WO2002040539A2 - Nouvelle proteine de type recepteur couple a la proteine g et acides nucleiques codant pour cette nouvelle proteine - Google Patents

Nouvelle proteine de type recepteur couple a la proteine g et acides nucleiques codant pour cette nouvelle proteine Download PDF

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WO2002040539A2
WO2002040539A2 PCT/US2001/032256 US0132256W WO0240539A2 WO 2002040539 A2 WO2002040539 A2 WO 2002040539A2 US 0132256 W US0132256 W US 0132256W WO 0240539 A2 WO0240539 A2 WO 0240539A2
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amino acid
polypeptide
nucleic acid
seq
ofthe
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PCT/US2001/032256
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WO2002040539A3 (fr
Inventor
Ramesh Kekuda
Kimberly A. Spytek
Stacie J. Casman
Bryan D. Zerhusen
Li Li
Velizar T. Tchernev
Steven D. Colman
Robert A. Ballinger
Muralidhara Padigaru
Adam R. Wolenc
Suresh G. Shenoy
Schlomit R. Edinger
Valerie Gerlach
Esha A. Gangolli
John R. Macdougall
Glennda Smithson
John A. Peyman
David J. Stone
Erik Gunther
Karen Ellerman
William M. Grosse
John P. Alsobrook
Denise M. Lepley
Catherine E. Burgess
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Curagen Corporation
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Priority to AU3923502A priority Critical patent/AU3923502A/xx
Application filed by Curagen Corporation filed Critical Curagen Corporation
Priority to AU2002239235A priority patent/AU2002239235A1/en
Publication of WO2002040539A2 publication Critical patent/WO2002040539A2/fr
Publication of WO2002040539A3 publication Critical patent/WO2002040539A3/fr

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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
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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 invention generally relates to novel GPCRl, GPCR2, GPCR3, GPCR4, GPCR5,
  • GPCR6, GPCR7, GPCR8, GPCR9, GPCR10, GPCRl 1 and GPCR12 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 generally relates to nucleic acids and polypeptides. More particularly, the invention relates to nucleic acids encoding novel G-protein coupled receptor (GPCR) polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
  • GPCR G-protein coupled receptor
  • the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
  • novel nucleic acids and polypeptides are referred to herein as GPCRl, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7, GPCR8, GPCR9, GPCRl 0, GPCRl 1 and GPCRl 2 nucleic acids and polypeptides.
  • 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, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.
  • 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:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.
  • 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:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.
  • an oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a GPCRX nucleic acid (e.g., SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96) or a complement of said oligonucleotide.
  • GPCRX nucleic acid e.g., SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96
  • substantially purified GPCRX polypeptides SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96
  • 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 bind 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 includes a method of detecting the presence of a GPCRX polypeptide in a sample, hi 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. hi 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.
  • a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., developmental diseases; MHCII and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal transduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus (NIDDM1); infectious disease; bacterial, fungal, protozoal and viral infections
  • NIDDM1 noninsulin-dependent diabetes mellitus
  • cancer including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer); cancer-associated cachexia; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeodysfrophy; angina pectoris; myocardial infarction; ulcers; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders; including anxiety; schizophrenia; manic depression; delirium; dementia; neurodegenerative disorders;
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders listed above and/or other pathologies and disorders.
  • 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 cDNA encoding GPCRX may be useful in gene therapy, and GPCRX may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering the diseases and disorders listed above and/or other pathologies and disorders.
  • the invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., diseases and disorders listed above and/or other pathologies and disorders and those 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 the diseases and disorders listed above and/or other pathologies and 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.
  • 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 diseases and disorders listed above and/or other pathologies and 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 the diseases and disorders listed above and/or other pathologies and 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 GPCRl, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7, GPCR8, GPCR9, GPCRl 0, GPCRl 1 and GPCR12.
  • GPCRX The nucleic acids, and their encoded polypeptides, are collectively designated herein as "GPCRX”.
  • the novel GPCRX nucleic acids ofthe invention include the GPCRl, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7, GPCR8, GPCR9, GPCRl 0, GPCRl 1 and GPCRl 2 nucleic acids, or a fragment, derivative, analog or homolog thereof.
  • the novel GPCRX proteins ofthe invention include the GPCRl, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7, GPCR8, GPCR9, GPCR10, GPCRl 1 and GPCR12 proteins, or a , derivative, analog or homolog thereof.
  • 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.
  • the GPCRX proteins ofthe invention have a high homology to the 7tm_l domain (PFam Ace. No. pfamOOOOl).
  • the 7tm_l domain is from the 7 transmembrane receptor family, which includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: 5-hydroxytryptamine receptors (See, e.g., PMIM 112821, 8488960, 112805, 231454, 1168221, 398971, 112806); rhodopsin (129209); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • 5-hydroxytryptamine receptors See, e.g., PMIM 112821, 8488960, 112805, 231454, 1168221,
  • proteins that are homologous to any one member ofthe family are also largely homologous to the other members, except where the sequences are different as shown below.
  • GPCRX proteins and nucleic acids disclosed herein suggest that GPCRl -GPCR12 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.
  • 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.
  • GPCRs G-Protein Coupled Receptor proteins
  • Human GPCR generally do not contain introns and belong to four different gene subfamilies, displaying great sequence variability. These genes are dominantly expressed in olfactory epithelium. See, e.g., Ben-Arie et al., Hum. Mol. Genet. 1994 3:229-235; and, Online Mendelian Inheritance in Man (“OMIM") entry # 164342 (http://www.ncbi.nlm.nih.gov/ entrez/ dispomim.cgi?).
  • OMIM Online Mendelian Inheritance in Man
  • the olfactory receptor (“OR") gene family constitutes one ofthe largest GPCR multigene families and is distributed among many chromosomal sites in the human genome. See Rouquier et al., Hum. Mol. Genet. 7(9):1337-45 (1998); Malnic et al., Cell 96:713-23 (1999). Olfactory receptors constitute the largest family among G protein-coupled receptors, with up to 1000 members expected. See Nanderhaeghen et al., Genomics 39(3):239-46 (1997); Xie et al., Mamm. Genome 11 (12): 1070-78 (2000); Issel-Tarver et al., Proc. Natl. Acad. Sci.
  • chemoreceptors Other examples of seven membrane spanning proteins that are related to GPCRs are chemoreceptors. See Thomas et al., Gene 178(1-2): 1-5 (1996). Chemoreceptors have been identified in taste, olfactory, and male reproductive tissues. See id.; Walensky et al., J. Biol.
  • the GPCRX nucleic acids ofthe invention encoding GPCR-like proteins include the nucleic acids whose sequences are provided herein, or fragments thereof.
  • the invention also includes mutant or variant nucleic acids any of whose bases may be changed from the corresponding base shown herein while still encoding a protein that maintains its GPCR-like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any ofthe nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • modifications include, byway of nonlimiting 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.
  • the GPCRX proteins ofthe invention include the GPCR-like proteins whose sequences are provided herein.
  • the invention also includes mutant or variant proteins any of whose residues may be changed from the corresponding residue shown herein while still encoding a protein that maintains its GPCR-like activities and physiological functions, or a functional fragment thereof.
  • the invention further encompasses antibodies and antibody fragments, such as F a b or (F ab ) 2 , that bind immunospecifically to any ofthe proteins ofthe invention.
  • GPCRX nucleic acids and proteins 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 GPCR (or olfactory- receptor) like protein may be useful in gene therapy, and the receptor -like protein may be useful when administered to a subject in need thereof.
  • the nucleic acids and proteins ofthe invention are also useful in potential therapeutic applications used in the treatment of developmental diseases; MHCII and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal fransduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus (NIDDM1); infectious disease; 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); cancer-associated cachexia; anorexia; bulimia; asthma; Parkinson's disease; acute
  • 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 cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • the anti-GPCRX antibody compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders listed above, as well as other related or associated pathologies.
  • the novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein ofthe invention, 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.
  • GPCRl is an Olfactory Receptor ("OR")-like protein.
  • OR Olfactory Receptor
  • Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCRl proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.
  • GPCRl nucleic acids and encoded polypeptides are provided, namely GPCRla, GPCRlb, GPCRlc, GPCRld, GPCRle and GPCRlf.
  • the GPCRl proteins are predicted to be a likely Type Illb membrane protein.
  • the disclosed GPCRl variant is the novel GPCRl a (alternatively referred to as GMAC073079_A), which includes the 964 nucleotide sequence (SEQ ID NO:l) shown in Table 1A.
  • the disclosed GPCRla open reading frame (“ORF") begins at an ATG initiation codon at nucleotides 19-21 and terminates at a TGA codon at nucleotides 958-960. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 1 A, and the start and stop codons are in bold letters. Table 1A.
  • GPCRl nucleotide sequence SEQ ID NO:l).
  • GPCRla target sequence was subjected to the exon linking process to confirm the sequence. These procedures provide the sequences reported below, which are designated GPCRlb (also refered to as AC073079_dal), GPCR2 (also refered to as AC073079_da2), and GPCRlc (also refered to as AC073079_da3).
  • the sequence of GPCRla was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence.
  • In silico prediction was based on sequences available in CuraGen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.
  • the disclosed GPCRl of this invention maps to chromosome 1. Chromosome localization information was assigned using OMIM, the electronic northern bioinfo-rmatic tool implemented by CuraGen Corporation, public ESTs, public literature references and/or genomic clone homologies. This was executed to derive the chromosomal mapping ofthe
  • the disclosed GPCRla polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:l has 313 amino acid residues, has a molecular weight of 34900.65 Daltons, and is presented in Table IB using the one-letter amino acid code.
