CA2404541A1 - G-protein coupled receptors and nucleic acids encoding same - Google Patents

G-protein coupled receptors and nucleic acids encoding same Download PDF

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Publication number
CA2404541A1
CA2404541A1 CA002404541A CA2404541A CA2404541A1 CA 2404541 A1 CA2404541 A1 CA 2404541A1 CA 002404541 A CA002404541 A CA 002404541A CA 2404541 A CA2404541 A CA 2404541A CA 2404541 A1 CA2404541 A1 CA 2404541A1
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nucleic acid
amino acid
polypeptide
seq
protein
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French (fr)
Inventor
Kumud Majumder
Corine A. M. Vernet
Stacie J. Casman
Adam R. Wolenc
Steven K. Spaderna
Muralidhara Padigaru
Vishnu S. Mishra
Velizar T. Tchernev
Kimberly A. Spytek
Li Li
Jason C. Baumgartner
Vladimir Y. Gusev
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CuraGen Corp
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

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

Description

NOVEL PROTEINS AND NUCLETC ACIDS ENCODING SAME
BACKGROUND OF THE INVENTION
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.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as LO GPCRX, or GPCRl, GPCR2, GPCR3, GPCR4, GPCRS, GPCR6, GPCR7, GPCRB, and GPCR9 nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "GPCRX" nucleic acid or polypeptide sequences.
In one aspect, the invention provides an isolated GPCRX nucleic acid molecule encoding a GPCRX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82. In some embodiments, the GPCRX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a GPCRX
nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a GPCRX
polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83. 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 m NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a GPCRX nucleic acid (e.g., SEQ
m NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82) or a complement of said oligonucleotide.

Also included in the invention are substantially purified GPCRX polypeptides (SEQ ID
NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83). In certain embodiments, the GPCRX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human GPCRX
polypeptide.
The invention also features antibodies that immunoselectively bind to GPCRX
polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable Garner. The therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX
0 polypeptide, or an antibody specific for a GPCRX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a GPCRX nucleic acid, under conditions allowing for expression 5 of the GPCRX polypeptide encoded by the DNA. If desired, the GPCRX
polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence of a GPCRN polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex !0 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
>.5 nucleic acid molecule in a sample by contacting the sample with a GPCRX
nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a GPCRX nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the activity of a GPCRX polypeptide by contacting a cell sample that includes the GPCRX
polypeptide with a 30 compound that binds to the GPCRX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, or other disorders related to cell signal processing and metabolic pathway modulation. The therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or a GPCRX-specific antibody, or biologically-active derivatives or fragments thereof.
For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), taste and scent detectability Disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, Retinal diseases including those involving photoreception, Cell growth rate disorders; cell shape disorders, feeding disorders; control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (NIDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation.
Dentatorubro-pallidoluysian atrophy (DRPLA) Hypophosphatemic rickets, autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders of the like.
The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding GPCRX may be useful in gene therapy, and GPCRX may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
The invention fixrther includes a method for screening for a modulator of disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation. The method includes contacting a test compound with a GPCRX polypeptide and determining if the test compound binds to said GPCRX polypeptide. Binding of the 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 of the invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a GPCRX nucleic acid. Expression or activity of GPCRX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses GPCRX polypeptide and is not at increased risk fox the disorder or syndrome. Next, the expression of GPCRX polypeptide in both the test animal and the control animal is compared. A change in the activity of GPCRX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.

In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a GPCRX
polypeptide, a GPCRX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the GPCRX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the GPCRX
polypeptide present in a control sample. An alteration in the level of the GPCRX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
Also, the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a GPCRX polypeptide, a GPCRX nucleic acid, or a GPCRX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors of the 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 of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based, in part, upon the discovery of novel nucleic acid sequences that encode novel polypeptides. The novel nucleic acids and their encoded polypeptides are referred to individually as GPCRl, GPCR2, GPCR3, GPCR4, GPCRS, GPCR6, GPCR7, GPCRB, and GPCR9, The nucleic acids, and their encoded polypeptides, are collectively 0 designated herein as "GPCRX".
The novel GPCRX nucleic acids of the invention include the nucleic acids whose sequences are provided in Tables 1A, 1D, 1E, 2A, 3A, 4A, 4F, 4J, SA, SD, SF, 6A, 6D, 7A, 7F, 7I, 8A, 8D, 8F, 8H, 8J, 9A, 9D, and 9F, inclusive ("Tables 1A - 9F"), or a fragment, derivative, analog or homolog thereof. The novel GPCRX proteins of the invention include 5 the protein fragments whose sequences are provided in Tables 1B, 1F, 2B, 3B, 4B, 4G, SB, SE, SG, 6B, 6E, 7C, 7G, 7J, 8B, 8E, 8G, 8I, 8K, 9B, 9E, and 9G, inclusive ("Tables 1B - 9G").
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.
'0 G-Protein Coupled Receptor proteins (GPCRs) have been identified as a large family of G protein-coupled receptors in a number of spacies. These receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated transduction of various signals.
Human GPCR generally do not contain introns and belong to four different gene subfamilies, 'S 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.nhn.nih.gov/entrez/
dispomim.cgi?).
The olfactory receptor (OR) gene family constitutes one of the largest GPCR
30 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 Vanderhaeghen et al., Genomics 39(3):239-(1997); Xie et al., Mamm. Genome 11(12):1070-78 (2000); Issel-Tarver et al., Proc. Natl.
Acad. Sci. USA 93(20):10897-902 (1996). The recognition of odorants by olfactory receptors is the first stage in odor discrimination. See Krautwurst et al., Cell 95(7):917-26 (1998); Buck et al., Cell 65(1):175-87 (1991). Many ORs share some characteristic sequence motifs and have a central variable region corresponding to a putative ligand binding site. See Issel-Tarver et al., Proc. Natl. Acad. Sci. USA 93:10897-902 (1996).
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.
Chem. 273(16):9378-87 (1998); Parmentier et al., Nature 355(6359):453-55 (1992); Asai et al., Biochem. Biophys. Res. Common. 221(2):240-47 (1996).
GPCRl GPCRl includes a family of three novel G-protein coupled receptor ("GPCR") proteins disclosed below. The disclosed proteins have been named GPCRla, GPCRlb, and GPCRlc and are related to olfactory receptors.
GPCRl a A disclosed GPCRla nucleic acid of 1050 nucleotides is shown in Table 1A. The disclosed GPCRla open reading frame ("ORF") begins at the ATG initiation codon at nucleotides 6-8, shown in bold in Table 1A. The encoded polypeptide is alternatively referred to herein as GPCRla or as rpl l-507n20 A. The disclosed GPCRla ORF terminates at a TGA
codon at nucleotides 1044-1046. As shown in Table 1A, putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters.
Table 1A. GPCRIa nucleotide sequence (SEQ ID NO:1).
GCCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCCCAG
CACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGACTATTACC
TCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGGCTCTTCACGTTGGCCATGAACAGGGCCGGG
AGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTTCAAAGTGGTCCACCCCCACCACGCGGTGAACACTAT
CTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTTTGCTGG
AGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCCTGTGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGAC
ATCATGTTCCAGCTGGAGTTCTTTATGCCCCTCGGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAG
GCGGAGGCAGCAGCTGGCCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATCATGGTGGTGGCAATTGTGTTCA
TCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCTATTTCCTCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCT
GTCCATGGGGCCCTGCACATAACCCTCAGCTTCACCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTTC
AAGCCCCTCCTTTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACACTCAAAAA
CACAAAGGCCGGAAGAGATGCCAATTTCGAACCTCGGTCGCAGGAGTTGCATCAGTGTGGCAAATAGTTTCCAAAGC
CAGTCTGATGGGCAATGGGATCCCCACATTGTTGAGTGGCACTGAACAA
A disclosed encoded GPCRl a protein has 346 amino acid residues, referred to as the GPCRl a protein. The GPCRl a protein was analyzed for signal peptide prediction and cellular localization. SignalP results predict that GPCRl a is cleaved between position 32 and 33 of SEQ m N0:2, i.e., in the amino acid sequence ALG-NG. Psort and Hydropathy profiles also predict that GPCRl contains a signal peptide and is likely to be localized in the endoplasmic reticulum (Certainty 0.6850) or the plasma membrane (Certainty=0.6400). The disclosed GPCR1 polypeptide sequence with a molecular weight of 39294.8 Daltons is presented in Table 1B using the one-letter amino acid code.
Table 1B. Encoded GPCRla protein sequence (SEQ ID N0:2).
MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLR
RRHWAFGDIPCRVGLFTLAMNRAGSIVFLTWAADRYFKWHPHHAVNTISTRVAAGTVCTLWALVILGTVYLLLEN
HLCVQETAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMWAIVFIT
CYLPSVSARLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKLKTCSLKPKQPGHSKTQ
RPEEMPISNLGRRSCISVANSFQSQSDGQWDPHIVEWH
GPCRla was initially identified on chromosome 12 with a TblastN analysis of a proprietary sequence file for a G-protein coupled receptor probe or homolog, which was run against the Genomic Daily Files made available by GenBank. A proprietary software program (GenScanTM) was used to further predict the nucleic acid sequence and the selection of exons.
The resulting sequences were further modified by means of similarities using BLAST
searches. The sequences were then manually corrected for appaxent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
A region of the GPCRla nucleic acid sequence has 517 of 746 bases (69%) identical to a Homo Sapiens GPCR mRNA (GENBANK-m: HUMHM74~acc:D10923), with an E-value of 1.9x10-71. In all BLAST alignments herein, the "E-value" or "Expect" value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, the probability that the subject ("Sbjct") retrieved from the GPCRIa BLAST
analysis, e.g., the Homo sapiehs GPCR, matched the Query GPCRla sequence purely by chance is 1.9x10'71.
A BLASTX search was performed against public protein databases. The full amino acid sequence of the protein of the invention was found to have 178 of 339 amino acid residues (52%) identical to, and 227 of 339 residues (66%) positive with, the 387 amino acid residue protein from species (ptnr: SWISSPROT-ACC:P49019).
The amino acid sequence of GPCRl a also had high homology to other proteins as shown in table 1 C.
Table 1C. BLASTX alignments of Sequences producing High-scoring Segment Smallest Pairs:

Sum ReadingHigh Prob.

Frame Score P(N) ptnr:SWISSPROT-ACC:000270 PROBABLE G PROTEIN-COUPLED 447 2.6e-41 R... +3 ptnr:TREMBLNEW-ACC:AAF26668 G PROTEIN COUPLED.. 441 1.1e-40 RECEPTOR. +3 ptnr:SWISSPROT-ACC:P34996 P2Y PURINOCEPTOR.. 357 8.8e-32 1 (ATP RECE. +3 ptnr:SWTSSPROT-ACC:P49652 P2Y PURINOCEPTOR.. 357 8.8e-32 1 (ATP RECE. +3 patp:W94654 G-protein coupled receptor +3 887 4.7e-88 HM74A protein -patp:Y90672 Human mutant G protein-coupled+3 883 1.2e-87 receptor HM

patp:Y90637 Human G protein-coupled receptor+3 882 1.6e-87 HM74 - Ho.

patp:W88460 Human 7-transmembrane receptor+3 524 1.4e-49 HEOAD54 - H.

patp:Y94339 Human cell surface receptor +3 524 1.4e-49 protein #6 - H.

GPCR1 b In the present invention, the target sequence identified previously, Accession Number rpl l-507n20 A, was subjected to the exon linking process to confirm the sequence. PCR
primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer.
In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: 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, Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp..
In addition, sequence traces were evaluated manually and edited for corrections if appropriate.
These procedures provide the sequence reported below, which is designated Accession Number rpl 1-507n20 A dal.
A disclosed GPCRlb (also referred to as rpl l-507n20 A dal) nucleic acid of nucleotides is shown in Table 1D. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 6-8 and ending with a TGA codon at nucleotides 1044-1046. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table ID, and the start and stop codons are in bold letters.
Table 1D. GPCRlb Nucleic acid sequence (SEQ ID N0:3).
TCGCCATGTACAACGGGTCGTGCTGCCGCATCGAGGGGGACACCATCTCCCAGGTGATGCCGCCGCTGCTCATTGTGG
CCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCCCAGCA
CTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGACTATTACCTCA
GACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGGCTCTTCACGTTGGCCATGAACAGGGCCGGGAGCA
TCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTTCAAAGTGGTCCACCCCCACCACGCGGTGAACACTATCTCCA
CCCGGGTGGCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTTTGCTGGAGAACC
ATCTCTGCGTGCAAGAGACGGCCGTCTCCTGTGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGACATCATGT
TCCAGCTGGAGTTCTTTATGCCCCTCGGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGC
AGCAGCTGGCCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCT
ACCTGCCCAGCGTGTCTGCTAGACTCTATTTCCTCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGG
CCCTGCACATAACCCTCAGCTTCACCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTTCAAGCCCCTCCT
TTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACACTCAAAAACACAAAGGCCGG
AAGAGATGCCAATTTCGAACCTCGGTCGCAGGAGTTGCATCAGTGTGGCAAATAGTTTCCAAAGCCAGTCTGATGGGC
AATGGGATCCCCACATTGTTGAGTGGCACTGAACAA
The encoded protein is the same as for GPCRla and is disclosed above in Table 1B.
GPCRl c A disclosed GPCRlc (also referred to as AC011711 dal) nucleic acid of I I04 nucleotides found by exon linking is shown in Table IE. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 60-62 and ending with a TGA codon at nucleotides 1098-1100. Putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined in Table 1E and the start and stop codons are in bold letters.
In a search of sequence databases, it was found, for example, that the nucleic acid sequence has 530 of 770 bases (68%) identical to a gb:GENBANK-m:HUMHM74~acc:D10923.1 mRNA from Homo Sapiens (Human mRNA for HM74).

Table 1E. GPCRlc Nucleic acid sequence (SEQ ID N0:4).
GTGCCATTGTGGGGACTCCCTGGGCTGCTCTGCACCCGGACACTTGCTCTGTCCCCGCCATGTACAACGGGTCGTGCTG

CCGCATCGAGGGGGACACCATCTCCCAGGTGATGCCGCCGCTGCTCATTGTGGCCTTTGTGCTGGGCGCACTAGACAAT

GGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCCCAGCACTGTTTACCTTTTCAATTTGGCCGTGG

CTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGACTATTACCTCAGACGTAGACACTGGGCTTTTGGGGACAT

TCCCTGCCGAGTGGGGCTCTTCACGTTGGCCATGAACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGGC

AGGTATTTCAAAGTGGTCCACCCCCACCACGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCC

TGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCCTG

TGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTTTATGCCCCTCGGCATC

ATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGGCCAGACAGGCTCGGATGAAGAAGG

CGACCCGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCTATTTCCT

CTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGGCCCTGCACATAACCCTCAGCTTCACCTACATGAAC

AGCATGCTGGATCCCCTGGTGTATTATTTTTCAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTC

TGAAACCCAAGCAGCCAGGACACTCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCGAACCTCGGTCGCAGGAGTTG

CATCAGTGTGGCAAATAGTTTCCAAAGCCAGTCTGATGGGCAATGGGATCCCCACATTGTTGAGTGGCACTGAACAA
The disclosed GPCRIc protein having 346 amino acid residues is presented using the one-letter code in Table 1F. An analysis using the PSORT program predicts that the AC011711 dal protein localizes in the plasma membrane with a certainty=0.6400.
It is also predicted that protein has a signal peptide whose most likely cleavage site is between residues 36 and 37,: GVA-LC in Table 1F.
Table 1F. Encoded GPCRlc protein sequence (SEQ ID N0:5).
MYNGSCCRIEGDTISQVMPPLLIVAFVLGALDNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFR
TDYYLRRRHWAFGDIPCRVGLFTLAMNRAGSIVFLTVVAAGRYFKVVHPHHAVNTISTRVAAGIVCTLWAL
VILGTVYLLLENHLCVQETAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQA
RMKKATRFIMVVAIVFITCYLPSVSARLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPS
FPKFYNKLKICSLKPKQPGHSKTQRPEEMPISNLGRRSCISVANSFQSQSDGQWDPHIVEWH
A BLASTX search was performed against public protein databases. The full amino acid sequence of the protein of the invention was found to have 270 of 317 amino acid residues 176 of 339 amino acid residues (51%) identical to, and 225 of 339 amino acid residues (66%) similar to, the 387 amino acid residue ptnr:SWISSPROT-ACC:P49019 protein from Homo sapiens (Human) (PROBABLE G PROTEIN-COUPLED RECEPTOR HM74). In the following positions, one or more consensus positions of the nucleotide sequence have been identified as single nucleotide polymorphisms (SNPs). "Depth" represents the number of clones covering the region of the SNP. The Putative Allele Frequency (PAF) is the fraction of all the clones containing the SNP. The sign ">" means "is changed to".
:Possible SNPs found for GPCRlc are listed in Table 1G.

Table 1G: SNPs ConsensusDepth Base PAF
Position Change 334 71 T>C 0.025 677 92 T>C 0.022 719 79 T>C 0.025 The amino acids differences between the three GPCRl proteins are shown in Table 1H. Deletions are marked by a delta (0). The differences between the three proteins appear to be localized to a few distinct regions. Thus, these proteins may have similar functions, such as serving as olfactory or chemokine receptors (see below).
Table 1H.
Differences for GPCRl Proteins Position32 112 GPCRIa G D

GPCRIc D G

A ClustalW analysis comparing disclosed proteins of the invention with related OR
protein sequences is given in Table 1I, with GPCRla shown on line 1, and GPCRlc on line 2.
In the ClustalW alignment of the GPCRl a protein, as well as all other ClustalW
analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function. Unless specifically addressed as GPCRla GPCRlb, or GPCRlc, 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 and the residue position with respect to the "a" variant.
GPCR residues in all following sequence alignments that differ between the individual GPCR
variants are highlighted with a box and marked with the (o) symbol above the variant residue in all alignments herein. All GPCRl proteins have significant homology to olfactory receptor (OR) proteins:.

Table 1I. ClustalW Analysis of GPCRl 1) Novel GPCRla (SEQ ID N0:2) 2) Novel GPCRlc (SEQ 1D NO: 5) 3). gi~21353861pir~1I69202 G protein-coupled receptor HM74 - human) (SEQ ID
N0:43) S 4) g~11558404IembICAC17790.11 (AJ300198) putative seven transmembrane spanning receptor [Mus musculus] (SEQ ID N0:44) 5) giJ67165091~bIAAF26668.11AF140708 1 (AF140708) G protein coupled receptor [Mus musculus]) (SEQ ID N0:45) 6) giJ4643271spIP349961P2YR CHICK P2Y PURII'TOCEPTOR 1 (ATP RECEPTOR) (P2Y1) .0 (PURINERGIC RECEPTOR) (SEQ ID N0:46) 7) gi~4972611gbIAAA18784.11 (LT09842) P2Y purinergic receptor [Meleagris gallopavo] (SEQ ID
N0:47) .S GPCRIa GPCRIc gi121353861 gi1115584041 )O gi167165091 gi14643271 gi14972611 ~S GPCRla gi121353861 gi1115584041 gi167165091 gi14643271 gi14972611 SS GPCRIa gi121353861 gi1115584041 gi167165091 gi14643271 gi(4972611 I . .I.. .1....I.. I . I.. .I . .I. .I.. I .-I QQ-I~S GPCRIa LCVQ ET;VS E~ IMESANG- HI; Q~F F ~L ~. E
GPCRIc LCVQ ET VS E IMESANG- G4HI, Q ; F LGF ~W~ , QQ
gi121353861 LIQN GP~NV I SICHTF- ~H L E L~, LG~ SA ~WQ
gi I 11558404 I MTKN GE 'YL S SICYNF- HLf ,: L E F~L'LA~G ~;W Q
gi167165091 TTQN -~TE P YPTGGT TATCQ1", F ~VL FGS SGR~C' #~K,~'LSES
gi14643271 TGVRRNKTIT YDTTADEYL~SYFV'SMCTTVF C 'FI YGL IY D--gi14972611 TGVRRNKTIT YDTTADEYLSXFVSMCTTVFI~C,'FI, YGL~K~. IY~D--I ~I .I ~ ~I~~ .I~~~ I ~.1....1~.~~I.. 1...~I ..~I
)S GPCRla AQARL~~TRF M"~ IT S 'YFLWTVPSSACD--- -PHG LH
GPCRIc ~QAR~~TRF ~ IT S ;YFLWTVPSSACD--- P HG LH
gi121353861 ~HAK~~,'ITF~ , I<RIFWLLHTSGTQNCEVY-R~ DL FF
gi1115584041 ~'HAIC~~INF IV ~RIFWLLYKYNVRNCDTY S DL~FFT
i I 6716509 I QPT RVL ~1L~.L Gt~ LT LVHIFQEFKSCS--- VQQ~~I~2 ~ SDs' )O gi14643271 NSPL _~iIYLwII LT S'~ 'FHVH~LNLRARLDFQTPQMCAFND YATYQ1 gi14972611 NSPLR~:~IYL'~~i~LT ~5; 'FHVM~TLNLRARLDFQTPQMCAFNDK~YATYQ~~

)S GPCRla GPCRlc gi121353861 gi1115584041 gi167165091 gi14643271 gi14972611 ...
GPCRla I. ANQSQSDGQWDPHIVEWH--------------------------GPCRlc I~AN~QSQSDGQWDPHIVEWH--------------------------giI21353861 RGAPE LMANSGEPWSPSYLGPTSNNHSKKGHCHQEPASLEKQLGCCIE
gi~11558404~ ~ PG~LMADPSEPGSPPYLASTSR--gi~67165091 ~~~RD~S-__________-____________________________-gi~464327~ L ~LTE~'KQN____GDTSL-_______-_______________,____-gi~497261~ L~~LTE~KQN-___GDTSL-_______________________-_____ The presence of identifiable domains in GPCR1, as well as all other GPCRX
proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (h ~ttp./lwww.ebi.ac.uk/interpro).
DOMAIN results, e.g., for GPCRl as disclosed in Table 1J, were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST analyses.
This BLAST analysis software samples domains found in the Smart and Pfam collections. For Table 1J 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 of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
Table 1J lists the domain description from DOMAIN analysis results against GPCR1.
This indicates that the GPCRl sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain itself.
Table 1J. Domain Analysis of GPCR1 gnl~Pfam~pfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin family) (SEQ ID N0:71) Length: 254 Score = 114 bits (284), Expect = 1e-26 ° 10 20 30 40 50 60 ConsensusQ7tm lOdomain ~LVLVILRT~LR-T~fNL~L FAT ~PWAL VGGD V AIL ~
70 80° 90 100 110 120 Consensus 7tm 1 domain VGALF~GYAL~SI ' L~~LRYRRIRP KVLLn VT L~~PP
~:

GPCR1 EH CVQ TAV------ ESFI S2~NGWH---DIMF E---- F ' GT SF
Consensus 7tm 1 domain ~FW~RTV~EGN~!----TV~LIDFP~EVK-----RSYVL~STLVG~L~LV~YT

Consensus 7tm 1 domain ~~R~~1~SA~~RSSSE--_-_ ~T~? ~ LYHIVLBLDSLC~L

GPCR1 ~SACD------PSHG~H~FO~
Consensus 7tm 1 domain ~IWR--------VPT~~L~L C ~IT

The 7 transmembrane receptor family 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:
rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); 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);
GPCRl is expressed in at least the following tissues: Apical microvilli of the 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, MHC II abnd III
expressing cells, nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells of the tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to proprietary database sources, Public EST sources, Literature sources, and/or RACE sources.
The nucleic acids and proteins of GPCRl axe useful in potential therapeutic applications implicated in various GPCR- or olfactory receptor (OR)-related pathologies and/or disorders. For example, a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof. The novel nucleic acid encoding GPCRl protein, or fragments thereof, may filrther 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 of the invention for use in therapeutic or diagnostic methods. The GPCRX nucleic acids and proteins are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneurogenic disease, Signal Transduction pathway disorders, Retinal diseases including those involving photoreception, Cell Growth rate disorders; Cell Shape disorders, Feeding disorders;control of feeding;
potential obesity due to over-eating; potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (NIDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm;
adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation.
Dentatorubro-pallidoluysian atrophy(DRPLA) Hypophosphatemic rickets, autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders of the like. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional GPCR-related diseases and disorders are mentioned throughout the Specification.
Further, the protein similarity information, expression pattern, and map location for GPCRl suggests that GPCRl may have important structural and/or physiological functions characteristic of the GPCR family. The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding GPCRl may be useful in gene therapy, and GPCR1 may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. The novel nucleic acid encoding OR-like protein, and the OR-like protein of the 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 of the invention for use in therapeutic or diagnostic methods.
These materials are further useful in the generation of antibodies that bind immuno-specifically to the novel GPCRl substances for use in therapeutic or diagnostic methods.
These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies"
section below.
The disclosed GPCRl protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCRl epitope is from about amino acids 1 to 10. In another embodiment, a GPCRl epitope is from about amino acids 75 to 100.
In additional embodiments, GPCRl epitopes are from amino acids 130 to 140, 210-230, and from amino acids 270 to 330. These novel proteins can also be used to develop assay system for functional analysis.

An additional GPCR-like protein of the invention, referred to herein as GPCR2, is an Olfactory Receptor ("OR")-like protein. The novel nucleic acid of 1149 nucleotides, mapped to chromosome 5, (80250319 EXT, SEQ ID N0:6) encoding a novel G-protein coupled receptor-like protein is shown in Table 2A.

Table 2A. GPCR2 Nucleotide Sequence (SEQ ID N0:6) ATGGCCGATGCAGCCACGATAGCCACCATGAATAAGGCAGCAGGCGGGGACAAGCTAGCAGAACTCTTCAGTCTGGT
CCCGGACCTTCTGGAGGCGGCCAACACGAGTGGTAACGCGTCGCTGCAGCTTCCGGACTTGTGGTGGGAGCTGGGGC
TGGAGTTGCCGGACGGCGCGCCGCCAGGACATCCCCCGGGCAGCGGCGGGGCAGAGAGCGCGGACACAGAGGCCCGG
GTGCGGATTCTCATCAGCGTGGTGTACTGGGTGGTGTGCGCCCTGGGGTTGGCGGGCAACCTGCTGGTTCTCTACCT
GATGAAGAGCATGCAGGGCTGGCGCAAGTCCTCTATCAACCTCTTCGTCACCAACCTGGCGCTGACGGACTTTCAGT
TTGTGCTCACCCTGCCCTTCTGGGCGGTGGAGAACGCTCTTGACTTCAAATGGCCCTTCGGCAAGGCCATGTGTAAG
ATCGTGTCCATGGTGACGTCCATGAACATGTACGCCAGCGTGTTCTTCCTCACTGCCATGAGTGTGACGCGCTACCA
TTCGGTGGCCTCGGCTCTGAAGAGCCACCGGACCCGAGGACACGGCCGGGGCGACTGCTGCGGCCGGAGCCTGGGGG
ACAGCTGCTGCTTCTCGGCCAAGGCGCTGTGTGTGTGGATCTGGGCTTTGGCCGCGCTGGCCTCGCTGCCCAGTGCC
ATTTTCTCCACCACGGTCAAGGTGATGGGCGAGGAGCTGTGCACTGGTGCGTTTCCCGGACAAGTTGCTGGGCCGCG
ACAGGCAGTTCTGGCTGGGCCTCTACCACTCGCAGAAGAAGCTGCTGGGGTACCGGCTTACTTAGCATATATTTTTA
TTCCAAAACAATTCTTTAGATCACTACCTCTTTCTTACGACCTCTTGTATTTTCCGCCCCTCTCTTACCCTTCCGTT
ATCCGCAACATTTCCTCCTTACCGCCACAACACGATAAACCGCGTAGGACCTGGTGTCCACCCCCATGGACTGGACC
CGCCAGTCCAGACCAGATTGAAAATACGTATAGATTTGCTACCTGCTATGTACATCACTATGAATTTCTGGCATTTA
AATCAAACAGATTTTCAGGAACTAGCCTGGGGACTCAGACACCATTTAAACCTTGGGAAAGCATGTTTTGA
An open reading frame (ORF) for GPCR2 was identified from nucleotides 1 to 1146.
The disclosed GPCR2 polypeptide (SEQ ID N0:7) encoded by SEQ ID N0:6 is 382 amino acid residues with a molecular weight of 41669.7 I~altons and is presented using the one-letter code in Table 2B. The GPCR2 protein was analyzed for signal peptide prediction and cellular localization. Psort and Hydropathy profiles predict that GPCR2 contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000).
Table 2B. Encoded GPCR2 protein sequence (SEQ ID NO:'n.
MADAATIATMNKAAGGDKLAELFSLVPDLLEAANTSGNASLQLPDLWWELGLELPDGAPPGHPPGSGGAESADTEAR
VRILISWYWVVCALGLAGNLLVLYLMKSMQGWRKSSINLFVTNLALTDFQFVLTLPFWAVENALDFKWPFGKAMCK
IVSMVTSMNMYASVFFLTAMSVTRYHSVASALKSHRTRGHGRGDCCGRSLGDSCCFSAKALCVWIWALAALASLPSA
IFSTTVKVMGEELCTGAFPGQVAGPRQAVLAGPLPLAEEAAGVPAYLAYIFIPKQFFRSLPLSYDLLYFPPLSYPSV
IRNISSLPPQHDKPRRTWCPPPWTGPASPDQIENTYRFATCYVHHYEFLAFKSNRFSGTSLGTQTPFKPWESMF
The GPCR2 nucleic acid sequence has 405 of 648 nucleotides (62%) identical to Sequence 9 from patent US 5436155 (GENBANK-ID:I13406) The full amino acid sequence of the protein of the invention was found to have 62 of 170 amino acid residues (36%) identical to, and 96 of 170 amino acid residues (56%)positive with, the 359 amino acid residue protein from Rattus norvegicus (SWISSPROT-ACC:P29089). The protein encoded by GPCR2 (SEQ ID N0:6) has significant homology to olfactory, odorant, and taste chemoreceptors and belongs to the family of G-Protein coupled receptors (GPCRs). This family of genes has been used as a target for small molecule drugs and GPCRs are expressed on the plasma membrane and are also a suitable target for protein drugs like therapeutic antibodies, cytotoxic antibodies and diagnostic antibodies.

