al- 1993. oncogene3l
1693-1696; and Brent WO 94/10300). to identify other proteins that bind to or interact with GPCRX ("GPCRX-binding proteins" or "GPCRX-bp") and modulate GPCRX activity. Such GPCRX-binding proteins are also likely to be involved in the propagation of signals by the GPCRX proteins as, for example, upstream or downstream elements ofthe GPCRX pathway. 5 The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for GPCRX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g- GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an 0 unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. If the "bait" and the "prey" proteins are able to interact. \n , forming an GPCRX-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g- LacZ) that is operably linked to a transcriptional 5 regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with GPCRX.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
-0- Detection Assays
Portions or fragments ofthe cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: ( ) map their respective genes on a chromosome: and, thus, locate gene regions associated with genetic disease; (/ ) identify an individual from a minute biological sample (tissue typing); and ( ) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below. Chromosome Mapping
Once the sequence (or a portion ofthe sequence) of a gene has been isolated, this sequence can be used to map the location ofthe gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments ofthe GPCRX sequences, SEQ 3D NOS:l, 3, 5, 7, 9, 11. 13. 15. 17, 19 and 21, or fragments or derivatives thereof, can be used to map the location ofthe GPCPvX genes, respectively, on a chromosome. The mapping ofthe GPCPvX sequences to chromosomes is an important first step in coreelating these sequences with genes associated with disease.
Briefly, GPCPvX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the GPCRX sequences. Computer analysis ofthe GPCRX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the GPCRX sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals
(e.g- human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzvme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustacliio, ef a 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the GPCRX sequences to design oligonucleotide primers, sub- localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence jn sjfU hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, seeι Verma, et α/- iuMAN CHROMOSOMES Δ MANUAL OF SASIC TECHNIQUES (Pergamon Press, New York 1988). Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents conesponding to noncoding regions ofthe genes actually are prefened for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. Such data are found. e g in McKusick, MENDELIAN INHERITANCE IN AN- available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, β.g, Egeland, et al- 1987. Natures 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the GPCRX gene, can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing
The GPCRX sequences ofthe invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences ofthe invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Patent No. 5.272,057). Furthermore, the sequences ofthe invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the GPCRX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of coreesponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences ofthe invention can be used to obtain such identification sequences from individuals and from tissue. The GPCRX sequences ofthe invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEO 3D NOS:l. 3. 5, 7. 9. 11. 13. 15. 17. 19 and 21. are used, amore appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect ofthe invention relates to diagnostic assays for determining GPCRX protein and/or nucleic acid expression as well as GPCRX activity, in the context of a biological sample (e.g- blood. serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with abenant GPCRX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease. Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with GPCPvX protein, nucleic acid expression or activity. For example, mutations in an GPCPvX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with GPCPvX protein, nucleic acid expression, or biological activity.
Another aspect ofthe invention provides methods for determining GPCPvX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (refereed to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g- drugs) for therapeutic or prophylactic treatment of an individual based on the genotype ofthe individual (e.g- the genotype ofthe individual examined to determine the ability ofthe individual to respond to a particular agent.)
