Novel G protein-coupled receptors

The present invention provides a gene encoding a G protein-coupled receptor termed nGPCR-1025; constructs and recombinant host cells incorporating the genes; the nGPCR-1025 polypeptides encoded by the gene; antibodies to the nGPCR-1025 polypeptides; and methods of making and using all of the foregoing.

EXAMPLES 
 Example 1 
 Identification of nGPCR-1025 A. Database Search The Celera database was searched using known GPCR receptors as query sequences to find patterns suggestive of novel G protein-coupled receptors. Positive hits were further analyzed with the GCG program BLAST to determine which ones were the most likely candidates to encode G protein-coupled receptors, using the standard (default) alignment produced by BLAST as a guide. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410, which is incorporated herein by reference in its entirety). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M&equals;5, N&equals;4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a GPCR gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a GPCR nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Homology searches are performed with the program BLAST version 2.08. A collection of 340 query amino acid sequences derived from GPCRs was used to search the genomic DNA sequence using TBLASTN and alignments with an E-value lower than 0.01 were collected from each BLAST search. The amino acid sequences have been edited to remove regions in the sequence that produce non-significant alignments with proteins that are not related to GPCRs. Multiple query sequences may have a significant alignment to the same genomic region, although each alignment may not cover exactly the same DNA region. A procedure is used to determine the region of maximum common overlap between the alignments from several query sequences. This region is called the consensus DNA region. The procedure for determining this consensus involves the automatic parsing of the BLAST output files using the program MSPcrunch to produce a tabular report. From this tabular report the start and end of each alignment in the genomic DNA is extracted. This information is used by a PERL script to derive the maximum common overlap. These regions are reported in the form of a unique sequence identifier, a start and the end position in the sequence. The sequences defined by these regions were extracted from the original genomic sequence file using the program fetchdb. The consensus regions are assembled into a non-redundant set by using the program phrap. After assembly with phrap a set of contigs and singletons were defined as candidate DNA regions coding for nGPCRs. These sequences were then submitted for further sequence analysis. Further sequence analysis involves the removal of sequences previously isolated and removal of sequences that are related to olfactory GPCR's. nGPCR-1025 cDNAs were sequenced directly using an ABI377 fluorescence-based sequencer (Perkin-Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI PRISM™ Ready Dye-Deoxy Terminator kit with Taq FS™ polymerase. Each ABI cycle sequencing reaction contained about 0.5 &mgr;g of plasmid DNA. Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 minute, followed by 50 cycles using the following parameters: 98° C. for 30 seconds, annealing at 50° C. for 30 seconds, and extension at 60° C. for minutes. Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler. Extension products were purified using Centriflex™ gel filtration cartridges (Advanced Genetic Technologies Corp., Gaithersburg, Md.). Each reaction product was loaded by pipette onto the column, which is then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 minutes at room temperature. Column-purified samples were dried under vacuum for about 40 minutes and then dissolved in 5 &mgr;l of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran). The samples were then heated to 90° C. for three minutes and loaded into the gel sample wells for sequence analysis using the ABI377 sequencer. Sequence analysis was performed by importing ABI377 files into the Sequencer program (Gene Codes, Ann Arbor, Mich.). Generally, sequence reads of 700 bp were obtained. Potential sequencing errors were minimized by obtaining sequence information from both DNA strands and by re-sequencing difficult areas using primers annealing at different locations until all sequencing ambiguities were removed. The following Table 5 contains the sequences of the polynucleotides and polypeptides of the invention. The transmembrane domains within the polypeptide sequence are identified by underlining, and start and stop sites are identified by bold text. 5 TABLE 5 The following DNA sequence nGPCR-1025 <SEQ ID NO. 1> was identified in H. sapiens : TTCCTTCCCGCGCTCGCTCGCCCGGCCCCCAGCCCCCTCATGAGGGTGTCCGTGCCGGGTCCGGCGG CCGCTGCCGCCCCCGCAGCCGGCCGCGAGCCCTCCACGCCCGGCGGGGGCAGCGGAGGCGGAGGCGC CGTCGCTGCAGCCTCAGGCGCCGCGGTGCCGGGCTCCGTGCAGTTGGCGCTGAGCGTCCTGCACGCC CTGCTCTACGCCGCGCTGTTCGCCTTTGCCTACCTGCAGCTGTGGCGGCTGCTCCTGTACCGCGAGC GGCGGCTGAGTTACCAGAGCCTCTGCCTCTTCCTCTGTCTCCTGTGGGCAGCGCTCAGGACCACCCT CTTCTCCGCCGCCTTCTCGCTCAGCGGCTCCCTGCCCTTGCTCCGGCCGCCCGCTCACCTGCACTTC TTCCCCCACTGGCTGCTCTACTGCTTCCCCTCCTGTCTCCAGTTCTCCACGCTCTGTCTCCTCAACC TCTACCTGGCGGAGGTTATATGTAAAGTCAGATGTGCCACTGAACTTGACAGACACAAAATTCTACT GCATTTGGGCTTTATAATGGCAAGCCTGCTCTTTTTAGTGGTGAACTTGACTTGCGCAATGCTAGTT CATGGAGATGTCCCAGAAAATCAGTTGAAGTGGACTGTGTTTGTTCGAGCATTAATTAATGATAGCC TGTTTATTCTTTGTGCCATCTCTTTAGTGTGTTACATATGCAAAATTACAAAAATGTCATCAGCTAA TGTCTACCTCGAATCAAAGGGTATGTCTCTGTGCCAGACTGTCGTCGTGGGCTCTGTAGTCATTCTT CTGTACTCTTCCAGAGCTTGTTATAATTTGGTGGTGGTCACCATATCTCAGGATACATTAGAAAGTC CATTTAATTATGGCTGGGATAATCTTTCAGATAAGGCTCATGTAGAAGACATAAGTGCAGAAGAGTA TATAGTATTTGGAATGGTCCTCTTTCTGTGGGAACATGTGCCAGCATGGTCGGTGGTACTGTTTTTC CGGGCACAGAGATTAAACCAGAATTTCGCACCTGCTGGCATGATAAATAGTCACAGTTATAGTTCCA GAGCTTACTTTTTCGACAATCCAAGACGATATGATAGTGATGATGACCTGCCAGGACTGGGAAGTTC AAGAGAAGGAAGTTTACCAAATTCGCAAAGTTTGCGCTGGTATGGCACCATGACTGGGTGTGGCAGC AGCAGTTACACAGTCACTCCCCACCTGAATGGACCTATGACAGATACTGCTCCTTTGCTCTTTACTT GTAGTAATTTAGATTTGAACAAICATCATAGCTTATATGTGACACCACAAAACTGACAGCATCACCA AGTCATGATTCTTGAGTTGTTTTTCATAAATGTGTATATTCAATGTGTTTAAATTCCATCTACATAA ACATTCCATTATCTGTTGCAACTGAAAACAAAATCTGGAAGTGTGGCTGTGTTTGGTAAATAACACA GCTATTATTTTTGACCTCTTCATAGTAAAATGAAGTAAAATGGAAAGTTTGGAGTAGGAGAAAAGAG AGATTAGATCTTAAGGCACTTGATGGCCTCCAAAAATCCTGACTTTGGAACATCAAATGCATATGTG CACTTTTATCTTTGTTCTGAGTCACTGCAGTCCCCAAAGTCATATGCCAATGTTCACACTGAAATAC TGTATTGTACACCAAACTGGAAGGCAATTTTCCTATCAAAATCAAAGCCGGTATATTCATTGGTATG CTCTATACAGATATCTTAATAAAAATTTTATAGTGTGAACAGTGCACAGAGTTAAGGCATAAAAATG TATCATTCTTTATAAAATCTACTGAAAATGTGTAATCATTGAAGACAGTTCTTTTAAGCATGATTT TAAAATAGCAACTGAAATTCAATCATTTTAAACAAATGATGGTAGTAATCCATTAGTTATGGCCAGC