Another BLAST against GenBank Accession Number: ~ 003874.1, a 471 amino acid G-protein coupled receptor SALPR; somatostatin and angiotensin-like peptide receptor protein from Homo Sapiens, produced 100% identity, between a 245 fragment and amino acids 1-247 of GPCRZ (Table 2C).
Table 2C: Alignment of GPCR2 with giJ11416964JrefJXP 003874.11 G-protein coupled receptor SALPR; somatostatin and angiotensin-like peptide receptor [Homo Sapiens] (SEQ ID N0:48) Score = 396 bits (1018), Expect = e-109 Identities = 207/255 (81%), Positives = 209/255 (81~) .1....1....1....1.. .I....~....I....J....I....I....I.. .I
GPCR2 _ . ~ . . , . i~ ~ ~ ~ ~ .
gi111416964~ MQ ~~~. , ., i ., ., .

....I....I....I....I....I....1....1....1....1....1....1....1 gi111416964J

....I....I....I....I....I....~....J....I....I....I....I....J

gi1114169641 ....I....I....I....I....I....1....1....1....1....1....1....1 gi111416964J

.I.. .1....I.. .I....J....I....J....I....I....I....I....I
GPCR2 TG G~__________ P~, GP ~ _-__________________ gi1114169641 ~LVR~DL~RDRQFWLGLYHS ~ GFV~GTIILCYLLLVRFIADRRAA

.J....J ...J. I . I I I....I....I....~....I
gi111416964J GTKGG~AV~RPTG~SAR~SKVTSVTI~VF~C~NQALTTWSILIKFNAV~F~

I I....I....I....I....I... 1....I ...I. I... I .I
giC114169641 QEYF~CQ~YF~18VCLAHSNS~LN~VLYCLVRRFRKAL.,~~"SLLWR~<AS--PSIT~~r"~PF

.1....1....I.. .~.. .1....1....1....1....1....1.
GPCR2 --FSGTS~TK~EMF-__________________________ giIlI4169641 TAyxT,PEHED~L~P~HAAEPDI,LYYPPGVVVYSGGRYDLLPSSSAY
Other BLAST results including the sequences used for ClustalW analysis are presented in Table 2D. Other BLAST alignment data is shown in Table 2E.

Table 2D. BLAST
results for Gene Index/ Protein/ Zength Identity PositivesExpect Identifier Organism (aa) (%) (%) Gi~12188901~emb~CACangiotensin359 46/109 61/109 6e-16 21550.1 (A)301623)II type (42%) (55%) receptor [Cavia porcellus]

>gi~89279951spIQ9WVTYPE-1 359 45/l09 61/109 le-15 26~AG2R_CAVPO ANGTOTENSIN (41%) (55%) II RECEPTOR

(AT1) [Cavia porcellus]

Gi~90297~piry angiotensin359 44/109 61/109 2e-15 1 II receptor (40%) (55%) 1A - mouse Gi~249946~gb~AAB222Angiotensin359 44/109 61/109 2e-15 69.1 (537484) II receptor (40%) (55%) isoform 1a, AT-1a receptor Balb/c, liver Table 2E. BLASTX alignments Sequences producing High-scoring Segment Pairs: Smallest Sum Reading High Prob.
Frame Score P(N) patp:Y92934 Human G-protein coupled receptor clone hOT +1 1297 6.1e-145 patp:Y92932 Rat G-protein coupled receptor clone rOT7T +1 1134 1.7e-125 patp:Y92933 Rat G-protein coupled receptor clone rOT7T +1 1134 1.7e-125 patp:W92645 Human membrane penetrating type receptor p +1 314 2.4e-27 This information is presented graphically in the multiple sequence alignment given in Table 2F (with GPCR2 being shown on line 1) as a ClustalW analysis comparing GPCR2 with related protein sequences.
Table ZF. Information for the ClustalW proteins:
1) Novel GPCRZ (SEQ ID N0:6) 2) giJ1I4I6964JrefJXP 003874.1) G-protein coupled receptor SALPR; somatostatin and angiotensin-like peptide receptor [Homo sapiens~ (SEQ ID N0:48) 3) giJ 12188901 JembJCAC2I550.1 J (A)301623) angiotensin II type I receptor [Cavia porcellus]
(SEQ ID N0:49) 4) giJ8927995JspJQ9WV26JAG2R CAVPO TYPE-1 ANGIOTENSIN II RECEPTOR (ATI) (SEQ D7 NO:50) 5) giJ90297JpirJJJH0621 angiotensin II receptor 1A - mouse (SEQ ID NO:51) 6) giJ249946JgbJAAB22269.1J (537484) angiotensin II receptor isoform la, AT-la receptor (SEQ
ID N0:52) GPCR2 --MADAATTAT K'~';AGGDKLAELFSLVPDLI,EAANTSGNASLQLPDLWW L~ LPDGA
gi~11416964~ MQMADAATTAT KAGGDKLAELFSLVPDLI,EAANTSGNASLQLPDLWW L ~LPDGA
ai1121889011 _________~I-________________________________ . .., w gi18927995~ _-_______ I ________________________________ , gi190297~ ________ ________________________________ , gi~249946~ ________ ________________________________ , ....I....I....I....I....I....I....i....l....i....l....t....l GPCR2 P ~GHPP SGGAESADTE E~R~~ SV~? WCA, ~' L '~tY~KSMQGW ~SI
gi ~ 11416964 ~ P GHPP SGGAESADTE~RR~ SV~ ~'~'CA~ A L .L~Y~,I~IKSMQGW SI
gi~12188901~ ~...:-D__ ___ I .
_ y_ 9i18927995~ ______ gi190297~ ______ gi1249946~ ______ gi~11416964~
gi~12188901~
gi~8927995~
gi~90297~
gi~249946~

gi~11416964~
gi~12188901~
gi~8927995~
gi~90297~
gi~249946~

GPCR2 MGEE CTG~ PGVAG-----------PR~ GPL L----~---------------gi~11416964~ GEE~CV PDLLGRDRQFW LYHSQ V~~ ~.L LG ~ELC LLLVRFIADRRAA
gi~12188901~ _ __,_________ gi~8927995~ _ ____________ gi~90297~ _ __,_________ gi~249946~ - ____________ GPCR2 -----AEE;:~yAAG- PA"yY~LAYIFIPKFFl~ P 5~,~',DLL('L' --------- -----PLS
gi ~ 11416964 ~ KGGA ~ AGGRPTGAA LSK~T ~ S~TI~ S LC ~IiL ~LiT L TWSi ~~~FN
~,~PFS
gi~12188901j ~9 ~ 'i~ ~ m n ~~ ~ mn gi~8927995~ ~ v gi~90297~ v m ~ v v gi~249946~ v ~ ~ v ~ v -gi~11416964~
gi~12188901~
gi~8927995~
gi~90297~
gi~249946~

GPCR2 --,r~*NRSGj,SLG.!QTP ESMF---------------------------gi~11416964~ T~~ TKPEHE QGLQ~PAP H P~3LLYYPPGVVVYSGGRYDLZPSSSAY
gi~12188901~ $~ ~V F ________________________ gi~8927995~ ~ S~ VQ F ________________________ gi~90297~ ~ ~ g g ________________________ gi~2499461 ~ S~S _________________-______ DOMAIN results for GPCR2 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The 7tm l, a seven transmembrane receptor (rhodopsin family), was shown to have homology to GPCR2. (E= 8e-20). This indicates that the GPCR2 sequence has properties similar to those of other proteins known to contain this domain as well as to the 7tm 1 domain itself (Table 2G).
Table 2G. Domain Analysis of GPCRZ
gnllPfamlpfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin family) (SEQ ID N0:72) Length: 254 CD-Length = 254 residues, only 50.0% aligned Score = 91.3 bits (225), Expect = 8e-20 ....I....I....I.....I...-I....I....~.. .I.-.
GPCR2 (SEQ ID N0:7) Consensus 7tm 1 domain 0 .I..
I...
I

I
I.
.1..1.11...1...1 GPCR2 SMTS: SHT GHG GDCCGRSLGDSCCFS' F ~ ~
T' ~ --HAS ----~
P
~
~
~

Consensus 7tm L YRI TPR
1 domain , ------G ---' ~,LLD
H
GA~FV

I....I..
.1....1....1....1.
I
..1....1....1....1....1 . GPCR2 ~C'~~ LST~ P
P
~~
D~
~

Consensus 7tm . L LESVK
1 domain I --L: Y
EGNT----TLI
'PLLR

....I....I....I....I....I....1....1....1....1....1....1.

i LAEEAAFLPL~VI'VCYT~II ~yNIKMBLVQVVDVFVLC
~
7 ~RK~ARSQRSLKRRSS~EI
1 d oma ~ t n Consensus tm .
..1....I..
I
I....I....I.
.I..
I
I
..1....I....

GPCR2 C PPWTGPASPDQ ------FATCY%1HHYE~ ~
RFGTSLGTQTPFKP
E ~V~
T
~

Consensus 7tm L YHIVLLLDSLCLI --------VLPTASLITL CLPIIY--------1 domain Based on information available on expression of SWISSPROT-ACC:P29089 TYPE-1B ANGIOTENS1N II RECEPTOR (AT1B) (AT3) - Rattus horvegicus (Rat), the closest G-Protein Coupled Receptor family member it is likely that GPCR2 is expressed in cardiac tissue, renal tissue, and vascular tissue as angiotensin is expressed in these tissues.
GPCR2 includes the nucleic acid whose sequence is provided in Table 2A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 2A while still encoding a protein that maintains its G-Protein Coupled Receptor-like activities and physiological functions, or a fragment of such a nucleic acid. GPCR2 further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. GPCR2 additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way 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 of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in WO 01/74904 ,; PCT/USO1/10241 therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 38 percent of the bases may be so changed.
The nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further below. For example, a cDNA encoding the olfactory receptor -like protein may be useful in gene therapy, and the olfactory receptor -like protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from Cardiovascular disorders, Hypertension, Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Cardiomyopathy, Atherosclerosis,Hypertension, Congenital heart defects, Aortic stenosis ,Atrial septal defect (ASD),Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Cell signalling disorders, Cancer, Muscular degeneration, Metabolic and Endocrine disorders, Respiratory disorders, Tissue/Cell growth regulation disorders, and Developmental disorders. Other GPCR-related diseases and disorders are contemplated.
The novel nucleic acid encoding the GPCR-like protein of the 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 of the 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. In one embodiment, a contemplated GPCR2 epitope is from about amino acids 50 to 90. In another embodiment, a GPCR2 epitope is from about amino acids 180 to 230. In an additional embodiment, GPCR2 epitopes are from amino acids 310 to 360.
These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

An additional GPCR-like protein of the invention, referred to herein as GPCR3, is an Olfactory Receptor ("OR")-like protein. The novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Corporation's sequence file fox GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank. The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein. The novel nucleic acid of 970 nucleotides (AC020597, SEQ ID
N0:8) LO encoding a novel olfactory receptor-like protein is shown in Table 3A. An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 27-29 and ending with a TAA codon at nucleotides 947-949. 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.
Table 3A. GPCR3 Nucleotide Sequence (SEQ ID N0:8) AAAAAGTTCCCAGAAGAACGGCCTCAATGAATACCACTCTATTTCATCCTTACTCTTTCCTTCTTCTGGGAATTC
CTGGGCTGGAAAGTATGCATCTCTGGGTTGGTTTTCCTTTCTTTGCTGTGTTCCTGACAGCTGTCCTTGGGAATA
TCACCATCCTTTTTGTGATTCAGACTGACAGTAGTCTCCATCATCCCATGTTCTACTTCCTGGCCATTCTGTCAT
CTATTGACCCGGGCCTGTCTACATCCACCATCCCTAAAATGCTTGGCACCTTCTGGTTTACCCTGAGAGAAATCT
CCTTTGAAGGATGCCTTACCCAGATGTTCTTCATCCACCTGTGCACTGGCATGGAATCAGCTGTGCTTGTGGCCA
TGGCCTATGATTGCTATGTGGCCATCTGTGACCCTCTTTGCTACACGTTGGTGCTGACAAACAAGGTGGTGTCAG
TTATGGCACTGGCCATCTTTCTGAGACCCTTAGTCTTTGTCATACCCTTTGTTCTATTTATCCTAAGGCTTCCAT
TTTGTGGACACCAAATTATTCCTCATACTTATGGTGAGCACATGGGCATTGCCCGCCTGTCTTGTGCCAGCATCA
GGGTTAACATCATCTATGGCTTATGTGCCATCTCTATCCTGGTCTTTGACATCATAGCAATTGTCATTTCCTATG
TACAGATCCTTTGTGCTGTATTTCTACTCTCTTCACATGATGCACGACTCAAGGCATTCAGCACCTGTGGCTCTC
ATGTGTGTGTCATGTTGACTTTCTATATGCCTGCATTTTTCTCATTCATGACCCATAGGTTTGGTCGGAATATAC
CTCACTTTATCCACATTCTTCTGGCTAATTTCTATGTAGTCATTCCACCTGCTCTCAACTCTGTAATTTATGGTG
TCAGAACCAAACAGATTAGAGCACAAGTGCTGAAAATGTTTTTCAATAAATAAAACATAGCTCATTTATA
LS
In a search of sequence databases, it was found, for example, that the nucleic acid sequence of GPCR3 has 627 of 904 bases (69%) identical to a Mus musculus odorant receptor S46 gene (GENBANK-ID: AF121979).
The disclosed GPCR3 polypeptide (SEQ ID N0:9) encoded by SEQ 1D N0:9 is 308 ?0 amino acid residues, a molecular weight of 34713.3 Daltons and is presented using the one-letter code in Table 3B. The GPCR3 protein were analyzed for signal peptide prediction and cellular localization. SignalP results predict that GPCR3 is cleaved between position 40 and 41 of SEQ m NO:10, i.e., at the slash in the amino acid sequence VLG-NI. Psort and Hydropathy profiles also predict that GPCR3 contains a signal peptide and is likely to be ?5 localized at the plasma membrane (certainty of 0.6400).

Table 3B. Encoded GPCR3 protein sequence (SEQ ID N0:9).
MNTTLFHPYSFLLLGIPGLESMHLWVGFPFFAVFLTAVLGNITILFVIQTDSSLHHPMFYFLAILSSIDPGLSTS
TIPKMLGTFWFTLREISFEGCLTQMFFIHLCTGMESAVLVAMAYDCYVAICDPLCYTLVLTNECWSVMALAIFLR
PLVFVIPFVLFILRLPFCGHQIIPHTYGEHMGIARLSCASIRVNIIYGLCAISILVFDIIAIVISYVQILCAVFL
LSSHDARLKAFSTCGSHVCVMLTFYMPAFFSFMTHRFGRNIPHFIHILLANFYVVIPPALNSVIYGVRTECQIRAQ
VLKMFFNFC
The full amino acid sequence of the protein of the invention was found to have 178 of 307 amino acid residues (57%) identical to, and 231 of 307 residues (75%) positive with, the 318 amino acid residue protein ODORANT RECEPTOR S46 from - Mus musculus (Mouse) (ptnr:SPTREMBL-ACC: Q9WU93), and 146 of 306 amino acid residues (47%) identical to, and 208 of 306 residues (67%) positive with, the 312 amino acid residue protein Olfactory Receptor HPFH1 OR from Homo Sapiens (TREMBLNEW-ACC:AAD51279).
The disclosed GPCR3 protein (SEQ ID N0:9) also has good identity with a number of olfactory receptor proteins, as shown in Table 3C and 3D.
"Table 3C. BLAST
results for Gene Tndex/ Protein/ LengthIdentity PositivesExpect Identifier Organism (aa) (%) (%) gi199354421refINPodorant 318 178/307 231/307 7e-86 064688.11 receptor S46 (57%) (74%) gene [Mus musculus]

gi165320011gbIAADodorant 339 159/291 211/292 1e-75 27596.21AF121976receptor S19 (54%) (71%) l (AF121976) [Mus musculus]

Gi1119082111gbIAAHOR 5'Betal4 318 157/299 214/299 5e-73 641676.11 [Homo Sapiens] (52%) (71%) (AF137396) Gi~99380141ref~NPodorant 321 155/304 213/304 5e-73 064686.11 receptor S18 (50%) (69%) gene [Mus musculus]

Gi173053491refINPolfactory 326 158/297 213/297 6e-71 038647.11 receptor 67 (53%) (71%) [Mus musculus]

Table 3D. BLASTX alignments Sequences Smallest producing High-scoring Segment Pairs:

Sum Reading HighProb.

Frame ScoreP(N) Patp:W01730Human G-protein receptor HPRAJ70Homo sa 721 1.8e-70 - +3 Patp:W56641G-protein coupled prostate eceptor 721 1.8e-70 tissue r +3 Patp:Y92365G protein-coupled receptor 5 - Hom 718 3.8e-70 protein +3 Patp:R27875Odorant receptor clone Il4 rattus +3 486 1.5e-45 - Rattus This information is presented graphically in the multiple sequence alignment given in Table 3E (with GPCR3 being shown on line 1) as a ClustalVV analysis comparing GPCR3 with related protein sequences.
Table 3E. Information for the ClustalW proteins:
1) Novel GPCR3 (SEQ ID N0:9) 2) gi~9935442~ref~NP_064688.1 ~ odorant receptor S46 gene [Mus musculus] (SEQ
ID N0:53) 3) gi~6532001~gb~AAD27596.2~AF121976 1 (AF121976) odorantreceptor S19 [Mus musculus] (SEQ ID N0:54) 1 ~ 4) gi~11908211~gb~AAG41676.1~ (AF137396) HOR 5'Betal4 [Homo Sapiens] (SEQ
ID NO:55) 5) gi~9938014~ref~NP_064686.1~ odorant receptor S18 gene [Mus musculus] (SEQ
ID NO:56) 6) gi~7305349~ref~NP 038647.1 olfactory receptor 67 [Mus musculus] (SEQ ID
N0:57) GPCR3 __________________-________ T .LFH~YS SIN~L VGF~ F
gi~9935442~ --------------------------MSPG~S~WIH~SS~ ~ ELQ G~ G
gi~6532001~ MPEKMLSKLIAYLLLIESCRQTAQLVKGRRIWVDS ~HWPNTTHYRE D T,., gi~11908211~ -------------------------MSDSNLSDNHL DT~F T 1, gi~9938014) --------------------MNSKASMLGT F~~!IIH TV I - QY T ~
gi~7305349~ ----------------w---MKVASSFH~D!~?NPQDVWY~T DL S T ~I

I_I_~___I_I_.I__I___I____I___I____I____I
GPCR3 ~~''T ~ 7~ IT',~. , Q~SH ~'Y I SI PG S I ~ GT TLR
V V~
gi ~ 9935442 ~ TI Jx' I~ YLH ,' I~YL 1 ~~ ~ G G GFH , gi ~ 6532001 ~ IL' .. GT ~YIS1 ~ L C ~.T ~ V Cy,'~ L ~ LRHV
gi ~ 11908211 ~ I,~, 'L,. ~L N L C ~~; ~ ~T ' L LHG
gi~9938014~ L GAEL LI~R ~~ V 8 G yL T Y' T ' HAG
gi ~ 7305349 ~ ~; ' I VL~~'~; V~RS F ~ ~ L HRG

gi~9935442~
gi~6532001~
gi~11908211~
gi~99380I4~
gi~7305349~

gi~9935442~
gi~6532001~
gi~11908211~
gi~9938014~
gi~7305349~

gi~9935442~
gi~6532001~
gi~11908211~
gi~9938014~
gi~7305349~

GPCR3 1): ~' ~ S .. ~ ~A'Q~T~ FFNK--_______ gi~9935442~ I~' v D ~TLCNEVGQLADD--gi~6532001~ I ' L ' N ~ L II;DFFVKRR--------gi ~ 11908211 ~ L E ~S~iKw~LHLGKTSI-----gi~9938014~ , ~ ~DSTR~LSVVWKS------gi~7305349~ s~ ~' V ' ~ 'D F~FSSVSTCQHDSRC

DOMAIN results for GPCR3 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The 7tm l, a seven transmembrane receptor (rhodopsin family), was shown to have significant homology to GPCR3. (E=7e-11). This indicates that the GPCR3 sequence has properties similar to those of other proteins known to contain this domain as well as to the 7tm 1 domain itself.
Table 3F. Domain Analysis of GPCR3 gnljPfamjpfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin family) (SEQ ID N0:73) Length: 254 CD-Length = 254 residues, 100.0o aligned Score = 61.2 bits (147), Expect = 7e-11 .j....j....~....j....j.. .j....j....j....~....j....j....j 10 ConsensusQ7tm lOdomain ~L~L~LQRTKIf~R---T'~PNI~LNVA~LLFLL~LP~WA~YYLVGG--DWVFGDA

....j....~....j....j....j....j....~....j....j....j.. .j.. .j ~Y
C
CD' C
TL
L
VS
diRP
F
LA~
~
~
~P~V
=
~
~
~
~
f Consensus7tm 1 R
domain RF.t R
RAK
~qI
L
L
KLVGALFV
NGYASI
I
VH
R
9~

.j....j ....j....j....j....j....j....j....j....j....j....j ~P~ ~IIPHTYGET~C~
V
~
V
B
~

Consensus7tm 1 S-W IDFPEE
domain S RSY
TVEET------------L

...j..
GPCR3 .j..
5 .j....j....j....j....~....j....j....j....j CA~,.SILFDS
V
C
___________________________-_________ ~
~
~
~

Consensus7tm 1 T
domain STjIGFLP
~
C
R--------------------------------------j j GPCR3 ....j....
....j....j....j....j....j....j....~....j....j....
___,________________________________________________________ 3 consensus7tm 1 ____________________________________________________________ 0 domain ....j....j.. .j.. ....j....j....~....j....j....j....~....j GPCR3 ---AVFLL '-.j CGS------H
5 AHD ~ C
~ ~R~ TF

FFSF
THRFGRN----~
~
~~
' Consensus7tm 1 ----TLRK S YHIVL
domain RS LDSLCLLSIWR
SERKAAKMLL
V
SFVLC~T~, ....j....j ....j ....~....j.. .j..
GPCR3 ------- HFH LN S' BIPP ~
~
~
C

40 Consensus7tm 1 --------V TA~L TWLA P
domain NS--The nucleic acids and proteins of the invention axe useful in potential therapeutic applications implicated in various GPCR-related pathological disorders andlor OR-related pathological disorders, described further below. For example, a cDNA encoding the olfactory 45 receptor -like protein may be useful in gene therapy, and the olfactory receptor -like protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from nfections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm;
50 adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. Other GPCR-related diseases and disorders are contemplated.
The novel nucleic acid encoding the GPCR-like protein of the 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 fixrther useful in the generation of antibodies that bind immunospecifically to the novel substances of the 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 imrnunogen. In one embodiment, a contemplated GPCR3 epitope is from about amino acids 160 to 190. In another embodiment, a GPCR3 epitope is from about amino acids 260 to 270. In an additional embodiment, GPCR3 epitopes are from amino acids 280 to 300.

GPCR4 includes a family of three nucleic acids disclosed below. The disclosed nucleic acids encode a GPCR-like protein.
GPCR4a The disclosed GPCR4a (also referred to herein as AC020597 B) is encoded by a nucleic acid, 994 nucleotides long (SEQ ID NO:10). An open reading frame was identified beginning with an ATG initiation codon at nucleotides 23-25 and ending with a TAA codon at nucleotides 968-970. 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 encoded protein having 315 amino acid residues is presented using the one-letter code in Table 4B (SEQ 1D N0:11).

Table 4A. GPCR4a Nucleotide Sequence (SEQ ID NO:10).
TGCTGAATTACTCAAAGTCACTATGGGAGACTGGAATAACAGTGATGCTGTGGAGCCCATATTTATCCTGAGGGG
TTTTCCTGGACTGGAGTATGTTCATTCTTGGCTCTCCATCCTCTTCTGTCTTGCATATTTGGTAGCATTTATGGG
TAATGTTACCATCCTGTCTGTCATTTGGATAGAATCCTCTCTCCATCAGCCCATGTATTACTTTATTTCCATCTT
AGCAGTGAATGACCTGGGGATGTCCCTGTCTACACTTCCCACCATGCTTGCTGTGTTATGGTTGGATGCTCCAGA
GATCCAGGCAAGTGCTTGCTATGCTCAGCTGTTCTTCATCCACACATTCACATTCCTGGAGTCCTCAGTGTTGCT
GGCCATGGCCTTTGACCGTTTTGTTGCTATCTGCCATCCACTGCACTACCCCACCATCCTCACCAACAGTGTAAT
TGGCAAAATTGGTTTGGCCTGTTTGCTACGAAGCTTGGGAGTTGTACTTCCCACACCTTTGCTACTGAGACACTA
TCACTACTGCCATGGCAATGCCCTCTCTCACGCCTTCTGTTTGCACCAGGATGTTCTAAGATTATCCTGTACAGA
TGCCAGGACCAACAGTATTTATGGGCTTTGTGTAGTCATTGCCACACTAGGTGTGGATTCAATCTTCATACTTCT
TTCTTATGTTCTGATTCTTAATACTGTGCTGGATATTGCATCTCGTGAAGAGCAGCTAAAGGCACTCAACACATG
TGTATCCCATATCTGTGTGGTGCTTATCTTCTTTGTGCCAGTTATTGGGGTGTCAATGGTCCATCGCTTTGGGAA
GCATCTGTCTCCCATAGTCCACATCCTCATGGCAGACATCTACCTTCTTCTTCCCCCAGTCCTTAACCCTATTGT
CTATAGTGTCAGAACAAAGCAGATTCGTCTAGGAATTCTCCACAAGTTTGTCCTAAGGAGGAGGTTTTAAGTAAC
CTCTGTCCTCCAACTTTTC
The disclosed nucleic acid GPCR4a sequence has 571 of 868 bases (65%) identical to a Rattus norvegicus GPCR mRNA (GENBANK-m: AF079864).
The GPCR4a polypeptide (SEQ m NO:11) encoded by SEQ ID NO:10 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 is likely to be localized at the plasma membrane with a certainty of 0.6000. The most likely cleavage site for a GPCR4a peptide is between amino acids 40 and 41, at: LVA-FM., based on the SignalP result. GPCR4a has a molecular weight of 35368.7.
Table 4B. GPCR4a protein sequence (SEQ ID NO:11) MGDWNNSDAVEPIFILRGFPGLEYVHSWLSILFCLAYLVAFMGNVTILSVIWIESSLHQPMYYFISILA
VNDLGMSLSTLPTMLAVLWLDAPEIQASACYAQLFFIHTFTFLESSVLLAMAFDRFVAICHPLHYPTIL
TNSVIGKIGLACLLRSLGVVLPTPLLLRHYHYCHGNALSHAFCLHQDVLRLSCTDARTNSIYGLCVVIA
TLGVDSIFILLSYVLILNTVLDIASREEQLKALNTCVSHICVVLIFFVPVIGVSMVHRFGKHLSPIVHI
LMADIYLLLPPVLNPIVYSVRTKQIRLGILHKFVLRRRF
The full amino acid sequence of the disclosed GPCR4a polypeptide has 159 of amino acid residues (52%) identical to, and 217 of 301 residues (72%) positive with, the 319 amino acid residue protein from Gallus gallus (ptnr:SPTREMBL-ACC: Q9YH55), (E
=
1.0x10-$4) BLASTP (Non-Redundant Composite database) analysis of the best hits for alignments with GPCR4a are listed in Table 4C. BLASTX analysis was also performed to determine which proteins have significant identity with GPCR4a, as shown in Table 4D.

Table 4C.
BLASTP
results for GPCR4a Gene Index/Protein/ Organism Length Identity PositivesExpect Tdentifier (aa) (~) (%) ACC:Q9WVN4 MOR 5'BETA1 - Mus 311 160/303 219/303 1.6e-83 musculus (52%) (72%) ACC:088628 PUTATIVE G-PROTEIN320 152/296 204/296 5.3e-83 COUPLED RECEPTOR (51%) (68%) - Rattus norvegicus ACC:Q9Y5P1 HOR 5'BETA3 - Homo312 145/312 221/312 1.9e-78 Sapiens (46%) (70%) Table 4D. BLASTX results for GPCR4a Smallest Sum Reading High Prob Sequences producing High-scoringPairs: Frame ScoreP(N) N
Segment Ptnr:SPTREMBL-ACC:Q9YH55 OLFACTORY 856 1.2e-84 RECEPTOR-LIKE PROTE.+2 1 ptnr:SPTREMBL-ACC:Q9WVN4 MOR Mus musculus 838 9.5e-83 5'BETA1 - (M.+2 1 ptnr:SPTREMBL-ACC:088628 PUTATIVE 833 3.2e-82 G-PROTEIN COUPLED RE.+2 1 ptnr:SPTREMBL-ACC:Q9WVN5 MOR Mus musculus 830 6.7e-82 5'BETA2 - (M.+2 1 ptnr:SPTREMBL-ACC:Q9WVN6 MOR Mus musculus 826 1.8e-81 5'BETA3 - (M.+2 1 ptnr:SPTREMBL-ACC:Q9Y5P1 HOR Homo Sapiens 790 1.2e-77 5'BETA3 - (H.+2 1 ptnr:SPTREMBL-ACC:Q9WU90 ODORANT 764 6.6e-75 RECEPTOR S19 - Mus mu.+2 1 patp:Y92365 G protein-coupledprotein 5 - Hom 829 6.5e-82 receptor +2 1 patp:W01730 Human G-protein 818 9.6e-81 receptor HPRAJ70 - Homo sa 1 +2 patp:W56641 G-protein coupledtissue receptor 818 9.6e-81 prostate +2 1 patp:R27874 Odorant receptor - Rattus rattus,440 1.1e-40 clone I9 +2 1 patp:R27875 Odorant receptor - Rattus rattus 440 1.1e-40 clone I14 +2 1 Possible SNPs found for GPCR4a are listed in Table4E.
Table 4E: SNPs Base Base Base Position Before After 11 S A G(4) 140 ' G gap(2) 141 C gap(2) 174 A gap(17) 175 C gap(17) 176 A gap(6) 177 C gap(6) 178 A gap(5) 179 C gap(5) 180 gap A(22) 181 gap C(22) 182 gap A(21) 183 C gap(20) 185 C gap(10) 229 gap A(2) 252 A gap(2) 256 gap A(2) 548 T C(2) 553 G gap(2) 614 G A(2) 1445 C A(2) 1446 C G(2) 1447 A G(2) 1448 A C(2) 1453 G A(2) 1454 G T(2) 1455 A G(2) 1456 C T(2) 1460 G C(2) 1462 G T(2) GPCR4b The disclosed GPCR4b (also referred to herein as AC020597B1) is encoded by a nucleic acid, 994 nucleotides long (SEQ m N0:12). An open reading frame was identified beginning with an ATG initiation codon at nucleotides 23-25 and ending with a TAA codon at nucleotides 968-970. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4F, and the start and stop codons are in bold letters. The encoded protein having 312 amino acid residues is presented using the one-letter code in Table 4G (SEQ ID N0:13).
Table 4F. GPCR4b Nucleotide Sequence (SEQ ID N0:12) TGCTGAATTACTCAAAGTCACTATGGGAGACTGGAATAACAGTGATGCTGTGGAGCCCATATTTATC
CTGAGGGGTTTTCCTGGACTGGAGTATGTTCATTCTTGGCTCTCCATCCTCTTCTGTCTTGCATATT
TGGTAGCATTTATGGGTAATGTTACCATCCTGTCTGTCATTTGGATAGAATCCTCTCTCCATCAGCC
CATGTATTACTTTATTTCCATCTTGGCAGTGAATGACCTGGGGATGTCCCTGTCTACACTTCCCACC
ATGCTTGCTGTGTTATGGTTGGATGCTCCAGAGATCCAGGCAAGTGCTTGCTATGCTCAGCTGTTCT
TCATCCACACATTCACATTCCTGGAGTCCTCAGTGTTGCTGGCCATGGCCTTTGACCGTTTTGTTGC
TATCTGCCATCCACTGCACTACCCCACCATCCTCACCAACAGTGTAATTGGCAAAATTGGTTTGGCC
TGTTTGCTACGAAGCTTGGGAGTTGTACTTCCCACACCTTTGCTACTGAGACACTATCACTACTGCC
ATGGCAATGCCCTCTCTCACGCCTTCTGTTTGCACCAGGATGTTCTAAGATTATCCTGTACAGATGC
CAGGACCAACAGTATTTATGGGCTTTGTGTAGTCATTGCCACACTAGGTGTGGATTCAATCTTCATA
CTTCTTTCTTATGTTCTGATTCTTAATACTGTGCTGGATATTGCATCTCGTGAAGAGCAGCTAAAGG
CACTCAACACATGTGTATCCCATATCTGTGTGGTGCTTATCTTCTTTGTGCCAGTTATTGGGGTGTC
AATGGTCCATCGCTTTGGGAAGCATCTGTCTCCCATAGTCCACATCCTCATGGCAGACATGTACCTT
CTTCTTCCCCCAGTCCTTAACCCTATTGTCTATAGTGTCAGAACAAAGCAGATTCGTCTAGGAATTC
TCCACAAGTTTGTCCTAAGGAGGAGGTTTTAAGTAACCTCTGTCCTCCAACTTTTC
The SignalP, Psort and/or Hydropathy profile for the disclosed GPCR4b Olfactory Receptor-like protein predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The SignalP shows a signal sequence is coded for in the first 36 amino acids with a cleavage site at between amino acids 40 and 41, between LVA/FM in Table 4G. The molecular weight of GPCR4b is 35386.7 Da.
This is typical of this type of membrane protein.