Yet another aspect ofthe invention pertains to monitoring the influence of agents (e.g- drugs, compounds) on the expression or activity of GPCPvX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of GPCRX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting GPCRX protein or nucleic acid
(e.g- mRNA, genomic DNA) that encodes GPCRX protein such that the presence of GPCRX is detected in the biological sample. An agent for detecting GPCRX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to GPCRX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length GPCRX nucleic acid, such as the nucleic acid ofSEO 3D NOS: 1. 3, 5, 7. 9, 11, 13, 15, 17.- 19 and 21, or a portion thereof, such as an oligonucleotide of at least 15. 30. 50. 100. 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to GPCPvX mPvNA or genomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein. An agent for detecting GPCPvX protein is an antibody capable of binding to GPCPvX protein, preferably an antibody with a detectable label. Antibodies can be polyclonal. or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g- Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling ( g.. physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently- labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is. the detection method ofthe invention can be used to detect GPCRX mRNA, protein, or genomic DNA in a biological sample jn vffro as well as m Vjvo. For example, ? ? vitro techniques for detection of GPCRX mRNA include Northern hybridizations and m sf u hybridizations. jn v?γr(? techniques for detection of GPCRX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. jn rγro techniques for detection of GPCRX genomic DNA include Southern hybridizations. Furthermore, jn V]-Vc techniques for detection of GPCRX protein include introducing into a subject a labeled anti-GPCRX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A prefened biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. hi another embodiment, the methods further involve obtaining a control biological ° sample from a control subject, contacting the control sample with a compound or agent capable of detecting GPCRX protein, mRNA, or genomic DNA, such that the presence of GPCRX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of GPCRX protein. mRNA or genomic DNA in the control sample with the presence of GPCRX protein. mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of GPCRX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting GPCRX protein or mRNA in a biological sample; means for determining the amount of GPCPvX in the sample; and means for comparing the amount of GPCPvX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect GPCPvX protein or nucleic acid.
Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with abenant GPCRX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with GPCRX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with abenant GPCRX expression or activity in which a test sample is obtained from a subject and GPCRX protein or nucleic acid (e.g- mRNA, genomic DNA) is detected, wherein the presence of GPCRX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abenant GPCRX expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g- serum), cell sample, or tissue- Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g- an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abenant GPCRX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder- Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abenant GPCRX expression or activity in which a test sample is obtained and GPCRX protein or nucleic acid is detected (e.g- wherein the presence of GPCRX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with abenant GPCRX expression or activity). The methods ofthe invention can also be used to detect genetic lesions in an GPCRX gene, thereby determimng if a subject with the lesioned gene is at risk for a disorder characterized by abenant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an GPCPvX-protein, or the misexpression ofthe GPCPvX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: ( ) a deletion of one or more nucleotides from an GPCPvX gene; (/ ) an addition of one or more nucleotides to an GPCRX gene; (;//) a substitution of one or more nucleotides of an GPCRX gene, (7- ) a chromosomal reanangement of an GPCRX gene; ( ) an alteration in the level of a messenger RNA transcript of an GPCRX gene, ( ) abenant modification of an GPCRX gene, such as of the methylation pattern ofthe genomic DNA. (v /) the presence of a non- wild-type splicing pattern of a messenger RNA transcript of an GPCRX gene. (Vf ) a non-wild-type level of an GPCRX protein. (fx) allelic loss of an GPCRX gene, and ( ) inappropriate post-translational modification of an GPCRX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an GPCRX gene. A prefened biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells. In certain embodiments, detection ofthe lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g- U-S- Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) ( gg e.g^ Landegran, et al- 1988. Science 241: 1077-1080; and Nakazawa, et al- 1994. proc. Natl Acad. Sci. USA 1: 360-364), the latter of which can be particularly useful for detecting point mutations in the GPCRX- gene (see, Abravaya, et al- 1995. Nucl Acids Res. ^'- 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g- genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an GPCRX gene under conditions such that hybridization and amplification ofthe GPCRX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein. Alternative amplification methods include: self sustained sequence replication (See, Guatelli, et al- 1990. proc. Natl Acad. Sci. USA 87: 1874-1878). transcriptional amplification system (see, Kwoh, et α/., 1989. pr0c. Natl Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, βt al- 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an GPCRX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases. and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (sβe, e g > U.S. Patent No. 5.493.531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. hi