  • the Signal P, Psort and/or Hydropathy results predict that GPCRla has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the GPCRla protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a GPCRla peptide is between amino acids 47 and 48, i.e., at the dash in the sequence VIA-DT.
  • the disclosed GPCRl variant is the novel GPCRlb (alternatively referred to as AC073079_dal), which includes the 971 nucleotide sequence (SEQ ID NO:3) shown in Table IC.
  • the disclosed GPCRlb open reading frame (“ORF") begins at an ATG initiation codon at nucleotides 30-32 and tenninates at a TGA codon at nucleotides 963-965. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table IC, and the start and stop codons are in bold letters.
  • the disclosed GPCRlb polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has 311 amino acid residues, a molecular weight of 34751.56 Daltons, and is presented in Table ID using the one-letter amino acid code.
  • the Signal P, Psort and/or Hydropathy results predict that GPCRlb has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the GPCRlb protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a GPCRlb peptide is between amino acids 47 and 48, i.e., at the dash in the sequence NIA-DT.
  • the GPCRlb nucleic acid sequence of this invention has 543 of 882 bases (61%) identical to a gb.-GENBANK- ID:AF102523
  • the full amino acid sequence ofthe protein ofthe invention was found to have 137 of 301 amino acid residues (45%) identical to, and 199 of 301 amino acid residues (66%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlN0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).
  • the disclosed GPCRl variant is the novel GPCRlc (alternatively referred to as AC073079_da3), which includes the 971 nucleotide sequence (SEQ ID ⁇ O:5) shown in Table IE.
  • the disclosed GPCRlc open reading frame (“ORF") begins at an ATG initiation codon at nucleotides 30-32 and terminates at a TGA codon at nucleotides 963-965. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table IE, and the start and stop codons are in bold letters.
  • the disclosed GPCRlc polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 has 311 amino acid residues, a molecular weight of 34733.52 Daltons, and is presented in Table IF using the one-letter amino acid code.
  • the Signal P, Psort and/or Hydropathy results predict that GPCRlc has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the GPCRlc protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a GPCRlc peptide is between amino acids 47 and 48, i.e., at the dash in the sequence VIA-DT.
  • the GPCRlc nucleic acid sequence of this invention has 589 of 932 bases (63%) identical to a gb:GENBANK- ID:AF101760
  • the full amino acid sequence ofthe protein ofthe invention was found to have 137 of 301 amino acid residues (45%) identical to, and 199 of 301 amino acid residues (66%) similar to, the 313 amino acid residue ptnr:SPTREMBL- ACC:Q9Z1V0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).
  • the disclosed GPCRl variant is the novel GPCRld (alternatively referred to as BA113A10_B_dal), which includes the 992 nucleotide sequence (SEQ ID NO:7) shown in Table 1G.
  • the disclosed GPCRld open reading frame (“ORF") begins at an GTC codon at nucleotides 3-5 and terminates at a TGA codon at nucleotides 987-989. Putative untranslated region downstream from the termination codon is underlined in Table 1G, and the initial and stop codons are in bold letters. Table 1G. GPCRl nucleotide sequence (SEQ ID NO:7).
  • the open reading frame ofthe disclosed GPCRld nucleic acid is an incomplete cDNA fragment, and it is contemplated that the ORF extends upstream (i.e., in the 5' direction) ofthe sequence provided in SEQ ID NO:7. It is further contemplated that a complete ORF would include an in-frame ATG codon as the start codon.
  • the disclosed GPCRld polypeptide (SEQ ID NO: 8) encoded by SEQ ID NO:7 has 327 amino acid residues, a molecular weight of 36526.60 Daltons, and is presented in Table IH using the one-letter amino acid code.
  • the Signal P, Psort and/or Hydropathy results predict that GPCRld has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • the GPCRld protein is localized to the Golgi body with a certainty of 0.4600, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the microbody (peroxisome) with a certainty of 0.1000.
  • the most likely cleavage site for a GPCRld peptide is between amino acids 63 and 64, i.e., at the dash in the sequence VIA-DT.
  • the GPCRld nucleic acid sequence of this invention has 555 of 904 bases (61%) identical to a gb:GENBANK- ID:AF 102523
  • the full amino acid sequence ofthe protein ofthe invention was found to have 140 of 308 amino acid residues (45%) identical to, and 203 of 308 amino acid residues (65%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlN0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).
  • the disclosed GPCRl variant is the novel GPCRl e (alternatively referred to as BA113A10_B_da3), which includes the 971 nucleotide sequence (SEQ ID ⁇ O:9) shown in Table II.
  • the disclosed GPCRle open reading frame (“ORF") begins at an ATG initiation codon at nucleotides 24-26 and terminates at a TGA codon at nucleotides 936- 938. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table II, and the start and stop codons are in bold letters.
  • the disclosed GPCRle polypeptide (SEQ ID NO: 10) encoded by SEQ ID NO:9 has 313 amino acid residues, a molecular weight of 34928.71 Daltons, and is presented in Table IJ using the one-letter amino acid code.
  • the Signal P, Psort and/or Hydropathy results predict that GPCRle has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the GPCRle protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a GPCRle peptide is between amino acids 49 and 50, i.e., at the dash in the sequence VIA-DT. Table IJ. Encoded GPCRle protein sequence (SEQ ID NO:10).
  • the GPCRle nucleic acid sequence of this invention has 555 of 904 bases (61%) identical to a gb:GENBANK- ID:AF102523
  • the full amino acid sequence ofthe protein ofthe invention was found to have 139 of 308 amino acid residues (45%) identical to, and 202 of 308 amino acid residues (65%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlN0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).
  • the disclosed GPCRl variant is the novel GPCRlf (alternatively referred to as CG50303_02), which includes the 992 nucleotide sequence (SEQ ID ⁇ O:l 1) shown in Table IK.
  • the disclosed GPCRl open reading frame (“ORF") begins at an ATG initiation codon at nucleotides 21-23 and terminates at a TAG codon at nucleotides 954-956. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table IK, and the start and stop codons are in bold letters.
  • CTGTCTTTTGTTTCTCTTGC CAAGGCCCCATACTGTGGATCATGGCAAATCTGAGCCAGCCCTCCG AATTTGTCCTCTTGGGCTTCTCCTCCTTTGGTGAGCTGCAGGCCCTTCTGTATGGCCCCTTCCTCATGC TTTATCTTCTCGCCTTCATGGGAAACACCATCATCATAGTTATGGTCATAGCTGACACCCACCTACATA CACCCATGTACTTCTTCCTGGGCAATTTTTCCCTGCTGGAGATCTTGGTAACCATGACTGCAGTGCCCA GGATGCTCTCAGACCTGTTGGTCCCACAAAGTCATTACCTTCACTGGCTGCATGGTCCAGTTCTACT TCCACTTTTCCCTGGGGTCCACCTCCTTCCTCATCCTGACAGACATGGCCCTTGATCGCTTTGTGGCCA TCTGCCACCCACTGCGCTATGGCACTCTGATGAGCCGGGCTATGTGTCCAGCTGGCTGGCTGGCTGCCT GGGCAGCTCCTTTCCTAGCCATGGTACCCACTGTCCTCTCTC
  • the disclosed GPCRlf polypeptide (SEQ ID NO:12) encoded by SEQ ID NO:ll has 311 amino acid residues, a molecular weight of 34741.50 Daltons, and is presented in Table 1L using the one-letter amino acid code.
  • the Signal P, Psort and/or Hydropathy results predict that GPCRlf has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • the GPCRlf protein is localized to the Golgi body with a certainty of 0.4600, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the microbody (peroxisome) with a certainty of 0.1000.
  • the most likely cleavage site for a GPCRlf peptide is between amino acids 57 and 58, i.e., at the dash in the sequence NIA-DT.
  • the full amino acid sequence ofthe protein ofthe invention was found to have 139 of 301 amino acid residues (46%) identical to, and 199 of 301 amino acid residues (66%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlV0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).
  • any reference to GPCRl 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 any one ofthe "b” through “f ' variants.
  • Example 3 Sequence differences between the GPCRl clones are shown in the ClustalW alignment in Table 1L, with variant positions marked with a "o" above the variant sequence.
  • 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 sequence e.g., patp ace no.
  • AAG71691 Homo sapiens olfactory receptor polypeptide retrieved from the GPCRl BLAST analysis ofthe proprietary PatP database matched the Query GPCRl sequence purely by chance is 7.2xl0 "166 , as shown in Table 1M.
  • the Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences of a database of comparable complexity. Essentially, the E value describes the random background noise that exists for matches between sequences.
  • the E value is used as a convenient way to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001. In BLAST 2.0, the E value is also used instead ofthe P value (probability) to report the significance of matches.
  • an E value of one assigned to a hit can be interpreted as meaning that in a database ofthe current size one might expect to see one match with a similar score simply by chance.
  • An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.mh.gov/Education BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering ofthe query for low-complexity sequence that is performed to prevent artifactual hits.
  • the filter substitutes any low-complexity sequence that it finds with the letter "N” in nucleotide sequence (e.g., "NN-N ⁇ N -N-NNN”) or the letter "X” in protein sequences (e.g., "XXX”).
  • Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment (Wootton and Federhen, Methods Enzymol 266:554-511, 1996).
  • the amino acid sequence of GPCRl had high homology to other proteins as shown in Table 1M.
  • nucleic acid sequence of this invention has 555 of 904 bases (61%) identical to a gb:GENBANK- ID:AF102523
  • the full amino acid sequence ofthe protein ofthe invention was found to have 140 of 308 amino acid residues (45%) identical to, and 203 of 308 amino acid residues (65%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlV0 protein from Mus musculus (Mouse) (Olfactory Receptor C6).
  • GPCRl also has homology to the proteins shown in the BLASTP data in Table IF.
  • GPCRl protein contains the following protein domain (as defined by friterpro): domain name 7tm_l 7 transmembrane receptor (rhodopsin family).
  • DOMAIN results for GPCRl were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections.
  • SEQ ID NO: 19 signature consensus sequence shown in Table 1Q below.
  • Table IR lists the domain description from DOMAIN analysis results against GPCRl. This indicates that the GPCRl sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO:39).
  • SEQ ID NO:39 For Table IR and all successive DOMAIN sequence alignments, fully conserved single residues are indicated by black shading and "strong" semi-conserved residues are indicated by grey shading.
  • the "strong" group of conserved amino acid residues may be any one ofthe following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the DOMAIN results are listed in Table IR with the statistics and domain description.
  • GPCRs guanine nucleotide-binding proteins
  • G-protein-coupled receptors constitute a vast protein family that encompasses a wide range of functions (including various autocrine, paracrine and endocrine processes). They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups.
  • the term clan is use to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence.
  • the currently known clan members include the rhodopsin-like GPCRs, the secretin- like GPCRs, the cAMP receptors, the fungal mating pheromone receptors, and the metabotropic glutamate receptor family.
  • the Olfactory Receptor-like GPCRl disclosed in this invention is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, coipus 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 lymph
  • This information was derived by determimng the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCRl is provided in Example 2.
  • the nucleic acids and proteins of GPCRl are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described above and further herein.
  • the novel GPCRl nucleic acid encoding the GPCR-like 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.
  • 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.
  • the disclosed GPCRl protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • GPCRla, GPCRlb and GPCRle have similar hydropathy plots, and hence are predicted to have similar epitope locations.
  • a contemplated GPCRl epitope for these variants is from about amino acids 1 to 20.
  • these GPCRl variants have epitopes that are from about amino acids 155 to 195, from about amino acids 230 to 240, from about amino acids 255 to 275 and from about amino acids 290 to the C- terminus.
  • a contemplated GPCRlc epitope is from about amino acids 1 to 25, from about amino acids 50 to 65, from about amino acids 120 to 130, from about amino acids 155 to 190, from about amino acids 220 to 240, from about amino acids 250 to 275 and from about amino acids 280 to the C-terminus.
  • a contemplated GPCRld epitope is from about amino acids 1 to 20, from about amino acids 170 to 210, from about amino acids 245 to 255, from about amino acids 270 to 285 and from about amino acids 305 to the C-terminus.
  • a contemplated GPCRlf epitope is from about amino acids 1 to 20, from about amino acids 165 to 210, from about amino acids 235 to 250, from about amino acids 260 to 280 and from about amino acids 300 to the C-terminus.
  • the disclosed novel GPCR2 (alternatively referred to herein as AC073079_da2) includes the 990 nucleotide sequence (SEQ ID NO:20) shown in Table 2 A.
  • a GPCR2 ORF begins with a Kozak consensus ATG initiation codon at nucleotides 7-9 and ends with a TAG codon at nucleotides 937-939. 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.
  • GPCR2 A GPCR-like protein ofthe invention, referred to herein as GPCR2, is an Olfactory Receptor ("OR")-like protein. Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR2 proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.
  • the GPCR2 polypeptide (SEQ ID NO:21) encoded by SEQ ID NO:20 is 310 amino acids in length, has a molecular weight of 35240.98 Daltons, and is presented using the one- letter amino acid code in Table 2B.
  • GPCR2 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the GPCR2 protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a GPCRla peptide is between amino acids 40 and 41, i.e., at the dash in the sequence LMG-NT.
  • GPCR2 is predicted to be a likely Type Illb membrane protein. Additional SNP variants of GPCR2 are disclosed in Example 3.
  • nucleic acid sequence of this invention has 302 of 343 bases (88%) identical to a gb:GENBANK- ID:HSHTPRX06
  • the full amino acid sequence ofthe protein ofthe invention was found to have 134 of 306 amino acid residues (43%) identical to, and 189 of 306 amino acid residues (61%) similar to, the 313 amino acid residue ptnr: SPTREMBL- ACC.-Q9Z1 NO protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).
  • the amino acid sequence of GPCR2 had high homology to other proteins as shown in
  • nucleic acid sequence of this invention has 543 of 882 bases (61%) identical to a gb:GE ⁇ BA- ⁇ K- ID:AF102523
  • the full amino acid sequence ofthe protein ofthe invention was found to have 137 of 301 amino acid residues (45%) identical to, and 199 of 301 amino acid ⁇ residues (66%) similar to, the 313 amino acid residue ptnr: SPTREMBL- ACC:Q9Z1V0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).
  • GPCR2 also has homology to the proteins shown in the BLASTP data in Table 2F.
  • SEQ ID NO 21 GPCR2 2.
  • SEQ ID NO 22 TREMBLNEW-ACC:AAK70859 ODORANT RECEPTOR ORZ6 3.
  • SEQ ID NO 23 SPTREMBL-ACC:Q9Z1V0 OLFACTORY RECEPTOR C6 4.
  • SEQ ID NO 24 SPTREMBL-ACC:Q9EPV0 M50 OLFACTORY RECEPTOR 5.
  • SEQ ID NO 25 SPTREMBL-ACC:Q9EPG1 M50 OLFACTORY RECEPTOR 6.
  • SEQ ID NO 26 SPTREMBL-ACC:Q9EPG2 M51 OLFACTORY RECEPTOR
  • Table 2H lists the domain description from DOMAIN analysis results against GPCR2. This indicates that the GPCR2 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.
  • the Olfactory Receptor-like GPCR2 disclosed in this invention 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 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
  • 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 herein.
  • the novel GPCR2 nucleic acid encoding the GPCR-like 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. 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.
  • the disclosed GPCR2 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR2 epitope is from about amino acids 5 to 25. In additional embodiments, GPCR2 epitopes are from about amino acids 50 to 60, from about amino acids 80 to 100, from about amino acids 130 to 145, from about amino acids 230 to 240, from about amino acids 260 to 270 and from about amino acids 290 to 310.
  • GPCR3 is from about amino acids 50 to 60, from about amino acids 80 to 100, from about amino acids 130 to 145, from about amino acids 230 to 240, from about amino acids 260 to 270 and from about amino acids 290 to 310.
  • the disclosed novel GPCR3 (alternatively referred to herein as sggc_draft_ba656o22_ 2000073 l_da4) includes the 971 nucleotide sequence (SEQ ID NO:27) shown in Table 5 A.
  • a GPCR3 ORF begins with a Kozak consensus ATG initiation codon at nucleotides 3-5 and ends with a TAG codon at nucleotides 963-965. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 3A, and the start and stop codons are in bold letters.
  • the GPCR3 polypeptide (SEQ ID NO:28) encoded by SEQ ID NO:27 is 320 amino acids in length, has a molecular weight of 35321.4 Daltons, and is presented using the one- letter amino acid code in Table 3B.
  • the Psort profile for GPCR3 predicts that these sequences have a signal peptide and are likely to be localized at the endoplasmic reticulum (membrane) with a certainty of 0.6850.
  • a GPCR3 polypeptide is located to the plasma membrane with a certainty of 0.6400, the Golgi body with a certainty of 0.4600, or the endoplasmic reticulum (lumen) with a certainty of 0.1000.
  • the Signal P predicts a likely cleavage site for a GPCR3 peptide is between positions 44 and 45, i.e., at the dash in the sequence GNG-II.
  • Example 3 Additional SNP variants of GPCR3 are disclosed in Example 3.
  • the amino acid sequence of GPCR3 had high homology to other proteins as shown in Table 3C.
  • nucleic acid sequence of this invention has 602 of 906 bases (66%) identical to a gb:GENBANK- ID:AF098664
  • the full amino acid sequence ofthe protein ofthe invention was found to have 182 of 305 amino acid residues (59%) identical to, and 233 of 305 amino acid residues (76%) similar to, the 312 amino acid residue ptnr:SWISSPROT- ACC:P23275 protein from-M ⁇ musculus (Mouse) [OLFACTORY RECEPTOR 15 (O-R3)].
  • Table 3F lists the domain description from DOMAIN analysis results against GPCR3.
  • This protein contains domain IPR000276 at amino acid positions 42 to 291. This indicates that the GPCR3 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.
  • bits bits
  • GPCR3 A GPCR-like protein ofthe invention, referred to herein as GPCR3, is an Olfactory Receptor ("OR")-like protein.
  • OR Olfactory Receptor
  • Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR3 proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.
  • the GPCR3 disclosed in this invention is expressed in at least some ofthe following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. This information was derived by determining the tissue sources ofthe sequences that were included in the invention. Further expression data for GPCR3 is provided in Example 2.
  • the nucleic acids and proteins of GPCR3 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.
  • the novel nucleic acid encoding the GPCR-like protein ofthe invention, 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. 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.