Table 4G. GPCR4b Amino Acid Sequence (SEQ ID N0:13) MGDWNNSDAVEPIFILRGFPGLEYVHSWLSILFCLAYLVAFMGNVTILSVIWIESSLHQPMYYFISILAVNDL
GMSLSTLPTMLAVLWLDAPEIQASACYAQLFFIHTFTFLESSVLLAMAFDRFVAICHPLHYPTILTNSVIGKI
GLACLLRSLGWLPTPLLLRHYHYCHGNALSHAFCLHQDVLRLSCTDARTNSIYGLCWIATLGVDSIFILLS
YVLILNTVLDIASREEQLKALNTCVSHICWLIFFVPVIGVSMVHRFGKHLSPIVHILMADMYLLLPPVLNPI
VYSVRTKQIRLGILHKFVLRRRF
BLASTP alignments also showed high homology between GPCR4b and other proteins as shown in Table 4H.
Table 4H.
BLASTP
results for GPCR4b Gene Index/Protein/ Organism Length Identity PositivesExpect Identifier (aa) (%) (%) SPTREMBL- OLFACTORY RECEPTOR-319 159/301 217/301 2.7e-85 ACC:Q9YH55 LIKE PROTEIN COR3'BETA (52%) (72%) - Gallus gallus (Chicken), SPTREMBL- MOR 5'BETA1 - Mus 311 160/303 219/303 2,2e-83 ACC:Q9WVN4 musculus (Mouse) (52%) (72%) ACC:Q9Y5P1 HOR 5'BETA3 - Homo312 144/312 221/312 3.4e-78 Sapiens (Human) (46%) (70%) The disclosed GPCR4b protein is homologous to a number of olfactory receptor proteins, as shown in Table 4I.
Table 4I. BLASTX results for GPCR4b Smallest Sum ReadingHigh Prob Sequences producingcoring Segment Pairs:Frame ScoreP(N) N
High-s ptnr:SPTREMBL-ACC:Q9YH55OLFACTORY RECEPTOR-LIKE 854 1.6e-84 PROTE.+2 1 ptnr:SPTREMBL-ACC:Q9WVN4MOR 5'BETA1 - Mus (M 836 1.3e-82 musculus +2 1 ptnr:SPTREMBL-ACC:088628PUTATIVE G-PROTEIN RE.+2 831 4.5e-82 ptnr:SPTREMBL-ACC:Q9WVN5MOR 5'BETA2 - Mus (M.+2 828 9.3e-82 musculus 1 ptnr:SPTREMBL-ACC:Q9WVN6.MOR 5'BETA3 - Mus (M,+2 823 3.1e-81 musculus 1 ptnr:SPTREMBL-ACC:Q9Y5P1HOR 5'BETA3 - Homo (H,+2 787 2.1e-77 Sapiens 1 ptnr:SPTREMBL-ACC:Q9WU90ODORANT RECEPTOR mu.+2 760 1.5e-74 S19 - Mus 1 ptnr:SPTREMBL-ACC;Q9WVD9MOR 3'BETA1 - Mus (M,+2 751 1.3e-73 musculus 1 ptnr:SPTREMBL-ACC;Q9WU89ODORANT RECEPTOR mu,+2 749 2.2e-73 S18 - Mus 1 ptnr:SPTREMBL-ACC;Q9Y5P0HOR 5'BETA1 - Homo (H,+2 745 5.8e-73 Sapiens 1 ptnr:SPTREMBL-ACC;Q9WVD7MOR 3'BETA3 - Mus (M,+2 710 3.0e-69 musculus 1 ptnr:SPTREMBL-ACC;Q9WVD8MOR 3'BETA2 - Mus (M.+2 705 1.0e-68 musculus 1 ptnr:SPTREMBL-ACC;Q9WU93ODORANT RECEPTOR mu.+2 703 1.6e-68 S46 - Mus 1 ptnr:TREMBLNEW-ACC:CAB89291 532 1.8e-54' - Homo +2 GPCR4c l0 The disclosed GPCR4c (also referred to herein as AC020597B2) is encoded by a nucleic acid, 994 nucleotides long (SEQ TD N0:14. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 23-25 and ending with a TAA codon at nucleotides 968-970. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4J, and the start and stop codons are in bold letters. The encoded protein having 312 amino acid residues is presented using the one-letter code in Table 4J (SEQ 1D N0:12).
Table 4J. GPCR4c Nucleotide Sequence (SEQ ID N0:14).
TGCTGAATTACTCAAAGTCACTATGGGAGACTGGAATAACAGTGATGCTGTGGAGCCCATATTTATCCTGAGGGG
TTTTCCTGGACTGGAGTATGTTCATTCTTGGCTCTCCATCCTCTTCTGTCTTGCATATTTGGTAGCATTTATGGG
TAATGTTACCATCCTGTCTGTCATTTGGATAGAATCCTCTCTCCATCAGCCCATGTATTACTTTATTTCCATCTT
GGCAGTGAATGACCTGGGGATGTCCCTGTCTACACTTCCCACCATGCTTGCTGTGTTATGGTTGGATGCTCCAGA
GATCCAGGCAAGTGCTTGCTATGCTCAGCTGTTCTTCATCCACACATTCACATTCCTGGAGTCCTCAGTGTTGCT
GGCCATGGCCTTTGACCGTTTTGTTGCTATCTGCCATCCACTGCACTACCCCACCATCCTCACCAACAGTGTAAT
TGGCAAAATTGGTTTGGCCTGTTTGCTACGAAGCTTGGGAGTTGTACTTCCCACACCTTTGCTACTGAGACACTA
TCACTACTGCCATGGCAATGCCCTCTCTCACGCCTTCTGTTTGCACCAGGATGTTCTAAGATTATCCTGTACAGA
TGCCAGGACCAACAGTATTTATGGGCTTTGTGTAGTCATTGCCACACTAGGTGTGGATTCAATCTTCATACTTCT
TTCTTATGTTCTGATTCTTAATACTGTGCTGGATATTGCATCTCGTGAAGAGCAGCTAAAGGCACTCAACACATG
TGTATCCCATATCTGTGTGGTGCTTATCTTCTTTGTGCCAGTTATTGGGGTGTCAATGGTCCATCGCTTTGGGAA
GCATCTGTCTCCCATAGTCCACATCCTCATGGCAGACATCTACCTTCTTCTTCCCCCAGTCCTTAACCCTATTGT
CTATAGTGTCAGAACAAAGCAGATTCGTCTAGGAATTCTCCACAAGTTTGTCCTAAGGAGGAGGTTTTAAGTAAC
CTCTGTCCTCCAACTTTTC
The encoded protein is the same as for GPCR4a and is disclosed above in Table 4B.
Unless specifically addressed as GPCR4a or GPCR4b, 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 and the residue position with respect to the "a" variant. In all following sequence alignments, the GPCR4a protein sequence was used.
The disclosed GPCR4 protein (SEQ ID N0:12) also has good identity with a number of olfactory receptor proteins. The identity information used for ClustalW
analysis is presented in Table 4K.
Table 4K. BLAST
results for GPCR4 Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) gi~11908220~gb~AAG41MOR 3'Beta4 303 174/307 223/307 2e-81 [Mus 684.1 (AF133300) musculus] (56%) (71%) gi~11908225~gb~AAG41Mor 5'Beta5 317 160/294 215/294 3e-79 [Mus 688.1 (AF071080) musculus] (54%) (72%) gi~11908214~gb~AAG41HOR5'Betall 314 152/294 226/294 9e-79 [Homo 679.1 (AF137396) Sapiens] (51%) (76%) gi~11908213~gb~AAG41HOR5'Betal2 312 160/296 211/296 2e-78 [Homo 678.1 (AF137396) Sapiens] (54%) (71%) gi111908218~gbIAAG41HOR5'Beta5 [Homo312 160/300 220/300 2e-77 I

683.1 (AF137396) Sapiens] (53%) (73%) This information is presented graphically in the multiple sequence alignment given in Table 4L (with GPCR4 being shown on line 1) as a ClustalW analysis comparing GPCR4 with related OR sequences.

Table 4L Information for the ClustalW proteins:
1) GPCR4A (SEQ ID NO:11) 2) GPCR4B (SEQ ID N0:13) 3) >gi~11908220~gb~AAG41684.1~ (AF133300) MOR 3'Beta4 [Mus musculus] (SEQ ID
NO:SB) 4) >gi~1190822S~gb~AAG41688.1~ (AF071080) Mor S'BetaS [Mus musculus]gi~4826S21~emb~CAB428S3.1~ dJ88J8.1 (novel 7 transmembrane receptor (rhodopsin family) (OR like) pro (SEQ ID NO:S9) 5)>gi~11908214~gb~AAG41679.1i (AF137396) HORS'Betal l [Homo Sapiens]tein) (hs6M1-1S)) Homo Sapiens (SEQ ID N0:60) 6) >gi~11908213~gb~AAG41678.1~ (AF137396) HORS'Betal2 [Homo Sapiens]gi~120S4431~emb~CAC20S23.1~ olfactory receptor 1 ~ Homo Sapiens (SEQ ID N0:61) 7) >gi~11908218~gb~AAG41683.1~ (AF137396) HORS'BetaS [Homo Sapiens]gi~120S4429~emb~CAC20S22.1~ olfactory receptor Homo Sapiens (SEQ ID N0:62) GPCR4a GPCR4b gi~11908220~gb~AAG41684.1) gi~119082251gbIAAG41688.1~
gi~119082141gbIAAG41679.1~
gi111908213~gb~AAG41678.1~
gi~11908218~gb~AAG41683.1~

GPCR4a GPCR4b gi~11908220~gb~AAG41684.1~
gi~11908225~gb~AAG41688.1~
3 ~ gi~11908214~gb~AAG41679.1~
gi~119082131gbIAAG41678.1~
gi~11908218I9bIAAG41683.1~

GPCR4a ~Fm kl~ H P ~ NS~ GK~ C~L' LGVL~TPLL RH'H~~~HG
GPCR4b ~F~' ' H P T NSVEJGK C L' LGV~'L~TPLL RH HY HG
gi~11908220~gb~AAG41684.1~~ ~Lm , 1_ PKLr,~~rn~K T ' SIL~I LIA FPe GS~V
gi~11908225~gb~AAG41688.1~~ ~Lm L~~ R ' GPRAL TVLG v ITAAPSLH LT'D~ HPG
E
4Q gi~11908214~gb~AAG41679.1~~ L~ ~~ ' V~ HN L GI~T" FTTFPFV KRLP KG~V H
gi ~ 11908218 ~ gb ~ AAG41683 1 ~ ~ Y ~' ~'~ D' ' T~,;: NTl~,yV ~ GV ' GFVS~V~PIRP~YF"LI HSU
GPCR4a GPCR4b gi~11908220~gb~AAG41684.1~
gi~11908225~gb~AAG41688.1~
gi~11908214~gb~AAG41679.1~
gi111908213~gb~AAG41678.1~
gi~11908218~gb~AAG41683.1~
GPCR4a GPCR4b gi~11908220~gb~AAG41684.1~
gi~11908225~gb~AAG41688.1~
gi~119082141gbIAAG41679.1~
gi~11908213~gb~AAG41678.11 gi~11908218~gb~AAG41683.1~

GPCR4a ~~~ LG 'H L RF----GPCR4b ~ 'L HK~VLRF----gi1119082201gbIAAG41684.1~ ~ 'E HLLCSPISSITM
gi111908225IgbIAAG41688.1~: ~ ' KVISLG .IQ====
gi~11908214~gb~AAG41679.1~ 'KG ~K HKSA-gi~119082131gbIAAG41678.1~ E ' F HHI I ----gi~11908218~gb~AAG41683.1~ ~ ~1, HL TTHIGT---DOMAIN results for GPCR4 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. Two regions of GPCR4 have identity to the 377 amino acid 7TM
domain, as described above. The 7tm 1, a seven transmembrane receptor (rhodopsin family), was shown to have homology to GPCR4 (E= 4e-17) (Table 4M).
Table 4M: Domain Alignment between GPCR4 and 7tm 1, 7 transmembrane receptor (rhodopsin family) (SEQ ID N0:74).
CD-Length = 254 residues, 100.0% aligned Score = 82.0 bits 201 Ex ect = 4e-17 .I.. .1....1....1....1....1.. .I....1.. .1....1....1....1 Consensus 7tm 1 domain ~L~L~LRTKK~R-Q~TNILN ~~A~LFL~LP~N1A~YY~VGGDWVFQGD L KL

....1....1....1....1....1....1.. .1....1....1....1....1....1 Consensus 7tm 1 domain ~GALFVVNG~ASIL~TI~ V~R~RR~R~PRRAKVI~LVW~LLS~P

.1....I.. .1....1....1....1....1....1.. .1....1....1.
GPCR4 P LLRHYHYCH --LSHAFCLH~V LSC DARTa~T~Y LC. ~. T~'iG DSIF
Consensus 7tm 1 domain LBFSWLRTVEE~T----TVCLIDFP'~SR~YVLLTV~FLP~.LVL~CYTR

.I....1....1....I....I....1....1....1....1....1....1....1 GPCR4 L'~~,YVLILN-_________________________________________________ Consensus 7tm 1 domain ~RLRKSAR-----'------------------------------_-------------....1....I....1....I....1....1....1....1....1....1.. .I
GPCR4 _________________________________________________=_LDA~E
Consensus 7tm 1 domain -------------------------------_----------------- - QRS
K S
310 320 330 340 350 ° 360 .1....I.. .1....1....1....1....1....1....1....1....1....1 GPCR4 E ~~NTCSH C~VF'~IG~S~HRFGKHLSPI--------~HILMAD~Y~B
Consensus 7tm 1 domain SS - ~~ KMLVV~ F~t~,C; YH2 LDSLCLLSIWR-------- LPTALL T

....1.. .1..

Consensus 7tm 1 domain AYVNSC
An alignment of GPCR4a and b amino acid sequences is shown in Table 4N.
Table 4N. Alignment between GPCR4a and b.

.1....1....I.. .1....I....1....1....1....1....1....1....1 GPCR4a n GPCR4b a. ~~ i- '1~

....I....I....I....I....I....1....1....1....1....1....1....1 GPCR4a GPCR4b .I....1.. .I....1....1....1....1....1....1.. .i....1....1 SS GPCR4a GPCR4b , m ~

....I....I....I....I....I....1....1....1....1....1....1....1 GPCR4a GPCR4b ....I....I....I.....I....I....I....I....I....I....I....I....I
GPCR4a GPCR4b .I.. .I
GPCR4a GPCR4b 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 below. For example, a cDNA encoding the olfactory l5 receptor-like protein may be useful in gene therapy, and the olfactory receptor-like protein may be useful when administered to a subject in need thereof. The protein similarity information, expression pattern, and map location for the Olfactory Receptor-like protein and nucleic acid disclosed herein suggest that this Olfactory Receptor may have important structural and/or physiological functions characteristic of the Olfactory Receptor family.
?0 Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the 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, ~5 (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
The nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or 30 other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, 35 osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de Ia Tourette and/or other pathologies and 1.0 disorders of the like. The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a 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. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome. Other GPCR-4 diseases and disorders are contemplated.
The novel nucleic acid encoding the GPCR-like protein of the 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 of the 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 GPCR4 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR4 epitope is from about amino acids 160 to 200. Tn another embodiment, a GPCR4 epitope is from about amino acids 230 to 250. In an additional embodiment, GPCR4 epitopes are from amino acids 290 to 305.
GPCRS
GPCRS includes a family of three similar nucleic acids and three similar proteins disclosed below. The disclosed nucleic acids encode GPCR, OR-like proteins.
GPCRSa The disclosed novel GPCRSa nucleic acid of 9~5 nucleotides (also referred to as 1 AC020597 C) is shown in Table SA. An ORF begins with an ATG initiation codon at nucleotides 27-29 and ends with a TAA codon at nucleotides 960-962. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table SA, and the start and stop codons are in bold letters.
Table SA. GPCRSa Nucleotide Sequence (SEQ ID N0:15) GTTCTCCTACACTGTGATTTGGAAAAATGTTTTATCACAACAAGAGCATATTTCACCCAGTCACATTTTTCCTCA
TTGGAATCCCAGGTCTGGAAGACTTCCACATGTGGATCTCCGGGCCTTTCTGCTCTGTTTACCTTGTGGCTTTGC
TGGGCAATGCCACCATTCTGCTAGTCATCAAGGTAGAACAGACTCTCCGGGAGCCCATGTTCTACTTCCTGGCCA
TTCTTTCCACTATTGATTTGGCCCTTTCTGCAACCTCTGTGCCTCGCATGCTGGGTATCTTCTGGTTTGATGCTC
ACGAGATTAACTATGGAGCTTGTGTGGCCCAGATGTTTCTGATCCATGCCTTCACTGGCATGGAGGCTGAGGTCT
TACTGGCTATGGCTTTTGACCGTTATGTGGCCATCTGTGCTCCACTACATTACGCAACCATCTTGACATCCCTAG
TGTTGGTGGGCATTAGCATGTGCATTGTAATTCGTCCCGTTTTACTTACACTTCCCATGGTCTATCTTATCTACC
GCCTACCCTTTTGTCAGGCTCACATAATAGCCCATTCCTACTGTGAGCACATGGGCATTGCAAAATTGTCCTGTG
GAAACATTCGTATCAATGGTATCTATGGGCTTTTTGTAGTTTCTTTCTTTGTTCTGAACCTGGTGCTCATTGGCA
TCTCGTATGTTTACATTCTCCGTGCTGTCTTCCGCCTCCCATCACATGATGCTCAGCTAAAAGCCCTAAGCACGT
GTGGCGCTCATGTTGGAGTCATCTGTGTTTTCTATATCCCTTCAGTCTTCTCTTTCCTTACTCATCGATTTGGAC
ACCAAATACCAGGTTACATTCACATTCTTGTTGCCAATCTCTATTTGATTATCCCACCCTCTCTCAACCCCATCA
TTTATGGGGTGAGGACCAAACAGATTCGAGAGCGAGTGCTCTATGTTTTTACTAAAAAATAAGACTCTTACCATG
TTATTTTACT
The GPCRSa protein encoded by SEQ ID NO:15 has 311 amino acid residues and is presented using the one-letter code in Table SB. The Psort profile for GPCRSa predicts that this sequence 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 peptide is between amino acids 49 and 50: between ILL-VI based on the SignalP result. The molecular weight of GPCRSa is 35059.6 Daltons.
Table SB. Encoded GPCRSa protein sequence (SEQ ID N0:16) MFYHNKSIFHPVTFFLIGIPGLEDFHMWISGPFCSVYLVALLGNATILLVIKVEQTLREPMFYFLAILSTIDL
ALSATSVPRMLGIFWFDAHEINYGACVAQMFLIHAFTGMEAEVLLAMAFDRYVAICAPLHYATILTSLVLVGI
SMCTVIRPVLLTLPMVYLIYRLPFCQAHIIAHSYCEHMGIAKLSCGNIRINGIYGLFWSFFVLNLVLIGTSY
VYILRAVFRLPSHDAQLKALSTCGAHVGVICVFYIPSVFSFLTHRFGHQIPGYIHILVANLYLITPPSLNPII
YGVRTKQIRERVLYVFTKK
The disclosed nucleic acid sequence for GPCRSa has 633 of 989 bases (64%) identical to a Mus musculus GPCR mRNA (GENBANK-ID: AF121979) (E= 7.2e 6°).
The full GPCRSa amino acid sequence has 174 of 303 amino acid residues (57%) identical to, and 231 of 303 residues (76%) positive with, the 318 amino acid residue odorant receptor 546 protein from Mus musculus (ptnr:SPTREMBL-ACC: Q9WU93) (E=1.9e~92) arid 146 of 310 amino acid residues (47%) identical to, and 2I2 of 310 residues (68%) positive with, the 312 amino acid residue Olfactory Receptor HPFH10R from Homo Sapiens (ptnr:
TREMBLNEW-ACC:AAD51279) (E= 2.1e 77).
GPCRSa also has homology to other proteins as shown in BLAST alignment results in Table SC.

Table 5C. BLASTX results for GPCRSa Smallest Sum ReadingHighProb Sequences Frame ScoreP(N) N
producing High-scoring Segment Pairs:

92365G protein-coupled receptor protein +3 778 1.7e-76 1 - Hom...

01730Human G-protein receptor HPRAJ70 . +3 735 6.0e-72 1 - Homo sa..

56641G-protein coupled prostate tissue +3 735 6.0e-72 1 receptor...

27875Odorant receptor clone I14 - Rattus+3 460 8.3e-43 1 rattus...

27874Odorant receptor clone I9 - Rattus +3 438 1.8e-40 1 rattus,...

GPCRSb GPCRSa (AC020597_C) was subjected to an exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer.
In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR
amplification based on a wide range of cDNA libraries. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide GPCRSb, which is also referred to as CG53668-02.
The nucleotide sequence for GPCRSb (947 bp, SEQ ID N0:17) is presented in Table SD.
Table SD. GPCRSb Nucleotide Sequence (SEQ ID N0:17) TGAAAAATGTTTTATCACAACAAGAGCATATTTCACCCAGTCACATTTTTCCTCATTGGAATCCCAGGTCTGGAA
GACTTCCACATGTGGATCTCCGGGCCTTTCTGCTCTGTTTACCTTGTGGCTTTGCTGGGCAATGCCACCATTCTG
CTAGTCATCAAGGTAGAACAGACTCTCCGGGAGCCCATGTTCTACTTCCTGGCCATTCTTTCCACTATTGATTTG
GCCCTTTCTACAACCTCTGTGCCTCGCATGCTGGGTATCTTCTGGTTTGATGCTCACGAGATTAACTATGGAGCT
TGTGTGGCCCAGATGTTTCTGATCCATGCCTTCACTGGCATGGAGGCTGAGGTCTTACTGGCTATGGCTTTTGAC
CGTTATGTGGCCGTCTGTGCTCCACTACATTACGCAACCATCTTGACATCCCAAGTGTTGGTGGGCATTAGCATG
TGCATTGTAATCCGTCCCGTTTTACTTACACTTCCCATGGTCTATCTTATCTACCGCCTACCCTTTTGTCAGGCT
CACATAATAGCCCATTCCTACTGTGAGCACATGGGCATTGCAAAATTGTCCTGTGGAAACATTCGTATCAATGGT
ATCTATGGGCTTTTTGTAGTTTCCTTCTTTGTTCTGAACCTGGTGCTCATTGGCATCTCGTATGTTTACATTCTC
CGTGCTGTCTTCCGCCTCCCATCACATGATGCTCAGCTAAAAGCCCTAAGCACGTGTGGCGCTCATGTTGGAGTC
ATCTGTGTTTTCTATATCCCTTCAGTCTTCTCTTTCCTTACTCATCGATTTGGACACCAAATACCAGGTTACATT
CACATTCTTGTTGCCAATCTCTATTTGATTATCCCACCCTCTCTCAACCCCATCATTTATGGGGTGAGGACCAAA
CAGATTCGAGAACGAGTGCTCTATGTTTTTACTAAAAAATAAGACTA
The encoded GPCRSb protein is presented in Table SE. The disclosed protein is amino acids long and is denoted by SEQ ID N0:18. GPCRSb differs from GPCRSa by amino acid changes in positions 77 A->T, 128 I->V and 141 L->Q. Like GPCRSa, the Psort profile for GPCRSb predicts that this sequence 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 peptide is between amino acids 49 and 50, i.e., at the slash in the amino acid sequence ILL-VI based on the SignalP result.
Table 5E. Encoded GPCRSb protein sequence (SEQ ID N0:18) MFYHNKSIFHPVTFFLIGIPGLEDFHMWISGPFCSVYLVALLGNATILLVIKVEQTLREPMFYFLAILSTIDLAL
STTSVPRMLGIFWFDAHETNYGACVAQMFLIHAFTGMEAEVLLAMAFDRYVAVCAPLHYATILTSQVLVGISMCI
VIRPVLLTLPMVYLIYRLPFCQAHIIAHSYCEHMGIAKLSCGNIRINGIYGLFWSFFVLNLVLIGISYVYILRA
VFRLPSHDAQLKALSTCGAHVGVICVFYIPSVFSFLTHRFGHQIPGYIHILVANLYLIIPPSLNPIIYGVRTKQI
RERVLYVFTKK
The disclosed nucleic acid sequence for GPCRSb has 598 of 921 (64%) identical to a Mus musculus odorant receptor S46 mRNA (GENBANK-ID: AF121979) (E= 9.5e 6°).
The full GPCRS amino acid sequence has 174 of 303 amino acid residues (57%) identical to, and 233 of 303 (76%) positive with, the 318 amino acid residue odorant receptor S46 protein from Mus musculus (ptnr:SPTREMBL-ACC: Q9WU93) (E= 9.7e'92).
GPCRSb is expressed in at least the following tissues: Apical microvilli of the 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, MHC II abnd III
expressing cells, nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells of the tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST
sources, Literature sources, and/or RACE sources.
GPCRSc Another nucleotide sequence resulted when GPCRSa (AC020597_C) was subjected to an exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a wide range of cDNA
libraries. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the sequence reported below, which is designated as Accession Number AC020597B
dal, or GPCRSc.
The nucleotide sequence for GPCRSc (945 bp, SEQ ID N0:19) is presented in Table SF
Table 5F. GPCRSc Nucleotide Sequence (SEQ ID N0:19) GAAAAATGTTTTATCACAACAAGAGCATATTTCACCCAGTCACATTTTTCCTCATTGGAATCCCAGGTCT
GGAAGACTTCCACATGTGGATCTCCGGGCCTTTCTGCTCTGTTTACCTTGCGGCTTTGCTGGGCAATGCC
ACCATTCTGCTAGTCATCAAGGTAGAACAGACTCTCCGGGAGCCCATGTTCTACTTCCTGGCCATTCTTT
CCACTATTGATTTGGCCCTTTCTACAACCTCTGTGCCTCGCATGCTGGGTATCTTCTGGTTTGATGCTCA
CGAGATTAACTATGGAGCTTGTGTGGCCCAGATGTTTCTGATCCATGCCTTCACTGGCATGGAGGCTGAG
GTCTTACTGGCTATGGCTTTTGACCGTTATGTGGCCGTCTGTGCTCCACTACATTACGCAACCATCTTGA
CATCCCAAGTGTTGGTGGGCATTAGCATGTGCATTGTAATTCGTCCCGTTTTACTTACACTTCCCATGGT
CTATCTTATCTACCGCCTACCCTTTTGTCAGGCTCACATAATAGCCCATTCCTACTGTGAGCACATGGGC
ATTGCAAAATTGTCCTGTGGAAACATTCGTATCAATGGTATCTATGGGCTTTTTGTAGTTTCTTTCTTTG
TTCTGAACCTGGTGCTCATTGGCATCTCGTATGTTTACATTCTCCGTGCTGTCTTCCGCCTCCCATCACA
TGATGCTCAGCTAAAAGCCCTAAGCACGTGTGGCGCTCATGTTGGAGTCATCTGTGTTTTCTATATCCCT
TCAGTCTTCTCTTTCCTTACTCATCGATTTGGACACCAAATACCAGGTTACATTCACATTCTTGTTGCCA
ATCTCTATTTGATTATCCCACCCTCTCTCAACCCCATCATTTATGGGGTGAGGACCAAACAGATTCGAGA
ACGAGTGCTCTATGTTTTTACTAAAAAATAAGACT
The coding region of GPCRSc is from nucleotide 6 to 938, giving the encoded GPCRSc protein, as presented in Table SG. The disclosed protein is 311 amino acids long and is denoted by SEQ ID NO: 20. The Psort profile for GPCRSc predicts that this sequence has a signal peptide and is likely to be localized at the endoplasmic reticulum with a certainty of 0.6850 or plasma membrane with a certainty of 0.6400. The most likely cleavage site for a peptide is between amino acids 49 and 50, ILL-VI based on the SignalP result.
The molecular weight of GPCRSc is 35062.6 Daltons.
Table 5G. Encoded GPCRSc protein sequence (SEQ ID N0:20) MFYHNKSIFHPVTFFLIGIPGLEDFHMWISGPFCSVYLAALLGNATILLVIKVEQTLREPMFYFLAILSTI
DLALSTTSVPRMLGIFWFDAHEINYGACVAQMFLIHAFTGMEAEVLLAMAFDRYVAVCAPLHYATILTSQV
LVGISMCIVIRPVLLTLPMVYLIYRLPFCQAHIIAHSYCEHMGIAKLSCGNIRINGIYGLFWSFFVLNLV
LIGISYVYILRAVFRLPSHDAQLKALSTCGAHVGVICVFYIPSVFSFLTHRFGHQIPGYIHILVANLYLII
PPSLNPIIYGVRTKQIRERVLYVFTKK
The full GPCRSc amino acid sequence has 174 of 303 amino acid residues (57%) identical to, and 232 of 303 residues (76%) positive with, the 318 amino acid residue odorant receptor S46 protein from Mus musculus (ptnr:SPTREMBL-ACC: Q9WU93) (E= 2.2e 92) and 146 of 310 amino acid residues (47%) identical to, and 212 of 310 residues (68%) positive with, the 312 amino acid residue Olfactory Receptor HPFH10R from Homo sapiehs (ptnr:
SPTREMBL-ACC:Q9UKL2) (E= 2.1e 77).
Possible SNPs found for GPCRSc are listed in Table SH.
Table 5H: SNPs Base Base Base Position Before After 63 T C(3) 94 C T(4) 110 C T(4) 114 A G(4) 157 G T(2) 170 T C(2) 197 G C(2) 242 T C(3) 262 G A(2) 290 G A(3) 299 G C(3) 314 G T(2) 316 A C(2) 329 C T(2) 332 A C(3) 333 T A(3) 356 A C(3) 376 A G(3) 377 C T(2) 396 A G(3) 428 C T(2) 453 A ! G(2) The disclosed GPCRS protein (SEQ ID N0:19) has good identity with a number of olfactory receptor proteins. The identity information used for ClustalW
analysis is presented in Table SJ. Unless specifically addressed as GPCRSa GPCRSb, or GPCRSc, any reference to GPCRS is assumed to encompass all variants. All GPCRS proteins have significant homology to olfactory receptor (OR) proteins: The homology information from BLASTX
alignments for the proteins in the ClustalW is presented in Table SI.