other embodiments, genetic mutations in GPCRX can be identified by hybridizing a sample and control nucleic acids, e „., DNA or RNA. to high-density anays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin. et al- 1996. Human Mutation 7: 244-255; Kozal, et a 19 6. ^al Med. 2: 753-759. For example, genetic mutations in GPCRX can be identified in two dimensional anays containing light-generated DNA probes as described in Cronin, e #/., supra- Briefly, a first hybridization anay of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear anays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization anay that allows the characterization of specific mutations by using smaller, specialized probe anays complementary to all variants or mutations detected. Each mutation anay is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the GPCPvX gene and detect mutations by comparing the sequence ofthe sample GPCPvX with the conesponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. proC. Natl. Acad. Sci. USA 74-' 560 or Sanger, 1977. proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (seβι e σ, Naeve, e aj., 1995. pfat fmjφffH, 19: 448). including sequencing by mass spectrometrv (see. g g.. PCT International Publication No. WO 94/16101; Cohen. βt al- 1996. Adv. Chromatography^l 127-162; and Griffin, g; ah 1993. Appl Biochem. Biotechnol. 38: 147-159). Other methods for detecting mutations in the GPCRX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA RNA or RNA/DNA heteroduplexes. See, e.g., Myers, βt al- 1985. Science 30'- 1242. hi general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type GPCRX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions ofthe duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S; nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al, I988- Proc. Natl. Acad. Sci. USA l 4397; Saleeba. et al., 922 .Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection. h still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in GPCRX cDNAs obtained from samples of cells. For example, the mutY enzyme of p;. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et a 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on an GPCRX sequence, e.g- a wild-type GPCRX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459.039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in GPCPvX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Ori a, et al- I989- Proc. Natl. Acad. Sci. USA- 86: 2766; Cotton, -Mutat. Res. 285: 125-144; Hayashi, 1992. Qenet. Anal. Tech. Appl. 9: 73~79- Single-stranded DNA fragments of sample and control GPCPvX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity ofthe assay may be enhanced by using RNA (rather than DNA). in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, e qh 1991. Trends Genet. 7'- 5-
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al, 1985. Nature 3 : 495. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. 3n a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner. 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the lmown mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, e aj.. 1986. Nature 324: 163; Saiki, et a 1989. Proc_ Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may cany the mutation of interest in the center ofthe molecule (so that amplification depends on differential hybridization; See, e.g., Gibbs, et a ' 1989. Nucl Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see> e g > Prossner. 1993. Tibtech. 11 •' 238). In addition it may be desirable to introduce a novel restriction site in the region ofthe mutation to create cleavage-based detection. See, e.g., Gasparini, et al, l" - o/. Cell Probes : 1- I is anticipated that in certain embodiments amplification may also be performed using qg ligase for amplification. See, e.g., Barany. 1991. P oc. Natl. Acad. Sci. USA : 189- hi such cases, ligation will occur only if there is a perfect match at the 3'-terminus ofthe 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used. e.g- in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an GPCRX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which GPCRX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on GPCRX activity (e.g- GPCRX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) hi conjunction with such treatment, the pharmacogenomics (/ e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drug) ofthe individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration ofthe pharmacologically active dnig. Thus, the pharmacogenomics ofthe individual pennits the selection of effective agents (e.g- drugs) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of GPCRX protein, expression of GPCRX nucleic acid, or mutation content of GPCRX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See g-g., Eichelbaum, 1996. clin. Exp. Pharmacol. Physio 23: 983-985; Linder, 1997. clin. Chem. - 43: 254-266. In general, two types of pharmaco genetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmaco genetic conditions can occur either as rare defects or as polymorptiisms. For example, glucose-6-phosρhate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolvsis after ingestion of oxidant drugs (anti-malarials. sulfonamides. analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g- N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicitv after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotvpes in the population, the extensive metabohzer (EM) and poor metabohzer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM. which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety. PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification. Thus, the activity of GPCRX protein, expression of GPCRX nucleic acid, or mutation content of GPCRX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmaco genetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an GPCRX modulator, such as a modulator identified by one ofthe exemplary screening assays described herein. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g- drugs, compounds) on the expression or activity of GPCRX (e.g- the ability to modulate abenant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase GPCPvX gene expression, protein levels, or upregulate GPCPvX activity, can be monitored in clinical trails of subjects exhibiting decreased GPCPvX gene expression, protein levels, or downregulated GPCPvX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease GPCPvX gene expression, protein levels, or downregulate GPCPvX activity, can be monitored in clinical trails of subjects exhibiting increased GPCRX gene expression, protein levels, or upregulated GPCRX activity. In such clinical trials, the expression or activity of GPCRX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers ofthe immune responsiveness of a particular cell- By way of example, and not of limitation, genes, including GPCRX, that are modulated in cells by treatment with an agent (e.g- compound, drug or small molecule) that modulates GPCRX activity (e.g- identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of GPCRX and other genes implicated in the disorder. The levels of gene expression (?- g.. a gene expression pattern) can be quantified by Northern blot analysis or
RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe methods as described herein, or by measuring the levels of activity of GPCRX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment ofthe individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g- an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (/) obtaining a pre-administration sample from a subject prior to administration ofthe agent; (; ) detecting the level of expression of an GPCRX protein, mRNA. or genomic DNA in the preadministration sample; (/ /) obtaining one or more post-administration samples from the subject; (?v) detecting the level of expression or activity ofthe GPCRX protein, mRNA. or genomic DNA in the post-administration samples: ( ) comparing the level of expression or activity ofthe GPCRX protein, mRNA, or genomic DNA in the pre-administration sample with the GPCRX protein, mRNA, or genomic DNA in the post administration sample or samples; and (v ) altering the administration ofthe agent to the subject accordingly. For example, increased administration ofthe agent may be desirable to increase the expression or activity of GPCRX to higher levels than detected, e- to increase the effectiveness ofthe agent. Alternatively, decreased administration ofthe agent may be desirable to decrease expression or activity of GPCRX to lower levels than detected, ;- g„ to decrease the effectiveness ofthe agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abenant GPCRX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASP), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD). valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions ofthe like. These methods of treatment will be discussed more fully, below
Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (j e. - reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: ( ) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (/ ) antibodies to an aforementioned peptide; (z-# nucleic acids encoding an aforementioned peptide; (?y) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" ( e. t due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endoggenous function of an aforementioned peptide by homologous recombination (see- e Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( g , inhibitors, agonists and antagonists, including additional peptide mimetic ofthe invention or antibodies specific to a peptide ofthe invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase ( g , are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., frot biopsy tissue) and assaying it m vjtro for RNA or peptide levels, structure and/or activity ofthe expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, jn situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an abenant GPCRX expression or activity, by administering to the subject an agent that modulates GPCRX expression or at least one GPCRX activity. Subjects at risk for a disease that is caused or contributed to by abenant GPCRX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe GPCRX abenancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of GPCRX abenancy, for example, an GPCRX agonist or GPCRX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods ofthe invention are further discussed in the following subsections. Therapeutic Methods
Another aspect ofthe invention pertains to methods of modulating GPCRX expression or activity for therapeutic purposes. The modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of GPCRX protein activity associated with the cell. An agent that modulates GPCRX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturallv-occurring cognate ligand of an GPCPvX protein, a peptide, an GPCPvX peptidomimetic. or other small molecule. hi one embodiment, the agent stimulates one or more GPCPvX protein activity. Examples of such stimulatory agents include active GPCPvX protein and a nucleic acid molecule encoding GPCPvX that has been introduced into the cell. In another embodiment, the agent inhibits one or more GPCPvX protem activity. Examples of such inhibitory agents include antisense GPCPvX nucleic acid molecules and anti-GPCRX antibodies. These modulatory methods can be performed jn vjtr (e g- bv culturing the cell with the agent) or, alternatively, m vivoSe.g^. by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by abenant expression or activity of an GPCRX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g- an agent identified by a screening assay described herein), or combination of agents that modulates (e.g- up-regulates or down-regulates) GPCRX expression or activity, hi another embodiment, the method involves administering an GPCRX protein or nucleic acid molecule as therapy to compensate for reduced or abenant GPCRX expression or activity.
Stimulation of GPCRX activity is desirable in situations in which GPCRX is abnormally downregulated and/or in which increased GPCRX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized bv abenant cell proliferation and/or differentiation (e.g- cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g- preclampsia).