  • the disclosed GPCR3 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR3 epitope is from about amino acids 220 to 245.
  • a GPCR3 epitope is from about amino acids 255 to 270.
  • GPCR3 epitopes are from about amino acids 275 to 315.
  • a fourth GPCR-like protein ofthe invention is an Olfactory Receptor ("OR")-Hke protein.
  • OR Olfactory Receptor
  • GPCR4a Two alternative novel GPCR4 nucleic acids and encoded polypeptides are provided, namely GPCR4a and GPCR4b.
  • GPCR4a Two alternative novel GPCR4 nucleic acids and encoded polypeptides are provided, namely GPCR4a and GPCR4b.
  • a GPCR4 variant is the novel GPCR4a (alternatively referred to herein as AC0170103_A_dal), which includes the 1025 nucleotide sequence (SEQ ID NO:34) shown in Table 4A.
  • a GPCR4a ORF begins with a Kozak consensus ATG initiation codon at nucleotides 33-35 and ends with a TGA codon at nucleotides 969-971. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4A, and the start and stop codons are in bold letters.
  • the sequence of GPCR4a was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence.
  • In silico prediction was based on sequences available in CuraGen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.
  • the cDNA coding for the GPCR4a sequence was cloned by the polymerase chain reaction (PCR). Primers were designed based on in silico predictions ofthe full length or some portion (one or more exons) ofthe cDNA/protein sequence ofthe invention. The DNA sequence and protein sequence for a novel Olfactory Receptor-like gene were obtained by exon linking and are reported here as GPCR4a.These primers and methods used to amplify GPCR4a cDNA are described in the Examples.
  • the GPCR4a polypeptide (SEQ ID NO:35) encoded by SEQ ID NO:34 is 312 amino acids in length, has a molecular weight of 34688.2 Daltons, and is presented using the one- letter amino acid code in Table 4B.
  • the Psort profile for GPCR4a predicts that this sequence has a signal peptide and are likely to be localized at the plasma membrane with a certainty of 0.6000.
  • a GPCR4a polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or a microbody (peroxisome) with a certainty of 0.3000.
  • the Signal P predicts a likely cleavage site for a GPCR4a peptide is between positions 47 and 48, i.e., at the dash in the sequence LMANS.
  • a GPCR4 variant is the novel GPCR4b (alternatively referred to herein as CG54212-03), which includes the 917 nucleotide sequence (SEQ ID NO:36) shown in Table 4C.
  • the GPCR4b ORF was identified at nucleotides 3-5 with a CTC codon and ends with a TGA codon at nucleotides 861-863. Putative untranslated regions upstream from the initiation codon and downsfream from the termination codon are underlined in Table 4C, and the start and stop codons are in bold letters.
  • the GPCR4b protein (SEQ ID NO:37) encoded by SEQ ID NO:36 is 286 amino acids in length, has a molecular weight of 31693.8 Daltons, and is presented using the one-letter code in Table 4D.
  • the Psort profile for GPCR4b predicts that this sequence has a signal peptide and is likely to be localized at the endoplasmic reticulum (membrane) with a certainty of 0.6850.
  • a GPCR4b polypeptide is located to the plasma membrane with a certainty of 0.6400, the Golgi body with a certainty of 0.4600, or to the endoplasmic reticulum (lumen) with a certainty of 0.1000.
  • the Signal P predicts a likely cleavage site for a GPCR4a peptide is between positions 21 and 22, i.e., at the dash in the sequence MAN ' S.
  • any reference to GPCR4 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.
  • the amino acid sequence of GPCR4 has high homology to other proteins as shown in Table 4E.
  • the GPCR4a nucleic acid sequence has 852 of 1010 bases (84 %) identical to aMus musculus orl7c gene mRNA (GENBANK-ID: MMU133429
  • the full amino acid sequence ofthe GPCR4a protein ofthe invention was found to have 256 of 312 amino acid residues (82%) identical to, and 275 of 312 residues (88 %) positive with, the 317 amino acid residue OLFACTORY RECEPTOR-LIKE PROTEIN protein froml- ⁇ musculus, 312 aa (ptnr: SPTREMBL- ACC:Q9QZ18).
  • the GPCR4b nucleic acid sequence of this invention has 781 of 920 bases (84%) identical to a gb:GENBANK-ID:MMU133429
  • the full amino acid sequence ofthe GPCR4b protein ofthe invention was found to have 234 of 286 amino acid residues (81%) identical to, and 253 of 286 amino acid residues (88%) similar to, the 312 amino acid residue ptnr:SPTREMBL-ACC:Q9QZ18 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR).
  • bits bits
  • the cDNA coding for the GPCR4b sequence was cloned by the polymerase chain reaction (PCR). Primers were designed based on in silico predictions ofthe full length or some portion (one or more exons) ofthe cDNA protein sequence ofthe invention. The DNA sequence and protein sequence for a novel Olfactory Receptor-like gene were obtained by exon linking and are reported here as GPCR4b. These primers and methods used to amplify GPCR4b cDNA are described in the Examples.
  • the GPCR4 disclosed in this invention 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
  • tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. This is by no way limiting in that olfactory receptors are a class of G protein-coupled receptor which are known to be expressed in all tissue types. Further tissue expression analysis is provided in Example 2.
  • the nucleic acids and proteins of GPCR4 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.
  • novel nucleic acid encoding the GPCR-like protein ofthe invention, 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. 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.
  • the disclosed GPCR4b protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR4b epitope is from about amino acids 220 to 250. In other specific embodiments, GPCR4b epitopes are from about amino acids 255 to 290.
  • GPCR5 The disclosed novel GPCR5 (alternatively referred to herein as 21629632.0.20) includes the 2028 nucleotide sequence (SEQ ID NO:43) shown in Table 5A.
  • a GPCR5 ORF begins with a Kozak consensus ATG initiation codon at nucleotides 469-471 and ends with a TAG codon at nucleotides 1447-1449. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 5A, and the start and stop codons are in bold letters. Table 5A.
  • GPCR5 Nucleotide Sequence SEQ ID NO:43
  • the GPCR5 polypeptide (SEQ ID NO:44) encoded by SEQ ID NO:43 is 326 amino acids in length, has a molecular weight of 35715.1 Daltons, and is presented using the one- letter amino acid code in Table 5B.
  • the Psort profile for GPCR5 predicts that these sequences have a signal peptide and are likely to be localized at the plasma membrane with a certainty of 0.0.6000.
  • a GPCR5 polypeptide is located to the Golgi body with a certainty of 0.0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial innpr membrane with a certainty of 0.3000.
  • Example 3 Additional SNP variants of GPCR5 are disclosed in Example 3.
  • the amino acid sequence of GPCR5 had high homology to other proteins as shown in Table 5C.
  • Table 5C Table 5C.
  • nucleic acid sequence of this invention has 1024 of 1351 bases (75%) identical to a gb:GENBANK- ID:-yi-MU133430
  • the full amino acid sequence ofthe protein ofthe invention was found to have 230 of 314 amino acid residues (73%) identical to, and 261 of 314 amino acid residues (83%) similar to, the 315 amino acid residue ptnr:SPTREMBL-ACC:Q9QZ17 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR).
  • Table 5F lists the domain description from DOMAIN analysis results against GPCR5. This protein contains domain IPR000276 at amino acid positions 41 to 287. This indicates that the GPCR5 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.
  • bits bits
  • pfam00001 7tm_ 1 7 transmembrane receptor (rhodopsin family) 114 3.3 e-35
  • GPCR5 88 TQRTISFPGCALQMYLTLALGSTECLLLAVMAYDRYVAICQPLRYPEL 135
  • GPCR5 A GPCR-like protein ofthe invention, referred to herein as GPCR5, is an Olfactory
  • OR Olfactory Receptor
  • Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR5 proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.
  • the GPCR disclosed in this invention is expressed in at least in some of the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the nucleic acids and proteins of GPCR5 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.
  • novel nucleic acid encoding the GPCR-like protein ofthe invention, 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. 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.
  • the disclosed GPCR5 protein has multiple hydrophilic regions, each of which can be used as an immunogen. hi one embodiment, a contemplated GPCR5 epitope is from about amino acids 1 to 25. In another embodiment, a GPCR5 epitope is from about amino acids 120 to 140. hi further specific embodiments,
  • GPCR5 epitopes are from about amino acids 230 to 245 and from about amino acids 252 to 280.
  • GPCR6 GPCR6
  • OR Olfactory Receptor
  • the disclosed novel GPCR6 (alternatively referred to herein as CG 50177-01) includes the 766 nucleotide sequence (SEQ ID NO:50) shown in Table 6A.
  • a GPCR6 ORF begins with a Kozak consensus ATG initiation codon at nucleotides 2-4 and ends with a TAG codon at nucleotides 761-763. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 6A, and the start and stop codons are in bold letters.
  • the GPCR6 protein (SEQ ID NO:51 encoded by SEQ ID NO50 is 253 aa in length, has a molecular weight of 28233.48 Daltons, and is presented using the one-letter amino acid code in Table 6B.
  • the Psort profile for GPCR6 predicts that these sequences have a signal peptide and are likely to be localized at the plasma membrane with a certainty of 0.640.
  • the Signal P predicts a likely cleavage site for a GPCR6 peptide is between positions 54 and 55, i.e., at the dash in the sequence ADA-AL.