Table SI. BLAST
results for GPCRS

Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) gi1119082111gbIAAG4HOR 5'Betal4 318 166/297 217/297 7e-81 1676.1 (AF137396)[Homo Sapiens] (55%) (72%) gi199354421ref~NP_0odorant receptor318 168/303 218/303 1e-80 64688.1 S46 gene [Mus (55%) (71%) musculus]

gi~6532001~gbIAAD27odorant receptor339 162/291 208/291 1e-79 596.2~AF121976_1S19 [Mus (55%) (70%) (AF121976) musculus]

gi~9938014~ref~NPodorant receptor321 158/299 218/299 1e-79 _ 518 gene [Mus (52%j (72%) 64686.11 musculus]

gi~7305349~refINPolfactory 326 164/309 222/309 5e-78 38647.1 receptor 67 (53%) (71%) [Mus musculus]

This information is presented graphically in the multiple sequence alignment given in Table SJ (with GPCRSa being shown on line 1 and GPCRSb on line 2) as a ClustalW analysis comparing GPCRS with related protein sequences.
Table SJ Information for the ClustalW proteins:
1) GPCRSa (SEQ ID N0:16) 2) GPCRSb (SEQ ID N0:18) 3) gi~11908211~gb~AAG41676.1~ (AF137396) HOR 5'Betal4 [Homo Sapiens] (SEQ ID
NO:55) 4) gi~9935442~re~NP_064688.1 ~ odorant receptor S46 gene [Mus musculus] (SEQ
ID N0:63) 5) gi~6532001~gb~AAD27596.2~AF121976_1 (AF121976) odorant receptor S19 [Mus musculus] (SEQ
ID NO:54) 6) gi~9938014~ref~NP_064686.1~ odorant receptor S18 gene [Mus musculus] (SEQ
ID N0:56) 7) gi~7305349~refJNP 038647.1 olfactory receptor 67 [Mus musculus] (SEQ ID
N0;57) ....I....I....I....1....1....1....1....1....1...~....I....I
GPCR5a --------------------------MFYHK;IFH~VTFI ~' ~G

GPCRSb --------------------------MFYH~KIFH'VT _ ;'F ~G~

gi1119082111-------------------------MSDSNLSDNHL ~DT T _ gi199354421__________________________MSPGmSWIH 'SSL ~LQ G~,' C

gi~65320011MPEKMLSKLIAYLLLIESCRQTAQLVKGRRIWVDS ~HWPNTTHYRE Q I ' V

gi I 9938014____________________~S~SMLGTF~jIIH'TV~I T, Y~T
I

gi173053491----------------------MKVASSFH~ ~ AL ~ 'I
D'NPQDVW~"V 1 S

GPCRSa GPCR5b gi~11908211~
gi199354421 gi165320011 gi I 993807.4 I
gi173053491 GPCRSa GPCRSb gi1119082111 gi199354421 ....I....I....I....I....I....1....1....1....1....1....1....1 ~i I 6532001 I '~H~T~ F VFAT A R~~TAG~GLACT~G
gi I 9938014 l PD 1 ~ v F VAFV !G~ m T .,~~SA P~',~,,~~GIKC~~~(TLAIWG(yS
iI73053491 GS a F FIFV A~ ~ Y T SS G GTAA~I S

.I....I.."~...I....I....I.. .I....I....I....I....I....I....I
Y .e GPCRSa TL ~ 1:Y' P: QA S S G~ G~ FVFFV T
GPCRSb ~l~xT.~TL Y' P Q . _~ S ~ Gu G~ FV'rFFV Ta' V
gi ~ 11908211 I DVS E'c~ ' ' P GHR;'~I~T T3 I~8 TA~LAMG nS

gi I 9935442 I F~T,V LPL' S3 GH ~ P ~ ~ V = K~ ~ ~L MIL ~
t' v v gi I 6532001 I FV F~ :I ~E' P~ GH P '~ . KP TT VTG n gi 1 9938014 l )~,~GTI I~ T,~ ' S3 RTE P T~ IW FS~P ' SVL, ~ 'A
gi I 7305349 I FTC F~Y' L(, GK~ P ~ ~ Dy, T~;, IT,~ y ~LSTG

GPCR5a GPCRSb giI11908211~
gi~99354421 giI65320011 gi199380141 giI7305349~

.I.. .I....~....I.. .I.. .I....I....I....I...
GPCRSa ~ ~' " S ~ E~YFTKK---------v' ~ '!;x .L Y
GPCRSb I=TT "S v ______________-_ giI119082111 ~" F~ ~,' " V ' L V E S K LHLGKTSI-----gi199354421 ~T X Y,. ~ (~D~T~LCNEVGQLADD--giI6532001~ T ~ ' 'I~ 1N L ~DFFVKRR--------giI99380141 i, ~ ADS TR~LSVVWKS------giI73053491 ~~'~ " DGtFFSSVSTCQHDSRC
DOMAIN results for GPCRS were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table SK with the statistics and domain S description.
Table 5K.
DOMAIN results for GPCRS
against the consnensus 7tm 1 domain (SEQ
ID
N0:75) CD-Length residues, = 254 100.0%
aligned Score = 60.5 bits (145), Expect =
1e-10 .I.. I....I....I....I..
GPCRS ~ T~ . .I....I....I....I....~....I....I
~ (SEQ ~ KVE~~---E~FY~I~T~LS~SV~~GIFWFD--AHEINYG