Determination of the Biological Effect of the Therapeutic
In various embodiments ofthe invention, suitable jn vjf o or in vivo assays are performed to detennine the effect of a specific Therapeutic and whether its administration is indicated for treatment ofthe affected tissue.
In various specific embodiments. jn vjtro assays may be performed with representative cells ofthe type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for jn vfvo testing, any ofthe animal model system known in the art may be used prior to administration to human subjects.
Prophylactic and Therapeutic Uses of the Compositions o the Invention
The GPCPvX nucleic acids and proteins ofthe invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dvslipidemias metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
As an example, a cDNA encoding the GPCRX protein ofthe invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. Bv way of non-limiting example, the compositions ofthe invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders. Alzheimer's Disease. Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dvslipidemias.
Both the novel nucleic acid encoding the GPCRX protein, and the GPCRX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule (j e some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies which immunospecifically-bind to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
EXAMPLES
The following examples illustrate by way of non-limiting example various aspects of the invention.
The following examples illustrate by way of non-limiting example various aspects of the invention. Example 1 : Method of Identifying the Nucleic Acids
The novel nucleic acids ofthe invention were identified by TblastN using a proprietary sequence file, run against the Genomic Daily Files made available by GenBank. The nucleic acids were further predicted by the proprietary software program GenScan™. including selection of exons. These were further modified bv means of similarities using BLAST searches. The sequences were then manually conected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length proteins.
Example 2. Quantitative expression analysis of GPCR2 in various cells and tissues
The quantitative expression of clone GPCRl was assessed in a large number of normal and tumor sample cells and cell lines (Panel 1), as well as in surgical tissue samples
(Panel 2), by real time quantitative PCR (TaqMan®) performed on a Perkin-Elmer Biosystems ABI PRISM® 7700 Sequence Detection System- First, 96 RNA samples were normalized to β-actin and GAPDH. RNA (~50 ng total or ~1 ng polyA+) was converted to cDNA using the TaqMan® Reverse Transcription Reagents Kit (PE Biosystems, Foster City, CA; Catalog No. N808-0234) and random hexamers according to the manufacturer's protocol. Reactions were performed in 20 ul and incubated for 30 min. at 48°C. cDNA (5 ul) was then transfened to a separate plate for the TaqMan® reaction using β-actin and GAPDH TaqMan® Assay Reagents (PE Biosvstems; Catalog Nos. 4310881E and 4310884E, respectively) and TaqMan® universal PCR Master Mix (PE Biosvstems; Catalog No. 4304447) according to the manufacturer's protocol. Reactions were performed in 25 ul using the following parameters: 2 min. at 50°C; 10 min. at 95°C; 15 sec, at 95°C/1 min. at 60°C (40 cycles). Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100. The average CT values obtained for 13-actin and GAPDH were used to normalize RNA samples. The RNA sample generating the highest CT value required no further diluting, while all other samples were diluted relative to this sample according to their β-actin /GAPDH average CT values. Normalized RNA (5 ul) was converted to cDNA and analyzed via TaqMan® using
One Step RT-PCR Master Mix Reagents (PE Biosvstems: Catalog No. 4309169) and gene- specific primers according to the manufacturer's instructions. Probes and primers were designed for each assay according to Perkin Elmer Biosystem's prjmer Express Software package (version I for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input. Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration = 250 nM, primer melting temperature (T ) range = 58°-60° C, primer optimal Tm = 59° C, maximum primer difference = 2° C, probe does not have 5' G, probe T must be 10° C greater than primer T , amplicon size 75 bp to 100 bp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, TX, USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends ofthe probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM each, and probe, 200nM.