AGTGTTCTTTGGAGAGCCACAATAATTTCAAGAGGAAAATATACCAGTGAAAATTGTGTGGCTATTT TGAGTAGAATTGGTCAGTTGATTATTTTGTGTAATTGAGATATATGTAGTAGTTTAAGCATGATTCT TGAAGAAAGCAATAGTGACTTTTGCATAGGGAGATTTTGGTAGAAACTTCTTGGGACTAAACAAGTT TAGAGATGCATTTAAGAATTATTCACAAAATGTGTCATTCTAAATTAAAACATAAATATATTTTCAA AAGCATTTGATTTCTCTGAAGCATGATATACCTGGTCTTACCTAGTGAATCAGGATTGTCCTCAGGT AAATGAAATCATGATACATTATTGCAGTGAACTCAAGTGCAATACTTTGTAAGACATATAATTCCTA TGATTTTCACATTTTTATATCTTATATATGGGAAAAGCCAAATTAAATTGAATTCAGATTAATTCCA GCATTAGACTAAATGAGCAAACTTAAGTAAATGTACAAACTAGGTAAGTATAAAACCACAGGTTAAC AATATTGGAGTACTTTTAGAATTACATTAAAACTGTCTTAAATGTCCTATCCCAAATCTAAAAAAAA AAAAAAA The following amino acid sequence <SEQ ID NO. 2> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO. 1: MRVSVPGPAAAAAAPAAGREPSTPGGGSGGGGAVAAASGAAVPGSVQLALSVLHALLYAALFAFAYLQLWRLL LYRERRLSYQSLCLFLCLLWAALRTTLFSAAPSLSGSLPLLRPPAHLHFFPHWLLYCFPSCLQFSTLCLLNL YLAEVTCKVRCATELDRHKILLHLGFIMASLLFLVVNLTCAMLVHGDVPENQLKWTVFVRALINDSLFILCA ISLVCYICKITKMSSANVYLESKCMSLCQTVVVGSVVILLYSSRACYNLVVVTISQDTLESPFNYGWDNLSD KAHVEDISGEEYIVFGMVLFLWEHVPAWBVVLFFRAQRLNQNLAPAGMINSHSYSSRAYFFDNPRRYDSDDD LPRLCSSREGSLPNSQSLGWYGTMTGCGSSSYTVTPHLNGPMTDTAPLLFTCSNLDLNNHHSLYVTPQN 
 Example 2 
 Cloning of nGPCR-1025 The nucleotide sequence of Incyte clone &num;2967969 was used to design RACE primers for cloning of the remaining part of the cDNA: 6 5′RACE primer 1 5′-CTGGCACAGAGACATACCCTTTGAT-3′ &lsqb;SEQ ID NO: 3&rsqb; 5′RACE primer 2 5′-AGACAGGAGGGGAAGCAGTAGAGCA-3′ &lsqb;SEQ ID NO: 4&rsqb; 5′RACE primer 3 5′-AGTGGGGGAAGAAGTGCAGGTGAGC-3′ &lsqb;SEQ ID NO: 5&rsqb; Nested 5′RACE primer 5′-ATGACTACAGAGCCCACGACGACAG-3′ &lsqb;SEQ ID NO: 6&rsqb; RACE was performed using SMART™ RACE cDNA Amplification Kit from Clontech (&num;K1811-1). Human fetal brain polyA&plus; RNA (&num;6525-1, Clontech) was used as starting material. The manufacturer's manual was followed completely with the following modifications: (1) The first RACE-PCR utilized the 5′RACE primer1, 2 or 3; (2) PCR cycling in 0.2 ml tubes in MJ research Tetrad Thermocycler using the following program: 7 94° C. 30 s &verbar; 72° C. 3 min &verbar; 5 cycles 94° C. 30 s &verbar; 70° C. 30 s &verbar; 5 cycles 72° C. 3 min &verbar; 94° C. 30 s &verbar; 68° C. 30 s &verbar; 25 cycles 72° C. 3 min &verbar; Nested PCR was performed using Nested Universal Primer (NUP, provided in the kit) and the 5′RACE primers or nested 5′RACE primer (shown above) resulted in bands of approximately 350, 600, and 700 base-pairs in length. Gel purification of reamplified products using QIAquick Gel Extraction Kit (&num;1.6122-50, Qiagen) was performed following the provided protocol. After purification, the fragments were immediately ligated to pCRII-TOPO vector and transformed into TOP10-cells, using the TOPO TA cloning kit from Promega (&num;K4600-01). Transformants were picked with a toothpick into 100 &mgr;l water. The toothpicks were then transferred to Terrific Broth (&num;22711-022, Life Technologies) containing 0.06 mg/ml ampicillin (&num;Q100-16, Invitrogen) for making glycerol stocks. The colonies that were transferred to water were boiled for 5 min and 10 &mgr;l were used in colony PCR with vector specific primers as follows: Forward primer (na 11): 5′-CACAGGAAACAGCTATGAC-3′ &lsqb;SEQ ID NO:7&rsqb; Reverse primer (na 10): 5′-CCAGTCACGACGTTGTAAA-3′ &lsqb;SEQ ID NO:8&rsqb; 8 10 &mgr;l boiled colony 2 &mgr;l fwd vector primer (10 &mgr;M) 2 &mgr;l rev vector primer (10 &mgr;M) 10 &mgr;l 10 × PCR buffer (provided with the enzyme, Amersham Pharmacia Biotech) 1 &mgr;l 10 mM dNTP (&num;1 969 064 from Boehringer Mannheim) 0.5 &mgr;l Taq polymerase (&num;27-0799-62, Amersham Pharmacia Biotech) 74.5 &mgr;l water Cycle conditions in MJ research Tetrad 9 94° C. 5 min &verbar; 94° C. 1 min &verbar; 50° C. 1 min &verbar; 35 cycles 72° C. 1 min &verbar; 72° C. 5 min &verbar; This resulted in PCR amplification of 500, 750, and 850 base-pair fragments, and the positive clones were subject to preparation of plasmid using QIAprep Spin Miniprep Kit (&num;1.6103-50) from Qiagen. The complete nucleotide sequences of the fragments could be assembled with the nucleotide sequence of Incyte clone &num;2967969 with perfect sequence match to the sequence of nGPCR-1025 (SEQ ID NO:1). However, 45 amino acids in the N-terminal end of nGPCR-1025 have not yet been identified. 
 Example 3 
 Subcloning of the Coding Region of nGPCR-1025 via PCR Additional experiments may be conducted to subclone the coding region of nGPCR-1025 and place the isolated coding region into a useful vector. Two additional PCR primers are designed based on the coding region of nGPCR, corresponding to either end. To protect against exonucleolytic attack during subsequent exposure to enzymes, e.g., Taq polymerase, primers are routinely synthesized with a protective run of nucleotides at the 5′ end that were not necessarily complementary to the desired target. PCR is performed in a 50 &mgr;l reaction containing 34 &mgr;l H 2 O, 5 &mgr;l 10×TT buffer (140 mM ammonium sulfate, 0.1% gelatin, 0.6 M Tris-tricine, pH 8.4), 5 &mgr;l 15 mM MgSO 4 , 2 &mgr;l dNTP mixture (dGTP, dATP, dTTP, and dCTP, each at 10 mM), 3 &mgr;l genomic phage DNA (0.25 &mgr;g/&mgr;l), 0.3 &mgr;l Primer 1 (1 &mgr;g/&mgr;l), 0.3 &mgr;l Primer 2 (1 &mgr;g/&mgr;l), 0.4 &mgr;l High Fidelity Taq polymerase (Boehringer Mannheim). The PCR reaction was started with 1 cycle of 94° C. for 2 minutes; followed by 25 cycles at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1.3 minutes; The contents from the PCR reaction are loaded onto a 2% agarose gel and fractionated. The DNA band of expected size is excised from the gel, placed in a GenElute Agarose spin column (Supelco) and spun for 10 minutes at maximum speed in a microfuge. The eluted DNA is precipitated with ethanol and resuspended in 6 &mgr;l H 2 O for ligation. The PCR-amplified DNA fragment containing the coding region is cloned into pCR2.1 using a protocol standard in the art. In particular, the ligation reaction consists of 6 &mgr;l of GPCR DNA, 1 &mgr;l 10×ligation buffer, 2 &mgr;l pCR2.1 (25 ng/&mgr;l, Invitrogen), and 1 &mgr;l T4 DNA ligase (Invitrogen). The reaction mixture is incubated overnight at 14° C. and the reaction is then stopped by heating at 65° C. for 10 minutes. Two microliters of the ligation reaction are transformed into One Shot cells (Invitrogen) and plated onto ampicillin plates. A single colony containing a recombinant pCR2. 1 bearing an insert is used to inoculate a 5 ml culture of LB medium. Plasmid DNA is purified using the Concert Rapid Plasmid Miniprep System (GibcoBRL) and sequenced. Following confirmation of the sequence, a 50 ml culture of LB medium is inoculated with the transformed One Shot cells, cultured, and processed using a Qiagen Plasmid Midi Kit to yield purified pCR-GPCR. 
