Abstract:
A new galanin receptor, GALR3, is described. Also provided are nucleic acids encoding same and various assays to identify ligands particular to said receptor. Ligands so identified are useful for the treatment of obesity, treatment of pain, and treatment of cognitive disorders.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of PCT/US98/26812, international filing date of Dec. 17, 1998, which claims priority to U.S. Serial No. 60/069,725, filed Dec. 17, 1997, now abandoned. The entirety of these applications are hereby incorporated by reference herein. 
    
    
     STATEMENT REGARDING FEDERALLY-SPONSORED R&amp;D 
     Not applicable 
     REFERENCE TO MICROFICHE APPENDIX 
     Not applicable 
     FIELD OF THE INVENTION 
     This invention relates to a novel galanin receptor, designated GALR3, to nucleotides encoding same, and to assays making use thereof. 
     BACKGROUND OF THE INVENTION 
     Although first isolated from porcine intestine, galanin is widely distributed in the central and peripheral nervous system. Galanin in most species is a 29 amino acid peptide with an amidated carboxyl terminus. Human galanin is unique in that it is longer, 30 amino acids, and is not amidated. There is strong conservation of the galanin sequence with the amino terminal fifteen residues being absolutely conserved in all species. Galanin immunoreactivity and binding is abundant in the hypothalamus, the locus coeruleus, the hippocampus and the anterior pituitary, as well as regions of the spinal cord, the pancreas and the gastrointestinal tract. 
     Like neuropeptide Y (NPY), injection of galanin into the paraventricular nucleus (PVN) of the hypothalamus produces a dose-dependent increase in feeding in satiated rats. While galanin, like norepinephrine, enhances carbohydrate ingestion, some studies have shown that it profoundly increases fat intake. It has been suggested that galanin shifts macronutrient preference from carbohydrate to fat. The same injections that increase feeding reduce energy expenditure and inhibit insulin secretion. There is enhanced galanin expression in the hypothalamus of genetically obese rats compared with their lean littermate controls. Injection of peptide receptor antagonists into the PVN blocks the galanin-specific induction of increased fat intake. Specific galanin antisense oligonucleotides when injected into the PVN produce a specific decrease in galanin expression associated with a decrease in fat ingestion and total caloric intake while hardly affecting either protein or carbohydrate intake. Thus galanin appears to be one potential neurochemical marker related to the behavior of fat ingestion. 
     Galanin inhibits cholinergic function and impairs working memory in rats. Lesions that destroy cholinergic neurons result in deficits in spatial learning tasks. While locally administered acetylcholine (ACh) reverses some of this deficit, galanin blocks this ACh-mediated improvement. Evidence from autopsy samples from Alzheimer&#39;s disease-afflicted brains suggests an increased galinergic innervation of the nucleus basilis. Thus, if galinergic overactivity contributes to the decline in cognitive performance in Alzheimer&#39;s disease, galanin antagonists may be therapeutically useful in alleviating cognitive impairment. 
     In the rat, administration of galanin intracerebroventricu-larly, subcutaneously or intravenously increases plasma growth hormone. Infusion of human galanin into healthy subjects also increases plasma growth hormone and potently enhances the growth hormone response to GHRH. 
     Galanin levels are particularly high in dorsal root ganglia. Sciatic nerve resection dramatically up-regulates galanin peptide and mRNA levels. Chronic administration of galanin receptor antagonists (M35, M15) after axotomy results in a marked increase in self mutilation behavior in rats, generally considered to be a response to pain. Application of antisense oligonucleotides specific for galanin to the proximal end of a transected sciatic nerve suppressed the increase in galanin peptide levels with a parallel increase in autotomy. Galanin injected intrathecally acts synergistically with morphine to produce analgesia, this antinociceptive effect of morphine is blocked by galanin receptor antagonists. Thus, galanin agonists may have some utility in relieving neural pain. 
     The actions of galanin are mediated by high affinity galanin receptors that are coupled by pertussis toxin sensitive G i /G o  proteins to inhibition of adenylate cyclase activity, closure of L-type Ca ++  channels and opening of ATP-sensitive K +  channels. Specific binding of  125 I-galanin (Kd approximately 1 nM) has been demonstrated in areas paralleling localization of galanin immunoreactivity: hypothalamus, ventral hippocampus, basal forebrain, spinal cord, pancreas and pituitary. In most tissues the amino terminus (GAL 1-15) is sufficient for high affinity binding and agonist activity. 