PCR conditions: Normalized RNA from each tissue and each cell line was spotted in each well of a 96 well PCR plate (Perkin Elmer Biosystems). PCR cocktails including two probes (a probe specific for the target clone and another gene-specific probe multiplexed with the target probe) were set up using IX TaqMan PCR Master Mix for the PE Biosvstems 7700. with 5 mM MgC12, dNTPs (dA, G, C, U at 1:1:1:2 ratios), 0.25 U/ml AmpliTaq GoldτM (PE Biosystems), and 0.4 U/.tl RNase inhibitor, and 0.25 U/μl reverse transcriptase. Reverse transcription was performed at 48° C for 30 minutes followed by amplification/PCR cycles as follows: 95° C 10 min, then 40 cycles of 95° C for 15 seconds, 60° C for 1 minute.
The results for various cells and cell lines that constitute Panel 1 are shown in Table 10. In Table 10, the following abbreviations are used: ca. = carcinoma; * = established from metastasis; met = metastasis; s cell var= small cell variant; non-s = non-sm =non-small; squam = squamous; pi. eff = pi effusion = pleural effusion; glio = glioma; astro = astrocytoma; and neuro = neuroblastoma.
Panel 2 consists of a 96 well plate (2 control wells, 94 test samples) composed of PvNA/cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI). The tissues procured are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins". The tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologists at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage ofthe patient. These matched margins are taken from the tissue sunounding (i.e. immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue. in Tables 10 and 11). In addition. RNA/cDNA was obtained from various human tissues derived from human autopsies performed on deceased elderly people or sudden death victims (accidents, etc.). These tissue were ascertained to be free of disease and were purchased from various high quality commercial sources such as Clontech. Research Genetics, and Invitrogen.
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electrophoresis using 28s and 18s ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the presence of low molecular weight RNAs indicative of degradation products. Samples are quality controlled for genomic DNA contamination by reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
Example 3. Quantitative expression analysis of GPCRl in various cells and tissues.
The quantitative expression of GPCRl was assessed in a large number of normal and tumor sample cells and cell lines (Panel 1). as well as in surgical tissue samples (Panel 2), by real time quantitative PCR (TaqMan®) performed on a Perkin-Elmer Biosvstems ABI PRISM® 7700 Sequence Detection System as described above in Example 2, with the following primers (Table 9).
Table 9. Probe set Ag2695.
Figure imgf000121_0001
The TaqMan results for panels 1 and 2 are shown in Table 10.
Table 10. TaqMan results for GPCRl
Figure imgf000121_0002
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
The quantitative expression of GPCRl was also assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR; TaqMan®). RTQ PCR was performed on a Perkin-Elmer Biosvstems ABI PRISM® 7700 Sequence Detection System. In this
Example, samples are refened to as Panel 4 and contain cells and cell lines from normal cells and cells related to inflammatory conditions.
Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4r) or cDNA (Panel 4d) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene ,La Jolla, CA) and thymus and kidney (Clontech) were employed. Total RNA from liver tissue from cinhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, hie, Hayward, CA). Intestinal tissue for IvNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDIvI) (Philadelphia, PA).
Astrocytes, lung fibroblasts, dermal fibroblasts. coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothehal cells, human pulmonary aortic endothehal cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as^ indicated. The following cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, 3L-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum.
Mononuclear cells were freshly prepared from normal human blood using standard methods known in the art. Monocvtes were isolated and differentiated by methods well known in the art. CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Nario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56. CD14 and CD19 cells using CD8. CD56. CD14 and CD19 Miltenyi beads and +ve selection. Then CD45RO beads were used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. To obtain B cells, tonsils were procured from ΝPRI and dissected to isolate B cells which were activated using PWM at 5 μg/ml or anti-CD40 (Pharmingen) at approximately 10 ng/ml and IL-4 at 5-10 ng/ml. Primary and secondary Thl/Th2 and Trl cells were cultured using a standard method well known in the art. Activated Thi and Th2 lymphocytes were maintained in this way for a maximum of three cycles. R A was prepared from primary and secondary Thi. Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.
The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1. KU-812. EOL cells were further differentiated bv culture: keratinocvte line CCD 106 and an airway epithelial tumor line ΝCI-H292 were also obtained from the ATCC. CCDl 106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml 3FN gamma.