 Example 4 
 Hybridization Analysis to Demonstrate nGPCR-1025 Expression in Brain The expression of nGPCR-1025 in mammals, such as the rat, may be investigated by in situ hybridization histochemistry. To investigate expression in the brain, for example, coronal and sagittal rat brain cryosections (20 &mgr;m thick) are prepared using a Reichert-Jung cryostat. Individual sections are thaw-mounted onto silanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.), and stored at −80° C. Sections are processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline (PBS), acetylated using acetic anhydride in triethanolamine buffer, and dehydrated through a series of alcohol washes in 70%, 95%, and 100% alcohol at room temperature. Subsequently, sections are delipidated in chloroform, followed by rehydration through successive exposure to 100% and 95% alcohol at room temperature. Microscope slides containing processed cryosections are allowed to air dry prior to hybridization. Other tissues may be assayed in a similar fashion. A nGPCR-1025-specific probe is generated using PCR. Following PCR amplification, the fragment is digested with restriction enzymes and cloned into pBluescript II cleaved with the same enzymes. For production of a probe specific for the sense strand of nGPCR-1025, the nGPCR-1025 clone in pBluescript II is linearized with a suitable restriction enzyme, which provides a substrate for labeled run-off transcripts (i.e., cRNA riboprobes) using the vector-borne T7 promoter and commercially available T7 RNA polymerase. A probe specific for the antisense strand of nGPCR-1025 is also readily prepared using the nGPCR-1025 clone in pBluescript II by cleaving the recombinant plasmid with a suitable restriction enzyme to generate a linearized substrate for the production of labeled run-off cRNA transcripts using the T3 promoter and cognate polymerase. The riboprobes are labeled with &lsqb; 35 &rsqb;-UTP to yield a specific activity of about 0.40×10 6 cpm/pmol for antisense riboprobes and about 0.65×10 6 cpm/pmol for sense-strand riboprobes. Each riboprobe is subsequently denatured and added (2 pmol/ml) to hybridization buffer which contained 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 1×Denhardt's Solution, and 10 mM dithiothreitol. Microscope slides containing sequential brain cryosections are independently exposed to 45 &mgr;l of hybridization solution per slide and silanized cover slips are placed over the sections being exposed to hybridization solution. Sections are incubated overnight (15-18 hours) at 52° C. to allow hybridization to occur. Equivalent series of cryosections are exposed to sense or antisense nGPCR-1025-specific cRNA riboprobes. Following the hybridization period, coverslips are washed off the slides in 1×SSC, followed by RNase A treatment involving the exposure of slides to 20 &mgr;g/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5M EDTA, and 0.5M NaCl for 45 minutes at 37° C. The cryosections are then subjected to three high-stringency washes in 0.1×SSC at 52° C. for 20 minutes each. Following the series of washes, cryosections are dehydrated by consecutive exposure to 70%, 95%, and 100% ammonium acetate in alcohol, followed by air drying and exposure to Kodak BioMax™ MR-1 film. After 13 days of exposure, the film is developed. Based on these results, slides containing tissue that hybridized, as shown by film autoradiograms, are coated with Kodak NTB-2 nuclear track emulsion and the slides are stored in the dark for 32 days. The slides are then developed and counterstained with hematoxylin. Emulsion-coated sections are analyzed microscopically to determine the specificity of labeling. The signal is determined to be specific if autoradiographic grains (generated by antisense probe hybridization) are clearly associated with cresyl violate-stained cell bodies. Autoradiographic grains found between cell bodies indicates non-specific binding of the probe. As discussed above, GPCR-1025 is expressed in many different tissues and regions, including in the brain. Expression of nGPCR-1025 in the brain provides an indication that modulators of nGPCR-1025 activity have utility for treating neurological disorders, including but not limited to, mental disorder, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia. Some other diseases for which modulators of nGPCR-1025 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like. Use of nGPCR-1025 - modulators, including nGPCR-1025 ligands and anti-nGPCR-1025 antibodies, to treat individuals having such disease states is intended as an aspect of the invention. 
 Example 5 
 Tissue Expression Profiling A PCR-based system was used to generate a comprehensive expression profile of nGPCR-1025 in human tissue, and in human brain regions. Analysis of which tissues significantly express human nGPCR-1025 was performed by hybridization on a Human Multiple Tissue Expression (MTE) Array (&num;7775-1) containing polyA&plus; RNA from 76 different human tissues and control RNAs and DNAs that was obtained from Clontech Laboratories. As a hybridization probe, PCR fragments encoding amino acids 241-396 of human nGPCR-1025 were labeled with &lsqb;&agr;- 32 P&rsqb;-dATP using the Strip-EZ kit (&num;1470) from Ambion used according to the manufacturer's protocol. The probes were then purified on ProbeQuant™ G-50 Micro Columns (&num;27-5335-01, Amersham Pharmacia Biotech). The hybridizations were carried out using ExpressHyb solution (Clontech Laboratories). An aliquot of 15 ml hybridization solution was prewarmed at 60° C. and mixed with 1.5 mg heat-denatured sheared salmon sperm DNA. The array was prehybridized in 10 ml of the prewarmed hybridization solution for 30 min at 65° C. The nGPCR-1025 probe was denatured for 5 min at 80° C. and added to the remaining 5 ml hybridization solution The array was hybridized over night at 65° C. with continuous agitation. The hybridization solution was carefully removed and array washed 5×20 min at 65° C. in wash solution 1 (2×SSC, 1%SDS, (20×SSC&equals;3M NaCl, 0.3M Na 3 Citrate.2H 2 O, pH7.0)) and 2×20 min at 55° C. in wash solution 2 (0.1×SSC, 0.5% SDS). The MTE array was then immediately wrapped in plastic wrap and exposed on phosphorimager screen (Molecular Dynamics) overnight and scanned in a STORM phosphorimager (Molecular Dynamics). The MTE arrays were quantified by calculating pixel volume using ImageQuaNT software (Molecular Dynamics). The results showed that human nGPCR-1025 is strongly expressed in cerebellum, with a lower but significant expression in cerebrum. Expression of nGPCR-1025 was also seen in testis. 
 Example 6 
 Northern Blot Analysis Northern blots are performed to examine the expression of nGPCR-1025 mRNA. The sense orientation oligonucleotide and the antisense-orientation oligonucleotide, described above, are used as primers to amplify a portion of the GPCR-x cDNA sequence of SEQ ID NO:1. Multiple human tissue northern blots from Clontech (Human II &num;7767-1) are hybridized with the probe. Pre-hybridization is carried out at 42 C. for 4 hours in 5×SSC, 1×Denhardt's reagent, 0.1% SDS, 50% formamide, 250 mg/ml salmon sperm DNA. Hybridization is performed overnight at 42° C. in the same mixture with the addition of about 1.5×10 6 cpm/ml of labeled probe. The probe is labeled with &agr;- 32 P-dCTP by Rediprime™ DNA labeling system (Amersham Pharmacia), purified on Nick Column™ (Amersham Pharmacia) and added to the hybridization solution. The filters are washed several times at 42° C. in 0.2×SSC, 0.1% SDS. Filters are exposed to Kodak XAR film (Eastman Kodak Company, Rochester, N.Y., USA) with intensifying screen at −80° C. 