  • Example 3 The amino acid sequence of GPCR6 had high homology to other proteins as shown in
  • nucleic acid sequence of this invention has 278 of 406 bases (68%) identical to a gb:GENBANK-
  • GPCR6 also has homology to the proteins shown in the BLASTP data in Table 6D.
  • Table 6F lists the domain description from DOMAIN analysis results against GPCR6.
  • GPCR6 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.
  • bits bits
  • GPCR6 56 LHEPMYLFLAMLAAIDLVLSSSALPKMLAIFWFRDREINFFACLAQMFFLHSFSIMESAV 115 7tm_l: 15 LRTPTNIFLLNLAVADLLFLLTLPPWALYYLVGGDWVFGDALCKLVGALFWNGYASILL 74
  • GPCR6 116 LLAMAFDRYVAICKPLHYTKVLT 138 (SEQ ID NO.51) 7tm_l: 75 LTAISIDRYLAIVHPLRYRRIRT 97 (SEQ ID NO: 18)
  • the GPCR6 disclosed in this invention 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
  • the nucleic acids and proteins of GPCR6 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.
  • the novel nucleic acid encoding the GPCR-like protein ofthe invention, 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. 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.
  • the disclosed GPCR6 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR6 epitope is from about amino acids 2 to 50. In another embodiment, a GPCR6 epitope is from about amino acids 50 to 80. In further specific embodiments, GPCR6 epitopes are from about amino acids 90 to 100, from about amino acids 100 to 175, from about amino acids 180 to 210 and from about amino acids 220 to 230.
  • GPCR7 is an Olfactory Receptor ("OR")-like protein.
  • OR Olfactory Receptor
  • the novel GPCR7 nucleic acid sequences were identified on chromosome 9 as described in Example 1. Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR7 proteins are available at the appropriate sub- cellular localization and hence accessible for the therapeutic uses described in this application.
  • Two alternative novel GPCR7 nucleic acids and encoded polypeptides are provided, namely GPCR7a and GPCR7b.
  • a GPCR7 variant is the novel GPCR7a (alternatively referred to herein as CG50201-01), which includes the 1000 nucleotide sequence (SEQ ID NO:57) shown in Table 7A.
  • a GPCR7a ORF begins with a Kozak consensus ATG initiation codon at nucleotides 3-5 and ends with a TGA codon at nucleotides 951-953. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 7 A, and the start and stop codons are in bold letters.
  • the GPCR7 protein (SEQ ID NO:58) encoded by SEQ ID NO:57 has 316 amino acid residues and is presented using the one-letter code in Table 7B.
  • the predicted molecular weight of GPCR7 protein is approximately 34266.42 Daltons.
  • the Psort profile for GPCR7 predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.4600.
  • the Signal P predicts a likely cleavage site between positions 42 and 43, i.e., at the dash in the sequence LLG-NG.
  • the DNA sequence and protein sequence of GPCR7a was obtained by exon linking as described in the Example 1.
  • a GPCR7 variant is the novel GPCR7b (alternatively referred to herein as CG50257-01 or CG40267-01), which includes the 991 nucleotide sequence (SEQ ID NO:59) shown in Table 7C.
  • the GPCR7b ORF begins with a Kozak consensus ATG initiation codon at nucleotides 3-5 and ends with a TGA codon at nucleotides 951-953, which are in bold letters in Table 4C.
  • the GPCR7b protein (SEQ ID NO:60) encoded by SEQ ID NO:59 is 316 amino acids in length, has a molecular weight of 34266.42 Daltons, and is presented using the one-letter code in Table 7D.
  • the most likely cleavage site for a GPCR7b peptide is between amino acids 42 and 43, i.e., at the dash in the sequence LLG-NG, based on the SignalP result.
  • the DNA sequence and protein sequence of GPCR7a was obtained by exon linking as described in the Example 1.
  • the amino acid sequence of GPCR7 had high homology to other proteins as shown in Table 7e.
  • nucleic acid sequence of this invention has 592 of 880 bases (67%) identical to a gb:GENBANK- ID:RATOLFPROC
  • the full amino acid sequence ofthe GPCR7 was found to have 174 of 309 amino acid residues (56%) identical to, and 223 of 309 amino acid residues (72%) similar to, the 313 amino acid residue ptnr:pir-id:B23701 protein from rat (olfactory receptor F5).
  • GPCR7 also has homology to the proteins shown in the BLASTP data in Table 7F.
  • Table 7H lists the domain description from DOMAIN analysis results against GPCR7. This indicates that the GPCR7 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.
  • bits (bits ) value gnl
  • GPCR7 42 GNGLIV-AAIQASPALHAPMYFLLAHLSFADLCFASVTVPKMLANLLAHDHSISLAGCLTQ 101 7tm 1: 1 GNLLVILVILRTKKLRTPTNIFLLNLAVADLLFLLTLPPWALYYLVGGD VFGDALCKLV 60
  • GPCR7 222 IAAAV LQLPSASGRLRAVSTCGSHLAWSLFYGTVIAVYFQA TSRRE 268 7tm 1: 172 ILRTLRKRARSQRSLKRRSSSERKAAKMLLVWVVFVLCWLPYHIVLLLDSLCLLSI RV 231
  • GPCR7 269 AE GRVATVMYTWTP LNPIIY 291 (SEQ ID NO: 58, 60) 7tm 1: 232 LPTALLITLWLAYVNSCLNPIIY 254 (SEQ ID NO: 18)
  • the GPCR7 protein predicted here is similar to the "Olfactory Receptor-Like Protein Family", some members of which end up localized at the cell surface where they exhibit activity. Therefore, it is likely that this novel GPCR7 protein is available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.
  • the Olfactory Receptor-like GPCR7 proteins disclosed 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 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
  • olfactory receptors are a class of G protein-coupled receptor which are known to be expressed in all tissue types. Further tissue expression analysis is provided in the Examples.
  • the nucleic acids and proteins of GPCR7 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further herein. 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.
  • the disclosed GPCR7 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR7 epitope is from about amino acids 10 to 80.
  • GPCR7 epitopes are from about amino acids 100 to 125, from about amino acids 130 to 160, from about amino acids 180 to 240 and from about amino acids 270 to 285.
  • GPCR8 The disclosed GPCR8 (also referred to as CG50193-01) includes the 1022 nucleotide sequence (SEQ ID NO:66) shown in Table 8A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 34-36 and ending with a TAG codon at nucleotides 1013-1015. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 8 A, and the start and stop codons are in bold letters. O 02 4
  • the disclosed GPCR8 nucleic acid sequence of this invention has 574 of 908 bases (63%) identical to a Canis familiaris olfactory receptor (CfOLF3) gene (gb:GENBANK- ID:CFU53681
  • acc:U53681.1)(E 1.3e-45).
  • CfOLF3 Canis familiaris olfactory receptor
  • the GPCR8 protein (SEQ ID NO:67) encoded by SEQ ID NO:66 is 323 aa in length and is presented using the one-letter amino acid code in Table 8B.
  • the Psort, SignalP and/or Hydropathy results predict that GPCR8 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • a GPCR8 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the SignalP shows a signal sequence is coded for in the first 45 amino acids with a likely cleavage site at between positions 45 and 46, at TLT-IL.
  • the full amino acid sequence ofthe protein ofthe invention was found to have 137 of 305 amino acid residues (44%) identical to, and 205 of 305 amino acid residues (67%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC.O76000 protein from Homo sapiens (Human) (DJ80I19.1 OLFACTORY RECEPTOR-LIKE PROTEIN (HS6M1-1)).
  • Example 3 Additional SNP variants of GPCR8 are disclosed in Example 3.
  • the amino acid sequence of GPCR3 has high homology to other proteins as shown in Table 8C.
  • Table 8C BLASTX results for GPCR8
  • GPCR8 also has homology to the proteins shown in the BLASTP data in Table 8D.
  • Table 8F lists the domain description from DOMAIN analysis results against GPCR8.
  • the results indicate that GPCR8 contains the protein domain 7tm_l (InterPro)7 transmembrane receptor (rhodopsin family) at amino acid positions 41 to 289. This indicates that the GPCR8 sequence has properties similar to those ofotherproteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO:18) itself.
  • GPCR8 182 PAVVKLVCG-DITVYETTVYISSILLLLPIFLISTSYVFILQSVIQMRSS 230
  • GPCR8 231 GSKRNAFATCGSHLTVVSLWFGACIFSYMRPRSQCT LL 268
  • the GPCR8 disclosed in this invention 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 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,
  • the nucleic acids and proteins of GPCR8 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.
  • the novel nucleic acid encoding the GPCR-like protein ofthe invention, 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. 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.
  • the disclosed GPCR8 protein has multiple hydrophilic regions, each of which can be used as an immunogen. hi one embodiment, a contemplated GPCR8 epitope is from about amino acids 5 to 15. In another embodiment, a GPCR8 epitope is from about amino acids 88 to 94. In further specific embodiments, GPCR8 epitopes are from about amino acids 130 to 135, from about amino acids 172 to 178, from about amino acids 220 to 235, from about amino acids 260 to 275 and from about amino acids 290 to 32
  • GPCR9 The disclosed GPCR9 (also referred to as CG50203-01) includes the 932 nucleotide sequence (SEQ ID NO:73) shown in Table 9A.
  • SEQ ID NO:73 An open reading frame was identified beginning with an ACA which codes for the amino acid Threonine at nucleotides 3-5 and ending with a TGA codon at nucleotides 900-902.
  • Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon and are underlined in Table 8A. The start and stop codons are in bold letters.