The primer-probe set used for expression analysis of clone GPCRl is shown in Table 9.
The results of two replicate runs assessing the expression of GPCRl on Panel 3 are shown in Table 11. GPCRl is expressed in normal tissues, such as kidney, thymus, lung and colon. It is most highly expressed on resting monocytes. Surprising results relating to inflammation indicate that the expression of GPCRl is reduced greater than 100 fold (virtually eliminated) on immune-activated monocytes.
Table 11. TaqMan results for clone GPCRl on Panel 3.
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
EQUIVALENTS
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope ofthe appended claims which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope ofthe invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge ofthe embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope ofthe following claims.

Claims

WHAT IS CLAIMED IS:
L_ An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting of SEO 3D NOS-.2. 4. 6. 8. 10. 12. 14. 16. 18. 20. and 22;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEO 3D NOS:2. 4, 6, 8, 10, 12. 14. 16. 18. 20. and 22. wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% ofthe amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEO 3D NOS:2, 4, 6, 8, 10. 12, 14. 16, 18. 20, and 22: and
(d) a variant of an amino acid sequence selected from the group consisting of SEQ 3D NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence.
2 The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence selected from the group consisting of SEO 3D NOS:2. 4. 6. 8. 10. 12. 14. 16. 18. 20. and 22.
3 The polypeptide of claim 2. wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from anucleic acid sequence selected from the group consisting of SEQ 3D NOS:l, 3, 5, 7, 9, 11, 13, 15, 17. 19, and 21.
4. The polypeptide of claim 1, wherein the amino acid sequence of said variant comprises a conservative amino acid substitution. b An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting of SEO 3D NOS:2, 4, 6. 8. 10. 12. 14. 16. 18. 20. and 22;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEO ID NOS:2. 4. 6. 8. 10. 12. 14. 16. 18, 20. and 22. wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% ofthe amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ 3D NOS:2. 4. 6. 8, 10. 12. 14. 16. 18. 20. and 22:
(d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2. 4. 6. 8, 10, 12, 14, 16, 18, 20, and 22, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence chosen from the group consisting of SEO 3D NOS:2, 4, 6. 8, 10, 12, 14, 16, 18, 20, and 22, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence; and
(f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).
6. The nucleic acid molecule of claim 5. wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant.
7. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant.
8^ The nucleic acid molecule of claim 5. wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEO 3D NOS-.l. 3, 5. 7. 9. 11. 13. 15. 17. 19. and 21.
9. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3. 5. 7. 9. 11. 13. 15. 17. 19. and 21:
(b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEO ID NOS.T, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, provided that no more than 20% ofthe nucleotides differ from said nucleotide sequence;
(c) a nucleic acid fragment of (a); and
(d) a nucleic acid fragment of (b).
10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence chosen from the group consisting of SEQ 3D NOS:l. 3, 5. 7. 9, 11. 13. 15, 17, 19. and 21, or a complement of said nucleotide sequence.
11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
(a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20% ofthe nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence;
(b) an isolated second polynucleotide that is a complement ofthe first polynucleotide; and
(c) a nucleic acid fragment of (a) or (b).
12. A vector comprising the nucleic acid molecule of claim 11.
13. The vector of claim 12, further comprising a promoter operably-linked to said nucleic acid molecule.
14. A cell comprising the vector of claim 12.
15. An antibody that binds immunospecifically to the polypeptide of claim 1.
16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.
17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. A method for determining the presence or amount ofthe polypeptide of claim 1 in a sample, the method comprising:
(a) providing the sample:
(b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and
(c) determining the presence or amount of antibody bound to said polypeptide, thereby detennining the presence or amount of polypeptide in said sample.
19. A method for determining the presence or amount ofthe nucleic acid molecule of claim 5 in a sample, the method comprising:
(a) providing the sample:
(b) contacting the sample with a probe that binds to said nucleic acid molecule; and
(c) detennining the presence or amount ofthe probe bound to said nucleic acid molecule, thereby determining the presence or amount ofthe nucleic acid molecule in said sample.