     Recently, a galanin receptor cDNA was isolated by expression cloning from a human Bowes melanoma cell line. (Habert-Ortoli, et al. 1994.  Proc. Nat. Acad. Sci, USA  91: 9780-9783). This receptor, GALR1, is expressed in human fetal brain and small intestine, but little else is known of its distribution. Gal(1-16) is at least 1000 times more active than pGAL(3-29) as an inhibitor of  125 I-porcine galanin binding to this receptor transiently expressed in COS cells. It remains to be determined whether this receptor subtype represents the hypothalamic receptor that mediates the galanin specific feeding behavior. 
     It would be desirable to identify further galanin receptors so that they can be used to further characterize this biological system and to identify galanin receptor subtype selective agonists and antagonists. 
     SUMMARY OF THE INVENTION 
     This invention relates to a novel galanin receptor, designated GALR3, substantially free from associated proteins, and to GALR3-like receptors which are at least about 40% homologous and which have substantially the same biological activity. In preferred embodiments of this invention, the GALR3-like receptors are at least about 60%, and more preferably at least about 75%, and even more preferably at least about 85% homologous to a GALR3 receptor. This invention also relates specifically to rat, human and mouse GALR3, substantially free from associated proteins, and to receptors which are at least about 50% homologous and which have substantially the same biological activity. 
     Another aspect of this invention are primate and non-primate GALR3 proteins which are encoded by substantially the same nucleic acid sequences, but which have undergone changes in splicing or other RNA processing-derived modifications or mutagenesis-induced changes, so that the expressed protein has a homologous, but different amino acid sequence from the native forms. These variant forms may have different and/or additional functions in human and animal physiology or in vitro in cell based assays. 
     A further aspect of this invention are nucleic acids which encode a GALR3 receptor, a GALR3-like receptor or a functional equivalent of a GALR3 receptor from rat, human, mouse, swine, or other species. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. The nucleic acids which encode a receptor of this invention may be any type of nucleic acid. Preferred forms are DNAs, including genomic and cDNA, although this invention specifically includes RNAs as well. Nucleic acid constructs may also contain regions which control transcription and translation such as one or more promoter regions, termination regions, and if desired enhancer regions. The nucleic acids may be inserted into any known vector including plasmids, and used to transfect suitable host cells using techniques generally available to one of ordinary skill in the art. 
     Another aspect of this invention are vectors comprising nucleic acids which encode GALR3, and host cells which contain these vectors. Still another aspect of this invention is a method of making GALR3 comprising introducing a vector comprising nucleic acids encoding GALR3 into a host cell under culturing conditions. 
     Yet another aspect of this invention are assays for GALR3 ligands which utilize the receptors and/or nucleic acids of this invention. Preferred assays of this embodiment compare the binding of the putative GALR3 ligand to the binding of galanin to GALR3. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is the DNA sequence of human GALR3 gene, clone GALR3—3 (SEQ ID NO:1). 
     FIG. 2 is the deduced amino acid sequence of human GALR3, clone GALR3—3 (SEQ ID NO:2). 
     FIG. 3 is the DNA sequence (open reading frame only) of human GALR3, clone GALR3-2 (SEQ ID NO:3). 
     FIG. 4 is the deduced amino acid sequence of GALR3, clone GALR3-2 (SEQ ID NO:4) 
     FIG. 5 is a comparison of the open reading frame protein sequences of human and rat GALR3 with the corresponding sequences of GALR1 (mouse—SEQ ID NO:5, rat—SEQ ID NO:6, and human—SEQ ID NO:7) and GALR2 (mouse—SEQ ID NO:8, rat—SEQ ID NO:9, and human—SEQ ID NO:10). 
     FIG. 6 is a phylogenetic analysis of the putative GALR3 protein sequence. 
     FIG. 7 illustrates the competition curves for  125 I-porcine glanin against human and porcine galanin. 
     FIG. 8 is the DNA sequence of rat GALR3 from region TM4 to region TM7 (SEQ ID NO:11). 
     FIG. 9 is the deduced amino acid sequence of rat GALR3 from region TM4 to region TM7 (SEQ ID NO:12). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     As used throughout the specification and claims, the following definitions apply: 
     “Substantially free from associated proteins” means that the receptor is at least about 90%, and preferably at least about 95% free from other cell membrane proteins which are normally found in a living mammalian cell which expresses a galanin receptor. 
     “Substantially free from associated nucleic acids” means that the nucleic acid is at least about 90%, and preferably at least about 95%, free from other nucleic acids which are normally found in a living mammalian cell which naturally expresses a galanin receptor gene. 
     “Substantially the same biological activity” means that the receptor-galanin binding constant is within 5-fold of the binding constant of GALR3 and galanin, and preferably within 2-fold of the binding constant of GALR3 and galanin. 
     “Stringent post-hybridizational washing conditions” means 0.1×standard saline citrate (SSC) at 65° C. 
     “Standard post-hybridizational washing conditions” means 6×SSC at 55° C. 