ID LP YYLVGGDWVFGDALC
N0:16) N V
L

Consensus ~ LL ~ ~
7tm ~LFL
1 LRT : --T~TNI~L
domain I I

I.
I
I
I....I..I..
I
I
I.
AC~Q
~~~T
GMSI
L
H~
~
P

~~
~
~
~
, ~
Consensus A
7tm PRRAKVIt domain L
~, L
V
R
R
L
T
I
KL
G

.i....I....1....i....l....~....1....1....1..

GPCR5 .I....I..
.I
P~C~AHIIA~~YC=-EH~GAKLSCG21~--YLI-------YRL-------~

Consensus -7tm LFS-----------------domain L
TVEEG---T
C~fIDFPEE~--....I.. ...I.. .I....I....I....I....I...I
GPCR5 .I....I....I....I. F____-____-________________ S INGIY~FV'SF~' ,L, G~.YS~

Consensus T1~RSARSQRS--------------------7tm -RSYVL

domain P
LvC
T

....I....~....I....I....~....I....I....I....I....I....I.

Consensus 7tm 1 domain -------------------------------LKRRSS~T~R~AKMLLVV~FV~C~!Y

S
GPCR5 ~FSF~THRFGHQ-----====-IPGYIH~~ANLYLI~PPS~
Consensus 7tm 1 domain H :VLL DSLCLLSIWR-- --VLPTA TLWLAYNSC
The nucleic acids and proteins of GPCRS are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further below. For example, a cDNA encoding the 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 of the 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 transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, potential obesity due to over-eating, potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (NIDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm;
adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, allergies, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease, multiple sclerosis, Albright hereditary ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, benign prostatic hypertrophy, psychotic and neurological disorders (including anxiety, schizophrenia, manic depression, delirium, dementia, and severe mental retardation), dentatorubro-pallidoluysian atrophy (DRPLA), hypophosphatemic rickets, autosomal dominant (2) acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. Other GPCR-related diseases and disorders are contemplated.
The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a 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. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from developmental diseases, MHCII and III
diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, potential obesity due to over-eating, potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (hTIDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm;
adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, allergies, Paxkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease, multiple sclerosis, Albright hereditary ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, benign prostatic hypertrophy, psychotic and neurological disorders (including anxiety, schizophrenia, manic depression, delirium, dementia, and severe mental retardation), dentatorubro-pallidoluysian atrophy (DRPLA), hypophosphatemic rickets, autosomal dominant (2) acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. The novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the 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 of the 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. For example the disclosed GPCRS protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCRS epitope is from about amino acids 170 to 200. In another embodiment, a GPCR5 epitope is from about amino acids 230 to 250. In additional embodiments, GPCRS
epitopes are from amino acids 270 to 310. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

GPCR6a The disclosed novel GPCR6a nucleic acid of 1012 nucleotides (also referred to as AC020597 D) is shown in Table 6A. An open reading begins with an ATG
initiation codon at nucleotides 16-18 and ends with a TGA codon at nucleotides 99I-993. A
putative untranslated region 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.

Table 6A. GPCR6a Nucleotide Sequence (SEQ ID N0:21) GCATTCACAAGCAGGATGTTCCTTCCCAATGACACCCAGTTTCACCCCTCCTCCTTCCTGTTGCTGGGGATCCCA
GGACTAGAAACACTTCACATCTGGATCGGCTTTCCCTTCTGTGCTGTGTACATGATCGCACTCATAGGGAACTTC
ACTATTCTACTTGTGATCAAGACTGACAGCAGCCTACACCAGCCCATGTTCTACTTCCTGGCCATGTTGGCCACC
ACTGATGTGGGTCTCTCAACAGCTACCATCCCTAAGATGCTTGGAATCTTCTGGATCAACCTCAGAGGGATCATC
TTTGAAGCCTGCCTCACCCAGATGTTTTTTATCCACAACTTCACACTTATGGAGTCAGCAGTCCTTGTGGCAATG
GCTTATGACAGCTATGTGGCCATCTGCAATCCACTCCAATATAGCGCCATCCTCACCAACAAGGTTGTTTCTGTG
ATTGGTCTTGGTGTGTTTGTGAGGGCTTTAATTTTCGTCATTCCCTCTATACTTCTTATATTGCGGTTGCCCTTC
TGTGGGAATCATGTAATTCCCCACACCTACTGTGAGCACATGGGTCTTGCTCATCTATCTTGTGCCAGCATCAAA
ATCAATATTATTTATGGTTTATGTGCCATTTGTAATCTGGTGTTTGACATCACAGTCATTGCCCTCTCTTATGTG
CATATTCTTTGTGCTGTTTTCCGTCTTCCTACTCATGAGCCCCGACTCAAGTCCCTCAGCACATGTGGTTCACAT
GTGTGTGTAATCCTTGCCTTCTATACACCAGCCCTCTTTTCCTTTATGACTCATTGCTTTGGCCGAAATGTGCCC
CGCTATATCCATATACTCCTAGCCAATCTCTATGTTGTGGTGCCACCAATGCTCAATCCTGTCATATATGGAGTC
AGAACCAAGCAGATCTATAAATGTGTAAAGAAAATATTATTGCAGGAACAAGGAATGGAAAAGGAAGAGTACCTA
ATACATACGAGGTTCTGAATGCAATTTTATGAAATTT
The disclosed nucleic acid sequence has 662 of 1005 bases (65%) identical to a Mus musculus odorant receptor S46 mRNA (GENBANI~-m: AF121979) (E value = 1.8e-7°).
Tha GPCR6a protein encoded by SEQ 1D N0:21 has 325 amino acid residues, and is presented using the one-letter code in Table 6B (SEQ ID N0:22). The Psort profile for GPCR6a predicts that this sequence has a signal peptide and is likely to be localized at the endoplasmic reticulum with a certainty of 0.6850 or plasma membrane with a certainty of 0.6400. The most likely cleavage site for a peptide is between amino acids 55 and 56, TDS-SL based on the SignalP result. The molecular weight of GPCR6a is 36602.5 Daltons.
Table 6B. Encoded GPCR6a protein sequence (SEQ ID N0:22).
MFLPNDTQFHPSSFLLLGIPGLETLHIWIGFPFCAVYMIALIGNFTILLVIKTDSSLHQPMFYFLAMLATTDVGL

FVRALIFVIPSILLILRLPFCGNHVIPHTYCEHMGLAHLSCASIKINTIYGLCAICNLVFDITVIALSYVHILCA
VFRLPTHEPRLKSLSTCGSHVCVILAFYTPALFSFMTHCFGRNVPRYIHILLANLYVVVPPMLNPVIYGVR
TKQIYKCVKKILLQEQGMEKEEYLIHTRF
The full amino acid sequence of GPCR6a was found to have 192 of 309 amino acid residues (62%) identical to, and 239 of 309 residues (77%) positive with, the 318 amino acid residue Odorant Reccptor S46 from Mus musculus (SPTREMBL-ACC:Q9WU93) (E value =
2.3e-lo3), and 152 of 302 amino acid residues (50%) identical to, and 211 of 302 residues (69%) positive with, the 312 amino acid residue Olfactory Receptor HPFHlOR
from Homo sapiens (TREMBLNEW-ACC:AAD51279) (E value =1.8e-8°) GPCR6a also had homology to the proteins shown in the BLAST alignments in Table 6C.

Table 6C. BLASTX results for GPCR6a Smallest Sum Reading High Prob Sequences Pairs:Frame ScoreP(N) N
producing High-scoring Segment patp:Y92365G protein-coupled protein5 - Hom..+1731 1.6e-71 receptor 1 patp:W01730Human G-protein receptor Homo sa..+1730 2.0e-71 patp:W56641G-protein coupled tissuereceptor..+1730 2.0e-71 prostate 1 patp:R27875Odorant receptor clone- 495 1.6e-46 I14 Rattus 1 rattus..+1 patp:R27868Odorant receptor cloneRattusrattus,. 494 2.1e-46 F5 - +1 1 GPCR6b In the present invention, the target sequence identified previously, Accession Number AC020597D, was subjected to the exon linking process to confirm the sequence.
PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a wide range of cDNA libraries. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the sequence reported below, which is designated Accession Number AC020597D1. The resulting nucleotide sequence differs in 5 of 1012 bases given by Accession Number AC020597D, which leads to a difference in the amino acid sequence at residues 234 and 264.
The disclosed novel GPCR6b nucleic acid of 1012 nucleotides (also referred to as AC020597 D1) is shown in Table 6D. An open reading begins with an ATG
initiation codon at nucleotides 16-18 and ends with a TGA codon at nucleotides 991-993. A
putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 6D, and the start and stop codons are in bold letters.

Table 6D. GPCR6b Nucleotide Sequence (SEQ ID N0:23) GCATTCACAAGCAGGATGTTCCTTCCCAATGACACCCAGTTTCACCCCTCCTCCTTCCTGTTGCTGGGGATCCCA
GGACTAGAAACACTTCACATCTGGATCGGCTTTCCCTTCTGTGCTGTGTACATGATCGCACTCATAGGGAACTTC
ACTATTCTACTTGTGATCAAGACTGACAGCAGCCTACACCAGCCCATGTTCTACTTCCTGGCCATGTTGGCCACC
ACTGATGTGGGTCTCTCAACAGCTACCATCCCTAAGATGCTTGGAATCTTCTGGATCAACCTCAGAGGGATCATC
TTTGAAGCCTGCCTCACCCAGATGTTTTTTATCCACAACTTCACACTTATGGAGTCAGCAGTCCTTGTGGCAATG
GCTTATGACAGCTATGTGGCCATCTGCAATCCACTCCAATATAGCGCCATCCTCACCAACAAGGTTGTTTCTGTG
ATTGGTCTTGGTGTGTTTGTGAGGGCTTTAATTTTCGTCATTCCCTCTATACTTCTTATATTGCGGTTGCCCTTC
TGTGGGAATCATGTAATTCCCCACACCTACTGTGAGCACATGGGTCTTGCTCATCTATCTTGTGCCAGCATCAAA
ATCAATATTATTTATGGTTTATGTGCCATTTGTAATCTAGTGTTTGACATCACAGTCATTGCCCTCTCTTATGTG
CATATTCTTTGTGCTGTTTTCCGTCTTCCTACTCATGAAGCCCGACTCAAGTCCCTCAGCACATGTGGTTCACAT
GTGTGTGTAATCCTTGCCTTCTATACACCAGCCCTCTTTTCCTTTATGACTCATCGCTTTGGCCGAAATGTGCCC
CGCTATATCCATATACTCCTAGCCAATCTCTATGTTGTGGTGCCACCAATGCTCAATCCTGTCATATATGGAGTC
AGAACCAAGCAGATCTATAAATGTGTGAAGAAAATATTATTGCAGGAACAAGGAATGGAAAAGGAAGAGTACCTA
ATACATACGAGGTTCTGAATGCAATTTTATGAAATTT
The GPCR6b protein encoded by SEQ ID N0:21 has 325 amino acid residues, and is presented using the one-letter code in Table 6E (SEQ TD N0:24). The Psort profile for GPCR6b predicts that this sequence has a signal peptide and is likely to be localized at the endoplasmic reticulum with a certainty of 0.6850 or plasma membrane with a certainty of 0.6400.. It seems to have an uncleavable N-terminal signal sequence. The most likely cleavage site for a peptide, if there was one, is between amino acids 55 and 56, TDS-SL based on the SignalP result. The molecular weight of GPCR6b is 36629.6 Daltons.
Table 6E. Encoded GPCR6b protein sequence (SEQ ID N0:24).
MFLPNDTQFHPSSFLLLGIPGLETLHIWIGFPFCAVYMIALIGNFTILLVIKTDSSLHQPMFYFLAMLATTDVGL
STATIPKMLGIFWINLRGIIFEACLTQMFFIHNFTLMESAVLVAMAYDSYVAICNPLQYSAILTNKWSVIGLGV
FVRALIFVIPSILLILRLPFCGNHVIPHTYCEHMGLAHLSCASIKINIIYGLCAICNLVFDITVIALSYVHILCA
VFRLPTHEARLKSLSTCGSHVCVILAFYTPALFSFMTHRFGRNVPRYIHILLANLYVWPPMLNPVIYGVRTKQI
YKCVKKILLQEQGMEKEEYLIHTRF
BLASTP results include those listed in Table 6F.
Table 6F BLASTP
results for GPCR6b Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) ($) SPTREMBL-ACC:Q9WU93ODORANT RECEPTOR318 194/309 241/309 1.6e-S46 - Mus (62g) (770) 105 musculus SPTREMBL-ACC:Q9WVD9MOR 3'BETA1 326 172/298 226/298 4.4e-- Mus .

musculus (Mouse) (57~) (75a) 94 SPTREMBL-ACC:Q9Y5P1HOR 5'BETA3 312 131/305 195/305 4.Oe--Homo Sapiens (42$) (63%) 68 (Human) The disclosed GPCR6 protein has good identity with a number of olfactory receptor proteins. The identity information used for ClustalW analysis is presented in Table 6G. The GPCR6 protein has significant identity to olfactory receptor (OR) proteins:

Table 6G. BLAST
results for Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) gi199354421ref~NP_0odorant receptor318 192/310 239/310 6e-98 64688.11 S46 gene [Mus (61%) (76%) musculus]

gi~73053491refINPO olfactory 326 70/298 224/298 1e-86 38647.1] receptor 67 (57%) (75%) [Mus musculus]

gi199380141refINP-0odorant receptor321 157/305 225/305 1e-83 64686.11 S18 gene [Mus (51%) (73%) musculus]

gi~65320011gb1AAD27odorant receptor339 158/280 209/280 4e-83 596.21AF121976 S19 [Mus (56%) (74%) (AF121976) musculus]

gi111908211~gb~AAG4HOR 5'Betal4 313 160/300 221/300 1e-82 1676.1 (AF137396)[Homo Sapiens] (53%) (73%) This information is presented graphically in the multiple sequence alignment given in Table 6H (with GPCR6a being shown on line 1 and GPCR6b being shown on line 2) as a ClustalW analysis comparing GPCR6 with related protein sequences.
Table 6H Information for the ClustalW proteins:
1) GPCR6a (SEQ ID N0:22) 2) GPCR6b (SEQ )D N0:24) 3) gi~9935442~ref~NP_064688.1 ~ odorant receptor S46 gene [Mus musculus] (SEQ
ID N0:53) 4) gi~7305349~ref~I~IP_038647.1~ olfactory receptor 67 [Mus musculus] (SEQ ID
N0:57) 5) gi~9938014~re~NP-064686.1 odorant receptor S18 gene [Mus musculus] (SEQ ID
N0:56) 6) gi~6532001~gb~AAD27596.2~AF121976_1 (AF121976) odorant receptor S19 [Mus musculus] (SEQ
ID N0:54) 7) gi~11908211~gb~AAG41676.1~ (AF137396) HOR 5'Betal4 [Homo sapiens] (SEQ ID
N0:55) ....1....1....)....)....1....1.. ...1...1....1....1...
GPCR6a --------------------------MFLP.1.SS T~ TL~IGE~
DQFH

GPCR6b --------------------------MFLPD'i#QFHSS TL~IGF
~
~

gi199354421--------------------------MSPGSWIHSS L ELQ G

gi173053491--------------------MKVASSFHD~.'NPQDVW I DL 'I
gi199380141--------------------MNSKASMLGTFIIH'TV~ ~, S '~S' QY
T

gi165320011MPEKMLSKLIAYLLLIESCRQTAQLVKGRRIWVDS DQ
'HWPNTTHYRE V
~

gi1119082111-------------------------MSDSNLSDNH
DT~F~T

GPCR6a GPCR6b gi199354421 gi173053491 gi199380141 gi165320011 gi1119082111 GPCR6a GPCR6b gi199354421 gi173053491 gi199380141 gi165320011 gi~11908211~

__..I.___I_I___.I._..I_...1....1....1....1....1....1....1 GPCR6a I 'LI<LP H KT C1,CNI~V
~~' S a . ~
S

GPCR6b ~ 'SL~L' P3H H S KI C W
'~i3..~ N ICN~'G
I ~ V
' ~
.t giI99354421F 'L 1" S Z ' ~ V K .
L L ra , i.L
S.
L

giI73053491~ ' Y' L,K~ ' I ' T~ T~LSTG n F ~ ~ D I
IC
~

giI99380141~GTHCF'IF. S>RT~t ' I D T~ FS~PASL, ~
' S

0 ~ ~IE' ~ _ ~~
~TP A
T T

11 I ~A P~'wR~7 T ~ .. T S
gi I 11908g L
. "
"G..

GPCR6a GPCR6b giI99354421 giI73053491 giI99380141 giI65320011 giI11908211~

.1....1....1....1....1....1.. ...1....1....1...
GPCR6a ~' '~~~ ~ .1. LQEQGMEKEEYLIHTRFRERVLY
n YKCKK~

GPCR6b Y'~ ' YKC~KKLQEQGMEKEEYLIHTRF------~

giI99354421;~~ ~~ 'X '~~ ~ ~DRAVT,CNEVGQLADD-------------gij73053491~3~ 'I ~ ~DL~VF~FSSVSTCQHDSRC-----------V . Y.

giI99380141~1 'T ~ iDS
TRSVVWKS-----------------giI65320011Q, I L ' '~SI,N~ L
LDFFVKRR-------------------F~

gi~11908211~ F 'I~ S
' LK~HLGKTSI----------------....I
GPCR6a VFTKK
GPCR6b -----gi~9935442~ _____ giI7305349~ -----giI99380141 _____ giI65320011 -----giI11908211~ __-__ The presence of identifiable domains in GPCR6 was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~.
DOMAIN results for GPCR6 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 6I with the statistics and domain description. The results indicate that GPCR6 has homology to the 7tm 1 (InterPro) 7 l0 transmembrane receptor (rhodopsin family) (as defined by Interpro) This indicates that the sequence of GPCR6 has properties similar to those of other proteins known to contain this/these domains) and similar to the properties of these domains.
Table 6I. DOMAIN results for GPCR6 against the consensus 7tm 1 domain (SEQ ID N0:76) CD-Length = 254 residues, 100.0% aligned Score = 77.4 bits (182), Expect = 1e-15 .J....J....J....J..
Q .J....J....J....I....J....J....J..

O .J
TT' S

Consensusdomain YYLVGG--7tm l DWF
DA
LLV~
LRTK
TNI
LN
LFLLTLP
R-Q

....J....J....J....J..
GPCR6 .
_J..
.J....J....l....J....J..
.J..
.J
FEAC(C~TQ
FNFTLMESA~
Y
S
C,1' S
L
IVS
~
G
I

Consensus7tm 1 domain~L~L~
LCKLGA~s7FTT--GYASIL~T
II~RV~RR~RBPRAK~

. J....J....J....J....J....J. ...J....J....J....J....J....J
~ ~ VIPH~ ~S
~ ~ ~
~ AH~
K
I
B

Consensus7tm 1 domainLS P TVEE EE
T----- S
V LIDFP--FSW K
-RSYVL

.. ...J....J....J....J....J....J....~....J....J
GPCR6 .J....J....J. C
_________________________,______________ CNLFDT ~
S
VH
~

Consensus7tm 1 domainL:~ R-----------------------------------------VGFLPL
LC
TR

J
J
J
J

GPCR6 ....
....
....
....J....J....J....J....~....J....J....~....

_-__________________________________________________________ Consensus7tm 1 domain-____________________________________________-______________ .. J....J....J.. ...J
GPCR6 . .J....J....J....J....J....J....J....~.
'N--AVF' LPTHEP'- ~
~ SL
~'CG--------SH
C
IAF'T
LFSF-----THCFG
BY
B
~
~
~
~

Consensus7tm 1 domain-TL F~LCL ---HIVLLDSLCLLSIW
KSARSQ
SERKAAKMLLVV
V
S
RR

....J.. ..J....J....J....J....J.
L
PP

Consensus7tm 1 domain-'---V
__ TAL~T~WL
NS
C I

The similarity information for the GPCR6 protein and nucleic acid disclosed herein suggest that GPCR6 may have important structural and/or physiological functions characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic ~.0 applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the 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 ~5 gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon. The novel nucleic acid encoding GPCR6, and the GPCR6 protein of the invention, or fragments thereof, may $uther be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
SO The nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders of the like.
The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a 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. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
The novel nucleic acid encoding OR-like protein, and the OR-like protein of the 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.
The novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the 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 of the 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. For example the disclosed GPCR6 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR6 epitope is from about amino acids 170 to 180. In another embodiment, a GPCR6 epitope is from about amino acids 230 to 240. In additional embodiments, GPCR6 epitopes are from amino acids 250 to 280 and 290-310. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

GPCR7a A novel GPCR nucleic acid was identified by TblastN using CuraGen Corporation's sequence file for GPCR probes or homologs, and run against the Genomic Daily Files made available by GenBank. The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein. The disclosed novel GPCR7 nucleic acid of 968 nucleotides (also referred to as AC025249 A) is shown in Table 7A. An open reading frame begins with an ATG initiation codon at nucleotides 6-8 and ends with a TAG codon at nucleotides 960-962. A putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 7A, and the start and stop codons are in bold letters.
Table 7A. GPCR7a Nucleotide Sequence (SEQ ID N0:25) TCTTCATGATGGTGGATCCCAATGGCAATGAATCCAGTGCTACATACTTCATCCTAATAGGCCTCCCTGGTTTAGAAG
AGGCTCAGTTCTGGTTGGCCTTCCCATTGTGCTCCCTCTACCTTATTGCTGTGCTAGGTAACTTGACAATCATCTACA
TTGTGCGGACTGAGCACAGCCTGCATGAGCCCATGTATATATTTCTTTGCATGCTTTCAGGCATTGACATCCTCATCT
CCACCTCATCCATGCCCAAAATGCTGGCCATCTTCTGGTTCAATTCCACTACCATCCAGTTTGATGCTTGTCTGCTAC
AGATGTTTGCCATCCACTCCTTATCTGGCATGGAATCCACAGTGCTGCTGGCCATGGCTTTTGACCGCTATGTGGCCA
TCTGTCACCCACTGCGCCATGCCACAGTACTTACGTTGCCTCGTGTCACCAAAATTGGTGTGGCTGCTGTGGTGCGGG
GGGCTGCACTGATGGCACCCCTTCCTGTCTTCATCAAGCAGCTGCCCTTCTGCCGCTCCAATATCCTTTCCCATTCCT
ACTGCCTACACCAAGATGTCATGAAGCTGGCCTGTGATGATATCCGGGTCAATGTCGTCTATGGCCTTATCGTCATCA
TCTCCGCCATTGGCCTGGACTCACTTCTCATCTCCTTCTCATATCTGCTTATTCTTAAGACTGTGTTGGGCTTGACAC
GTGAAGCCCAGGCCAAGGCATTTGGCACTTGCGTCTCTCATGTGTGTGCTGTGTTCATATTCTATGTACCTTTCATTG
GATTGTCCATGGTGCATCGCTTTAGCAAGCGGCGTGACTCTCCGCTGCCCGTCATCTTGGCCAATATCTATCTGCTGG
TTCCTCCTGTGCTCAACCCAATTGTCTATGGAGTGAAGACAAAGGAGATTCGACAGCGCATCCTTCGACTTTTCCATG
TGGCCACACACGCTTCAGAGCCCTAGGTGTCA
GPCR7a has high homology to several other nucleic acids including those in the BLASTN alignments described in Table 7B.

Table 7B. BLASTN
results for GPCR7a Gene Index/ Protein/ OrganismLengthIdentityExpect Identifier (bp) (%) gb:GENBANK- Rattus norvegicus2910 596/879 2.4e-68 ID:AF079864~acc:AFOputative G- (67%) 79864 protein coupled receptor RAlc mRNA, complete cds gb:GENBANK- Sequence 1 from1474 594/874 7.0e-69 ID:AR009514~acc:AROpatent US 5756309 (67%) patn:X40518 Human secreted 381 242/242 3.8e-49 protein 5' EST (1000 patn:X40528 Human secreted 399 280/286 4.1e-57 protein 5' EST (97s) gb:GENBANK- PT2.1_13_Hll.r 1000 165/173 5.5e-28 ID:AI557139~acc:AI5tumor2 Homo (95~) 57139 sapiens cDNA
3', mRNA sequence -Homo sapiens The GPCR7a protein encoded by SEQ m N0:25 has 318 amino acid residues, and is presented using the one-letter code in Table 7B (SEQ m N0:26). The SignalP, Psort and/or Hydropathy profile for GPCR7a predict that GPCR7a has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. The SignalP shows a signal sequence is coded for in the first 44 amino acids, i.e., with a cleavage site at VRT-EH, between amino acids 54 and 55. This is typical of this type of membrane protein. The molecular weight of GPCR7b is 35401.9 Daltons.
Table 7C. Encoded GPCR7 protein sequence (SEQ ID N0:26).
MMVDPNGNESSATYFILIGLPGLEEAQFWLAFPLCSLYLIAVLGNLTIIYIVRTEHSLHEPMYIFLCMLSGIDIL
ISTSSMPKMLAIFWFNSTTIQFDACLLQMFAIHSLSGMESTVLLAMAFDRYVAICHPLRHATVLTLPRVTKIGVA
AVVRGAALMAPLPVFTKQLPFCRSNILSHSYCLHQDVMKLACDDIRVNWYGLIVIISAIGLDSLLISFSYLLIL
KTVLGLTREAQAKAFGTCVSHVCAVFIFYVPFIGLSMVHRFSKRRDSPLPVILANIYLLVPPVLNPIVYGVKTKE
IRQRILRLFHVATHASEP
The full amino acid sequence of the protein of the invention was found to have high homology to a number of polypeptides including the ones in the BLASTX
alignments in Table 7D and 7E.

Table 7D. BLAST
results for GPCR7a Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) ptnr:SPTREMBL- PUTATIVE G- 320 183/305 235/305 1.5e-ACC:088628 PROTEIN COUPLED (60%) (77%) 98 -Rattus norvegicus (Rat) patp:W01730 Human G-protein320 181/305 235/305 1.8e-receptor HPRAJ70 (59%) (77%) 97 - Homo sapiens patp:Y11796 Human 5' EST 21 20/20 20/20 1.3e-secreted protein (100%) (100%) 05 gi~6532001~gb~AAD27Odorant receptor339 158/280 209/280 4e-83 596.2~AF121976_1S19 [Mus (56%) (74%) (AF121976) musculus]

Table 7E. BLASTX results for GPCR7a Smallest Sum Reading High Prob Sequencesoducing High-scoring Pairs: Frame ScoreP(N) N
pr Segment Patp:Y92365G protein-coupled protein 5 - Hom 1623 4.8e-166 receptor +3 1 Patp:W01730Human G-protein receptor 971 5.9e-97 HPRAJ70 - Homo sa.+3 1 Patp:W56641G-protein coupled tissue receptor.+3971 5.9e-97 prostate 1 patp:R27875Odorant receptor clone- Rattus rattus.+3425 4.2e-39 GPCR7b and c In the present invention, the target sequence identified previously, Accession Number AC025249 A, was subjected to the exon linking process to confirm the sequence.
PCR
primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer.
In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in PCR
amplifications based on a library containing a wide range of cDNA species. The resulting two amplicons were gel purified, cloned and sequenced to high redundancy to provide the sequences reported below, which are designated GPCR7b (Accession Numbers AC025249 Al) and GPCR7c (AC025249 A da3). The sequence of GPCR7b contains one amino acid difference from that of GPCR7a at position 247, wherein alanine is replaced by valine. The sequence of GPCR7c has one amino acid different at position 183, wherein leucine is replaced by proline.

GPCR7b A novel GPCR nucleic acid was identified by TblastN using CuraGen Corporation's sequence file for GPCR probes or homologs, and run against the Genomic Daily Files made available by GenBank. The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein. The disclosed novel GPCR7 nucleic acid of 969 nucleotides (also referred to as AC025249 Al) is shown in Table 7F. An open reading frame begins with an ATG initiation codon at nucleotides 7-9 and ends with a TAG codon at nucleotides 961-963. A putative untranslated region upstream from the.
initiation codon and downstream from the termination codon are underlined in Table 7F, and the start and stop codons are in bold letters.
Table 7F. GPCR7 Nucleotide Sequence (SEQ ID N0:27) TTCTTCATGATGGTGGATCCCAATGGCAATGAATCCAGTGCTACATACTTCATCCTAATAGGCCTCCCTGGTTTAGAA
GAGGCTCAGTTCTGGTTGGCCTTCCCATTGTGCTCCCTCTACCTTATTGCTGTGCTAGGTAACTTGACAATCATCTAC
ATTGTGCGGACTGAGCACAGCCTGCATGAGCCCATGTATATATTTCTTTGCATGCTTTCAGGCATTGACATCCTCATC
TCCACCTCATCCATGCCCAAAATGCTGGCCATCTTCTGGTTCAATTCCACTACCATCCAGTTTGATGCTTGTCTGCTA
CAGATGTTTGCCATCCACTCCTTATCTGGCATGGAATCCACAGTGCTGCTGGCCATGGCTTTTGACCGCTATGTGGCC
ATCTGTCACCCACTGCGCCATGCCACAGTACTTACGTTGCCTCGTGTCACCAAAATTGGTGTGGCTGCTGTGGTGCGG
GGGGCTGCACTGATGGCACCCCTTCCTGTCTTCATCAAGCAGCTGCCCTTCTGCCGCTCCAATATCCTTTCCCATTCC
TACTGCCCACACCAAGATGTCATGAAGCTGGCCTGTGATGATATCCGGGTCAATGTCGTCTATGGCCTTATCGTCATC
ATCTCCGCCATTGGCCTGGACTCACTTCTCATCTCCTTCTCATATCTGCTTATTCTTAAGACTGTGTTGGGCTTGACA
CGTGAAGCCCAGGCCAAGGCATTTGGCACTTGCGTCTCTCATGTGTGTGCTGTGTTCATATTCTATGTACCTTTCATT
GGATTGTCCATGGTGCATCGCTTTAGCAAGCGGCGTGACTCTCCACTGCCCGTCATCTTGGCCAATATCTATCTGCTG
GTTCCTCCTGTGCTCAACCCAATTGTCTATGGAGTGAAGACAAAGGAGATTCGACAGCGCATCCTTCGACTTTTCCAT
GTGGCCACACACGCTTCAGAGCCCTAGGTGTCA
The GPCR7b protein encoded by SEQ ID N0:27 has 318 amino acid residues, and is presented using the one-letter code in Table 7G (SEQ ID N0:28). The SignalP, Psort and/or Hydropathy profile for GPCR7a predict that GPCR7a has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. The SignalP shows a signal sequence is coded for in the first 44 amino acids, i.e., with a cleavage site at VRT-EH, between amino acids 54 and 55. This is typical of this type of membrane protein. The molecular weight og GPCR7b is 35385.8 Daltons.
Table 7G. Encoded GPCR7b protein sequence (SEQ ID N0:28).
MMVDPNGNESSATYFILTGLPGLEEAQFWLAFPLCSLYLIAVLGNLTIIYIVRTEHSLHEPMYIFLCMLSGIDIL
ISTSSMPKMLAIFWFNSTTIQFDACLLQMFAIHSLSGMESTVLLAMAFDRYVAICHPLRHATVLTLPRVTKIGVA
AWRGAALMAPLPVFIKQLPFCRSNILSHSYCPHQDVMKLACDDIRVNWYGLIVIISAIGLDSLLISFSYLLIL
KTVLGLTREAQAKAFGTCVSHVCAVFIFYVPFIGLSMVHRFSKRRDSPLPVILANIYLLVPPVLNPIVYGVKTKE

The full amino acid sequence of GPCR7b was found to have 152 of 301 amino acid residues (50%) identical to, and 213 of 301 residues (70%) positive with, the 319 amino acid residue Putative G-Protein Coupled Receptor RAlC from Rattus no~vegicus (SPTREMBL-ACC:088628) (E value = 3.3e-81), and 142 of 295 amino acid residues (48%) identical to, and 199 of 295 residues (67%) positive with, the 312 amino acid residue Olfactory Receptor HPFH1 OR from Homo sapiehs (sptrEMBL-Q9UKL2) (E value = 2.9e-7s) GPCR7b also has high homology to the following proteins found through BLASTX
alignments, shown in Table 7H.
Table 7H. BLASTX results for GPCR7b Smallest Sum ReadingHigh Prob Sequences producingcoring Segment Pairs:Frame ScoreP(N) N
High-s Ptnr:SPTREMBL-ACC:088628PUTATIVE G-PROTEIN RE.+1 981 6.6e-98 ptnr:SPTREMBL-ACC:Q9YH55OLFACTORY RECEPTOR-LIKE 826 2.0e-80 PROTE.+1 1 ptnr:SPTREMBL-ACC:Q9WVD9MOR 3'BETA1 - Mus (M.+1 812 5.3e-80 musculus 1 ptnr:SPTREMBL-ACC:Q9WU90ODORANT RECEPTOR mu.+1 799 1.3e-78 S19 - Mus 1 ptnr:SPTREMBL-ACC:Q9WVN5MOR 5'BETA2 - Mus (M.+1 777 2.7e-76 musculus 1 ptnr:SPTREMBL-ACC:Q9WVD8MOR 3'BETA2 - Mus (M +1 769 1.9e-75 musculus 1 ptnr:SPTREMBL-ACC:Q9WU89ODORANT RECEPTOR mu.+1 769 1.9e-75 S18 - Mus 1 ptnr:SPTREMBL-ACC:Q9WVD7MOR 3'BETA3 - Mus (M.+1 764 6.5e-75 musculus 1 ptnr:SPTREMBL-ACC:Q9UKL2OLFACTORY RECEPTOR - .+1 760 1.7e-74 ptnr:SPTREMBL-ACC:Q9WVN6MOR 5'BETA3 - Mus (M.+1 756 4.6e-74 musculus 1 ptnr:SPTREMBL-ACC:Q9Y5P1HOR 5'BETA3 - Homo (H.+1 723 1.4e-70 sapiens 1 ptnr:SPTREMBL-ACC:Q9WVN4MOR 5'BETA1 - Mus (M.+1 707 7.1e-69 musculus 1 ptnr:SPTREMBL-ACC:Q9WU93ODORANT RECEPTOR mu.+1 701 3.1e-68 S46 - Mus 1 ptnr:SPTREMBL-ACC:Q9Y5P0HOR 5'BETA1 - Homo (H.+1 630 1.0e-60 sapiens 1 ptnr:SPTREMBL-ACC:Q9WU94ODORANT RECEPTOR mu.+1 578 3.3e-55 S50 - Mus 1 GPCR7c The disclosed novel GPCR7c nucleic acid of 968 nucleotides (also referred to as AC025249_Ada3) is shown in Table 7I. An open reading frame begins with an ATG
initiation codon at nucleotides 7-9 and ends with a TAG codon at nucleotides 961-963. A
putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 7I, and the staxt and stop codons are in bold letters.
Table 7I. GPCR7 Nucleotide Sequence (SEQ ID N0:29) TTCTTCATGATGGTGGATCCCAATGGCAATGAATCCAGTGCTACATACTTCATCCTAATAGGCCTCCCTGGTTTAGAA
GAGGCTCAGTTCTGGTTGGCCTTCCCATTGTGCTCCCTCTACCTTATTGC'~GTGCTAGGTAACTTGACAATCATCTAC

ATTGTGCGGACTGAGCACAGCCTGCATGAGCCCATGTATATATTTCTTTGCATGCTTTCAGGCATTGACATCCTCATC
TCCACCTCATCCATGCCCAAAATGCTGGCCATCTTCTGGTTCAATTCCACTACCATCCAGTTTGATGCTTGTCTGCTA
CAGATGTTTGCCATCCACTCCTTATCTGGCATGGAATCCACAGTGCTGCTGGCCATGGCTTTTGACCGCTATGTGGCC
ATCTGTCACCCACTGCGCCATGCCACAGTACTTACGTTGCCTCGTGTCACCAAAATTGGTGTGGCTGCTGTGGTGCGG
GGGGCTGCACTGATGGCACCCCTTCCTGTCTTCATCAAGCAGCTGCCCTTCTGCCGCTCCAATATCCTTTCCCATTCC
TACTGCCCACACCAAGATGTCATGAAGCTGGCCTGTGATGATATCCGGGTCAATGTCGTCTATGGCCTTATCGTCATC
ATCTCCGCCATTGGCCTGGACTCACTTCTCATCTCCTTCTCATATCTGCTTATTCTTAAGACTGTGTTGGGCTTGACA
CGTGAAGCCCAGGCCAAGGCATTTGGCACTTGCGTCTCTCATGTGTGTGCTGTGTTCATATTCTATGTACCTTTCATT
GGATTGTCCATGGTGCATCGCTTTAGCAAGCGGCGTGACTCTCCACTGCCCGTCATCTTGGCCAATATCTATCTGCTG
GTTCCTCCTGTGCTCAACCCAATTGTCTATGGAGTGAAGACAAAGGAGATTCGACAGCGCATCCTTCGACTTTTCCAT
GTGGCCACACACGCTTCAGAGCCCTAGGTGTA

The GPCR7c protein encoded by SEQ m N0:29 has 318 amino acid residues, and is presented using the one-letter code in Table 7J (SEQ m N0:30). The SignalP, Psort and/or Hydropathy profile for GPCR7a predict that GPCR7a has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. The SignalP shows a signal sequence is coded for in the first 44 amino acids, i.e., with a cleavage site at VRT-EH, between amino acids 54 and 55. This is typical of this type of membrane protein. The molecular weight of GPCR7c is 35385.8 Daltons.
Table 7J. Encoded GPCR7c protein sequence (SEQ ID N0:30).
MMVDPNGNESSATYFILIGLPGLEEAQFWLAFPLCSLYLIAVLGNLTIIYTVRTEHSLHEPMYIFLCMLSGIDIL
ISTSSMPKMLAIFWFNSTTIQFDACLLQMFAIHSLSGMESTVLLAMAFDRYVAICHPLRHATVLTLPRVTKIGVA
AWRGAALMAPLPVFIKQLPFCRSNILSHSYCPHQDVMKLACDDIRVNWYGLIVIISAIGLDSLLISFSYLLIL
KTVLGLTREAQAKAFGTCVSHVCAVFIFYVPFTGLSMVHRFSKRRDSPLPVILANIYLLVPPVLNPIVYGVKTKE
IRQRILRLFHVATHASEP
Homologous proteins to GPCR7c were searched for using BLASTX. Some of the alignments are included in Table 7I~.
Table 7K. BLAST
results for GPCR7c Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (o) (%) ptnr:SPTREMBL- PUTATIVE G- 320 183/305 235/305 1.5e-ACC:088628 PROTEIN COUPLED (60%) (770) 98 -Rattus norvegicus (Rat) ptnr:SPTREMBL- OLFACTORY 319 152/301 213/301 3.3e-ACC:Q9YH55 RECEPTOR-LIKE (50s) (70~) 81 PROTEIN COR3'BETA

- Gallus gallus s ptnr:SPTREMBL- OLFACTORY 312 142/295 199/295 2.9e-ACC:Q9UKL2 RECEPTOR HPFH10R (48~) (67a) 75 - Homo sapiens GPCR7c also has high homology to the following proteins found through BLASTX
alignments, shown in Table 7L.
Table 7L. BLASTX results for GPCR7b Smallest Sum ReadingHigh Prob Sequences producingcoring Segment Pairs:Frame ScoreP(N) N
High-s ptnr:SPTREMBL-ACC:088628PUTATIVE G-PROTETN RE.+1 981 6.8e-98 ptnr:SPTREMBL-ACC:Q9YH55OLFACTORY RECEPTOR-LIKE 816 2.1e-80 PROTE.+1 1 ptnr:SPTREMBL-ACC:Q9WVD9MOR 3'BETA1 - Mus (M.+1 812 5.5e-80 musculus 1 ptnr:SPTREMBL-ACC:Q9WU90ODORANT RECEPTOR S19 mu.+1 799 1.3e-78 - Mus 1 Possible SNPs found for GPCR7c are listed in Table 7M.

Table 7M: SNPs ConsensusDepth Base PAF
Position Change 446 18 A>- 0.111 513 17 T>C 0.118 555 16 T>C 0.125 827 9 A>G 0.333 The disclosed GPCR7 protein has good identity with a number of olfactory receptor proteins. The identity information used for ClustalW analysis is presented in Table 70. The GPCR7 protein has significant identity to olfactory receptor (OR) proteins (Table 71~:
Table 7N. BLAST
results for Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) gi[3420759[gb~AADl2putative G- 320 173/299 221/299 3e-88 761.1[ (AF079864)protein coupled (57%) (73%) receptor RAlc [Rattus norvegicus]

gi[11875778[gb[AAG4prostate specific320 173/299 223/299 5e-88 0776.1[AF311306'1G-protein coupled (57%) (73%) (AF312306) receptor; PSGR

[Homo sapiens]

gi[11908223[gb[AAG4HOR5'Betal2 312 257/304 205/304 4e-76 [Homo 1678.1[ (AF137396)Sapiens] (51%) (66%) gi[11908220[gb[AAG4MOR 3'Beta4 319 156/308 207/308 6e-73 [Mus 1684.1[ (AF133300)musculus] (50%) (66%) gi[11908214[gb[AAG4HOR5'Betall 314 143/310 207/310 2e-69 [Homo 1679.1[ (AF137396)Sapiens] (46%) (66%) This information is presented graphically in the multiple sequence alignment given in Table 70 (with GPCR7 being shown on line 1) as a ClustalW analysis comparing with related protein sequences.
Table 70. Information for the ClustalW proteins:
1) GPCR7a (SEQ ID N0:26) 2) GPCR7b (SEQ )D N0:28) 2) gi~3420759~gb~AAD12761.1~ (AF079864) putative G-protein coupled receptor RAlc [Rattus norvegicus] (SEQ ID N0:64) 3) gi~11875778~gb~AAG40776.1~AF311306_1 (AF311306) prostate specific G-protein coupled receptor;
PSGR [Homo Sapiens] (SEQ ID N0:65) 4) gi~11908213~gb~AAG41678.1~ (AF137396) HORS'Betal2 [Homo Sapiens] (SEQ ID
N0:61) 5) gi~11908220~gb~AAG41684.1~ (AF133300) MOR 3'Beta4 [Mus musculus] (SEQ ID
N0:58) 6) gi~11908214~gb~AAG41679.1~ (AF137396) HORS'Betal l [Homo Sapiens] (SEQ ID
N0:60) ....I....I....I....~....I....I....I....I....~....I.. .~....I
GPCR7a MMVDP GNESSATY x I ~Q F' S 1 ~L LT Y H
GPCR7b MMVDP~GNESSATY I I AQ _~F' S T :L LT~Y H
gi134207591 -- MxSCNFTH T I H nFG ' LS3, ~ ~ ,CIF
gi1118757781 -- -MSCNFTH~T I H G ' LS ~ CI F
L.. r F 1*
gi1119082131 ---MGLFNVTHPT S3HS G' r TV "Q P
gi I 119082141 -MLGLGTPFQP~TLQ~T ' ~,TGLT ELI I3,. ~STV LS T FW~P;

GPCR7a GPCR7b gi~3420759~
giI118757781 gi1119082131 giI119082201 giI119082141 GPCR7a GPCR7b giI34207591 gi1118757781 gi~119082131 gi1119082201 gi111908214~
GPCR7a GPCR7b giI34207591 gi1118757781 gi1119082131 gi1119082201 gi1119082141 GPCR7a GPCR7b gi134207591 gi111875778~
gi~119082131 gi1119082201 giI119082141 .I.. .I.. .~....~....
GPCR7a ~'Q HVTHASEP-----GPCR7b 'Q HV~THASEP-----giI34207591 'TA KI~CDKDIEAGGNT
giI118757781 'T ~# KI CDKDLQAVGGK
giI119082131 ~ F HHIKI---------gi 1119082201 .EG~H' ~LCSPKISSITM----gi1119082141 KG KE'pHK~QA---------The presence of identifiable domains in GPCR7 was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website 5 (http:www.ebi.ac.uk/interpro~.
DOMAIN results for GPCR7 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 7P with the statistics and domain description. The results indicate that this protein contains homology to the following protein domains (as defined by Interpro) at the indicated positions: domain name 7tm I
(InterPro) 7 transmembrane receptor (rhodopsin family). This indicates that the sequence of GPCR7 has properties similar to those of other proteins known to contain this/these domains) and similar to the properties of these domains.
Table 7P. DOMAIN results for GPCR7 against the consensus 7tm 1 domain (SEQ ID NO:Tn CD-Length = 254 residues, 100.0% aligned Score = 77.4 bits (189), Expect = 1e-15 GPCR7 (SEQ ID N0:26) I0 Consensus 7tm 1 domain I5 i ~L
VVL'L
~~
~
R~
~L'~'~~~
~Y
~

Consensus7tm 1 L
doma K
n ITIV
R
R
L
FV~1~7 A

PFCRSNI----LSH B
Y
HQDV
LACDDI' N
YGL
IISAIG
~
~
~
~
g ~
E~
~S

Consensus7tm 1 LPLST VI
domain GFLLPL
RTVEEGNT------V
IDFP
SVK---Y
LFSW

GPCR7 ISFSY LI____________________________-___________________ Consensus7tm 1 CYTRI BRTKSAR----------------------------------------------domain GPCR7 _-_____________________-______________-_____________________ 3 consensus7tm 1 -___________________________________________________________ 0 domain ~
~
L~
B
~
~
~
' ~
~~

Consensus7tm 1 DS
domain AKML
VV
VVFV
Ca~, ,kHIV
QRS
K
------------------ER
R

..

KR--------RDS PPV
~ ~
~
~

40 Consensus7tm 1 CL ~~TLWLAY
domain IWR--------VL TA
I~ISC

The similarity information for the GPCR7 protein and nucleic acid disclosed herein suggest that GPCR7 may have important structural andlor physiological functions characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic 45 acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the 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 50 target (therapeutic, diagnostic, drug taxgeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon. The novel nucleic acid encoding GPCR7, and the GPCR7 protein of the 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.
The nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fimgal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome syndrome and/or other pathologies and disorders.
The disclosed GPCR7 polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a 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. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. The novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, rnay 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 of the invention for use in therapeutic or diagnostic methods.
The novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the 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 of the 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. For example the disclosed GPCR7 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR7 epitope is from about amino acids 50 to 60. In another embodiment, a GPCR7 epitope is from about amino acids 170 to 200. In additional embodiments, GPCR7 epitopes are from amino acids 260 to 280 and 290-310. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

GPCRBa A novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Corporation's sequence file for GPCR probes or homologs and run against the Genomic Daily Files made available by GenBank. The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein. The disclosed novel GPCRBa nucleic acid of 980 nucleotides (also referred to as AC025249 B) is shown in Table 8C. An open reading frame begins with an ATG initiation codon at nucleotides 3-5 and ends with a TGA codon at nucleotides 975-977. A putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8A, and the start and stop codons are in bold letters.

Table 8A. GPCRBa Nucleotide Sequence (SEQ ID N0:31) _TGATGCTGGGTCCAGCTTACAACCACACAATGGAAACCCCTGCCTCCTTCCTCCTTGTGGGTATCCCAGGACTGCA
ATCTTCACATCTTTGGCTGGCTATCTCACTGAGTGCCATGTACATCACAGCCCTGTTAGGAAACACCCTCATCGTG
ACTGCAATCTGGATGGATTCCACTCGGCATGAGCCCATGTATTGCTTTCTGTGTGTTCTGGCTGCTGTGGACATTG
TTATGGCCTCCTCCGTGGTACCCAAGATGGTGAGCATCTTCTGCTCGGGAGACAGCTCCATCAGCTTTAGTGCTTG
TTTCACTCAGATGTTTTTTGTCCACTTAGCCACAGCTGTGGAGACGGGGCTGCTGCTGACCATGGCTTTTGACCGC
TATGTAGCCATCTGCAAGCCTCTACACTACAAGAGAATTCTCACGCCTCAAGTGATGCTGGGAATGAGTATGGCCG