 Example 7 
 Recombinant Expression of nGPCR-1025 in Eukaryotic Host Cells A. Expression of nGPCR-1025 in Mammalian Cells To produce nGPCR-1025 protein, a nGPCR-1025-encoding polynucleotide is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques. For example, the nGPCR-1025-encoding sequence described in Example 1 is subcloned into the commercial expression vector pzeoSV2 (Invitrogen, San Diego, Calif.) and transfected into Chinese Hamster Ovary (CHO) cells using the transfection reagent FuGENE6™ (Boehringer-Mannheim) and the transfection protocol provided in the product insert. Other eukaryotic cell lines, including human embryonic kidney (HEK-293) and COS cells, are suitable as well. Cells stably expressing nGPCR-1025 are selected by growth in the presence of 100 &mgr;g/ml zeocin (Stratagene, LaJolla, Calif.). Optionally, nGPCR-1025 may be purified from the cells using standard chromatographic techniques. To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the nGPCR-1025 amino acid sequence, and the antisera is used to affinity purify nGPCR-1025. The nGPCR-1025 also may be expressed in-frame with a tag sequence (e.g., polyhistidine, hemagluttinin, FLAG) to facilitate purification. Moreover, it will be appreciated that many of the uses for nGPCR-1025 polypeptides, such as assays described below, do not require purification of nGPCR-1025 from the host cell. B. Expression of nGPCR-1025 in HEK-293 Cells For expression of nGPCR-1025 in mammalian cells HEK293 (transformed human, primary embryonic kidney cells), a plasmid bearing the relevant nGPCR-1025 coding sequence is prepared, using vector pSecTag2A (Invitrogen). Vector pSecTag2A contains the murine IgK chain leader sequence for secretion, the c-myc epitope for detection of the recombinant protein with the anti-myc antibody, a C-terminal polyhistidine for purification with nickel chelate chromatography, and a Zeocin resistant gene for selection of stable transfectants. The forward primer for amplification of this GPCR cDNA is determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce the HindIII cloning site and nucleotides matching the GPCR sequence. The reverse primer is also determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce an XhoI restriction site for cloning and nucleotides corresponding to the reverse complement of the nGPCR-1025 sequence. The PCR conditions are 55° C. as the annealing temperature. The PCR product is gel purified and cloned into the HindIII-XhoI sites of the vector. The DNA is purified using Qiagen chromatography columns and transfected into HEK-293 cells using DOTAP™ transfection media (Boehringer Mannheim, Indianapolis, Ind.). Transiently transfected cells are tested for expression after 24 hours of transfection, using western blots probed with anti-His and anti-nGPCR-1025 peptide antibodies. Permanently transfected cells are selected with Zeocin and propagated. Production of the recombinant protein is detected from both cells and media by western blots probed with anti-His, anti-Myc or anti-GPCR peptide antibodies. C. Expression of nGPCR-1025 in COS Cells For expression of the nGPCR-1025 in COS7 cells, a polynucleotide molecule having a sequence of SEQ ID NO:1 can be cloned into vector p3-Cl. This vector is a pUC18-derived plasmid that contains the HCMV (human cytomegalovirus) promoter-intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site. In addition, the plasmid contains the dhrf (dihydrofolate reductase) gene which provides selection in the presence of the drug methotrexane (MTX) for selection of stable transformants. The forward primer is determined by routine procedures and preferably contains a 5′ extension which introduces an XbaI restriction site for cloning, followed by nucleotides which correspond to a sequence of SEQ ID NO:1. The reverse primer is also determined by routine procedures and preferably contains 5′-extension of nucleotides which introduces a SalI cloning site followed by nucleotides which correspond to the reverse complement of a sequence of SEQ ID NO:1. The PCR consists of an initial denaturation step of 5 min at 95° C. 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and 30 sec extension at 72° C., followed by 5 min extension at 72° C. The PCR product is gel purified and ligated into the XbaI and SalI sites of vector p3-CI. This construct is transformed into E. coli cells for amplification and DNA purification. The DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using Lipofectamine™ reagent from BRL, following the manufacturer's protocols. Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression. nGPCR-1025 expressed from a COS cell culture can be purified by concentrating the cell-growth media to about 10 mg of protein/ml, and purifying the protein by, for example, chromatography. Purified nGPCR-1025 is concentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at −80° C. D. Expression of nGPCR-1025 in Insect Cells For expression of nGPCR-1025 in a baculovirus system, a polynucleotide molecule having a sequence of SEQ ID NO:1 can be amplified by PCR. The forward primer is determined by routine procedures and preferably contains a 5′ extension which adds the NdeI cloning site, followed by nucleotides which correspond to a sequence of SEQ ID NO:1. The reverse primer is also determined by routine procedures and preferably contains a 5′ extension which introduces the KpnI cloning site, followed by nucleotides which correspond to the reverse complement of a sequence of SEQ ID NO:1. The PCR product is gel purified, digested with NdeI and KpnI, and cloned into the corresponding sites of vector pACHTL-A (Pharmingen, San Diego, Calif.). The pAcHTL expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), and a 6×His tag upstream from the multiple cloning site. A protein kinase site for phosphorylation and a thrombin site for excision of the recombinant protein precede the multiple cloning site is also present. Of course, many other baculovirus vectors could be used in place of pAcHTL-A, such as pAc373, pVL941 and pAcIM1. Other suitable vectors for the expression of GPCR polypeptides can be used, provided that the vector construct includes appropriately located signals for transcription, translation, and trafficking, such as an in-frame AUG and a signal peptide, as required. Such vectors are described in Luckow et al., Virology 170:31-39, among others. The virus is grown and isolated using standard baculovirus expression methods, such as those described in Summers et al. (A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987)). In a preferred embodiment, pAcHLT-A containing nGPCR-1025 gene is introduced into baculovirus using the “BaculoGold™ transfection kit (Pharmingen, San Diego, Calif.) using methods established by the manufacturer. Individual virus isolates are analyzed for protein production by radiolabeling infected cells with 35 S-methionine at 24 hours post infection. Infected cells are harvested at 48 hours post infection, and the labeled proteins are visualized by SDS-PAGE. Viruses exhibiting high expression levels can be isolated and used for scaled up expression. For expression of a nGPCR-1025 polypeptide in a Sf9 cells, a polynucleotide molecule having a sequence of SEQ ID NO:1 can be amplified by PCR using the primers and methods described above for baculovirus expression. The nGPCR-1025 cDNA is cloned into vector pAcHLT-A (Pharmingen) for expression in Sf9 insect. The insert is cloned into the NdeI and KpnI sites, after elimination of an internal NdeI site (using the same primers described above for expression in baculovirus). DNA is purified with Qiagen chromatography columns and expressed in Sf9 cells. Preliminary Western blot experiments from non-purified plaques are tested for the presence of the recombinant protein of the expected size which reacted with the GPCR-specific antibody. These results are confirmed after further purification and expression optimization in HiG5 cells. 
 Example 8 
 Interaction Trap/Two-Hybrid System In order to assay for nGPCR-1025-interacting proteins, the interaction trap/two-hybrid library screening method can be used. This assay was first described in Fields et al., Nature, 1989, 340, 245, which is incorporated herein by reference in its entirety. A protocol is published in Current Protocols in Molecular Biology 1999, John Wiley & Sons, NY, and Ausubel, F. M. et al. 1992, Short protocols in molecular biology, Fourth edition, Greene and Wiley-interscience, NY, each of which is incorporated herein by reference in its entirety. Kits are available from Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System 3). A fusion of the nucleotide sequences encoding all or partial nGPCR-1025 and the yeast transcription factor GAL4 DNA-binding domain (DNA-BD) is constructed in an appropriate plasmid (i.e., pGBKT7) using standard subcloning techniques. Similarly, a GAL4 active domain (AD) fusion library is constructed in a second plasmid (i.e., pGADT7) from cDNA of potential GPCR-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al. 1989, Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The DNA-BD/nGPCR-1025 fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two-hybrid analysis. Similar controls are performed with the AD/library fusion construct to ensure expression in host cells and lack of transcriptional activity. Yeast cells are transformed (ca. 105 transformants/mg DNA) with both the nGPCR-1025 and library fusion plasmids according to standard procedures (Ausubel et al., 1992, Short protocols in molecular biology, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety). In vivo binding of DNA-BD/nGPCR-1025 with AD/library proteins results in transcription of specific yeast plasmid reporter genes (i.e., lacZ, HIS3, ADE2, LEU2). Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes. Colonies are dually assayed for &bgr;-galactosidase activity upon growth in Xgal (5-bromo-4-chloro-3-indolyl-&bgr;-D-galactoside) supplemented media (filter assay for &bgr;-galactosidase activity is described in Breeden et al., Cold Spring Harb. Symp. Quant. Biol., 1985, 50, 643, which is incorporated herein by reference in its entirety). Positive AD-library plasmids are rescued from transformants and reintroduced into the original yeast strain as well as other strains containing unrelated DNA-BD fusion proteins to confirm specific nGPCR-1025/library protein interactions. Insert DNA is sequenced to verify the presence of an open reading frame fused to GAL4 AD and to determine the identity of the nGPCR-1025-binding protein. 