     “Relaxed post-hybridizational washing conditions” means 6×SSC at 30° C., or 1 to 2×SSC at 55° C. 
     “Functional equivalent” means that a receptor which does not have the exact same amino acid sequence of a naturally occurring GALR3 protein due to alternative splicing, deletions, mutations, or additions, but retains at least 1%, preferably 10%, and more preferably 25% of the biological activity of the naturally occurring receptor. Such derivatives will have a significant homology with a natural GALR3 and can be detected by reduced stringency hybridization with a DNA sequence obtained from a GALR3. The nucleic acid encoding a functional equivalent has at least about 60% homology at the nucleotide level to a naturally occurring receptor nucleic acid. 
     It has been found, in accordance with this invention, that there is a third galanin receptor, which is designated GALR3. The human (clone 3—3 and 3-2) and rat GALR3 sequences are given in FIGS. 1,  3  and  8 , respectively, and are referenced in the Examples; however it is to be understood that this invention specifically includes GALR3 without regard to the species and, in particular, specifically includes rodent (including rat and mouse), rhesus, swine, chicken, cow and human. The galanin 3 receptors are highly conserved throughout species, and one of ordinary skill in the art, given the rat, human and/or mouse sequences presented herein, can easily design probes to obtain the GALR3 from other species. 
     GALR3 proteins contain various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. Thus this invention specifically includes modified functionally equivalent GALR3s which have deleted, truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent GALR3s which contain modifications and/or deletions in other domains, which are not accompanied by a loss of functional activity. 
     Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention. 
     The proteins of this invention were found to have structural features which are typical of the 7-transmembrane domain (TM) containing G-protein linked receptor superfamily (GPC-R&#39;s or 7-TM receptors). Thus GALR3 proteins make up new members of the GPC-R family of receptors. The intact GALR3 of this invention was found to have the general features of GPC-R&#39;s, including seven transmembrane regions, three intra- and extracellular loops, and the GPC-R protein signature sequence. The TM domains and GPC-R protein signature sequence are noted in the protein sequences of the GALR3. Not all regions are required for functioning, and therefore this invention also comprises functional receptors which lack one or more non-essential domains. 
     Determination of the nucleotide sequence indicated that the GALR3 belongs to the intron-containing class of GPC-R&#39;s. 
     The DNA sequence encoding the putative GALR3 is shown in FIGS. 1 and 3. The human putative GALR3 gene is organized similarly to human GALR2 with a single intron (˜1 kb) dividing the open reading into two exons with Exon 1 consisting of ˜350 bp, and Exon 2˜700 bp. Based on database searching, the open reading frame protein sequence for this novel gene (FIGS. 2 and 4) is most closely related to GALR2 and GALR1 with 58, 75% identity and similarity to human GALR2, and 37, 61% identity and similarity to rat GALR1 (FIG.  5 ). Differences in open reading frame DNA sequence and the resulting deduced amino acid sequence between clone GALR3-2 and GALR3—3 may be allelic in nature. Phylogenetic analysis of the putative GALR3 protein sequence supports the notion that this gene encodes a receptor for galanin (FIG.  6 ). 
     The human GALR3 protein bears strong sequence identity and similarity to the rat GALR3 ortholog. 
     This invention also relates to truncated forms of GALR3, particularly those which encompass the extracellular portion of the receptor, but lack the intracellular signaling portion of the receptor, and to nucleic acids encoding these truncated forms. Such truncated receptors are useful in various binding assays. Thus this invention specifically includes modified functionally equivalent GALR3s which have deleted, truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent GALR3s including receptor chimeras which contain modifications and/or deletions in other domains, which are not accompanied by a loss of functional activity. 
     Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention. 
     Assays which make up further aspects of this invention include binding assays (competition for  125 I-galanin binding), coupling assays (including galanin-mediated inhibition of forskolin-stimulated adenylate cyclase in cells expressing galanin receptors), measurement of galanin-stimulated calcium release in cells expressing galanin receptors (such as aequorin assays), stimulation of inward rectifying potassium channels (GIRK channels, measured by voltage changes) in cells expressing galanin receptors, and measurement of pH changes upon galanin stimulation of cells expressing galanin receptors as measured with a microphysiometer. 
     Host cells may be cultured under suitable conditions to produce GALR3. An expression vector containing DNA encoding the receptor may be used for expression of receptor in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as  E. coli,  fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila, Spodoptera, and silkworm derived cell lines. Cell lines derived from mammalian species which are suitable and which are commercially available include, but are not limited to, L cells L-M(TK − ) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171). 