20. The method of claim 19 wherein presence or amount ofthe nucleic acid molecule is used as a marker for cell or tissue type.
21. The method of claim 20 wherein the cell or tissue type is cancerous.
22. A method of identifying an agent that binds to a polypeptide of claim 1, the method comprising:
(a) contacting said polypeptide with said agent; and
(b) determining whether said agent binds to said polypeptide.
23. The method of claim 22 wherein the agent is a cellular receptor or a downstream effector.
24. A method for identifying an agent that modulates the expression or activity of the polypeptide of claim 1. the method comprising:
(a) providing a cell expressing said polypeptide;
(b) contacting the cell with said agent, and
(c) determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide.
25. A method for modulating the activity ofthe polypeptide of claim 1, the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity ofthe polypeptide.
26. A method of treating or preventing a GPCPvX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the polypeptide of claim 1 in an amount sufficient to treat or prevent said GPCPvX-associated disorder in said subject.
27. The method of claim 26 wherein the disorder is selected from the group consisting of cardiomyopathv, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASP), atrioventricular (A-N) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophv. congenital adrenal hypeφlasia, prostate cancer, neoplasm: adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hvpercoagulation. idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy. and other diseases, disorders and conditions ofthe like.
28. The method of claim 26 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
29. The method of claim 26. wherein said subject is a human.
30. A method of treating or preventing a GPCRX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the nucleic acid of claim 5 in an amount sufficient to treat or prevent said GPCRX-associated disorder in said subject.
31. The method of claim 30 wherein the disorder is selected from the group consisting of cardiomyopathv, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASP), atrioventricular (A-N) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hvpercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, A3DS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions ofthe like.
32. The method of claim 30 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
33. The method of claim 30, wherein said subject is a human.
34. A method of treating or preventing a GPCRX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the antibody of claim 15 in an amount sufficient to treat or prevent said GPCPvX-associated disorder in said subject
35. The method of claim 34 wherein the disorder is selected from the group consisting of diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dvslipidemias.
36. The method of claim 34 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
37. The method of claim 34, wherein the subiect is a human.
38. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically-acceptable carrier.
39. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically-acceptable carrier.
40. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically-acceptable carrier.
41. A kit comprising in one or more containers, the phannaceutical composition of claim 38.
42. A kit comprising in one or more containers, the pharmaceutical composition of claim 39.
43. A kit comprising in one or more containers, the pharmaceutical composition of claim 40.
44. A method for determining the presence of or predisposition to a disease associated with altered levels ofthe polypeptide of claim 1 in a first mammalian subject, the method comprising:
(a) measuring the level of expression ofthe polypeptide in a sample from the first mammalian subiect: and
(b) comparing the amount of said polypeptide in the sample of step (a) to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease; wherein an alteration in the expression level ofthe polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.
45. The method of claim 44 wherein the predisposition is to cancers.
46. A method for determining the presence of or predisposition to a disease associated with altered levels ofthe nucleic acid molecule of claim 5 in a first mammalian subiect, the method comprising:
(a) measuring the amount ofthe nucleic acid in a sample from the first mammalian subject; and
(b) comparing the amount of said nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level ofthe nucleic acid in the first subiect as compared to the control sample indicates the presence of or predisposition to the disease.
47. The method of claim 46 wherein the predisposition is to cancers.
48. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising an amino acid sequence of at least one of SEQ 3D NOS:2, 4, 6, 8. 10, 12, 14, 16. 18. 20 and 22, or a biologically active fragment thereof.
49. A method of treating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.
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WO2001077149A2 (en) * 2000-04-07 2001-10-18 Bayer Aktiengesellschaft Regulation of human cyslt2-like gpcr protein
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WO2003080097A3 (en) * 2002-03-22 2004-03-11 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with homo sapiens g - protein - coupled receptor 7 (gpr7)

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