 Example 9 
 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis A gel electrophoresis mobility shift assay can rapidly detect specific protein-DNA interactions. Protocols are widely available in such manuals as Sambrook et al. 1989, Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. and Ausubel, F. M. et al., 1992, Short Protocols in Molecular Biology, fourth edition, Greene and Wiley-interscience, NY, each of which is incorporated herein by reference in its entirety. Probe DNA (<300 bp) is obtained from synthetic oligonucleotides, restriction endonuclease fragments, or PCR fragments and end-labeled with 32 P. An aliquot of purified nGPCR-1025 (ca. 15 &mgr;g) or crude nGPCR-1025 extract (ca. 15 ng) is incubated at constant temperature (in the range 22-37 C.) for at least 30 minutes in 10-15 &mgr;l of buffer (i.e. TAE or TBE, pH 8.0-8.5) containing radiolabeled probe DNA, nonspecific carrier DNA (ca. 1 &mgr;g), BSA (300 &mgr;g/ml), and 10% (v/v) glycerol. The reaction mixture is then loaded onto a polyacrylamide gel and run at 30-35 mA until good separation of free probe DNA from protein-DNA complexes occurs. The gel is then dried and bands corresponding to free DNA and protein-DNA complexes are detected by autoradiography. 
 Example 10 
 Antibodies to nGPCR-1025 Standard techniques are employed to generate polyclonal or monoclonal antibodies to the nGPCR-1025 receptor, and to generate useful antigen-binding fragments thereof or variants thereof, including “humanized” variants. Such protocols can be found, for example, in Sambrook et al. (1989) and Harlow et al. (Eds.), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988). In one embodiment, recombinant nGPCR-1025 polypeptides (or cells or cell membranes containing such polypeptides) are used as antigen to generate the antibodies. In another embodiment, one or more peptides having amino acid sequences corresponding to an immunogenic portion of nGPCR-1025 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) are used as antigen. Peptides corresponding to extracellular portions of nGPCR-1025, especially hydrophilic extracellular portions, are preferred. The antigen may be mixed with an adjuvant or linked to a hapten to increase antibody production. A. Polyclonal or Monoclonal Antibodies As one exemplary protocol, recombinant nGPCR-1025 or a synthetic fragment thereof is used to immunize a mouse for generation of monoclonal antibodies (or larger mammal, such as a rabbit, for polyclonal antibodies). To increase antigenicity, peptides are conjugated to Keyhole Lympet Hemocyanin (Pierce), according to the manufacturer's recommendations. For an initial injection, the antigen is emulsified with Freund's Complete Adjuvant and injected subcutaneously. At intervals of two to three weeks, additional aliquots of nGPCR-1025 antigen are emulsified with Freund's Incomplete Adjuvant and injected subcutaneously. Prior to the final booster injection, a serum sample is taken from the immunized mice and assayed by western blot to confirm the presence of antibodies that immunoreact with nGPCR-1025. Serum from the immunized animals may be used as polyclonal antisera or used to isolate polyclonal antibodies that recognize nGPCR-1025. Alternatively, the mice are sacrificed and their spleen removed for generation of monoclonal antibodies. To generate monoclonal antibodies, the spleens are placed in 10 ml serum-free RPMI 1640, and single cell suspensions are formed by grinding the spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 &mgr;g/ml streptomycin (RPMI) (Gibco, Canada). The cell suspensions are filtered and washed by centrifugation and resuspended in serum-free RPMI. Thymocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a Feeder Layer. NS-1 myeloma cells, kept in log phase in RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged and washed as well. To produce hybridoma fusions, spleen cells from the immunized mice are combined with NS-1 cells and centrifuged, and the supernatant is aspirated. The cell pellet is dislodged by tapping the tube, and 2 ml of 37° C. PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) is stirred into the pellet, followed by the addition of serum-free RPMI. Thereafter, the cells are centrifuged, resuspended in RPMI containing 15% FBS, 100 &mgr;M sodium hypoxanthine, 0.4 &mgr;M aminopterin, 16 &mgr;M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×10 6 thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning N.Y.). On days 2, 4, and 6 after the fusion, 100 &mgr;l of medium is removed from the wells of the fusion plates and replaced with fresh medium. On day 8, the fusions are screened by ELISA, testing for the presence of mouse IgG that binds to nGPCR-1025. Selected fusion wells are further cloned by dilution until monoclonal cultures producing anti-nGPCR-1025 antibodies are obtained. B. Humanization of Anti-nGPCR-1025 Monoclonal Antibodies The expression pattern of nGPCR-1025 as reported herein and the proven track record of GPCRs as targets for therapeutic intervention suggest therapeutic indications for nGPCR-1025 inhibitors (antagonists). nGPCR-1025-neutralizing antibodies comprise one class of therapeutics useful as nGPCR-1025 antagonists. Following are protocols to improve the utility of anti-nGPCR-1025 monoclonal antibodies as therapeutics in humans by “humanizing” the monoclonal antibodies to improve their serum half-life and render them less immunogenic in human hosts (i.e., to prevent human antibody response to non-human anti-nGPCR-1025 antibodies). The principles of humanization have been described in the literature and are facilitated by the modular arrangement of antibody proteins. To minimize the possibility of binding complement, a humanized antibody of the IgG4 isotype is preferred. For example, a level of humanization is achieved by generating chimeric antibodies comprising the variable domains of non-human antibody proteins of interest with the constant domains of human antibody molecules. (See, e.g., Morrison et al., Adv. Immunol., 44:65-92 (1989)). The variable domains of nGPCR-1025-neutralizing anti-nGPCR-1025 antibodies are cloned from the genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA isolated from the hybridoma of interest. The V region gene fragments are linked to exons encoding human antibody constant domains, and the resultant construct is expressed in suitable mammalian host cells (e.g., myeloma or CHO cells). To achieve an even greater level of humanization, only those portions of the variable region gene fragments that encode antigen-binding complementarity determining regions (“CDR”) of the non-human monoclonal antibody genes are cloned into human antibody sequences. (See, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-36 (1988); and Tempest et al., Bio/Technology 9: 266-71 (1991)). If necessary, the &bgr;-sheet framework of the human antibody surrounding the CDR3 regions also is modified to more closely mirror the three dimensional structure of the antigen-binding domain of the original monoclonal antibody. (See Kettleborough et al., Protein Engin., 4:773-783 (1991); and Foote et al., J. Mol. Biol., 224:487-499 (1992)). In an alternative approach, the surface of a non-human monoclonal antibody of interest is humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all of the interior and contacting residues of the non-human antibody. See Padlan, Molecular Immunol., 28(4/5):489-98 (1991). The foregoing approaches are employed using nGPCR-1025-neutralizing anti-nGPCR-1025 monoclonal antibodies and the hybridomas that produce them to generate humanized nGPCR-1025-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein nGPCR-1025 expression or ligand-mediated nGPCR-1025 signaling is detrimental. C. Human nGPCR-1025-Neutralizing Antibodies from Phage Display Human nGPCR-1025-neutralizing antibodies are generated by phage display techniques such as those described in Aujame et al., Human Antibodies 8(4):155-168 (1997); Hoogenboom, TIBTECH 15:62-70 (1997); and Rader et al., Curr. Opin. Biotechnol. 8:503-508 (1997), all of which are incorporated by reference. For example, antibody variable regions in the form of Fab fragments or linked single chain Fv fragments are fused to the amino terminus of filamentous phage minor coat protein pIII. Expression of the fusion protein and incorporation thereof into the mature phage coat results in phage particles that present an antibody on their surface and contain the genetic material encoding the antibody. A phage library comprising such constructs is expressed in bacteria, and the library is screened for nGPCR-1025-specific phage-antibodies using labeled or immobilized nGPCR-1025 as antigen-probe. D. Human nGPCR-1025-neutralizing Antibodies from Transgenic Mice Human nGPCR-1025-neutralizing antibodies are generated in transgenic mice essentially as described in Bruggemann et al., Immunol. Today 17(8):391-97 (1996) and Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997). Transgenic mice carrying human V-gene segments in germline configuration and that express these transgenes in their lymphoid tissue are immunized with a nGPCR-1025 composition using conventional immunization protocols. Hybridomas are generated using B cells from the immunized mice using conventional protocols and screened to identify hybridomas secreting anti-nGPCR-1025 human antibodies (e.g., as described above). 