  • the disclosed GPCR9 nucleic acid sequence of this.invention has 550 of 848 bases (64%) identical to a Mus musculus gene for odorant receptor MOR10 (gb:GENBANK- ID:AB030893
  • acc:AB030893.1)(E 5.4e-54).
  • the GPCR9 protein (SEQ ID NO:74) encoded by SEQ ID NO:73 is 299 aa in length and is presented using the one-letter amino acid code in Table 9B.
  • the Psort, SignalP and/or Hydropathy results predict that GPCR9 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • a GPCR9 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial inner membrane with a certainty of 0.0300.
  • the SignalP shows a signal sequence is coded for in the first 35 amino acids with a likely cleavage site at between positions 35 and 36, at NFL-II.
  • the amino acid sequence of GPCR9 had high homology to other proteins as shown in Table 9C.
  • AAY92364 Human G protein-coupled receptor protein 4, 307 aa 1336 2.6e- -136
  • GPCR9 also has homology to the proteins shown in the BLASTP data in Table 9D.
  • Table 9F lists the domain description from DOMAIN analysis results against GPCR9. This indicates that the GPCR9 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO.T8) itself.
  • bits bits
  • GPCR9 79 EKKVISYRSCITQLFFLHFLGAGEMFLLVVMAFDRYIAICRPLHYSTI 126 RTSPRRAKVVILLV VLALLLSLPPLLFSWVKTVEEGNGTLN ++ ++ +++++++
  • GPCR9 215 RIRehssegkSKAISTCTTHIIIIFLMFGPAIFIYTCPFQaFP 257
  • GPCR9 258 ADKVVSLFHTVIF—PLMNPVIY 278
  • the GPCR9 disclosed in this invention 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 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
  • the sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:AB030893lacc:AB030893.1) a closely related Mus musculus gene for odorant receptor MOR10, complete eds homolog in species Mus musculus: Brain.
  • the nucleic acids and proteins of GPCR9 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.
  • novel nucleic acid encoding the GPCR-like protein ofthe invention, 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 O 02/40539 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.
  • the disclosed GPCR9 protein has multiple hydrophilic regions, each of which can be used as an immunogen. hi one embodiment, a contemplated GPCR9 epitope is from about amino acids 75 to 80.
  • a GPCR9 epitope is from about amino acids 118 to 123.
  • GPCR9 epitopes are from about amino acids 127 to 132, from about amino acids 165 to 172, from about amino acids 215 to 230 and from about amino acids 280 to 299.
  • GPCRl 0 includes two GPCR proteins disclosed below. The disclosed proteins have been named GPCRl 0a and GPCRl 0b, and are related to olfactory receptors.
  • GPCRl 0a The disclosed GPCRlOa nucleic acid (SEQ ID NO:80) of 984 nucleotides (also referred to as CG50197-01) is shown in Table 10A.
  • An open reading frame was identified beginning with an CTC initiation codon, which codes for leucine, at nucleotides 2-4 and ending with a TAA codon at nucleotides 941-943.
  • Putative untranslated regions found upstream from the first codon and downstream from the termination codon are underlined in Table 10A, and the start and stop codons are in bold letters.
  • the disclosed GPCRlOa polypeptide (SEQ ID NO:81) encoded by SEQ ID NO:80 has 313 amino acid residues and is presented using the one-letter amino acid code in Table 10B.
  • the SignalP, Psort and/or Hydropathy results predict that GPCRlOa 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 GPCRlOa peptide is between amino acids 38 and 39, at: NTL-IL.
  • GPCRlOb was isolated as described in Example 1. GPCRlOb differs from the previously identified sequence (Accession Number CG50197-01) in having 2 extra amino acid at N-terminal and 1 aminoacid changed at position 117 Y->S.
  • GPCRlOb nucleic acid (SEQ ID NO:82) of 1008 nucleotides (also refeired to as CG50197-02) is shown in Table IOC.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 20-22 and ending with a TAA codon at nucleotides 965-967.
  • Putative untranslated regions found upstream from the initiation codon and downstream from the termination codon are underlined in Table 10C, and the start and stop codons are in bold letters.
  • Table IOC GPCRlOb Nucleotide Sequence (SEQ ID NO-82).
  • the disclosed GPCRlOb nucleic acid sequence has 564 of 880 bases (64%) identical to aMus musculus odorant receptor S19 mRNA (gb:GENBANK-ID:AF121976
  • acc:AF121976.2) (E 1.9e "51 ).
  • the disclosed GPCRlOb polypeptide (SEQ ID NO:83) encoded by SEQ ID NO:82 has 315 amino acid residues and is presented using the one-letter amino acid code in Table 10D.
  • the SignalP, Psort and/or Hydropathy results predict that GPCRlOb 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 GPCRlOb peptide is between amino acids 49 and 50, at: TDA-TL.
  • the disclosed GPCRlOb amino acid sequence 164 of 295 amino acid residues (55%) identical to, and 223 of 295 amino acid residues (75%) similar to, the Mus musculus 319 amino acid residue protein; MOR 3 ⁇ ETA4 (ptnr:TREMBLNEW-ACC:AAG41684)(E 8.1e "
  • GPCRl 0 is used to refer to all GPCRl 0 variants or members ofthe GPCRl 0 family disclosed herein unless we identify a specific family member or variant.
  • GPCRl 0 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, med
  • the GPCRl 0 sequence is predicted to be expressed in brain tissue because ofthe expression pattern of a closely related Mus musculus MOR 5'beta3, MOR 5'beta2, and MOR 5'betal genes, complete eds; beta-globin (Hbb) gene, Hbb-D allele, locus control region homolog (GENBANK-ID: gb:GENBANK-ID:AF071080
  • GPCRlOa GPCRlOb -GGTCTAGAAGGTCTCCATGGCTGGATCTCTATTCCCTTCTGCTTCATCTACCTGACAG'-
  • GPCRlOa TTGCCATTCCTCCTGAAGCGCTTCCAATACTGCCACTCCCATGTGCTGGCTCATGCTTA
  • GPCRlOb TTGCCATTCCTCCTGAAGCGCTTCCAATACTGCCACTCCCATGTGCTGGCTCATGCTTA
  • the disclosed GPCRlOa has homology to the amino acid sequences shown in the BLASTP data listed in Table 10G.
  • GPCRlOa This indicates that the GPCRlOa sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain (SEQ ID NO: 18) itself.
  • Length 254 residues, 100.0% aligned
  • GPCRlOa 213 A ILRTVLSIASHQERLRALNTCVSHICAVLLFYIPMIGLS VHRFGEHLPRVVHLF SY 272
  • GPCRl 0 polypeptides are useful in the generation of antibodies that bind immunospecifically to the GPCRlO polypeptides ofthe invention. These antibodies maybe generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below.
  • the disclosed GPCRlO proteins have multiple hydrophilic regions, each of which can be used as an immunogen. h one embodiment, a contemplated GPCRlO epitope is from about amino acids 225 to 235. In another embodiment, a GPCRlO epitope is from about amino acids 280 to 305.
  • the GPCRlO proteins also have value in the 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.
  • GPCR11 The disclosed GPCR11 nucleic acid (SEQ ID NO:89) of 922 nucleotides (also referred to as CG50199-01) is shown in Table 11A .
  • An open reading frame was identified beginning with an AGT initiation codon, which codes for serine, at nucleotides 2-4 and ending with a TGA codon at nucleotides 920-922.
  • a putative untranslated region upstream from the initiation codon is underlined in Table 11 A, and the start and stop codons are in bold letters.
  • the disclosed GPCRl 1 nucleic acid sequence of this invention has 794 of 922 bases (86%) identical to a Mus musculus olfactory receptor P2 (Olfrl7) mRNA (gb:GENBANK- ID:AF247657
  • acc:AF247657.1) (E 1.7e "149 ).
  • the disclosed GPCRl 1 polypeptide (SEQ ID NO:90) encoded by SEQ ID NO:89 has 794 of 922 bases (86%) identical to a Mus musculus olfactory receptor P2 (Olfrl7) mRNA (gb:GENBANK- ID:AF247657
  • acc:AF247657.1) (E 1.7e "149 ).
  • the disclosed GPCRl 1 polypeptide (SEQ ID NO:90) encoded by SEQ ID NO:89 has
  • GPCRl 1 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • the most likely cleavage site for a GPCRl 1 peptide is between amino acids 45 and 46, at: ADS-AL.
  • the disclosed GPCRl 1 amino acid sequence has 268 of 305 amino acid residues
  • GPCRl 1 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 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
  • GPCRl 1 sequence is predicted to be expressed in brain tissue because ofthe expression pattern of a closely related Mus musculus olfactory receptor P2 (Olfrl7) gene homolog (GENBANK-ID: gb:GENBANK-ID:AF247657
  • the disclosed GPCRl 1 has homology to the amino acid sequences shown in the BLASTP data listed in Table 1 IC.
  • Table 1 IE lists the domain description from DOMAIN analysis results against GPCRl 1. This indicates that the GPCRl 1 sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain (SEQ IS NO: 18) itself.
  • GPCRll 33 GNVLIILVTIADSALQSP YFFLRNLSFLEIGFNLVIVSK LGTLIIQDTTISFLGCATQ 92 11 + 1 + 111 + I ++ 1 I I I I + ++ I I + I I + I I I
  • GPCRl 1 polypeptides are useful in the generation of antibodies that bind immunospecifically to the GPCRl 1 polypeptides ofthe invention.