TCACCATCAGAGCTGTCACATTCATGACTCCACTGAGTTGGATGATGAATCATCTACCTTTCTGTGGCTCCAATGT
GGTTGTCCACTCCTACTGTAAGCACATAGCTTTGGCCAGGTTAGCATGTGCTGACCCCGTGCCCAGCAGTCTCTAC
AGTCTGATTGGTTCCTCTCTTATGGTGGGCTCTGATGTGGCCTTCATTGCTGCCTCCTATATCTTAATTCTCAGGG
CAGTATTTGATCTCTCCTCAAAGACTGCTCAGTTGAAAGCATTAAGCACATGTGGCTCCCATGTGGGGGTTATGGC
TTTGTACTATCTACCTGGGATGGCATCCATCTATGCGGCCTGGTTGGGGCAGGATATAGTGCCCTTGCACACCCAA
GTGCTGCTAGCTGACCTGTACGTGATCATCCCAGCCACTTTAAATCCCATCATCTATGGCATGAGGACCAAACA
ATTGCTGGAGGGAATATGGAGTTATCTGATGCACTTCCTCTTTGACCACTCCAACCTGGGTTCATGAACA
The disclosed nucleic acid sequence has 537 of 858 bases (62%) identical to a Mus musculus odorant receptor S 19 gene, complete cds:(GENBANK-ID:AF121976~acc:AF121976) (E value = 3.5e-41).
The GPCRBa protein encoded by SEQ ID N0:31 has 324 amino acid residues, and is presented using the one-letter code in Table 8B (SEQ ID N0:32). The Signall', Psort andlor Hydropathy profile for GPCRBa predict that GPCRBa has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. The SignalP shows a signal sequence with a cleavage site at the slash in the sequence VTA-IW, between amino acids 52 and 53. This is typical of this type of membrane protein. The molecular weight of GPCR8a is 35385.6 Daltons.
Table 8B. Encoded GPCRBa protein sequence (SEQ ID N0:32).
MLGPAYNHTMETPASFLLVGIPGLQSSHLWLAISLSAMYITALLGNTLIVTAIWMDSTRHEPMYCFLCVLAAVDIV
MASSWPKMVSIFCSGDSSISFSACFTQMFFVHLATAVETGLLLTMAFDRYVAICKPLHYKRILTPQVMLGMSMAV
TIRAVTFMTPLSWMMNHLPFCGSNVVVHSYCKHIALARLACADPVPSSLYSLTGSSLMVGSDVAFIAASYTLILRA
VFDLSSKTAQLKALSTCGSHVGVMALYYLPGMASIYAAWLGQDIVPLHTQVLLADLYVIIPATLNPIIYGMRTKQL
LEGIWSYLMHFLFDHSNLGS
The full amino acid sequence of the protein of the invention was found to have 142 of 308 amino acid residues (46%) identical to, and 202 of 306 residues (65%) positive with, the 321 amino acid residue Odorant Receptor S 18 from Mus musculus (ptnr:SPTREMBL-ACC:Q9WU89) (E value =1.5e-71), and 123 of 293 amino acid residues (41%) identical to, and 192 of 293 residues (65%) positive with, the 321 amino acid residue G-protein coupled prostate tissue receptor designated HPRAJ70 from Homo sapiehs (patp:W56641) (E
value =
1.9e-6s).
GPCR8a also has homology to the following proteins shown in the BLAST
alignments in Table 8C.

Table $C. BLASTX results for GPCRBa Smallest Sum Reading High Prob Sequences Frame ScoreP(N) N
producing High-scoring Segment Pairs:

patp:W01730Human G-protein receptor Homo sa 676 1.1e-65 HPRAJ70 - +2 1 patp:W56641G-protein coupled prostate receptor.+2676 1.1e-65 tissue 1 patp:Y92365G protein-coupled receptor 5 - Hom.+2648 9.9e-63 protein 1 patp:Y90873Human G protein-coupled TAR14-3 425 4.2e-39 receptor G .+2 l GPCRBb A novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Corporation's sequence file for GPCR probes or homologs and run against the Genomic Daily Files made available by GenBank. The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein. The disclosed novel GPCRBb nucleic acid of 985 nucleotides (also referred to as AC025249 C) is shown in Table 8C. An open reading frame begins with an ATG initiation codon at nucleotides 5-7 and ends with a TGA codon at nucleotides 977-979. A putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8D, and the start and stop codons are in bold letters.
Table 8D. GPCRBb Nucleotide Sequence (SEQ ID N0:33) TGTGATGCTGGGTCCAGCTTATAACCACACAATGGAAACCCCTGCCTCCTTCCTCCTTGTGGGTATCCCAGGACTG
CAATCTTCACATCTTTGGCTGGCTATCTCACTGAGTGCCATGTACATCATAGCCCTGTTAGGAAACACCATCATCG
TGACTGCAATCTGGATGGATTCCACTCGGCATGAGCCCATGTATTGCTTTCTGTGTGTTCTGGCTGCTGTGGACAT
TGTTATGGCCTCCTCGGTGGTACCCAAGATGGTGAGCATCTTCTGCTCAGGAGACAGCTCAATCAGCTTTAGTGCT
TGTTTCACTCAGATGTTTTTTGTCCACTTAGCCACAGCTGTGGAGACGGGGCTGCTGCTGACCATGGCTTTTGACC
GCTATGTAGCCATCTGCAAGCCTCTACACTACAAGAGAATTCTCACGCCTCAAGTGATGCTGGGAATGAGTATGGC
CATCACCATCAGAGCTATCATAGCCATAACTCCACTGAGTTGGATGGTGAGTCATCTACCTTTCTGTGGCTCCAAT
GTGGTTGTCCACTCCTACTGTGAGCACATAGCTTTGGCCAGGTTAGCATGTGCTGACCCCGTGCCCAGCAGTCTCT
ACAGTCTGATTGGTTCCTCTCTTATGGTGGGCTCTGATGTGGCCTTCATTGCTGCCTCCTATATCTTAATTCTCAA
GGCAGTATTTGGTCTCTCCTCAAAGACTGCTCAGTTGAAAGCATTAAGCACATGTGGCTCCCATGTGGGGGTTATG
GCTTTGTACTATCTACCTGGGATGGCATCCATCTATGCGGCCTGGTTGGGGCAGGATGTAGTGCCCTTGCACACCC
AAGTCCTGCTAGCTGACCTGTACGTGATCATCCCAGCCACCTTAAATCCCATCATCTATGGCATGAGGACCAAACA
ACTGCGGGAGAGAATATGGAGTTATCTGATGCATGTCCTCTTTGACCATTCCAACCTGGGTTCATGAACACAA
The disclosed nucleic acid sequence has 533 of 858 bases (62%) identical to a Mus musculus odorant receptor S19 gene, complete cds:(GENBANK-m:AF121976~acc:AF121976) (E value = 3.5e-41).
The GPCR8b protein encoded by SEQ ID N0:33 has 324 amino acid residues, and is presented using the one-letter code in Table 8E (SEQ m N0:34). The SignaIP, Psort and/or Hydropathy profile for GPCRBb predict that GPCRBb has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. The SignalP shows a signal sequence with a cleavage site at the slash in the sequence VTA-IW, between amino acids 52 and 53. This is typical of this type of membrane protein. The molecular weight of GPCRBb is 35279.5 Daltons.
Table 8E. Encoded GPCRBb protein sequence (SEQ ID N0:34).
MLGPAYNHTMETPASFLLVGIPGLQSSHLWLAISLSAMYIIALLGNTIIVTAIWMDSTRHEPMYCFLCVLAAVDIV
MASSWPKMVSIFCSGDSSISFSACFTQMFFVHLATAVETGLLLTMAFDRYVAICKPLHYKRILTPQVMLGMSMAI
TIRAIIAITPLSWMVSHLPFCGSNWVHSYCEHIALARLACADPVPSSLYSLIGSSLMVGSDVAFIAASYILILKA
VFGLSSKTAQLKALSTCGSHVGVMALYYLPGMASIYAAWLGQDWPLHTQVLLADLYVIIPATLNPIIYGMRTKQL
RERIWSYLMHVLFDHSNLGS
The full amino acid sequence of the protein of the invention was found to have 146 of 306 amino acid residues (47%) identical to, and 202 of 306 residues (66%) positive with, the 321 amino acid residue Odorant Receptor S18 from Mus musculus (ptnr:SPTREMBL-ACC:Q9WU89) (E value = 8.2e-73), and 124 of 297 amino acid residues (41%) identical to, and 196 of 297 residues (65%) positive with, the 320 amino acid residue G-protein coupled prostate tissue receptor designated HPRAJ70 from Homo Sapiens (patp:W56641) (E
value =
3.4e-66).
GPCRBc In the present invention, the target sequence identified previously, Accession Number AC025249'C, was subj ected to the exon linking process to confirm the sequence. PCR
primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer.
In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR
amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the sequence reported below, which is designated Accession Number AC025249_C1. There are 13 bases that are different between the two sequences.
The disclosed novel GPCRBc nucleic acid of 985 nucleotides (also referred to as AC025249 C1) is shown in Table 8F. An open reading frame begins with an ATG
initiation codon at nucleotides 5-7 and ends with a TGA codon at nucleotides 977-979.
A.putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8F, and the start and stop codons are in bold letters.

Table 8F. GPCRBc Nucleotide Sequence (SEQ ID N0:35) TGTGATGCTGGGTCCAGCTTATAACCACACAATGGAAACCCCTGCCTCCTTCCTCCTTGTGGGTATCCCAGGACTG
CAATCTTCACATCTTTGGCTGGCTATCTCACTGAGTGCCATGTACATCACAGCCCTGTTAGGAAACACCATCATCG
TGACTGCAATCTGGATGGATTCCACTCGGCATGAGCCCATGTATTGCTTTCTGTGTGTTCTGGCTGCTGTGGACAT
TGTTATGGCCTCCTCGGTGGTACCCAAGATGGTGAGCATCTTCTGCTCAGGAGACAGCTCAATCAGCTTTAGTGCT
TGTTTCACTCAGATGTTTTTTGTCCACTTAGCCACAGCTGTGGAGACGGGGCTGCTGCTGACCATGGCTTTTGACC
GCTATGTAGCCATCTGCAAGCCTCTACACTACAAGAGAATTCTCACGCCTCAAGTGATGCTGGGAATGAGTATGGC
CATCACCATCAGAGCTATCATAGCCATAACTCCACTGAGTTGGATGGTGAGTCATCTACCTTTCTGTGGCTCCAAT
GTGGTTGTCCACTCCTACTGTGAGCACATAGCTTTGGCCAGGTTAGCATGTGCTGACCCCGTGCCCAGCAGTCTCT
ACAGTCTGATTGGTTCCTCTCTTATGGTGGGCTCTGATGTGGCCTTCATTGCTGCCTCCTATATCTTAATTCTCAG
GGCAGTATTTGATCTCTCCTCAAAGACTGCTCAGTTGAAAGCATTAAGCACATGTGGCTCCCATGTGGGGGTTATG
GCTTTGTACTATCTACCTGGGATGGCATCCATCTATGCGGCCTGGTTGGGGCAGGATATAGTGCCCTTGCACACCC
AAGTGCTGTTAGCTGACCTGTACGTGATCATCCCAGCCACTTTAAATCCCATCATCTATGGCATGAGGACCAAACA
ATTGCTGGAGGGAATATGGAGTTATCTGATGCACTTCCTCTTTGACCACTCCAACCTGGGTTCATGAACACAA
The GPCRBc protein encoded by SEQ ID N0:35 has 324 amino acid residues, and is presented using the one-letter code in Table 8G (SEQ ID N0:36). The SignalP, Psort and/or Hydropathy profile for GPCRBc predict that GPCRBc has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. The SignalP shows a signal sequence with a cleavage site at the slash in the sequence VTA-IW, between amino acids 52 and 53. This is typical of this type of membrane protein. The molecular weight of theGPCRBc protein is 35273.4 Daltons.
Table 8G. Encoded GPCRBc protein sequence (SEQ ID N0:36).
MLGPAYNHTMETPASFLLVGIPGLQSSHLWLAISLSAMYITALLGNTIIVTATWMDSTRHEPMYCFLCVLAAVDIV
MASSVVPKMVSIFCSGDSSISFSACFTQMFFVHLATAVETGLLLTMAFDRYVAICKPLHYKRILTPQVMLGMSMAI
TIRAIIAITPLSWMVSHLPFCGSNVWHSYCEHIALARLACADPVPSSLYSLIGSSLMVGSDVAFIAASYILILRA
VFDLSSKTAQLKALSTCGSHVGVMALYYLPGMASIYAAWLGQDIVPLHTQVLLADLYVIIPATLNPIIYGMRTKQL
LEGIWSYLMHFLFDHSNLGS
The full amino acid sequence of the protein of the invention was found to have 146 of 306 amino acid residues (47%) identical to, and 202 of 306 residues (66%) positive with, the 321 amino acid residue Odorant Receptor S18 from Mus musculus (ptnr:SPTREMBL-ACC:Q9WU89) (E value = 6.8e'3), 130 of 303 amino acid residues (42%) identical to, and 195 of 303 residues (64%) positive with, the 326 amino acid residue MOR
3'BETAl from Mus musculus (SPTREMBL-ACC:Q9WVD9) (E value = 2.1e-69), and 107 of 297 amino acid residues (36%) identical to, and 175 of 297 residues (58%) positive with, the 321 amino acid residue HOR 5'BETA3 from Homo Sapiens (SPTREMBL-ACC:Q9YSP1) (E value = 4.Oe-Sa) GPCRBd A novel nucleic acid was identified vy exon linking. The disclosed novel GPCRBd nucleic acid of 981 nucleotides (also referred to as CG53845-02) is shown in Table 8H. An open reading frame begins with an ATG initiation codon at nucleotides 3-5 and ends with a TGA codon at nucleotides 954-956. A putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8G, and the start and stop codons are in bold letters.
Table 8H. GPCRBe Nucleotide Sequence (SEQ ID N0:80) _TGATGCTGGGTCCAGCTTATAACCACACAATGGAAACCCCTGCCTCCTTCCTCCTTGTGGGTATCCCAGGACTGCA
ATCTTCACATCTTTGGCTGGCTATCTCACTGAGTGCCATGTACATCATAGCCCTGTTAGGAAACACCCTCATCGTG
ACTGCAATCTGGATGGATTCCACTCGGCATGAGCCCATGTATTGCTTTCTGTGTGTTCTGGCTGCTGTGGACATTG
TTATGGCCTCCTCCGTGGTACCCAAGATGGTGAGCATCTTCTGCTCGGGAGACAGCTCCATCAGCTTTAGTGCTTG
TTTCACTCAGATGTTTTTTGTCCACTTAGCCACAGCTGTGGAGACGGGGCTGCTGCTGACCATGGCTTTTGACCGC
TATGTAGCCATCTGCAAGCCTCTACACTACAAGAGAATTCTCACGCCTCAAGTGATGCTGGGAATGAGTATGGCCG
TCACCATCAGAGCTGTCACATTCATGACTCCACTGAGTTGGATGATGAATCATCTACCTTTCTGTGGCTCCAATGT
GGTTGTCCACTCCTACTGTAAGCACATAGCTTTGGCCAGGTTAGCATGTGCTGACCCCGTGCCCAGCAGTCTCTAC
AGTCTGATTGGTTCCTCTCTTATGGTGGGCTCTGATGTGGCCTTCATTGCTGCCTCCTATATCTTAATTCTCAGGG
CAGTATTTGATCTCTCCTCAAAGACTGCTCAGTTGAAAGCATTAAGCACATGTGGCTCCCATGTGGGGGTTATGGC
TTTGTACTATCTACCTGGGATGGCATCCATCTATGCGGCCTGGTTGGGGCAGGATATAGTGCCCTTGCACACCCAA
GTGCTGCTAGCTGACCTGTACGTGATCATCCCAGCCACTTTAAATCCCATCATCTATGGCATGAGGACCAAACAAT
TGCTGGAGGGAATATGGAGTTATCTGATGCACTGTCCTCTTTGACCACTCCAACCTGGGTTCATGAACA
S The disclosed nucleic acid sequence has 538 of 858 bases (62%) identical to a Mus musculus odorant receptor S19 gene, complete cds:(GENBANI~-ID:AF121976~acc:AF121976) (E value = 3.5e-41).
The GPCR8d protein encoded by SEQ ID N0:80 has 317 amino acid residues, and is presented using the one-letter code in Table 8I (SEQ ID N0:81). The SignalP, Psort and/or Hydropathy profile for GPCR8d predict that GPCRBd has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. The SignalP shows a signal sequence with a cleavage site at the slash in the sequence VTA-IW, between amino acids 52 and 53. This is typical of this type of membrane protein.
Table 8I. Encoded GPCRBd protein sequence (SEQ ID N0:81).
MLGPAYNHTMETPASFLLVGIPGLQSSHLWLAISLSAMYIIALLGNTLIVTAIWMDSTRHEPMYCFLCVLAAVDIV
MASSWPKMVSIFCSGDSSISFSACFTQMFFVHLATAVETGLLLTMAFDRYVAICKPLHYKRILTPQVMLGMSMAV
TIRAVTFMTPLSWMMNHLPFCGSNVWHSYCKHIALARLACADPVPSSLYSLIGSSLMVGSDVAFIAASYTLILRA
VFDLSSKTAQLKALSTCGSHVGVMALYYLPGMASIYAAWLGQDIVPLHTQVLLADLYVIIPATLNPIIYGMRTKQL
LEGIWSYLMHCPL
The full amino acid sequence of the protein of the invention was found tohave 142 of 308 amino acid residues (46%) identical to, and 202 of 308 residues (65%) positive with, the 321 amino acid residue Odorant Receptor S 18 from Mus rriusculus (ptnr:
SPTREMBL-ACC:Q9WU89) (E value = l.Se-7i) GPCR8d is expressed in at least the following tissues: Apical rnicrovilli of the 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, MIiC II abnd III
expressing cells, nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells of the tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST
sources, Literature sources, and/or RACE sources..
0 GPCRBe A novel nucleic acid was identified vy exon linking. The disclosed novel GPCRBe nucleic acid of 982 nucleotides (also referred to as CG53845-03) is shown in Table 8J. An open reading frame begins with an ATG initiation codon at nucleotides 4-6 and ends with a TGA codon,at nucleotides 978-980. A putative untranslated region upstream from the 5 initiation codon and downstream from the termination codon are underlined in Table 8J, and the start and stop codons are in bold letters.
Table 8J. GPCRBe Nucleotide Sequence (SEQ ID N0:82) _TTGATGCTGGGTCCAGCTTACAACCACACAATGGAAACCCCTGCCTCCTTCCTCCTTGTGGGTATCCCAGGACTGC
AATCTTCACATCTTTGGCTGGCTATCTCACTGAGTGCCATGTACATCATAGCCCTGTTAGGAAACACCCTCATCGT
GACTGCAATCTGGATGGATTCCACTCGGCATGAGCCCATGTATTGCTTTCTGTGTGTTCTGGCTGCTGTGGACATT
GTTATGGCCTCCTCGGTGGTACCCAAGATGGTGAGCATCTTCTGCTCGGGAGACAGCTCCATCAGCTTTAGTGCTT
GTTTCACTCAGATGTTTTTTGTCCACTTAGCCACAGCTGTGGAGACGGGGCTGCTGCTGACCATGGCTTTTGACCG
CTATGTAGCCATCTGCAAGCCTCTACACTACAAGAGAATTCTCACGCCTCAAGTGATGCTGGGAATGAGTATGGCC
GTCACCATCAGAGCTGTCACATTCATGACTCCACTGAGTTGGATGATGAATCATCTACCTTTCTGTGGCTCCAATG
TGGTTGTCCACTCCTACTGTAAGCACATAGCTTTGGCCAGGTTAGCATGTGCTGACCCCGTGCCCAGCAGCCTCTA
CAGTCTGATTGGTTCCTCTCTTATGGTGGGCTCTGATGTGGCCTTCATTGCTGCCTCCTATATCTTAATTCTCAGG
GCAGTATTTGATCTCTCCTCAAAGACTGCTCAGTTGAAAGCATTAAGCACATGTGGCTCCCATGTGGGGGTTATGG
CTTTGTACTATCTACCTGGGATGGCATCCATCTATGCGGCCTGGTTGGGGCAGGATATAGTGCCCTTGCACACCCA
AGTGCTGCTAGCTGACCTGTACGTGATCATCCCAGCCACTTTAAATCCCATCATCTATGGCATGAGGACCAAACAA
TTGCTGGAGGGAATATGGAGTTATCTGATGCACTTCCTCTTTGACCACTCCAACCTGGGTTCATGAACAA
The disclosed nucleic acid sequence has 538 of 858 bases (62%) identical to a Mus >.0 musculus odorant receptor S19 gene, complete cds:(GENBANK-ID:AF121976~acc:AF121976) (E value = 4.5e-42).
The GPCRBe protein encoded by SEQ ID N0:82 has 324 amino acid residues, and is presented using the one-letter code in Table 8K (SEQ ID N0:83). The SignalP, Psort and/or Hydropathy profile for GPCRBe predict that GPCRBe has a signal peptide and is likely to be ?5 localized at the plasma membrane with a certainty of 0.6400. The SignalP
shows a signal sequence with a cleavage site at the slash in the sequence VTA-IW, between amino acids 52 and 53. This is typical of this type of membrane protein.

Table 8K. Encoded GPCRBd protein sequence (SEQ ID N0:81).
MLGPAYNHTMETPASFLLVGIPGLQSSHLWLAISLSAMYIIALLGNTLIVTAIWMDSTRHEPMYCFLCVLAAVDIV
MASSWPKMVSIFCSGDSSISFSACFTQMFFVHLATAVETGLLLTMAFDRWAICKPLHYKRILTPQVMLGMSMAV
TIRAVTFMTPLSWMMNHLPFCGSNWVHSYCKHIALARLACADPVPSSLYSLIGSSLMVGSDVAFIAASYILILRA
VFDLSSKTAQLKALSTCGSHVGVMALYYLPGMASIYAAWLGQDIVPLHTQVLLADLYVTIPATLNPIIYGMRTKQL
LEGIWSYLMHFLFDHSNLGS
The full amino acid sequence of the protein of the invention was found tohave 142 of 308 amino acid residues (46%) identical to, and 202 of 308 residues (65%) positive with, the 321 amino acid residue Odorant Receptor S 18 from Mus musculus (ptnr: SPTREMBL-ACC:Q9WU89) (E value =1.5e-7i) GPCRBe is expressed in at least the following tissues: Apical microvilli of the 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, MHC II abnd III
expressing cells, nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells of the tongue, testis, thalamus, and thymus tissue. Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of CuraGen Acc. No. CG53845-03.
Further BLAST analysis produced the significant results listed in Table 8L.
The disclosed GPCR8 protein has good identity with a number of olfactory receptor proteins.
Table 8L. BLAST
results for GPCR8 Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) gi~9938014~ref~NP_06Odorant receptor321 142/308 202/308 2e-65 4686.1 S18 gene [Mus (46%) (65%) musculus]

gi~11908211~gb~AAG41HOR 5'Betal4 318 134/292 187/292 2e-64 676.1 (AF137396) [Homo Sapiens] (45%) (63%) gi~6532001~gb~AAD275Odorant receptor339 128/281 185/281 1e-63 96.2~AF121976_1 S19 [Mus (45%) (65%) (AF121976) musculus]

gi~7305349~ref~NPOlfactory 326 129/299 192/299 3e-62 8647.1 receptor 67 (43%) (64%) [Mus musculus]

gi~11875778~gb~AAG40Prostate specific320 124/293 190/293 1e-60 776.1~AF311306 G-protein coupled (42%) (64%) (AF311306) receptor; PSGR

[Homo Sapiens]

This information is presented graphically in the multiple sequence alignment given in Table 8M (with GPCR8 being shown on line 1) as a ClustalW analysis comparing with related protein sequences.
Table 8M. Information for the ClustalW proteins:
1) GPCRBa (SEQ ID N0:32) 2) GPCR8b (SEQ 1D N0:34) 3) GPCR8c (SEQ 1T7 N0:36) 2) giJ9938014Jref~NP_064686.1J odorant receptor S18 gene [Mus musculus] (SEQ
ID N0:56) 3) giJ11908211JgbJAAG41676.1J (AF137396) HOR 5'Betal4 [Homo sapiens] (SEQ ID
NO:55) 4) giJ6532001JgbJAAD27596.2JAF121976 1 (AF121976) odorant receptor S19 [Mus musculus] (SEQ ID
N0:54) 5) giJ7305349JrefJNP 038647.1 J olfactory receptor 67 [Mus musculus] (SEQ n7 N0:57) 6) giJ11875778JgbJAAG40776.1JAF311306 1 (AF311306) prostate specific G-protein coupled (SEQ ID
N0:65) ..
GPCRBa ---------------------HET - y ES n SASS
GPAY L
.

GPCR8b ---------------------HET ~J ~S L SS
~GPAY

GPCR8c ---------------------HET ~S~ L SOS
1~GPAY Y

gi~9938014~____________________~S~SMLGT IIH~ ~ QY T~'~FC
V

gi~11908211~SDSNLSDNHL D~FT ~ FC
--------------------- ' giI6532001~~ HWPN TTHYR E I FC
MPEKMLSKLIAYLLLIESCRQTAQ DQ
KGRRIWVDS

gi~7305349~--------------------- ~ DL SZ ~C
MK~ASSFH~D~NPQDVW ~
~

gi~11875778~_____________________________~SCNFTHA'~ -- K GFL

GPCR8a GPCR8b GPCRBc gi~9938014~
gi~11908211~
gi~6532001~
gi~7305349~
gi~11875778~
GPCR8a GPCR8b GPCR8c gi19938014~
gi~11908211~
gi~6532001~
gi~7305349~
gi~11875778~
GPCRSa GPCRBb GPCR8c gi~9938014~
gi~11908211~
gi~6532001~
gi~7305349~
gi~11875778~
GPCR8a GPCRBb GPCRBc gi~9938014~
gi~11908211~
gi~6532001~

gi ~ 7305349 ~ L II T T Q'I' A~Y ' C T' FF'~F' GKTmR~
gi ~ 11875778 ~ FL~FT~LQSE~V~~LIGLSVV' -NS'I3PI

GPCR8a TQ~
~'ym' T
LG~WSY
HFLFDHSNLGS--GPCR8b TQ~. ~~' T ~WSY HVLFDHSNLGS--GPCR8c TQ. L' T v L GWSYHFLFDHSNLGS--gi~9938014~.H ~ ST SVVWKS--------gi~11908211~H E S ~LKLHLGKTSI-------~

gi ~ 6532001~ T~ ' N ~ L IiLDFFVKRR----------~ ~

H

gi~7305349~H~ ~ ~ FLF SSVSTCQHDSRC--gi~11875778~R~~iGIcy " ,y 'T LAMF KISCDKDLQAVGGK

The presence of identifiable domains in GPCR8 was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~.
DOMAIN results for GPCR8 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 8N with the statistics and domain description. The results indicate that GPCR8 has homology to the 7tm 1 (InterPro) 7 transmembrane receptor (rhodopsin family)domain (as defined by Interpro). This indicates that the sequence of GPCR8 has properties similar to those of other proteins known to contain this/these domains) and similar to the properties of these domains.
Table 8N. DOMAIN results for GPCR8 against the consnensus 7tm 1 domain (SEQ ID N0:78) CD-Length = 254 residues, 100.0% aligned Score = 79.7 bits (195), Expect = 2e-16 ° 10 20 30 40 . 50 60 .i....
ConsensusQ7tm l0domain ~L~LV LRTKKLR-T~TNI~LN~VV T,FLL~P~WKF~YYLVG ,SVFGD~L~KL

....~..
.~....i..

GPCR8 QMFFVHTAVETG ~TTI ; : ~ ~
T~
~
~
~
LS
~

P
Consensus 7tm VGALFV ~
1 domain : VH
GYASIL R
RAKV
I C.s,L
LL
L
, 2$ ....~....~..
.~....~....~....~..
.~..
.~....~..
.~..
.~..
.I

GPCR8 SWMMNHLPFCGS V----VVHSYCKHIALARL 'SSL-----Y
DPV LGS L SD

Consensus 7tm LLFSWLRTVEEG ~T-----------------TV BEESVK---R~YLL~T~FL
1 domain LIDF

3 ..~....~..
0 .~..
.~....~....~....~....~....i....~....~....i GPCR8 S~AFS ~~,~~';F-_______________________,_________________ 1 ~ IL
t ~LV
i ~

Consensus 7 iPLLVTR RKSARSQRS----------------------------------m C
doma n 35 ....i....~....~....i....~....~....~....i....

GPCR8 _________________________________________________DLSS

~'.3~QL

Consensus 7tm -------------------------------------------------LKRRS

1 domain ,.....

.1....I.. 1....1....1....1....1....1....1 I....~. I
Consensus 7tm 1 domain AKMLLVH G F CYHIV~LLDS~C~LSIWR-------VV~P " ~TLWLAY~C
.I.

Consensus 7tm 1 domain The similarity information for the GPCR8 protein and nucleic acid disclosed herein suggest that GPCR8 may have important structural and/or physiological functions characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon. The novel nucleic acid encoding GPCRB, and the GPCR8 protein of the 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.
The disclosed GPCR8 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson°s disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. For example, 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. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome. The novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the 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 of the 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. For example the disclosed GPCRB protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR8 epitope is from about amino acids 50 to 60. In another embodiment, a GPCRB epitope is from about amino acids 130 to 140. In additional embodiments, GPCRB epitopes are from amino acids 230 to 240, and 280-310. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

GPCR9a A novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Corporation's sequence hle for GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank. The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein. The disclosed novel GPCR9a nucleic acid of 960 nucleotides (also referred to as AC025249 D) is shown in Table 9A. An open reading frame begins with an ATG initiation codon at nucleotides 4-6 and ends with a TAG codon at nucleotides 951-953. A putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 9A, and the start and stop codons are in bold letters.

The nucleic acid sequence has 567 of 880 bases (64%) identical to (E= 2.6e-53) a G-protein coupled prostate tissue receptor designated HPRAJ70 cDNA from Homo sapiehs (patn:V30143).
Table 9A. GPCR9a Nucleotide Sequence (SEQ ID N0:37) _GCCATGCTCACTTTTCATAATGTCTGCTCAGTACCCAGCTCCTTCTGGCTCACTGGCATCCCAGGGCTGGA
GTCCCTACACGTCTGGCTCTCCATCCCCTTTGGCTCCATGTACCTGGTGGCTGTGGTGGGGAATGTGACCA
TCCTGGCTGTGGTAAAGATAGAACGCAGCCTGCACCAGCCCATGTACTTTTTCTTGTGCATGTTGGCTGCC
ATTGACCTGGTTCTGTCTACTTCCACTATACCCAAACTTCTGGGAATCTTCTGGTTCGGTGCTTGTGACAT
TGGCCTGGACGCCTGCTTGGGCCAAATGTTCCTTATCCACTGCTTTGCCACTGTTGAGTCAGGCATCTTCC
TTGCCATGGCTTTTGATCGCTACGTGGCCATCTGCAACCCACTACGTCATAGCATGGTGCTCACTTATACA
GTGGTGGGTCGTTTGGGGCTTGTTTCTCTCCTCCGGGGTGTTCTCTACATTGGACCTCTGCCTCTGATGAT
CCGCCTGCGGCTGCCCCTTTATAAAACCCATGTTATCTCCCACTCCTACTGTGAGCACATGGCTGTAGTTG
CCTTGACATGTGGCGACAGCAGGGTCAATAATGTCTATGGGCTGAGCATCGGCTTTCTGGTGTTGATCCTG
GACTCAGTGGCTATTGCTGCATCCTATGTGATGATTTTCAGGGCCGTGATGGGGTTAGCCACTCCTGAGGC
TAGGCTTAAAACCCTGGGGACATGCGCTTCTCACCTCTGTGCCATCCTGATCTTTTATGTTCCCATTGCTG
TTTCTTCCCTGATTCACCGATTTGGTCAGTGTGTGCCTCCTCCAGTCCACACTCTGCTGGCCAACTTCTAT
CTCCTCATTCCTCCAATCCTCAATCCCATTGTCTATGCTGTTCGCACCAAGCAGATCCGAGAGAGCCTTCT
CCAAATACCAAGGATAGAAATGAAGATTAGATGATTA
The GPCR9a protein encoded by SEQ ID N0:37 has 317 amino acid residues, and is presented using the one-letter code in Table 9B (SEQ ID N0:38). The SignalP, Psort and/or Hydropathy profile for GPCR9a predict that GPCR9a has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The SignalP
predicts a cleavage site at the sequence VVG-NV between amino acids 43 and 44. The Molecular weight of GPCR9a is 35036.8 Daltons.
Table 9B. Encoded GPCR9 protein sequence (SEQ ID N0:38) MLTFHNVCSVPSSFWLTGIPGLESLHVWLSIPFGSMYLVAWGNVTILAWKIERSLHQPMYFFLCMLAAI
DLVLSTSTIPKLLGIFWFGACDIGLDACLGQMFLIHCFATVESGIFLAMAFDRYVAICNPLRHSMVLTYTV
VGRLGLVSLLRGVLYIGPLPLMIRLRLPLYKTHVISHSYCEHMAWALTCGDSRVNNVYGLSIGFLVLTLD
SVAIAASYVMTFRAVMGLATPEARLKTLGTCASHLCAILIFYVPIAVSSLIHRFGQCVPPPVHTLLANFYL
LIPPILNPIVYAVRTKQIRESLLQIPRIEMKIR
The full amino acid sequence of the protein of the invention was found to have 157 of 301 amino acid residues (52%) identical to, and 210 of 301 residues (69%) positive with, the 320 amino acid residue Putative G-Protein Coupled Receptor R.A1C from Rattus noYVegicus (ptnr:SPTREMBL-ACC:088628) (E value = 2.7e-81).
GPCR9 also has homology to the proteins shown in the BLAST alignments in Table 9C.

Table 9C. BLASTX results for GPCR9a Smallest Sum Reading High Prob Sequences Pairs;Frame ScoreP(N) N
producing High-scoring Segment patp:Y92365G protein-coupled protein5 - Hom.+1 831 4.0e-82 receptor 1 patp:W01730Human G-protein receptor Homo sa.+1 802 4.7e-79 patp:W56641G-protein coupled tissuereceptor.+~.802 4.7e-79 prostate 1 patp:R27869Odorant receptor - Rattusrattus,.+1 418 2.3e-38 clone F6 1 GPCR9b In the present invention, the target sequence identified previously, Accession Number AC079759 D, was subjected to the exon linking process to confirm the sequence.
PCR
primers were designed by starting at the most upstream sequence available, fox the forward primer, and at the most downstream sequence available for the reverse primer.
In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: 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. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp.
In addition, sequence traces were evaluated manually and edited for corrections if appropriate.
These procedures provide the sequence reported below, which is designated Accession Number AC079759 dal. This differs from the previously identified sequence (Accession Number AC079759 D) in having a single aminoacid change.
The disclosed novel GPCR9b nucleic acid of 997 nucleotides (also referred to as AC025249_dal) is shown in Table 9D. An open reading frame begins with an ATG
initiation codon at nucleotides 5-7 and ends with a TGA codon at nucleotides 956-95~. A
putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 9D, and the start and stop codons are in bold letters.
In a seaxch of sequence databases, it was found, for example, that the nucleic acid sequence has 587 of 896 bases (65%) identical to (E= 2.7e-53) a Rattus norvegicus putative G-protein coupled receptor RAIc mRNA, complete cds (GENBANK-ID:AF079864~acc:AF079864.1).
Table 9D. GPCR9b Nucleotide Sequence (SEQ ID N0:39) AGCCATGCTCACTTTTCATAATGTCTGCTCAGTACCCAGCTCCTTCTGGCTCACTGGCATCCCAGGGCTGG
AGTCCCTACACGTCTGGCTCTCCATCCCCTTTGGCTCCATGTACCTGGTGGCTGTGGTGGGGAATGTGACC
ATCCTGGCTGTGGTAAAGATAGAACGCAGCCTGCACCAGCCCATGTACTTTTTCTTGTGCATGTTGGCTGC
CATTGACCTGGTTCTGTCTACTTCCACTATACCCAAACTTCTGGGAATCTTCTGGTTCGGTGCTTGTGACA
TTGGCCTGGATGCCTGCTTGGGCCAAATGTTCCTTATCCACTGCTTTGCCACTGTTGAGTCAGGCATCTTC
CTTGCCATGGCTTTTGATCGCTATGTGGCCATCTGCAACCCACTACGTCATAGCATGGTGCTCACTTATAC
AGTGGTGGGTCGTTTGGGGCTTGTTTCTCTCCTCCGGGGTGTTCTCTACATTGGACCTCTGCCTCTGATGA
TCCGCCTGCGGCTGCCCCTTTATAAAACCCATGTTATCTCCCACTCCTACTGTGAGCACATGGCTGTAGTT
GCCTTGACATGTGGCGACAGCAGGGTCAATAATGTCTATGGGCTGAGCATCGGCTTTCTGGTGTTGATCCT
GGACTCAGTGGCTATTGCTGCATCCTATGTGATGATTTTCAGGGCCGTGATGGGGTTAGCCACTCCTGAGG
CTAGGCTTAAAACCCTGGGGACATGCGCTTCTCACCTCTGTGCCATCCTGATCTTTTATGTTCCCATTGCT
GTTTCTTCCCTGATTCACCGATTTGGTCAGTGTGTGCCTCCTCCAGTCCACACTCTGCTGGCCAACTTCTA
TCTCCTCATTCCTCCAATCCTCAATCCCATTGTCTATGCTGTTCGCACCAAGCAGATCCGAGAGAGGCTTC
TCCAAATACCAAGGATAGAAATGAAGATTAGATGATTACTATTTTCTTCTCTCTCAAATAAGCTCATGGAG
AAG
The GPCR9b protein encoded by SEQ ID N0:39 has 317 amino acid residues, and is presented using the one-letter code in Table 9E (SEQ 1D N0:40). The SignalP, Psort and/or Hydropathy profile for GPCR9 predict that GPCR9 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The SignalP
predicts a cleavage site at the sequence VVG-NV between amino acids 43 and 44.
Table 9E. Encoded GPCR9b protein sequence (SEQ ID N0:40) MLTFHNVCSVPSSFWLTGIPGLESLHVWLSIPFGSMYLVAVVGNVTILAWKIERSLHQPMYFFLCMLAAI
DLVLSTSTIPKLLGIFWFGACDIGLDACLGQMFLIHCFATVESGIFLAMAFDRYVAICNPLRHSMVLTYTV
VGRLGLVSLLRGVLYIGPLPLMIRLRLPLYKTHVISHSYCEHMAWALTCGDSRVNNVYGLSIGFLVLILD
SVAIAASYVMIFRAVMGLATPEARLKTLGTCASHLCAILIFYVPIAVSSLIHRFGQCVPPPVHTLLANFYL
LIPPILNPIVYAVRTKQIRERLLQIPRIEMKIR
The full amino acid sequence of the protein of the invention was found to have 158 of 301 amino acid residues (52%) identical to, and 211 of 301 residues (70%) positive with, the 320 amino acid residue Putative G-Protein Coupled Receptor RA1C from Rattus horvegicus (ptnr:SPTREMBL-ACC:088628) (E value = 2.7e-82) The Olfactory Receptor disclosed in this invention is expressed in at least the following tissues: Apical rnicrovilli of the 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, MHC
II abnd III expressing cells, nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells of the tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE
sources.
GPCR9c In the present invention, the target sequence identified previously, Accession Number AC079759 D, was subjected to the exon linking process to confirm the sequence.
PCR
primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer.
In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: 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. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp.
In addition, sequence traces were evaluated manually and edited for corrections if appropriate.
These procedures provide the sequence reported below, which is designated Accession Number AC079759 dal . This differs from the previously identified sequence (Accession Number AC079759 D) in having a single aminoacid change.
The disclosed novel GPCR9c nucleic acid of 997 nucleotides (also referred to as CG50223-O1) is shown in Table 9F An open reading frame begins with an ATG
initiation codon at nucleotides 5-7 and ends with a TGA codon at nucleotides 958-960. A
putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 9F, and the start and stop codons are in bold letters.
In a search of sequence databases, it was found, for example, that the nucleic acid sequence has 573 of 891 bases (64%) identical to (E= 3.7e-52) a Sequence 1 from patent US
5756309 (GENBANI~-ID:AR009514~acc:AR009514.1).
Table 9F. GPCR9c Nucleotide Sequence (SEQ ID N0:41) AGCCATGCTCACTTTTCATAATGTCTGCTCAGTACCCAGCTCCTTCTGGCTCACTGGCATCCCAGGGCTGG
AGTCCCTACACGTCTGGCTCTCCATCCCCTTTGGCTCCATGTACCTGGTGGCTGTGGTGGGGAATGTGACC
ATCCTGGCTGTGGTAAAGATAGAACGCAGCCTGCACCAGCCCATGTACTTTTTCTTGTGCATGTTGGCTGC
CATTGACCTGGTTCTGTCTACTTCCACTATACCCAAACTTCTGGGAATCTTCTGGTTCGGTGCTTGTGACA
TTGGCCTGGATGCCTGCTTGGGCCAAATGTTCCTTATCCACTGCTTTGCCACTGTTGAGTCAGGCATCTTC
CTTGCCATGGCTTTTGATCGCTACGTGGCCATCTGCAACCCACTACGTCATAGCATGGTGCTCACTTATAC
AGTGGTGGGTCGTTTGGGGCTTGTTTCTCTCCTCCGGGGTGTTCTCTACATTGGACCTCTGCCTCTGATGA
TCCGCCTGCGGCTGCCCCTTTATAAAACCCATGTTATCTCCCACTCCTACTGTGAGCACATGGCTGTAGTT
GCCTTGACATGTGGCGACAGCAGGGTCAATAATGTCTATGGGCTGAGCATCGGCTTTCTGGTGTTGATCCT
GGACTCAGTGGCTATTGCTGCATCCTATGTGATGATTTTCAGGGCCGTGATGGGGTTAGCCACTCCTGAGG
CTAGGCTTAAAACCCTGGGGACATGCGCTTCTCACCTCTGTGCCATCCTGATCTTTTATGTTCCCATTGCT
GTTTCTTCCCTGATTCACCGATTTGGTCAGTGTGTGCCTCCTCCAGTCCACACTCTGCTGGCCAACTTCTA
TCTCCTCATTCCTCCAATCCTCAATCCCATTGTCTATGCTGTTCGCACCAAGCAGATCCGAGAGAGGCTTC
TCCAAATACCAAGGATAGAAATGAAGATTAGATGATTACTATTTTCTTCTCTCTCAAATAAGCTCATGGAG
AAG
The GPCR9c protein encoded by SEQ ID N0:41 has 317 amino acid residues, and is presented using the one-letter code in Table 9G (SEQ ID N0:42). The SignalP, Psort and/or Hydropathy profile for GPCR9 predict that GPCR9 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The SignalP
predicts a cleavage site at the sequence VVG-NV between amino acids 43 and 44.
Table 9G. Encoded GPCR9c protein sequence (SEQ ID N0:42) MLTFHNVCSVPSSFWLTGIPGLESLHVWLSIPFGSMYLVAWGNVTILAWKIERSLHQPMYFFLCMLAAI
DLVLSTSTIPKLLGIFWFGACDIGLDACLGQMFLIHCFATVESGIFLAMAFDRYVAICNPLRHSMVLTYTV
VGRLGLVSLLRGVLYIGPLPLMIRLRLPLYKTHVISHSYCEHMAWALTCGDSGVNNVYGLSIGFLVLILD
SVAIAASYVMIFRAVMGLATPEARLKTLGTCASHLCAILIFYIPIAVSSLIHRFGQCVPPPVHTLLANFYL
LIPPILNPIVYAVRTKQIRERLLQIPRIEMKIR
The full amino acid sequence of the protein of the invention was found to have 157 of 301 amino acid residues (52%) identical to, and 211 of 301 residues (70%) positive with, the 320 amino acid residue Putative G-Protein Coupled Receptor RA1C from Rattus noYVegicus (ptnr:SPTREMBL-ACC:088628) (E value = 5.6e-8a) GPCR9c is expressed in at least the following tissues: Apical microvilli of the 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, MHC II abnd III
expressing cells, nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells of the tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST
sources, Literature sources, and/or RACE sources.
Possible SNPs found for GPCR9c are listed in Table 9H.
Table 9H: SNPs ConsensusDepth Base PAF
Position Change 298 18 C>T 0.333 445 22 G>A 0.091 Further BLAST analysis produced the significant results listed in Table 9T.
The disclosed GPCR9 protein has good identity with a number of olfactory receptor proteins.
Table 9I. BLAST
results for Gene Index/ Protein/ LengthIdentityPositivesExpect Identifier Organism (aa) (%) (%) gi~11991867~gb~AAG4odorant receptor316 154/309 193/309 2e-71 2368.11 (AF289204)HOR3'beta5 (49%) (61%) [Homo Sapiens]

gi~11908221~gb~AAG4MOR 3'Beta6 316 151/299 189/299 3e-70 [Mus 1685.1) (AF133300)musculus] (50%) (62%) gi~7305351~ref~NPOlfactory 315 148/309 188/309 7e-68 38648.1 receptor 68 (470) (59%) [Mus musculus]

gi~7305353~ref~NPOlfactory 316 147/309 187/309 1e-66 38649.1 receptor 69 (47%) (59%) [Mus musculus]

gi~3927808~gb~AAD03olfactory 319 145/296 196/296 1e-66 349.1 (L17432) receptor-like (48%) (65%) protein COR3'beta [Gallus gallus]

This information is presented graphically in the multiple sequence alignment given in Table 9J (with GPCR9a being shown on line 1, GPCR9b on line 2, and GPCR9c on line 3) as a ClustalW analysis comparing GPCR9 with related protein sequences.
Table 9J. Information for the ClustalW proteins:
1) GPCR9a (SEQ ID N0:38) 2) GPCR9b (SEQ B7 N0:40) 3) GPCR9c (SEQ ID N0:42) 4) gi~11991867~gb~AAG42368.1~ (AF289204) odorant receptor HOR3'beta5 [Homo sapiens] (SEQ 1D
L 0 N0:66) 3) gi~11908221~gb~AAG41685.1~ (AF133300) MOR 3'Beta6 [Mus musculus] (SEQ ID
N0:67) 5) gi~7305351~re~NP_038648.1~ olfactory receptor 68 [Mus musculus] (SEQ ID
N0:68) 6) gi~7305353~ref~NP_038649.1~ olfactory receptor 69 [Mus musculus] (SEQ )D
N0:69) 7) gi~3927808~gb~AAD03349.1~ (LI7432) olfactory receptor-like protein COR3'beta [Gallus gallus] (SEQ
l5 m N0:70) GPCR9a T "VCS;,:S T ~~'H GS I ' n~ r v y ~'S VT.

GPCR9b 'T VCS.S T H 'SGS VT
w ur a a GPCR9c T IVCS~S T H 5 ' VT ~ ' ~ GS

gi ~ 11991867PT I I VQC G S hG SLVIIY NI
~ G~'3,VF~ ~

gi~11908221~PHL ' T ,Q G CI ~~,v SL
S'IF VLT

~ ~ V II I
V ~

gi~3927808~YP ~ - QFHH F GL GT~~ R~HQ
AQ ~L

GPCR9a GPCR9b GPCR9c gi~11991867~
gi~11908221~
gi~7305351~
gi~7305353~
gi~3927808~
GPCR9a F ' aM3~L YT~G V G P L' PL H
Y~G

GPCR9b F ~ ~IL~YT ~ : P 1~L'PL H
' G V G~Y
G

GPCR9c F ~ ~ ~ V GV P FFLPL H
M L,,~~~~,, Y ' YT, G
VG

gi~11991867~L I' T FQQF~TH VT ' S wCCKH T
~It~IG ~

gi~11908221~ ~ F~QRiCTT~L..VT :C~FC T
t~ ~w K

gi ~ 7305351L ~' ~'PQTTC L S C KY;~"~T
~ E' .S

gi~7305353~L ~ 'T~ SL~TTI FC KY;T
F PQTTC , P

gi~3927808~ C r,S~ L~GAR~A IC CT~S~LC ~-TW; H~f, PFC~

GPCR9a GPCR9b GPCR9c gi~11991867~
gi~11908221~
gi~7305351~
gi~7305353~
gi~3927808~
GPCR9a GPCR9b GPCR9c gi~11991867) gi~11908221~
gi~7305351~
~2 _.._I__..I..__I..__I._._I__..I..__I.._.I..__I..._I....I..._I

gi ~ 7305353 ~ F~~QLAFE'~FF
gi~3927808~ L ~ I LPLAGLSI

..
GPCR9a ~S' :PRIMKIR-_____ GPCR9b ~ ~'PRIn KIR-_____ ~~

GPCR9c ~ PRI'a KIR-_____ ,~
~

gi~11991867~~V~y~FFF
VT-------gi~11908221~~'~S~LYCNYSY-------gi~73053511Q~,' LFS KH--------gi~7305353~DQ~FFS KPL-------gi~3927808~CKG ~', LCQRAAWPGHAQNC

The presence of identifiable domains in GPCR9 was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~.
DOMAIN results for GPCR9 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 9K with the statistics and domain description. The results indicate that GPCR9 has homology to the 7tm 1 (InterPro) 7 transmembrane receptor (rhodopsin family)domain (as defined by Interpro). This indicates that the sequence of GPCR9 has properties similar to those of other proteins known to contain this/these domains) and similar to the properties of these domains.
Table 9K. DOMAIN results for GPCR9 against the consnensus 7tm 1 domain (SEQ ID N0:79) CD-Length = 254 residues, 100.0% aligned Score = 63.9 bits (154), Expect = 1e-11 . . 10 . 20 . . 30 , . 40 . . 50 . 60 GPCR9 (SEQ ID N0:38) Consensus 7tm 1 domain ...(..
GPCR9 ~~~~,G~~LSGLT~I ~ ~ ~ V~'.~RF~~PRRAK~IVW~LALSL
Consensus 7tm 1 domain 130 140 150 160 °170 180 .~.. .(..
GPCR9 LP~~y',~,~~I'D~~ LPLYKTHVISH[,,,,~Y~ EHM----------- ALTCGDS ,LAN Y~LS
:G~
Consensus 7tm 1 domain ~PL~SW~TVEEGNT--- '1'VVJLIDFP-------EE~K---RSYV~T~~FL~P~
3 0 190 200 210 220 1230 , 240 GPCR9 ~iDSV YVMI A;VMG TPE-________________________________ Consensus 7tm 1 domain ~T~'~2'CYTLRKSA~QRS~KRRSS--------------------------------3 5 .. .250 . '260 270 280 290 300 .~....~....~....~....(....I....~....~....~
GPCR9 ___________________________________-______-_________________ Consensus 7tm 1 domain _______-______________________________________-_____________ 310 320 330 ° 340 350 360 .I....~....~....~....~