 Example 11 
 Assays to Identify Modulators of nGPCR-1025 Activity Set forth below are several nonlimiting assays for identifying modulators (agonists and antagonists) of nGPCR-1025 activity. Among the modulators that can be identified by these assays are natural ligand compounds of the receptor; synthetic analogs and derivatives of natural ligands; antibodies, antibody fragments, and/or antibody-like compounds derived from natural antibodies or from antibody-like combinatorial libraries; and/or synthetic compounds identified by high-throughput screening of libraries; and the like. All modulators that bind nGPCR-1025 are useful for identifying nGPCR-1025 in tissue samples (e.g., for diagnostic purposes, pathological purposes, and the like). Agonist and antagonist modulators are useful for up-regulating and down-regulating nGPCR-1025 activity, respectively, to treat disease states characterized by abnormal levels of nGPCR-1025 activity. The assays may be performed using single putative modulators, and/or may be performed using a known agonist in combination with candidate antagonists (or visa versa). A. cAMP Assays In one type of assay, levels of cyclic adenosine monophosphate (cAMP) are measured in nGPCR-1025-transfected cells that have been exposed to candidate modulator compounds. Protocols for cAMP assays have been described in the literature. (See, e.g., Sutherland et al., Circulation 37: 279 (1968); Frandsen et al., Life Sciences 18: 529-541 (1976); Dooley et al., Journal of Pharmacology and Experimental Therapeutics 283 (2): 735-41 (1997); and George et al., Journal of Biomolecular Screening 2 (4): 235-40 (1997)). An exemplary protocol for such an assay, using an Adenylyl Cyclase Activation FlashPlate® Assay from NEN™ Life Science Products, is set forth below. Briefly, the nGPCR-1025 coding sequence (e.g., a cDNA or intronless genomic DNA) is subcloned into a commercial expression vector, such as pzeoSV2 (Invitrogen), and transiently transfected into Chinese Hamster Ovary (CHO) cells using known methods, such as the transfection protocol provided by Boehringer-Mannheim when supplying the FuGENE 6 transfection reagent. Transfected CHO cells are seeded into 96-well microplates from the FlashPlate® assay kit, which are coated with solid scintillant to which antisera to cAMP has been bound. For a control, some wells are seeded with wild type (untransfected) CHO cells. Other wells in the plate receive various amounts of a cAMP standard solution for use in creating a standard curve. One or more test compounds (i.e., candidate modulators) are added to the cells in each well, with water and/or compound-free medium/diluent serving as a control or controls. After treatment, cAMP is allowed to accumulate in the cells for exactly 15 minutes at room temperature. The assay is terminated by the addition of lysis buffer containing &lsqb; 125 I&rsqb;-labeled cAMP, and the plate is counted using a Packard Topcount™ 96-well microplate scintillation counter. Unlabeled cAMP from the lysed cells (or from standards) and fixed amounts of &lsqb; 125 I&rsqb;-cAMP compete for antibody bound to the plate. A standard curve is constructed, and cAMP values for the unknowns are obtained by interpolation. Changes in intracellular cAMP levels of cells in response to exposure to a test compound are indicative of nGPCR-1025 modulating activity. Modulators that act as agonists of receptors which couple to the G s subtype of G proteins will stimulate production of cAMP, leading to a measurable 3-10 fold increase in cAMP levels. Agonists of receptors which couple to the G i/o subtype of G proteins will inhibit forskolin-stimulated cAMP production, leading to a measurable decrease in cAMP levels of 50-100%. Modulators that act as inverse agonists will reverse these effects at receptors that are either constitutively active or activated by known agonists. B. Aequorin Assays In another assay, cells (e.g., CHO cells) are transiently co-transfected with both a nGPCR-1025 expression construct and a construct that encodes the photoprotein apoaquorin. In the presence of the cofactor coelenterazine, apoaquorin will emit a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium. (See generally, Cobbold, et al. “Aequorin measurements of cytoplasmic free calcium,” In: McCormack J. G. and Cobbold P. H., eds., Cellular Calcium: A Practical Approach. Oxford: IRL Press (1991); Stables et al., Analytical Biochemistry 252: 115-26 (1997); and Haugland, Handbook of Fluorescent Probes and Research Chemicals. Sixth edition. Eugene Oreg.: Molecular Probes (1996).) In one exemplary assay, nGPCR-1025 is subcloned into the commercial expression vector pzeoSV2 (Invitrogen) and transiently co-transfected along with a construct that encodes the photoprotein apoaquorin (Molecular Probes, Eugene, Oreg.) into CHO cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided in the product insert. The cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 &mgr;g/ml streptomycin, at which time the medium is changed to serum-free MEM containing 5 &mgr;M coelenterazine (Molecular Probes, Eugene, Oreg.). Culturing is then continued for two additional hours at 37° C. Subsequently, cells are detached from the plate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in serum-free MEM. Dilutions of candidate nGPCR-1025 modulator compounds are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate at 50 &mgr;l/well. Plates are then loaded onto an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, Va.). The instrument is programmed to dispense 50 &mgr;l cell suspensions into each well, one well at a time, and immediately read luminescence for 15 seconds. Dose-response curves for the candidate modulators are constructed using the area under the curve for each light signal peak. Data are analyzed with SlideWrite, using the equation for a one-site ligand, and EC 50 values are obtained. Changes in luminescence caused by the compounds are considered indicative of modulatory activity. Modulators that act as agonists at receptors which couple to the G q subtype of G proteins give an increase in luminescence of up to 100 fold. Modulators that act as inverse agonists will reverse this effect at receptors that are either constitutively active or activated by known agonists. C. Luciferase Reporter Gene Assay The photoprotein luciferase provides another useful tool for assaying for modulators of nGPCR-1025 activity. Cells (e.g., CHO cells or COS 7 cells) are transiently co-transfected with both a nGPCR-1025 expression construct (e.g., nGPCR-1025 in pzeoSV2) and a reporter construct which includes a gene for the luciferase protein downstream from a transcription factor binding site, such as the cAMP-response element (CRE), AP-1, or NF-kappa B. Agonist binding to receptors coupled to the G s subtype of G proteins leads to increases in cAMP, thereby activating the CRE transcription factor and resulting in expression of the luciferase gene. Agonist binding to receptors coupled to the G q subtype of G protein leads to production of diacylglycerol that activates protein kinase C, which activates the AP-1 or NF-kappa B transcription factors, in turn resulting in expression of the luciferase gene. Expression levels of luciferase reflect the activation status of the signaling events. (See generally, George et al., Journal of Biomolecular Screening 2(4): 235-240 (1997); and Stratowa et al., Current Opinion in Biotechnology 6: 574-581 (1995)). Luciferase activity may be quantitatively measured using, e.g., luciferase assay reagents that are commercially available from Promega (Madison, Wis.). In one exemplary assay, CHO cells are plated in 24-well culture dishes at a density of 100,000 cells/well one day prior to transfection and cultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 &mgr;g/ml streptomycin. Cells are transiently co-transfected with both a nGPCR-1025 expression construct and a reporter construct containing the luciferase gene. The reporter plasmids CRE-luciferase, AP-1-luciferase and NF-kappaB-luciferase may be purchased from Stratagene (LaJolla, Calif.). Transfections are performed using the FuGENE 6 transfection reagent (Boehringer-Mannheim) according to the supplier's instructions. Cells transfected with the reporter construct alone are used as a control. Twenty-four hours after transfection, cells are washed once with PBS pre-warmed to 37° C. Serum-free MEM is then added to the cells either alone (control) or with one or more candidate modulators and the cells are incubated at 37° C. for five hours. Thereafter, cells are washed once with ice-cold PBS and lysed by the addition of 100 &mgr;l of lysis buffer per well from the luciferase assay kit supplied by Promega. After incubation for 15 minutes at room temperature, 15 &mgr;l of the lysate is mixed with 50 &mgr;l of substrate solution (Promega) in an opaque-white, 96-well plate, and the luminescence is read immediately on a Wallace model 1450 MicroBeta scintillation and luminescence counter (Wallace Instruments, Gaithersburg, Md.). Differences in luminescence in the presence versus the absence of a candidate