  • the antibodies are 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.
  • the disclosed GPCRl 1 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCRl 1 epitope is from about amino acids 160 to 170.
  • a GPCRl 1 epitope is from about amino acids 210 to 230.
  • GPCRl 1 epitopes are from about amino acids 245 to 260 and from about amino acids 280 to 300.
  • the GPCRl 1 protein also has value in the 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.
  • GPCR12 is an Olfactory Receptor ("OR")-like protein.
  • OR Olfactory Receptor
  • the novel GPCR12 nucleic acid (SEQ ID NO:96) of 1019 nucleotides encoding a novel Olfactory Receptor-like protein is shown in Table 12A.
  • An open reading frame for the mature protein was identified beginning with a GTG codon which codes for the amino acid Valine at nucleotides 1-3 and ending with a TGA codon at nucleotides 943-45. Putative untranslated regions downstream from the termination codon are underlined in Table 12A, and the start and stop codons are in bold letters.
  • GPCRl 2 nucleic acid sequence has 600 of 877 bases (68%) identical to a gb:GENBANK-ID:GGA012570
  • acc:AJ012570.1 mRNA from Gallus gallus (Gallus gallus DNA sequence downstream of beta- globin locus) (E 1.4e '73 )).
  • the disclosed GPCR12 ploypeptide (SEQ ID NO: 97) encoded by SEQ ID NO: 96 has 314 amino acid residues using the one-letter code in Table 12B.
  • the SignalP, Psort and/or Hydropathy results predict that GPCR12 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • a GPCR12 polypeptide is located to the Golgi body with a certainty of 0.4600, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the endoplasmic reticulum (lumen) with a certainty of 0.1000.
  • the most likely cleavage site for a GPCR12 peptide is between amino acids 40 and 41, i.e. at the dash in the sequence ANA-W.
  • Example 3 Additional SNP variants of GPRC12 are disclosed in Example 3.
  • Expression data for GPCR12 is provided in Example 2.
  • the amino acid sequence of GPCR12 was found to have high homology to other OR-like proteins, as shown in Table 12C.
  • J-AG71545 Human olfactory receptor polypeptide, 314 aa 924 1.2e-92 AAG72396 Human OR-like polypeptide query sequence, 314 aa 924 1 2e-92
  • Table 12F lists the domain description from DOMAIN analysis results against GPRC12. This protein contains domain 7tm_l, 7 transmembrane receptor at amino acid positions 43 to 255. This indicates that the GCPR12 sequence has properties similar to those of other proteins known to contain this domain as well as to the 255 amino acid 7tm domain (SEQ ID NO: 18) itself.
  • GPCR12 90 HAHEIQYHACLIQVFFIHAFSSVESGVLMAMALDCYVAICFPLRHSSI 137
  • the Olfactory Receptor-like GPCRl 2 disclosed in this invention 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, pon
  • tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCR12 is provided in Example 2.
  • the nucleic acids and proteins of GPCRl 2 are useful in potential diagnostic and therapeutic applications implicated in various GPCR-related pathological diseases and/or disorders, and/or in various other pathologies, as described above.
  • 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 cDNA encoding the OR -like protein may be useful in gene therapy, and the OR-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding OR-like protein, and the OR-like 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.
  • a contemplated GPCRl 2 epitope is from about amino acids 80 to 95.
  • a GPCR12 epitope is from about amino acids 170 to 185.
  • a GCPR12 epitope is from about 230 to 247.
  • a GPCRl 2 epitope is from about amino acids 290 to 325.
  • 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 of the 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, glycosylation, 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, e.g., 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 (i.e., 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, 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.).
  • 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • GPCRX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 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.
  • oligonucleotides corresponding to GPCRX nucleotide sequences can be prepared by standard synthetic techniques, e.g., 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, hi 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or a complement thereof.
  • Oligonucleotides may be 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, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, thereby fonning 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.
  • 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.g.
  • 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.
  • 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 SEQ ID NOS: 2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 or 97, as well as a polypeptide possessing GPCRX biological activity. Various biological activities ofthe GPCRX proteins are described below.
  • "identical" residues correspond to those residues in a comparison between two sequences where the equivalent nucleotide base or amino acid residue in an alignment of two sequences is the same residue. Residues are alternatively described as "similar” or “positive” when the comparisons between two sequences in an alignment show that residues in an equivalent position in a comparison are either the same amino acid or a conserved amino acid as defined 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.g., 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96; or an anti-sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96; or of a naturally occurring mutant of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.
  • 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, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • 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 invention, 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 SEQ ID NOS: 1, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 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 (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of GPCRX.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 due to degeneracy ofthe genetic code and thus encode the same GPCRX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.
  • 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.
  • ORF open reading frame
  • 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.
  • 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.
  • 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 i.e., nucleic acids encoding GPCRX proteins derived from species other than human
  • other related sequences e.g., 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.
  • 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.
  • 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.
  • 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 al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • 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% PNP, 0.02% FicoU, 0.02% BSA, and 500 mg/ml denatured salmon sperm D ⁇ A at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences of SEQ ID ⁇ OS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 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 hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 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.
  • a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PNP, 0.02% FicoU, 0.2% BSA, 100 mg/ml denatured salmon sperm D ⁇ A, 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).
  • 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 thereby leading to changes in the amino acid sequences ofthe encoded GPCRX proteins, without altering the functional ability of said GPCRX proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential” amino acid residues can be made in the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.
  • 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.
  • amino acid residues that are conserved among the GPCRX proteins of the 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.
  • GPCRX proteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 61, 74, 81, 83, 90 and 97 yet 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 SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.
  • the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; more preferably at least about 70% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; and most preferably at least about 95% homologous to SEQ ID NOS:2, 4,
  • An isolated nucleic acid molecule encoding an GPCRX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues.
  • a "conservative amirio 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.
  • 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
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • 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.
  • SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
  • 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, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other.
  • a mutant GPCRX protein can be assayed for (i) the ability to form proteimprotein interactions with other GPCRX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant GPCRX protein and an GPCRX ligand; or (in) the ability of a mutant GPCRX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, 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" of the 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 (i.e., 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 Hoogsteen 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, 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.
  • 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.g., 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-(carboxyhydroxylmethyl) 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-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., 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 in situ 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.
  • 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. , by 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 which 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, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641.
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (see, e.g., Inoue, et al. 1987. Nucl. Acids Res.
  • RNA-DNA analogue or a chimeric RNA-DNA analogue (see, e.g., Inoue, et al., 1987. FEBSLett. 215: 327-330.
  • 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 (i.e., SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96).
  • a derivative of a Tetrahymena L-19 IVS RNA 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.g., U.S. Patent 4,987,071 to Cech, et al.
  • GPCRX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel etal, (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., the
  • GPCRX promoter and/or enhancers to form triple helical structures that prevent transcription ofthe GPCRX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. NY. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
  • the GPCRX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23.
  • peptide nucleic acids 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 al., 1996. supra; Perry-O'Keefe, et al, 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.g., 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, e.g., S ⁇ nucleases (see, Hyrup, et al, 1996.supra); or as probes or primers for DNA sequence and hybridization (see, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra).
  • PNAs of GPCRX can be modified, e.g., 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.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, et al., 1996. supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 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. See, e.g.,
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al, 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemai
  • oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549).
  • the oligonucleotide maybe conjugated to another molecule, e.g., 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 SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 61, 74, 81, 83, 90 and 97.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97 while still encoding a protein that maintains its GPCRX activities and physiological functions, or a functional fragment thereof.
  • 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.
  • GPCRX proteins and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof.
  • polypeptide fragments suitable for use as immunogens to raise anti-GPCRX antibodies are provided.
  • native GPCRX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • GPCRX proteins are produced by recombinant DNA techniques.
  • 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.
  • 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.
  • non-GPCRX proteins also referred to herein as a "contaminating protein”
  • the GPCRX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., 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, hi 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 SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97) that include fewer amino acids than the full-length GPCRX proteins, and exhibit at least one activity of an GPCRX protein.
  • 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.
  • GPCRX protein has an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.
  • the GPCRX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, and retains the functional activity ofthe protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
  • the GPCRX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, and retains the functional activity ofthe GPCRX proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.
  • 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.
  • 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 (i.e., 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. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453.
  • 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 SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.
  • 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.
  • 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 (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical 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.
  • an GPCRX "chimeric protein” or “fusion protein” comprises an GPCRX polypeptide operatively- linked to a non-GPCRX polypeptide.
  • 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, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97), 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.
  • an GPCRX fusion protein can correspond to all or a portion of an GPCRX protein.
  • an GPCRX fusion protein comprises at least one biologically-active portion of an GPCRX protein.
  • an GPCRX fusion protein comprises at least two biologically-active portions of an GPCRX protein, hi yet another embodiment, an GPCRX fusion protein comprises at least three biologically-active portions of an GPCRX 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.
  • 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.
  • GST glutthione S-transferase
  • Such fusion proteins can facilitate the purification of recombinant GPCRX polypeptides.
  • the fusion protein is an GPCRX protein containing a heterologous signal sequence at its N-terminus.
  • GPCRX protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of GPCRX can be increased through use of a heterologous signal sequence.
  • the fusion protein is an GPCRX-immunoglobulin fusion protein in which the GPCRX sequences are fused to sequences derived from a member ofthe immunoglobulin 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 fransduction in vivo.