GPCR9 ------------ RL TLGTCASHCAI ~ I SS ~HRFGQCVPPP----Consensus 7tm 1 domain ------------ ~ER~AAKMLLVVfVVF~LC~Y~VLDSLCLLSIWR----....

Consensus 7tm Z domain ---VLPT ITLAYVN--SC
The similarity information for the GPCR9 protein and nucleic acid disclosed herein suggest that GPCR9 may have important structural and/or physiological functions characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon. The novel nucleic acid encoding GPCR9, and the GPCR9 protein of the 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.
The nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of developmental diseases, MHCII and III
diseases (immune diseases), Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneurogenic disease, Signal Transduction pathway disorders, Retinal diseases including those involving photoreception, Cell Growth rate disorders; Cell Shape disorders, Feeding disorders;control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (NIDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma;
prostate cancer;
uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation. Dentatorubro-pallidoluysian atrophy(DRPLA) Hypophosphatemic rickets, ~4 autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome 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. For example, 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. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from developmental diseases, MHCII and III
diseases (immune diseases), Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneurogenic disease, Signal Transduction pathway disorders, Retinal diseases including those involving photoreception, Cell Growth rate disorders; Cell Shape disorders, Feeding disorders;control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (NIDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma;
prostate cancer;
uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation. Dentatorubro-pallidoluysian atrophy(DRPLA) Hypophosphaternic rickets, autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders. The novel nucleic acid encoding GPCR-like protein, and the GPCR-Iike protein of the 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 immuno-specifically to the novel GPCR9 substances for use in therapeutic or diagnostic methods.
These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies"
section below. Fox example the disclosed GPCR9 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR9 epitope is from about amino acids 130 to 180. In another embodiment, a GPCR9 epitope is from about amino acids 280 to 320. These novel proteins can also be used to develop assay system for functional analysis.
A summary of the GPCRX nucleic acids and proteins of the invention is provided in Table 10.
TABLE 10: Summary Of Nucleic Acids And Proteins Of The Invention Name Tables Clone; Description of Homolog NucleicAmino Acid Acid SEQ SEQ
ID ID

NO NO

GPCRl 1A, 1B, GPCRla:, rpl l-507n20 A GPCR 1 2 1D GPCRlb:, 11-507n20 A dal, GPCR 3 1E, 1F GPCRlc: AX011711 dal, GPCR 4 5 GPCR2 2A, 2B GPCR2: 80250319 EXT, GPCR 6 7 GPCR3 3A, 3B GPCR3: AC020597, GPCR 8 9 GPCR4 4A, 4B, GPCR4a: AC020597_B, GPCR 10 11 4F, 4G GPCR4b: AC020597B1, GPCR 12 13 4J GPCR4c: AC020597B2, GPCR 14 GPCRS SA, SB, GPCRSa: AC020597_C, GPCR 15 16 SD, SE GPCRSb: CG53668-02, GPCR 17 18 SF, SG GPCRSc: AC020597B dal, GPCR 19 20 GPCR6 6A, 6B GPCR6a: AC020597 D, GPCR 21 22 6D, 6E GPCR6b: AC020597 D1, GPCR 23 24 GPCR7 7A, 7B GPCR7a: AC020597 A, GPCR 25 26 7F, 7G GPCR7b: AC020597 Al, GPCR 27 28 7I, 7J GPCR7c: AC025249 Ada3, GPCR 29 30 GPCR8 8A, 8B GPCRBa: AC025249 B, GPCR 31 32 8D, 8E GPCRBb: AC025249 C, GPCR 33 34 8F, 8G GPCR8c: AC025249 C1, GPCR 35 36 8H, 8I GPCR8d: CG53845-O2, GPCR 80 81 8J, 8K. GPCRBe: CG53845-03, GPCR 82 83 GPCR9 9A, 9B GPCR9a: AC025249_D, GPCR 37 38 9D, 9E GPCR9b: AC079759 D, GPCR 39 40 9F, 9G GPCR9c: CG50223-O1, GPCR 41 42 GPCRX Nucleic Acids and Polypeptides One aspect of the invention pertains to isolated nucleic acid molecules that encode GPCRX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify GPCRX-encoding nucleic acids (e.g., GPCRX mRNAs) and fragments for use as PCR
primers for the amplification and/or mutation of GPCRX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs 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.
An GPCRX nucleic acid can encode a mature GPCRX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.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 ~7 double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA
of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated GPCRX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 3I, 33, 35, 37, 39, 41, 80, and 82 as a hybridization probe, 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"d 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 of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to GPCRX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the 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, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, or a complement thereof.
Oligonucleotides may be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, 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:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the 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.
Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of GPCRX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an GPCRX
polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurnng allelic variations and mutations of the 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:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, as well as a polypeptide possessing GPCRX biological activity. Various biological activities of the GPCRX proteins are described below.
An GPCRX polypeptide is encoded by the open reading frame ("ORF") of an GPCRX
nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop colon. An ORF that represents the coding sequence for a full protein begins with an ATG
"start" colon and terminates with one of the three "stop" colons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start colon, a stop colon, or both. For an ORF to be considered as a good candidate for coding for a bona fide cellular protein, 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 of the 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:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82; or an anti-sense strand nucleotide sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 3S, 37, 39, 41, 80, and 82; or of a naturally occurnng mutant of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82.
Probes based on the human GPCRX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g.
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 of the 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, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 2S, 27, 29, 31, 33, 3S, 37, 39, 41, 80, and 82 that encodes a polypeptide having an GPCRX biological activity (the biological activities of the GPCRX
proteins are described below), expressing the encoded portion of GPCRX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of GPCRX.
GPCRX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82 due to degeneracy of the genetic code and thus encode the same GPCRX proteins as that encoded by the nucleotide sequences shown in SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 1S, 17, 19, 21, 23, 25, 27, 29, 31, 33, 3S, 37, 39, 41, 80, and 82.
In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID
NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83.
In addition to the human GPCRX nucleotide sequences shown in SEQ ID NOS:I, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 3S, 37, 39, 41, 80, and 82 it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the GPCRX polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the GPCRX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an GPCRX protein, preferably a vertebrate GPCRX protein.
Such natural allelic variations can typically result in 1-S% variance in the nucleotide sequence of the GPCRX genes. Any and all such nucleotide variations and resulting amino acid 2S polymorphisms in the GPCRX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the GPCRX polypeptides, are intended to be within the scope of the invention.
Moreover, 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:1, 3, 4, 6, 8, 10, 12, 14, 1S, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82 are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the GPCRX cDNAs of the invention can be isolated based on their homology to the human GPCRX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ B? NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding GPCRX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in ?0 different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about S
°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target ?5 sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or 30 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. Preferably, the conditions are such that sequences at least about 6S%, 70%, 7S%, 8S%, 90%, 9S%, 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, SO mM Tris-HCl (pH
7.S), 1 mM
S EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and S00 mg/ml denatured salmon sperm DNA
at 6S°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at SO°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, 33, 3S, 37, 39, 41, 80, and 82 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, 33, 3S, 37, 39, 41, 80, and 82 or fragments, analogs or ' 1 S derivatives thereof, under conditions of moderate stringency is provided.
A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, SX
Denhardt's solution, O.S% SDS and 100 mg/ml denatured salmon sperm DNA at SS°C, followed by one or more washes in 1X SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, 33, 3S, 37, 39, 41, 80, and 82 or fragments, analogs or derivatives thereof, 2S under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 3S% formamide, SX SSC, SO mM
Tris-HCl (pH
7.S), S mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 2S
mM Tris-HCl (pH 7.4), S mM EDTA, and 0.1% SDS at SO°C. Other conditions of low stringency that rnay be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND
EXPRESSION, A
LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad S'ci USA 78: 6789-6792.

Conservative Mutations In addition to naturally-occurring allelic variants of GPCRX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by S mutation into the nucleotide sequences of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, 33, 3S, 37, 39, 41, 80, and 82 thereby leading to changes in the amino acid sequences of the encoded GPCRX proteins, without altering the functional ability of said GPCRX proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID
NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83. A
"non-essential"
amino acid residue is a residue that can be altered from the wild-type sequences of the GPCRX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the GPCRX proteins of the invention are predicted to be particularly non-1 S amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding GPCRX
proteins that contain changes in amino acid residues that are not essential for activity. Such GPCRX proteins differ in amino acid sequence from SEQ ID NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83 yet retain biological activity. Tn one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 4S% homologous to the amino acid sequences of SEQ ID NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83. Preferably, the protein encoded by the nucleic acid 2S molecule is at least about 60% homologous to SEQ ID NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83; more preferably at least about 70%
homologous to SEQ ID NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83; still more preferably at least about 80% homologous to SEQ
m NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83; even more preferably at least about 90% homologous to SEQ ID NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40; 42, 81, and 83; and most preferably at least about 9S%
homologous to SEQ ID NOS:2, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83.

An isolated nucleic acid molecule encoding an GPCRX protein homologous to the protein of SEQ m NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82 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, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the GPCRX
protein is replaced with another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an GPCRX
coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GPCRX biological activity to identify mutants that retain activity.
Following mutagenesis of SEQ m NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, ~25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the 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. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY, wherein the letters within each group represent the single letter amino acid code.

In one embodiment, a mutant GPCItX protein can be assayed for (i) the ability to form protein:protein interactions with other GPCItX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant GPC1RX protein and an GPCItX ligand; or (iii) the ability of a mutant GPCItX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g.
avidin proteins).
In yet another embodiment, a mutant GPC1ZX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids Another aspect of the 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:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, or fragments, analogs or derivatives thereof.
An "antisense"
nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or S00 nucleotides or an entire GPC1RX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an GPCRX protein of SEQ 1D NOS:2, 5, 7, 9, 1 I, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, or antisense nucleic acids complementary to an GPCRX
nucleic acid sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an GPCRX
protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the GPCIZX protein. The term "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).
Given the coding strand sequences encoding the GPCRX protein disclosed herein, antisense nucleic acids of the 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 of the coding or noncoding region of GPCRX
mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GPCRX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurnng nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylinethyl) 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'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isoperltenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (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 of the invention are typically administered to a subject or generated ih situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an GPCRX protein to thereby inhibit expression of the 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 of the double helix. An example of a route of administration of antisense 9~

nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., 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.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-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., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a chimeric RNA-DNA analogue (see, e.g., moue, et al., 1987. FEBSLett. 2I5: 327-330.
Ribozymes and PNA Moieties Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozyrnes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave GPCRX
mRNA transcripts to thereby inhibit translation of GPCRX mRNA. A ribozyme having specificity for an GPCRX-encoding nucleic acid can be designed based upon the nucleotide sequence of an GPCRX cDNA disclosed herein (i.e., SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 2I, 23, 25, 27, 29, 3I, 33, 35, 37, 39, 41, 80, and 82). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the 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. and U.S. Patent 5,116,742 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 et al., (1993) Science 261:1411-1418.
Alternatively, GPCRX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the GPCRX nucleic acid (e.g., the GPCRX promoter and/or enhancers) to form triple helical structures that prevent transcription of the GPCRX gene in target cells. See, e.g., Helene, 1991. Ahticahcer Drug Des. 6a 569-84;
Helene, et al. 1992. Anh. N. Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the GPCRX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et 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., S1 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).
In another embodiment, 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. For example, PNA-DNA chimeras of GPCRX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA

chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, 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. For example, 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., Finn, et al., 1996. supra. Alternatively, 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.
In other embodiments, 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.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., I~rol, et al., 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988.
Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be 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.
GPCRX Polypeptides 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, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83.
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, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83 while still encoding a protein that maintains its GPCRX
activities and physiological functions, or a functional fragment thereof.
In general, an GPCRX variant that preserves GPCRX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated GPCRX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-GPCRX
antibodies. In one embodiment, native GPCR.X proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, GPCRX proteins are produced by recombinant DNA techniques.
Alternative to recombinant expression, an GPCRX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the GPCRX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of GPCRX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material" includes preparations of GPCRX proteins having less than about 30% (by dry weight) of non-GPCRX
proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-GPCRX proteins, still more preferably less than about 10% of non-GPCRX
proteins, and most preferably less than about 5% of non-GPCRX proteins. When the GPCRX
protein or biologically-active portion thereof is 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% of the 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 of the protein. In one,embodiment, the language "substantially free of chemical precursors or other chemicals"
includes preparations of GPCRX proteins having less than about 30% (by dry weight) of chemical precursors or non-GPCRX chemicals, more preferably less than about 20% chemical precursors or non-GPCRX chemicals, still more preferably less than about 10% chemical precursors or non-GPCRX chemicals, and most preferably less than about 5% chemical precursors or non-GPC1ZX chemicals.
Biologically-active portions of GPCRX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the GPCRX proteins (e.g., the amino acid sequence shown in SEQ B~ NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83) that include fewer amino acids than the full-length GPCRX proteins, and exhibit at least one activity of an GPCRX protein.
Typically, biologically-active portions comprise a domain or motif with at least one activity of the GPCRX protein. A biologically-active portion of an GPCR.X protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native GPCRX protein.
In an embodiment, the GPCRX protein has an amino acid sequence shown in SEQ ID
NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83. In other embodiments, the GPCRX protein is substantially homologous to SEQ ID
NOS:2, 5, 7, 9, 1 l, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, and retains the functional activity of the protein of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the GPCRX protein is a protein that comprises an amino acid sequence at least about 45%
homologous to the amino acid sequence SEQ ID NOS:2, 5, 7, 9, 1 l, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, and retains the functional activity of the GPCRX
proteins of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83.
Determining Homology Between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (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. JMoI Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension penalty of 0.3, the coding region of the 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 of the DNA sequence shown in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i. e., the window size), and multiplying the result by 100 to yield the percentage of sequence ,0 identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
~5 Chimeric and Fusion Proteins The invention also provides GPCRX chimeric or fusion proteins. As used herein, an GPCRX "chimeric protein" or "fusion protein" comprises an GPCRX polypeptide operatively-linked to a non-GPCRX polypeptide. An "GPCRX polypeptide" refers to a polypeptide 30 having an amino acid sequence corresponding to an GPCRX protein (SEQ ID
NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83), whereas a "non-GPCRX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the GPCRX
protein, e.g., a protein that is different from the GPCRX protein and that is derived from the same or a different organism. Within an GPCRX fusion protein the GPCRX polypeptide can correspond to all or a portion of an GPCRX protein. In one embodiment, an GPCRX fusion protein comprises at least one biologically-active portion of an GPCRX protein. In another embodiment, an GPCRX fusion protein comprises at least two biologically-active portions of an GPCRX protein. In yet another embodiment, an GPCRX fusion protein comprises at least three biologically-active portions of an GPCRX protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the GPCRX polypeptide and the non-GPCRX
polypeptide are fused in-frame with one another. The non-GPCRX polypeptide can be fused to the N-terminus or C-terminus of the GPCRX polypeptide.
In one embodiment, the fusion protein is a GST-GPCRX fusion protein in which the GPCRX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant GPCRX
polypeptides.
In another embodiment, the fusion protein is an GPCRX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of GPCRX can be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is an GPCRX-immunoglobulin fusion protein in which the GPCRX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The GPCRX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an GPCRX ligand and an GPCRX protein on the surface of a cell, to thereby suppress GPCRX-mediated signal transduction in vivo. The GPCRX-immunoglobulin fusion proteins can be used to affect the bioavailability of an GPCRX
cognate ligand. Inhibition of the 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. Moreover, the GPCRX-immunoglobulin fusion proteins of the 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 of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carned 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). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An GPCRX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GPCRX protein.
GPCRX Agonists and Antagonists The invention also pertains to variants of the GPCRX proteins that function as either GPCRX agonists (i.e., mimetics) or as GPCRX antagonists. Variants of the GPCRX
protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the GPCRX
protein). An agonist of the GPCRX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the GPC1RX
protein. An antagonist of the GPCRX protein can inhibit one or more of the activities of the naturally occurring form of the GPCRX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the GPCRX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the GPCRX proteins.
Variants of the 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) of the GPCRX proteins for GPCRX protein agonist or antagonist activity. In one embodiment, a variegated library of GPCRX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of 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. There are a variety of methods which can be used to produce libraries of potential GPCRX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the 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.
Tet~ahedroh 39: 3;
Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984.
Science 198: 1056;
Ike, et al., 1983. Nuel. Acids Res. 11: 477.
Polypeptide Libraries In addition, libraries of fragments of the 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. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an GPCRX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the GPCRX
proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and fox screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of GPCRX proteins.
The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify GPCRX variants.

See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815;
Delgrave, et al., 1993. Protein Ehgihee~ing 6:327-331.
Anti-GPCRX Antibodies The invention encompasses antibodies and antibody fragments, such as Fab or (Fab)z>
that bind immunospecifically to any of the GPCRX polypeptides of said invention.
An isolated GPCRX protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind to GPCRX polypeptides using standard techniques for polyclonal and monoclonal antibody preparation. The full-length GPCRX
proteins can be used or, alternatively, the invention provides antigenic peptide fragments of GPCRX proteins for use as immunogens. The antigenic GPCRX peptides comprises at least 4 amino acid residues of the amino acid sequence shown in SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83 and encompasses an epitope of GPCRX such that an antibody raised against the peptide forms a specific immune complex with GPCRX. Preferably, the antigenic peptide comprises at least 6, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on use and according to methods well known to someone skilled in the art.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of GPCRX that is located on the surface of the protein (e.g., a hydrophilic region). As a means 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 (see, e.g., Hopp and Woods, 1981. P~oc. Nat.
Acad. Sci. USA
78: 3824-3828; Kyte and Doolittle, 1982. J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety).
As disclosed herein, GPCRX protein sequences of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, or derivatives, fragments, analogs or homologs thereof, may be utilized as immunogens in the generation of antibodies that immunospecifically-bind these protein components. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i. e., molecules that contain an antigen binding site that specifically-binds (immunoreacts with) an antigen, such as GPCRX. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab and F~ab~~a fragments, and an Fab expression library. In a specific embodiment, antibodies to human GPCRX
proteins are disclosed. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to an GPCRX protein sequence of SEQ ID, NOS:2, 5, 7, 9, 1 l, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, or a derivative, fragment, analog or homolog thereof. Some of these proteins are discussed below.
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed GPCRX protein or a chemically-synthesized GPCRX polypeptide. The preparation can fiarther include an adjuvant.
Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Coryhebacterium parvum, or similar immunostimulatory agents. If desired, the antibody molecules directed against GPCRX can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG
fraction.
The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of GPCRX. A
monoclonal antibody composition thus typically displays a single binding affinity for a particular GPCRX
protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular GPCRX protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line ~ culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975. Natu~~e 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see, e.g., Kozbor, et al., 1983. Immunol.
Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. L1SS, Inc., pp. 77-96).
Human monoclonal antibodies may be utilized in the practice of the invention and may be produced by using human hybridomas (see, e.g., 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, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.
Liss, Inc., pp. 77-96). Each of the above citations is incorporated herein by reference in their entirety.

According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an GPCRX protein (see, e.g., U.S. Patent No. 4,946,778).
In addition, methods can be adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., 1989. Science 246: 1275-1281) to allow rapid and effective identification of S monoclonal Fab fragments with the desired specificity for an GPCRX protein or derivatives, fragments, analogs or homologs thereof. Non-human antibodies can be "humanized" by techniques well known in the art. See, e.g., U.S. Patent No. 5,225,539.
Antibody fragments that contain the idiotypes to an GPCRX protein may be produced by techniques known in the art including, but not limited to: (i) an F~ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F~ab~)a fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent; and (iv) F" fragments.
Additionally, recombinant anti-GPCRX antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made 1 S using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA
techniques known in the art, for example using methods described in International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT
International Publication No. WO 86/01533; U.S. Patent No. 4,816,567; U.S. Pat. No.
5,225,539; European Patent Application No. 125,023; Better, et al., 1988. Science 240: 1041-1043;
Liu, et al., 1987.
Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139:
3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987.
Cancer Res. 47:
999-1005; Wood, et al., 1985. Nature 314 :446-449; Shaw, et al., 1988. J.
Natl. CanceY Inst.
2S 80: 1553-1559); Morrison(1985) Science 229:1202-1207; Oi, et al. (1986) BioTechniques 4:214; Jones, et al., 1986. Nature 321: SS2-S2S; Verhoeyan, et al., 1988.
Science 239: 1534;
and Beidler, et al., 1988. J. Immunol. 141: 4053-4060. Each of the above citations are incorporated herein by reference in their entirety.
In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an GPCRX
protein is facilitated by generation of hybridomas that bind to the fragment of an GPCRX
protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an GPCRX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-GPCRX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an GPCRX protein (e.g., for use in measuring levels of the GPCRX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for GPCRX
proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
An anti-GPCRX antibody (e.g., monoclonal antibody) can be used to isolate an GPCRX polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-GPCRX antibody can facilitate the purification of natural GPCRX polypeptide from cells and of recombinantly-produced GPCRX polypeptide expressed in host cells. Moreover, an anti-GPCRX antibody can be used to detect GPCRX
protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the 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.
Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include lash isy' sss or 3H.
GPCRX Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an GPCRX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a S bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
1 S The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences) in a manner that allows for expression of the 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).
The term "regulatory sequence" is intended to includes promoters, enhancers and other 2S 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 of the 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 of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusibn proteins or peptides, encoded by nucleic acids as described herein (e.g., GPCRX
proteins, mutant forms of GPCRX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of GPCRX proteins in prokaryotic or eukaryotic cells. For example, GPCRX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed fiuther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the 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. Gene 67: 31-40), pMAL
(New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET l 1d (Studier 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 of the 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 of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the GPCRX expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et 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 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, GPCRX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, et al., 1987. EMBO
1 S J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, 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.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the 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 axe known in the art. Non-limiting examples of 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:
235-275), in particular 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. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the marine hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the 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 of the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA
as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer ZO 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 of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, GPCRX
protein ~5 can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and 30 "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et 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.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as 6418, 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 of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) GPCRX protein. Accordingly, the invention further provides methods for producing GPCRX protein using the host cells of the invention. In 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.
Transgenic GPCRX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which GPCRX protein-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic animals in which exogenous GPCRX sequences have been introduced into their genome or homologous recombinant animals in which endogenous GPCRX sequences have been altered. Such animals are useful for studying the function and/or activity of GPCRX protein and for identifying and/or evaluating modulators of GPCRX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the 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 of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous GPCRX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the 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 human GPCRX cDNA sequences of SEQ ID NOS:l, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human GPCRX
gene, such as a mouse GPCRX gene, can be isolated based on hybridization to the human GPCRX
cDNA (described fixrther supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequences) 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. In:
MA~PUL,ATnm 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 of the GPCRX transgene in its genome and/or expression of GPCRX mRNA in tissues or cells of the animals. A
transgenic founder animal can then be used to breed additional animals carrying the transgene.
Moreover, transgenic animals carrying a transgene-encoding GPCRX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an GPCRX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GPCRX gene. The GPCRX gene can be a human gene (e.g., the cDNA of SEQ ID NOS:l, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82), but more preferably, is a non-human homologue of a human GPCRX gene. For example, a mouse homologue of human GPCRX gene of SEQ
ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82 can be used to construct a homologous recombination vector suitable for altering an endogenous GPCRX gene in the mouse genome. In one embodiment, 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).