modulator compound are indicative of modulatory activity. Receptors that are either constitutively active or activated by agonists typically give a 3 to 20-fold stimulation of luminescence compared to cells transfected with the reporter gene alone. Modulators that act as inverse agonists will reverse this effect. D. Intracellular Calcium Measurement Using FLIPR Changes in intracellular calcium levels are another recognized indicator of G protein-coupled receptor activity, and such assays can be employed to screen for modulators of nGPCR-1025 activity. For example, CHO cells stably transfected with a nGPCR-1025 expression vector are plated at a density of 4×10 4 cells/well in Packard black-walled, 96-well plates specially designed to discriminate fluorescence signals emanating from the various wells on the plate. The cells are incubated for 60 minutes at 37° C. in modified Dulbecco's PBS (D-PBS) containing 36 mg/L pyruvate and 1 g/L glucose with the addition of 1% fetal bovine serum and one of four calcium indicator dyes (Fluo-3™ AM, Fluo-4™ AM, Calcium Green™-1 AM, or Oregon Green™ 488 BAPTA-1 AM), each at a concentration of 4 &mgr;M. Plates are washed once with modified D-PBS without 1% fetal bovine serum and incubated for 10 minutes at 37° C. to remove residual dye from the cellular membrane. In addition, a series of washes with modified D-PBS without 1% fetal bovine serum is performed immediately prior to activation of the calcium response. A calcium response is initiated by the addition of one or more candidate receptor agonist compounds, calcium ionophore A23187 (10 &mgr;M; positive control), or ATP (4 &mgr;M; positive control). Fluorescence is measured by Molecular Device's FLIPR with an argon laser (excitation at 488 nm). (See, e.g., Kuntzweiler et al., Drug Development Research, 44(1):14-20 (1998)). The F-stop for the detector camera was set at 2.5 and the length of exposure was 0.4 milliseconds. Basal fluorescence of cells was measured for 20 seconds prior to addition of candidate agonist, ATP, or A23187, and the basal fluorescence level was subtracted from the response signal. The calcium signal is measured for approximately 200 seconds, taking readings every two seconds. Calcium ionophore A23187 and ATP increase the calcium signal 200% above baseline levels. In general, activated GPCRs increase the calcium signal approximately 10-15% above baseline signal. E. Mitogenesis Assay In a mitogenesis assay, the ability of candidate modulators to induce or inhibit nGPCR-1025-mediated cell division is determined. (See, e.g., Lajiness et al., Journal of Pharmacology and Experimental Therapeutics 267(3): 1573-1581 (1993)). For example, CHO cells stably expressing nGPCR-1025 are seeded into 96-well plates at a density of 5000 cells/well and grown at 37° C. in MEM with 10% fetal calf serum for 48 hours, at which time the cells are rinsed twice with serum-free MEM. After rinsing, 80 &mgr;l of fresh MEM, or MEM containing a known mitogen, is added along with 20 &mgr;l MEM containing varying concentrations of one or more candidate modulators or test compounds diluted in serum-free medium. As controls, some wells on each plate receive serum-free medium alone, and some receive medium containing 10% fetal bovine serum. Untransfected cells or cells transfected with vector alone also may serve as controls. After culture for 16-18 hours, 1 &mgr;Ci of &lsqb; 3 H&rsqb;-thymidine (2 Ci/mmol) is added to and cells are incubated for an additional 2 hours at 37° C. The cells are trypsinized and collected on filter mats with a cell harvester (Tomtec); the filters are then counted in a Betaplate counter. The incorporation of &lsqb; 3 H&rsqb;-thymidine in serum-free test wells is compared to the results achieved in cells stimulated with serum (positive control). Use of multiple concentrations of test compounds permits creation and analysis of dose-response curves using the non-linear, least squares fit equation: A&equals;B×&lsqb;C/(D&plus;C)&rsqb;&plus;G where A is the percent of serum stimulation; B is the maximal effect minus baseline; C is the EC 50 ; D is the concentration of the compound; and G is the maximal effect. Parameters B, C and G are determined by Simplex optimization. Agonists that bind to the receptor are expected to increase &lsqb; 3 H&rsqb;-thymidine incorporation into cells, showing up to 80% of the response to serum. Antagonists that bind to the receptor will inhibit the stimulation seen with a known agonist by up to 100%. F. &lsqb; 35 S&rsqb;GTP&ggr;S Binding Assay Because G protein-coupled receptors signal through intracellular G proteins whose activity involves GTP binding and hydrolysis to yield bound GDP, measurement of binding of the non-hydrolyzable GTP analog &lsqb; 35 S&rsqb;GTP&ggr;S in the presence and absence of candidate modulators provides another assay for modulator activity. (See, e.g., Kowal et al., Neuropharmacology 37:179-187 (1998).) In one exemplary assay, cells stably transfected with a nGPCR-1025 expression vector are grown in 10 cm tissue culture dishes to subconfluence, rinsed once with 5 ml of ice-cold Ca 2&plus; /Mg 2&plus; -free phosphate-buffered saline, and scraped into 5 ml of the same buffer. Cells are pelleted by centrifugation (500×g, 5 minutes), resuspended in TEE buffer (25 mM Tris, pH 7.5, 5 mM EDTA, 5 mM EGTA), and frozen in liquid nitrogen. After thawing, the cells are homogenized using a Dounce homogenizer (one ml TEE per plate of cells), and centrifuged at 1,000×g for 5 minutes to remove nuclei and unbroken cells. The homogenate supernatant is centrifuged at 20,000×g for 20 minutes to isolate the membrane fraction, and the membrane pellet is washed once with TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA). The resuspended membranes can be frozen in liquid nitrogen and stored at −70° C. until use. Aliquots of cell membranes prepared as described above and stored at −70° C. are thawed, homogenized, and diluted into buffer containing 20 mM HEPES, 10 mM MgCl 2 , 1 mM EDTA, 120 mM NaCl, 10 &mgr;M GDP, and 0.2 mM ascorbate, at a concentration of 10-50 &mgr;g/ml. In a final volume of 90 &mgr;l, homogenates are incubated with varying concentrations of candidate modulator compounds or 100 &mgr;M GTP for 30 minutes at 30° C. and then placed on ice. To each sample, 10 &mgr;l guanosine 5′-O-(3&lsqb; 35 S&rsqb;thio) triphosphate (NEN, 1200 Ci/mmol; &lsqb; 35 S&rsqb;-GTP&ggr;S), was added to a final concentration of 100-200 pM. Samples are incubated at 30° C. for an additional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM MgCl 2 , at 4° C. is added and the reaction is stopped by filtration. Samples are filtered over Whatman GF/B filters and the filters are washed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl 2 . Filters are counted by liquid scintillation spectroscopy. Nonspecific binding of &lsqb; 35 S&rsqb;-GTP&ggr;S is measured in the presence of 100 &mgr;M GTP and subtracted from the total. Compounds are selected that modulate the amount of &lsqb;35S&rsqb;-GTP&ggr;S binding in the cells, compared to untransfected control cells. Activation of receptors by agonists gives up to a five-fold increase in &lsqb; 35 S&rsqb;GTP&ggr;S binding. This response is blocked by antagonists. G. MAP Kinase Activity Assay Evaluation of MAP kinase activity in cells expressing a GPCR provides another assay to identify modulators of GPCR activity. (See, e.g., Lajiness et al., Journal of Pharmacology and Experimental Therapeutics 267(3):1573-1581 (1993) and Boulton et al., Cell 65:663-675 (1991).) In one embodiment, CHO cells stably transfected with nGPCR-1025 are seeded into 6-well plates at a density of 70,000 cells/well 48 hours prior to the assay. During this 48-hour period, the cells are cultured at 37° C. in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 &mgr;g/ml streptomycin. The cells are serum-starved for 1-2 hours prior to the addition of stimulants. For the assay, the cells are treated with medium alone or medium containing either a candidate agonist or 200 nM Phorbol ester-myristoyl acetate (i.e., PMA, a positive control), and the cells are incubated at 37° C. for varying times. To stop the reaction, the plates are placed on ice, the medium is aspirated, and the cells are rinsed with 1 ml of ice-cold PBS containing 1 mM EDTA. Thereafter, 200 &mgr;l of cell lysis buffer (12.5 mM MOPS, pH 7.3, 12.5 mM glycerophosphate, 7.5 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM sodium vanadate, 1 mM benzamidine, 1 mM dithiothreitol, 10 &mgr;g/ml leupeptin, 10 &mgr;g/ml aprotinin, 2 &mgr;g/ml pepstatin A, and 1 &mgr;M okadaic acid) is added to the cells. The cells are scraped from the plates and homogenized by 10 passages through a 23¾ G needle, and the cytosol fraction is prepared by centrifugation at 20,000×g for 15 minutes. Aliquots (5-10 &mgr;l containing 1-5 &mgr;g protein) of cytosol are mixed with 1 mM MAPK Substrate Peptide (APRTPGGRR (SEQ ID NO:9), Upstate Biotechnology, Inc., N.Y.) and 50 &mgr;M &lsqb;&ggr; 32 P&rsqb;ATP (NEN, 3000 Ci/mmol), diluted to a final specific activity of ˜2000 cpm/pmol, in a total volume of 25 &mgr;l. The