  • the GPCRX- immunoglobulin 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 inhibiting) cell survival.
  • the GPCRX ligand/GPCRX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well
  • 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.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, fiUing-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • 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, e.g., Ausubel, et al (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • 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
  • 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.
  • the invention also pertains to variants o the GPCRX proteins that function as either GPCRX agonists (i.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 form ofthe GPCRX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the GPCRX protein.
  • 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 occurring form ofthe GPCRX proteins.
  • Variants ofthe GPCRX proteins that function as either GPCRX agonists (i.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.
  • 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 GPCRX 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., for phage display) containing the set of GPCRX sequences therein.
  • a degenerate set of potential GPCRX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GPCRX sequences therein.
  • 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.
  • 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. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al, 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al, 1984. Science 198: 1056; Ike, et al, 1983. Nucl. Acids Res. 11: 477.
  • libraries of fragments ofthe GPCRX protein coding sequences can be used to generate a variegated population of GPCRX fragments for screening and subsequent selection of variants of an GPCRX protein
  • 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 S ⁇ nuclease, and ligating the resulting fragment library into an expression vector.
  • expression libraries can be derived which encodes N-terminal and internal fragments of various sizes ofthe GPCRX proteins.
  • 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. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (Ig) molecules i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F a , F a ' and F (a y )2 fragments, and an F a expression library.
  • an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG IgG 2 , and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • An isolated GPCRX-related protein ofthe invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens.
  • An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of GPCRX-related protein that is located on the surface ofthe protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis ofthe human GPCRX-related protein sequence will indicate which regions of a GPCRX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.
  • a protein ofthe invention may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein ofthe invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
  • an appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated , to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • 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., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28). Monoclonal Antibodies
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell
  • hnmortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxantiiine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells ofthe invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for admimsfration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immi oglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen- binding subsequences of antibodies) that are principally comprised ofthe sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
  • Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; -Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol. , 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 11-96).
  • Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825;
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602).
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.
  • An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications.
  • the preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • U.S. Patent No. 5,939,598 An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F a b expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen may be produced -by techniques known in the art including, but not limited to: (i) an F (a ')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F (a b') fragment; (iii) an F a b fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one ofthe binding specificities is for an antigenic protein ofthe invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because ofthe random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMB ' OJ., 10:3655-3659.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One ofthe Fab'-TNB derivatives is then reconverted to the Fab '-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule. Each Fab' fragment was separately secreted from E.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen ofthe invention.
  • an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CDl 6) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.
  • antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.
  • Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
  • TF tissue factor
  • Heteroconjugate antibodies are also within the scope ofthe present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-merca ⁇ tobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980. Effector Function Engineering
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved intemalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989). Immunoconjugates
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-
  • a ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the antibody can be conjugated to a "receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient;, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
  • a "receptor” such streptavidin
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • 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.
  • 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., for use in measuring levels ofthe GPCRX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • 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
  • GPCRX polypeptide by standard techniques, such as affinity chromatography or in munoprecipitation.
  • An anti-GPCRX antibody can facilitate the purification of natural GPCRX polypeptide from cells and of recombinantly-produced GPCRX polypeptide expressed in host cells.
  • 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, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S
  • vectors preferably expression vectors, containing a nucleic acid encoding an GPCRX protein, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • a viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genomp.
  • 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.
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked.
  • expression vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • 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.
  • "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., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, 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 fo ⁇ ns of GPCRX proteins, fusion proteins, etc.).
  • proteins or peptides including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., GPCRX proteins, mutant fo ⁇ ns 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.
  • 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 TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • 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: (i) to increase expression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • 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 Inc; Smith and Johnson, 1988.
  • GST glutathione S-transferase
  • E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Siudier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli 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 TECHNOLOGY: METHODS IN ENZYMOLOGY 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 E. coli (see, 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.
  • the GPCRX expression vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invifrogen Corporation, San Diego, Calif), and picZ (InNitrogen Corp, San Diego, Calif).
  • GPCRX can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM-8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, et al, 1987. EMBO J. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • 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.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol 43:
  • promoters of T cell receptors Winoto and Baltimore, 1989. EMBO J. 8: 729-733 and immunoglobulins (Banerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al, 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat.
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat.
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation.
  • 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 GPCRX 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.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect ofthe invention pertains to host cells into which a recombinant expression vector ofthe invention has been introduced.
  • 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.
  • 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.
  • 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, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • 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.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin 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 stably 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 (i.e., express) GPCRX protein.
  • 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 GPCRX protein has been introduced) in a suitable medium such that GPCRX protein is produced. In another embodiment, the method further comprises isolating GPCRX protein from the medium or the host cell.
  • the host cells ofthe invention can also be used to produce non-human transgenic animals.
  • 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.
  • 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.
  • 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., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the O 02/40539 human GPCRX cDNA sequences of SEQ ID NOS-1, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 can be introduced as a transgene into the genome of a non-human animal.
  • a non-human homologue ofthe human GPCRX gene such as a mouse GPCRX gene, can be isolated based on hybridization to the human GPCRX cDNA (described further supra) and used as a transgene.
  • hitronic 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 GPCRX transgene to direct expression of GPCRX 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. hi: MANIPULATING THE MOUSE EMBRYO, Cold 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 GPCRX transgene in its genome and/or expression of GPCRX mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a transgene-encoding GPCRX protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of an GPCRX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GPCRX gene.
  • the GPCRX gene can be a human gene (e.g., the cDNA of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96), but more preferably, is a non-human homologue of a human GPCRX gene.
  • a mouse homologue of human GPCRX gene of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 can be used to construct a homologous recombination vector suitable for altering an endogenous GPCRX gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous GPCRX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous GPCRX 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 GPCRX protein).
  • the altered portion ofthe GPCRX 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.
  • flanking GPCRX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5'- and 3'-termini
  • the vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced GPCRX gene has homologously-recombined with the endogenous GPCRX gene are selected. See, e.g., Li, et al, 1992. Cell 69: 915.
  • the selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
  • an animal e.g., a mouse
  • 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.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression ofthe transgene.
  • a system is the cre/loxP recombinase system of bacteriophage Pl.
  • cre/loxP recombinase system See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236.
  • FLP recombinase system is the FLP recombinase system of
  • Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813.
  • a cell e.g., a somatic cell
  • 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.
  • compositions suitable for administration 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.
  • 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.
  • 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.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., 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.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under 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 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, hi many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the 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 GPCRX protein or anti-GPCRX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., an GPCRX protein or anti-GPCRX antibody
  • 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.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of 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.

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Abstract

Cette invention concerne une séquence d'acides nucléiques codant pour des polypeptides associés au récepteur couplé à la protéine G. L'invention concerne également des polypeptides codés par ces séquences d'acides nucléiques et des anticorps qui se lient de manière immunospécifique au polypeptide, ainsi que des dérivés, variants, mutants ou fragments dudit polypeptide, polynucléotide ou anticorps. L'invention concerne en outre des méthodes thérapeutiques, des méthodes diagnostiques et des méthodes de recherche servant à diagnostiquer, traiter ou prévenir des troubles dans lesquels intervient l'un quelconque des ces acides et protéines humains.
PCT/US2001/032256 2000-10-16 2001-10-16 Nouvelle proteine de type recepteur couple a la proteine g et acides nucleiques codant pour cette nouvelle proteine WO2002040539A2 (fr)

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AU3923502A AU3923502A (en) 2000-10-16 2001-10-11 Novel gpcr-like protein and nucleic acids encoding same
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US24548400P 2000-11-03 2000-11-03
US60/245,484 2000-11-03
US25501700P 2000-12-12 2000-12-12
US60/255,017 2000-12-12
US26215901P 2001-01-17 2001-01-17
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US26334001P 2001-01-22 2001-01-22
US26321601P 2001-01-22 2001-01-22
US60/263,340 2001-01-22
US60/263,216 2001-01-22
US26411801P 2001-01-25 2001-01-25
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US26822501P 2001-02-12 2001-02-12
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US28903101P 2001-02-15 2001-02-15
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US7250298B2 (en) 2004-04-07 2007-07-31 The University Of Chicago Monomeric red fluorescent proteins
US8664471B2 (en) 2001-12-19 2014-03-04 The University Of Chicago Rapidly maturing fluorescent proteins and methods for using the same
US8679749B2 (en) 2007-11-01 2014-03-25 The University Of Chicago Red fluorescent proteins with enhanced bacterial expression, increased brightness and reduced aggregation

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EP2146210A1 (fr) 2008-04-07 2010-01-20 Arena Pharmaceuticals, Inc. Procédés d'utilisation du récepteur couplé aux protéines A G pour identifier les secrétagogues de peptide YY (PYY) et composés utiles dans le traitement d'états modulés par PYY

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8664471B2 (en) 2001-12-19 2014-03-04 The University Of Chicago Rapidly maturing fluorescent proteins and methods for using the same
US7250298B2 (en) 2004-04-07 2007-07-31 The University Of Chicago Monomeric red fluorescent proteins
US7671185B2 (en) 2004-04-07 2010-03-02 The University Of Chicago Monomeric red fluorescent proteins
US7910714B2 (en) 2004-04-07 2011-03-22 The University Of Chicago Monomeric red fluorescent proteins
US8679749B2 (en) 2007-11-01 2014-03-25 The University Of Chicago Red fluorescent proteins with enhanced bacterial expression, increased brightness and reduced aggregation

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