Alternatively, 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). In the homologous recombination vector, the altered portion of the GPCRX gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the GPCRX gene to allow for homologous recombination to occur between the exogenous GPCRX gene carried by the vector and an endogenous GPCRX gene in an embryonic stem cell. The additional flanking GPCRX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of homologous recombination vectors.
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 a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 19$7. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152.
A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechhol. 2: 823-829; PCT
International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P 1. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Aced.
Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase system of 11$

Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Sciehce 251:1351-1355.
If a crelloxP
recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the 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 of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions The GPCRX nucleic acid molecules, GPCRX proteins, and anti-GPCRX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such Garners 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 of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (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.
The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under ?0 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 >5 the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifimgal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the 30 composition. Prolonged absorption of the 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. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable S solutions, 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.
They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic l0 administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, L S troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppernzint, ?0 methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For ?S transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into 30 ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories .(e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydndes, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
Patent No. 4,522,811.
It is especially advantageous to formulate oral or paxenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated;
each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical Garner. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Aced. Sci.
USA 91: 3054-3057).
The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Screening and Detection Methods The isolated nucleic acid molecules of the invention can be used to express GPCRX
protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect GPCRX mRNA (e.g., in a biological sample) or a genetic lesion in an GPC1RX gene, and to modulate GPCRX activity, as described further, below. In addition, the GPCRX
proteins can be used to screen drugs or compounds that modulate the GPCRX
protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of GPCRX protein or production of GPCItX protein forms that have decreased or aberrant activity compared to GPC1ZX wild-type protein (e.g.; diabetes (regulates insulin release);
obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-GPCRX antibodies of the invention can be used to detect and isolate GPCRX proteins and modulate GPCRX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
~0 Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to GPCRX proteins or have a stimulatory or inhibitory effect on, e.g., GPCRX protein expression or GPCRX
protein ?5 activity. The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of an GPC1ZX protein or polypeptide or biologically-active portion thereof. The test compounds of 30 the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909;
Erb, et al., 1994.
Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med.
Chem. 37: 2678;
Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int.
Ed. Engl. 33:
2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J.
Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of GPCRX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to an GPCRX protein determined. The cell, for example, can of mammalian origin or a yeast cell.
Determining the ability of the test compound to bind to the GPCRX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the GPCRX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with lzsh 35s~ 14C~ or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of GPCRX
protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds GPCRX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an GPCRX
protein, wherein determining the ability of the test compound to interact with an GPCRX
protein comprises determining the ability of the test compound to preferentially bind to GPCRX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of GPCRX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GPCRX
protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of GPCRX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the GPCRX protein to bind to or interact with an GPCRX target molecule. As used herein, a "target molecule" is a molecule with which an GPCRX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an GPCRX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An GPCRX target molecule can be a non-GPCRX
molecule or an GPCRX protein or polypeptide of the invention. In one embodiment, an GPCRX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound GPCRX molecule) through the cell membrane and into the cell.
The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with GPCRX.
Determining the ability of the GPCRX protein to bind to or interact with an GPCRX
target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the GPCRX
protein to bind to or interact with an GPCRX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the taxget molecule can be determined by detecting induction of a cellular second messenger of the target (i. e.
intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising an GPCRX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting an GPCRX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the GPCRX protein or biologically-active portion thereof. Binding of the test compound to the GPCRX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the GPCRX protein or biologically-active portion thereof with a known compound which binds GPCRX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an GPCRX
protein, wherein determining the ability of the test compound to interact with an GPCRX
protein comprises determining the ability of the test compound to preferentially bind to GPCRX or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting GPCRX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the GPCRX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of GPCRX can be accomplished, for example, by determining the ability of the GPCRX protein to bind to an GPCRX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of GPCRX
protein can be accomplished by determining the ability of the GPCRX protein further modulate an GPCRX target molecule. For example, the catalyticlenzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the GPCRX
protein or biologically-active portion thereof with a known compound which binds GPCRX
protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an GPCRX
protein, wherein determining the ability of the test compound to interact with an GPCRX protein comprises determining the ability of the GPCRX protein to preferentially bind to or modulate the activity of an GPCRX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of GPCRX protein. In the case of cell-free assays comprising the membrane-bound form of GPCRX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of GPCRX protein is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylinaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-I00, Triton~ X-I 14, Thesit~, Isotridecypoly(ethylene glycol ether)n, N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either GPCRX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to GPCRX protein, or interaction of GPCRX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-GPCRX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or GPCRX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra.
Alternatively, the complexes can be dissociated from the matrix, and the level of GPCRX
protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the GPCRX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated GPCRX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with GPCRX
protein or target molecules, but which do not interfere with binding of the GPCRX protein to its target molecule, can be derivatized to the wells of the plate, and unbound taxget or GPCRX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the GPCRX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity S associated with the GPCRX protein or target molecule.
In another embodiment, modulators of GPCRX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of GPCRX
mRNA or protein in the cell is determined. The level of expression of GPCRX
mRNA or protein in the presence of the candidate compound is compared to the level of expression of GPCRX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of GPCRX mRNA or protein expression based upon this comparison. For example, when expression of GPCRX mRNA or protein is greater (i. e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GPCRX
mRNA or protein expression. Alternatively, when expression of GPCRX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GPCRX mRNA or protein expression.
The level of GPCRX mRNA or protein expression in the cells can be determined by methods described herein for detecting GPCRX mRNA or protein.
In yet another aspect of the invention, the GPCRX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem.
268: 12046-12054;
Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
~rZevgene 8:
1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with GPCRX ("GPCRX-binding proteins" or "GPCRX-by") and modulate GPCRX activity.
Such GPCRX-binding proteins are also likely to be involved in the propagation of signals by the GPCRX proteins as, for example, upstream or downstream elements of the GPCRX
pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for GPCRX
is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to interact, in vivo, forming an GPCRX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with GPCRX.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the GPCRX sequences, SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, or fragments or derivatives thereof, can be used to map the location of the GPCRX genes, respectively, on a chromosome. The mapping of the GPCRX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, GPCRX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 by in length) from the GPCRX sequences. Computer analysis of the GPCRX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes.
Only those hybrids containing the human gene corresponding to the GPCRX
sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al., L 0 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using L 5 a single thermal cycler. Using the GPCRX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence ih situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in '0 metaphase by a chemical like colcemid that disrupts the mitotic spindle.
The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to a unique ~5 chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN
CHROMOSOMES: A
MANUAL OF BASIC TECHt~IIIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single 30 chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adj scent genes), described in, e.g., Egeland, et al., 1987.
Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the GPCRX gene, can be determined.
If a mutation L 0 is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing The GPCRX sequences of the invention can also be used to identify individuals from ?0 minute biological samples. In this technique, an individual's genomic DNA
is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA
markers for RFLP
("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative ?5 technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the GPCRX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, 30 can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The GPCRX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (ItFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining GPCRX
protein and/or nucleic acid expression as well as GPCRX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant GPCRX
expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with GPCRX protein, nucleic acid expression or activity. For example, mutations in an GPCRX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with GPCItX protein, nucleic acid expression, or biological activity.

WO 01/74904 PCT/USO1/10241.,33" ~",u ~r,;;;, ~~»ttn Another aspect of the invention provides methods for determining GPCRX
protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
Pharmacogenomics allows fox the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of GPCRX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays An exemplary method for detecting the presence or absence of GPCRX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting GPCRX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes GPCRX protein such that the presence of GPCRX
is detected in the biological sample. An agent for detecting GPCRX mRNA or genomic DNA
is a labeled nucleic acid probe capable of hybridizing to GPCRX mRNA or genomic DNA.
The nucleic acid probe can be, for example, a full-length GPCRX nucleic acid, such as the nucleic acid of SEQ m NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to GPCRX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
An agent for detecting GPCRX protein is an antibody capable of binding to GPCRX
protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')Z) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect GPCRX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
For example, ih vitro techniques for detection of GPCRX mRNA include Northern hybridizations and ih situ hybridizations. In vitro techniques for detection of GPCRX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of GPCR.X genomic DNA
include Southern hybridizations. Furthermore, in vivo techniques for detection of GPCRX protein include introducing into a subject a labeled anti-GPCRX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting GPCRX protein, mRNA, or genomic DNA, such that the presence of GPCRX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of GPCRX protein, mRNA or genomic DNA in the control sample with the presence of GPCRX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of GPCRX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting GPCRX protein or mRNA in a biological sample; means for determining the amount of GPCRX in the sample; and means for comparing the amount of GPCRX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect GPCRX protein or nucleic acid.
Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant GPCRX
expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subj ect having or at risk of developing a disorder associated with GPCRX protein, nucleic acid expression or activity.

Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant GPCRX expression or activity in which a test sample is obtained from a subject and GPCRX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of GPCRX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant GPCRX
expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant GPCRX expression or activity. For example, such methods can be used to determine whether a subj ect can be effectively treated with an agent for a disorder.
Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant GPCRX expression or activity in which a test sample is obtained and GPCRX protein or nucleic acid is detected (e.g., wherein the presence of GPCRX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant GPCRX
expression or activity).
The methods of the invention can also be used to detect genetic lesions in an GPCRX
gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subj ect, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an GPCRX-protein, or the misexpression of the GPCRX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an GPCRX gene; (ii) an addition of one or more nucleotides to an GPCRX gene; (iii) a substitution of one or more nucleotides of an GPCRX
gene, (iv) a chromosomal rearrangement of an GPCRX gene; (v) an alteration in the level of a messenger RNA transcript of an GPCRX gene, (vi) aberrant modification of an GPCRX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of an GPCRX gene, (viii) a non-wild-type level of an GPCRX protein, (ix) allelic loss of an GPCRX gene, and (x) inappropriate post-translational modification of an GPCRX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an GPCRX gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994.
Proc. Natl.
Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the GPCRX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682).
This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an GPCRX gene under conditions such that hybridization and amplification of the GPCRX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR andlor LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177);
Q(3 Replicase (see, Lizardi, et al, 1988. BioTechhology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an GPCRX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared.
Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in GPCRX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic S mutations in GPCRX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the GPCRX gene and detect mutations by comparing the sequence of the sample GPCRX with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl.
Acad. Sci. USA
74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995.
Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the GPCRX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type GPCRX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc. Natl.
Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in GPCRX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli l0 cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662.
According to an exemplary embodiment, a probe based on an GPCRX sequence, e.g., a wild-type GPCRX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be l5 detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in GPCRX genes. Fox example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86:
2766; Cotton, ?0 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9:
73-79.
Single-stranded DNA fragments of sample and control GPCRX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with 'S labeled probes. The sensitivity of the assay may be enhanced by using RNA
(rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility.
See, e.g., Keen, et al., 1991. Trends Geraet. 7: 5.
30 In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 by of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al., 1986. Nature 324: 163;
Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides l0 are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR
amplif cation may be used in conjunction with the instant invention.
Oligonucleotides used as L S primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11: 238). In addition it may be desirable to introduce a novel '0 restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of ~5 a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an GPCRX
30 gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which GPCRX is expressed may be utilized in the prognostic assays described herein.
However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on GPCRX
activity (e.g., GPC1ZX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parl~inson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of GPCRX
protein, expression of GPCRX nucleic acid, or mutation content of GPCRX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clih. Exp. Pharmacol. Physiol., 23: 983-985; Linder, 1997. Clin.
, Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taleing the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYPZD6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM
show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite mozphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of GPCRX protein, expression of GPCRX nucleic acid, or mutation content of GPCRX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subj ect with an GPCRX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of GPCRX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase GPCRX gene expression, protein levels, or upregulate GPCRX activity, can be monitored in clinical trails of subjects exhibiting decreased GPCRX gene expression, protein levels, or downregulated GPCRX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease GPCRX gene expression, protein levels, or downregulate GPCRX activity, can be monitored in clinical trails of subjects exhibiting increased GPCRX gene expression, protein levels, or upregulated GPCRX
activity. In such clinical trials, the expression or activity of GPCRX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including GPCRX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates GPCRX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of GPCRX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of GPCRX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an GPCRX protein, mRNA, or genomic DNA in the preadministration sample;
(iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the GPCRX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the GPCRX
protein, mRNA, or genomic DNA in the pre-administration sample with the GPCRX
protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. Fox example, increased administration of the agent may be desirable to increase the expression or activity of GPCRX
to higher levels than detected, i.e., to increase the effectiveness of the agent.
Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of GPCRX to lower levels than detected, i.e., to decrease the effectiveness of the agent.

Methods of Treatment The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant GPCRX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AmS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.
These methods of treatment will be discussed more fully, below.
Disease and Disorders Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endoggenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it iu vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant GPCRX expression or activity, by administering to the subject an agent that modulates GPCRX expression or at least one GPCRX
activity. Subjects at risk for a disease that is caused or contributed to by aberrant GPCRX
expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the GPCRX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of GPCRX
aberrancy, for example, an GPCRX agonist or GPCRX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections.
Therapeutic Methods Another aspect of the invention pertains to methods of modulating GPCRX
expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of GPCRX protein activity associated with the cell. An agent that modulates GPCRX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of an GPCRX protein, a peptide, an GPCRX peptidomimetic, or other small molecule.
In one embodiment, the agent stimulates one or more GPCRX protein activity.
Examples of such stimulatory agents include active GPCRX protein and a nucleic acid molecule encoding GPCRX that has been introduced into the cell. In another embodiment, the agent inhibits one or more GPCRX protein activity. Examples of such inhibitory agents include antisense GPCIZX nucleic acid molecules and anti-GPCIZX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an GPCRX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) LO GPCRX expression or activity. In another embodiment, the method involves administering an GPCRX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant GPCRX expression or activity.
Stimulation of GPCRX activity is desirable in situations in which GPCRX is abnormally downregulated and/or in which increased GPCRX activity is likely to have a LS beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic ~0 In various embodiments of the invention, suitable in vitro or ih vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.
In various specific embodiments, ira vitro assays may be performed with representative cells of the types) involved in the patient's disorder, to determine if a given Therapeutic exerts ?5 the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subj ects.
Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subj ects.
30 Prophylactic and Therapeutic Uses of the Compositions of the Invention The GPCRX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
As an example, a cDNA encoding the GPCRX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof.
By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hernatopoietic disorders, and the various dyslipidemias.
Both the novel nucleic acid encoding the GPCRX protein, and the GPCRX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A
further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
EQUIVALENTS
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims.

Claims (52)

WHAT IS CLAIMED IS:
1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83;

(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;

(c) an amino acid sequence selected from the group consisting of SEQ m NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83;
and (d) a variant of an amino acid sequence selected from the group consisting of SEQ
m NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence.
2 The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83.
3. The polypeptide of claim 2, wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82.
4. The polypeptide of claim 1, wherein the amino acid sequence of said variant comprises a conservative amino acid substitution.
5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83;

(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;

(c) an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83;

(d) a variant of an amino acid sequence selected from the group consisting SEQ
ID
NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence;

(e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence chosen from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence; and (f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).
6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant.
7. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occuring polypeptide variant.
8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82.
9. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82;

(b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, provided that no more than 20% of the nucleotides differ from said nucleotide sequence;

(c) a nucleic acid fragment of (a); and (d) a nucleic acid fragment of (b).
10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence chosen from the group consisting of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 80, and 82, or a complement of said nucleotide sequence.
11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

(a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20% of the nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence;

(b) an isolated second polynucleotide that is a complement of the first polynucleotide; and (c) a nucleic acid fragment of (a) or (b).
12. A vector comprising the nucleic acid molecule of claim 11.
13. The vector of claim 12, further comprising a promoter operably-linked to said nucleic acid molecule.
14. A cell comprising the vector of claim 12.
15. An antibody that binds immunospecifically to the polypeptide of claim 1.
16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.
17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:

(a) providing the sample;

(b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and (c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.
19. A method for determining the presence or amount of the nucleic acid molecule of claim 5 in a sample, the method comprising:

(a) providing the sample;

(b) contacting the sample with a probe that binds to said nucleic acid molecule; and (c) determining the presence or amount of the probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.
20. The method of claim 19 wherein presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type.
21. The method of claim 20 wherein the cell or tissue type is cancerous.
22. A method of identifying an agent that binds to a polypeptide of claim l, the method comprising:

(a) contacting said polypeptide with said agent; and (b) determining whether said agent binds to said polypeptide.
23. The method of claim 22 wherein the agent is a cellular receptor or a downstream effector.
24. A method for identifying an agent that modulates the expression or activity of the polypeptide of claim 1, the method comprising:

(a) providing a cell expressing said polypeptide;

(b) contacting the cell with said agent, and (c) determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide.
25. A method for modulating the activity of the polypeptide of claim 1, the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity of the polypeptide.
26. A method of treating or preventing a GPCRX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the polypeptide of claim 1 in an amount sufficient to treat or prevent said GPCRX-associated disorder in said subject.
27. The method of claim 26 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis.
28. The method of claim 26 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
29. The method of claim 26, wherein said subject is a human.
30. A method of treating or preventing a GPCRX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the nucleic acid of claim 5 in an amount sufficient to treat or prevent said GPCRX-associated disorder in said subject.
31. The method of claim 30 wherein the disorder is selected from the group consisting of caxdiomyopathy and atherosclerosis.
32. The method of claim 30 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
33. The method of claim 30, wherein said subject is a human.
34. A method of treating or preventing a GPCRX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the antibody of claim 15 in an amount sufficient to treat or prevent said GPCRX-associated disorder in said subject.
35. The method of claim 34 wherein the disorder is diabetes.
36. The method of claim 34 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
37. The method of claim 34, wherein the subject is a human.
38. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically-acceptable carrier.
39. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically-acceptable Garner.
40. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically-acceptable carrier.
41. A kit comprising in one or more containers, the pharmaceutical composition of claim 38.
42. A kit comprising in one or more containers, the pharmaceutical composition of claim 39.
43. A kit comprising in one or more containers, the pharmaceutical composition of claim 40.
44. A method for determining the presence of or predisposition to a disease associated with altered levels of the polypeptide of claim 1 in a first mammalian subject, the method comprising:

(a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and (b) comparing the amount of said polypeptide in the sample of step (a) to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease;

wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.
45. The method of claim 44 wherein the predisposition is to cancers.
46. A method for determining the presence of or predisposition to a disease associated with altered levels of the nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising:

(a) measuring the amount of the nucleic acid in a sample from the first mammalian subj ect; and (b) comparing the amount of said nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease;
wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
47. The method of claim 46 wherein the predisposition is to a cancer.
48. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising an amino acid sequence of at least one of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, or a biologically active fragment thereof.
49. A method of treating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.
50. A method for the screening of a candidate substance interacting with an olfactory receptor polypeptide selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, or fragments or variants thereof, comprises the following steps:

a) providing a polypeptide selected from the group consisting of the sequences of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, or a peptide fragment or a variant thereof;

b) obtaining a candidate substance;

c) bringing into contact said polypeptide with said candidate substance;

and d) detecting the complexes formed between said polypeptide and said candidate substance.
51. A method for the screening of ligand molecules interacting with an olfactory receptor polypeptide selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, wherein said method comprises:

a) providing a recombinant eukaryotic host cell containing a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptides comprising the amino acid sequences SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83;

b) preparing membrane extracts of said recombinant eukaryotic host cell;

c) bringing into contact the membrane extracts prepared at step b) with a selected ligand molecule; and d) detecting the production level of second messengers metabolites.
52. A method for the screening of ligand molecules interacting with an olfactory receptor polypeptide selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 1 l, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83, wherein said method comprises:

a) providing an adenovirus containing a nucleic acid encoding a polypeptide selected from the group consisting of polypeptides comprising the amino acid sequences SEQ ID NOS:2, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 81, and 83;

b) infecting an olfactory epithelium with said adenovirus;

c) bringing into contact the olfactory epithelium b) with a selected ligand molecule; and d) detecting the increase of the response to said ligand molecule.
CA002404541A 2000-03-31 2001-03-30 G-protein coupled receptors and nucleic acids encoding same Abandoned CA2404541A1 (en)

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US23961300P 2000-10-11 2000-10-11
US60/239,613 2000-10-11
US26250801P 2001-01-18 2001-01-18
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US26516101P 2001-01-30 2001-01-30
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US7361338B2 (en) 1999-10-05 2008-04-22 Agensys, Inc. Methods to inhibit growth of prostate cancer cells
US7208280B2 (en) 1999-10-05 2007-04-24 Agensys, Inc. Nucleic acid and corresponding protein entitled 101P3A41 useful in treatment and detection of cancer
AU2001246526A1 (en) * 2000-04-05 2001-10-23 Bayer Aktiengesellschaft Regulation of human hm74-like g protein coupled receptor
WO2002050117A2 (en) * 2000-12-18 2002-06-27 Curagen Corporation G-proteins coupled receptor proteins and nucleic acids encoding same
US20030171541A1 (en) * 2001-02-14 2003-09-11 Amgen Inc., A Corporation Of The State Of Delaware G-protein coupled receptor molecules and uses thereof
JP2005512509A (en) * 2001-05-15 2005-05-12 アジェンシス,インコーポレイテッド Nucleic acids and corresponding proteins given the names 101P3A11 or PHOR-1 useful in the treatment and detection of cancer
US6902902B2 (en) 2001-11-27 2005-06-07 Arena Pharmaceuticals, Inc. Human G protein-coupled receptors and modulators thereof for the treatment of metabolic-related disorders
US7189524B1 (en) 2002-11-25 2007-03-13 Amgen, Inc. Receptor ligands and methods of modulating receptors
US7893197B2 (en) 2004-08-25 2011-02-22 Janssen Pharmaceutica N.V. Relaxin-3 chimeric polypeptides and their preparation and use
GB201513921D0 (en) 2015-08-05 2015-09-23 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against prostate cancer and other cancers

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WO2001036473A2 (en) * 1999-11-16 2001-05-25 Pharmacia & Upjohn Company Human g protein-coupled receptors
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