samples are incubated for 5 minutes at 30° C., and reactions are stopped by spotting 20 &mgr;l on 2 cm 2 squares of Whatman P81 phosphocellulose paper. The filter squares are washed in 4 changes of 1% H 3 PO 4 , and the squares are subjected to liquid scintillation spectroscopy to quantitate bound label. Equivalent cytosolic extracts are incubated without MAPK substrate peptide, and the bound label from these samples are subtracted from the matched samples with the substrate peptide. The cytosolic extract from each well is used as a separate point. Protein concentrations are determined by a dye binding protein assay (Bio-Rad Laboratories). Agonist activation of the receptor is expected to result in up to a five-fold increase in MAPK enzyme activity. This increase is blocked by antagonists. H. &lsqb; 3 H&rsqb;Arachidonic Acid Release The activation of GPCRs also has been observed to potentiate arachidonic acid release in cells, providing yet another useful assay for modulators of GPCR activity. (See, e.g., Kanterman et al., Molecular Pharmacology 39:364-369 (1991).) For example, CHO cells that are stably transfected with a nGPCR-1025 expression vector are plated in 24-well plates at a density of 15,000 cells/well and grown in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 &mgr;g/ml streptomycin for 48 hours at 37° C. before use. Cells of each well are labeled by incubation with &lsqb; 3 H&rsqb;-arachidonic acid (Amersham Corp., 210 Ci/mmol) at 0.5 &mgr;Ci/ml in 1 ml MEM supplemented with 10 mM HEPES, pH 7.5, and 0.5% fatty-acid-free bovine serum albumin for 2 hours at 37° C. The cells are then washed twice with 1 ml of the same buffer. Candidate modulator compounds are added in 1 ml of the same buffer, either alone or with 10 &mgr;M ATP and the cells are incubated at 37° C. for 30 minutes. Buffer alone and mock-transfected cells are used as controls. Samples (0.5 ml) from each well are counted by liquid scintillation spectroscopy. Agonists which activate the receptor will lead to potentiation of the ATP-stimulated release of &lsqb; 3 H&rsqb;-arachidonic acid. This potentiation is blocked by antagonists. I. Extracellular Acidification Rate In yet another assay, the effects of candidate modulators of nGPCR-1025 activity are assayed by monitoring extracellular changes in pH induced by the test compounds. (See, e.g., Dunlop et al., Journal of Pharmacological and Toxicological Methods 40(1):47-55 (1998).) In one embodiment, CHO cells transfected with a nGPCR-1025 expression vector are seeded into 12 mm capsule cups (Molecular Devices Corp.) at 4×10 5 cells/cup in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10 &mgr;g/ml streptomycin. The cells are incubated in this medium at 37° C. in 5% CO 2 for 24 hours. Extracellular acidification rates are measured using a Cytosensor microphysiometer (Molecular Devices Corp.). The capsule cups are loaded into the sensor chambers of the microphysiometer and the chambers are perfused with running buffer (bicarbonate-free MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10 &mgr;g/ml streptomycin, 26 mM NaCl) at a flow rate of 100 &mgr;l/minute. Candidate agonists or other agents are diluted into the running buffer and perfused through a second fluid path. During each 60-second pump cycle, the pump is run for 38 seconds and is off for the remaining 22 seconds. The pH of the running buffer in the sensor chamber is recorded during the cycle from 43-58 seconds, and the pump is re-started at 60 seconds to start the next cycle. The rate of acidification of the running buffer during the recording time is calculated by the Cytosoft program. Changes in the rate of acidification are calculated by subtracting the baseline value (the average of 4 rate measurements immediately before addition of a modulator candidate) from the highest rate measurement obtained after addition of a modulator candidate. The selected instrument detects 61 mV/pH unit. Modulators that act as agonists of the receptor result in an increase in the rate of extracellular acidification compared to the rate in the absence of agonist. This response is blocked by modulators which act as antagonists of the receptor. 
 Example 12 
 Using nGPCR-1025 Proteins to Isolate Neurotransmitters Isolated nGPCR-1025 proteins of the present invention can be used to isolate novel or known neurotransmitters (Saito et al., Nature 400: 265-269, 1999). The cDNAs that encode the isolated nGPCR-1025 can be cloned into mammalian expression vectors and used to stably or transiently transfect mammalian cells including CHO, Cos or HEK293 cells. Receptor expression can be determined by Northern blot analysis of transfected cells and identification of an appropriately sized mRNA band (predicted size from the cDNA). Brain regions shown by mRNA analysis to express each of the nGPCR-1025 proteins could be processed for peptide extraction using any of several protocols ((Reinsheidk R. K. et al., Science 270: 243-247, 1996; Sakurai, T., et al., Cell 92; 573-585, 1998; Hinuma, S., et al., Nature 393: 272-276, 1998). Chromotographic fractions of brain extracts could be tested for ability to activate nGPCR-1025 proteins by measuring second messenger production such as changes in cAMP production in the presence or absence of forskolin, changes in inositol 3-phosphate levels, changes in intracellular calcium levels or by indirect measures of receptor activation including receptor stimulated mitogenesis, receptor mediated changes in extracellular acidification or receptor mediated changes in reporter gene activation in response to cAMP or calcium (these methods should all be referenced in other sections of the patent). Receptor activation could also be monitored by co-transfecting cells with a chimeric GI q/i3 to force receptor coupling to a calcium stimulating pathway (Conklin et al., Nature 363; 274-276, 1993). Neurotransmitter mediated activation of receptors could also be monitored by measuring changes in &lsqb; 35 S&rsqb;-GTPKS binding in membrane fractions prepared from transfected mammalian cells. This assay could also be performed using baculoviruses containing nGPCR-1025 proteins infected into SF9 insect cells. The neurotransmitter which activates nGPCR-1025 proteins can be purified to homogeneity through successive rounds of purification using nGPCR-1025 proteins activation as a measurement of neurotransmitter activity. The composition of the neurotransmitter can be determined by mass spectrometry and Edman degradation if peptidergic. Neurotransmitters isolated in this manner will be bioactive materials which will alter neurotransmission in the central nervous system and will produce behavioral and biochemical changes. 
 Example 13 
 Using nGPCR-1025 Proteins to Isolate and Purify G Proteins cDNAs encoding nGPCR-1025 proteins are epitope-tagged at the amino terminus end of the cDNA with the cleavable influenza-hemagglutinin signal sequence followed by the FLAG epitope (IBI, New Haven, Conn.). Additionally, these sequences are tagged at the carboxyl terminus with DNA encoding six histidine residues. (Amino and Carboxyl Terminal Modifications to Facilitate the Production and Purification of a G Protein-Coupled Receptor, B. K. Kobilka, Analytical Biochemistry, Vol. 231, No. 1, October 1995, pp. 269-271). The resulting sequences are cloned into a baculovirus expression vector such as pVL1392 (Invitrogen). The baculovirus expression vectors are used to infect SF-9 insect cells as described (Guan, X. M., Kobilka, T. S., and Kobilka, B. K. (1992) J. Biol. Chem. 267, 21995-21998). Infected SF-9 cells could be grown in 1000-ml cultures in SF900 11 medium (Life Technologies, Inc.) containing 5% fetal calf serum (Gemini, Calabasas, Calif.) and 0.1 mg/ml gentamicin (Life Technologies, Inc.) for 48 hours at which time the cells could be harvested. Cell membrane preparations could be separated from soluble proteins following cell lysis. nGPCR-1025 protein purification is carried out as described for purification of the &ngr;2 receptor (Kobilka, Anal. Biochem., 231 (1): 269-271, 1995) including solubilization of the membranes in 0.8-1.0% n-dodecyl-D-maltoside (DM) (CalBiochem, La Jolla, Calif.) in buffer containing protease inhibitors followed by Ni-column chromatography using chelating Sepharose™ (Pharmacia, Uppsala, Sweden). The eluate from the Ni-column is further purified on an M1 anti-FLAG antibody column (IBI). Receptor containing fractions are monitored by using receptor specific antibodies following western blot analysis or by SDS-PAGE analysis to look for an appropriate sized protein band (appropriate size would be the predicted molecular weight of the protein). This method of purifying G protein is particularly useful to isolate G proteins that bind to the nGPCR-1025 proteins in the absence of an activating ligand. Some of the preferred embodiments of the invention described above are outlined below and include, but are not limited to, the following embodiments. As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention. The entire disclosure of each publication cited herein is hereby incorporated by reference.