     The specificity of binding of compounds showing affinity for the receptor is shown by measuring the affinity of the compounds for cells transfected with the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that inhibit the binding of radiolabeled ligand to these cells provides a rational way for rapid selection of compounds with high affinity for the receptor. These compounds identified by the above assays may be agonists or antagonists of the receptor and may be peptides, proteins, or non-proteinaceous organic molecules. Alternatively, functional assays of the receptor may be used to screen for compounds which affect the activity of the receptor. Such functional assays range from ex vivo muscle contraction assays to assays which determine second messenger levels in cells expressing the receptor. The second messenger assays include, but are not limited to, assays to measure cyclic AMP or calcium levels or assays to measure adenyl cyclase activity. These compounds identified by the above assays may be agonists, antagonists, suppressors, or inducers of the receptor. The functional activity of these compounds is best assessed by using the receptor either natively expressed in tissues or cloned and exogenously expressed. 
     Using the assays of this invention, galanin agonists and antagonists may be identified. A galanin agonist is a compound which binds to the GALR3, such as a galanin mimetic, and produces a cellular response which is at least about equivalent to that of galanin, and which may be greater than that of galanin. Such compounds would be useful in situations where galanin insufficiency causes anorexia, or for treatment of pain. 
     Also using this embodiment of the assay, galanin antagonists may be identified. A galanin antagonist is a compound which can bind to the GALR3, but produces a lesser response than that of native galanin. Such compounds would be useful in the treatment of obesity. 
     One assay of this invention is a method of identifying a compound which modulates GALR3 receptor comprising: a) culturing cells expressing the GALR3 receptor in the presence of the compound and b) measuring GALR3 receptor activity or second messenger activity. If desired, the determined activity can be compared to a standard, such as that measured using galanin as the compound. In preferred embodiments, the cells are transformed and express the GALR3 receptor. 
     The consultant cDNA clone (or shorter portions of, for instance, only 15 nucleotides long) may be used to probe libraries under hybridization conditions to find other receptors which are similar enough so that the nucleic acids can hybridize, and is particularly useful for screening libraries from other species. In this step, one of ordinary skill in the art will appreciate that the hybridization conditions can vary from very stringent to relaxed. Proper temperature, salt concentrations, and buffers are well known. 
     The following non-limiting Examples are presented to better illustrate the invention. 
     EXAMPLE 1 
     Human GALR3 
     Identification and Cloning of Human GalR3 Gene, Sequence and Gene Structure 
     Automated searching of sequence data from GenBank (National Center For Biotechnology Information, Bethesda, Md.) were queried using sequences from known receptor clones. Using a list of 50-60 rhodopsin family amino acid sequences, the NEW division of GenBank was searched. The query algorithm is TFASTX and the output is placed in a file where alignments are sorted by query sequence and scored (cut-off based on the expectation value, set for example, at 0.01). A DNA sequence alignment of 300 bp to a portion of a large BAC clone (˜100,000 bp) with accession number Z97630 was identified from the high through-put genomic sequence (HTGS database, GenBank). The complete open reading frame (ORF) for the putative gene encoding GALR3 was then identified using sequence from BAC Z97630 and an additional BAC clone, with assession number Z82241, from the HTGS database. The Genbank assession numbers corresponded to the following HTGS BAC clones (HS entries): Z97630, HS466N1; Z82241, HS8112. 
     DNA sequences derived from these BACs were used to choose PCR primers. PCR primers begining at the predicted initiating Met and ending more 3′ than the predicted stop codon were utilized to PCR from human genomic DNA a fragment containing the predicted Exon I, the intervening intron, and predicted Exon II. This PCR product was subcloned and sequenced, resulting in expression plasmid GALR3—3. 
     In a parallel approach, a human genomic DNA library (Stratagene, La Jolla, Calif.) was screened to isolate the putative GALR3 gene. Primary screening under medium stringency resulted in 6 positive plaques using an Exon 2 probe. One hybridizing phage plaque was obtained upon secondary screening. A 13 kb EcoR1/EcoRV fragment was identified from the genomic clone by Southern blotting, transferred into pBluescript vector (Stratagene, La Jolla, Calif.), and confirmed to be GALR3 by sequencing. A intronless GALR3 expression construct was assembled in a similar manner to that described above using Pfu DNA polymerase (Stratagene, La Jolla, Calif.) resulting in expression plasmid GALR3-2. 
     EXAMPLE 2 
     Chromosomal Location 
     The BAC clones which were identified by the searches of the HTGS dataset have been mapped by the The Sanger Centre (Cambridge, UK) genome research laboratory to human chromosome 22. FISH analysis conducted herein has confirmed this assignment and refined it to 22q12.2-13.1. 
     EXAMPLE 3 
     Receptor Expression 
     Construction of Human GalR3 Expression Plasmid 
     The human GalR3 cDNA expression construct was assembled stepwise from PCR products amplified from human genomic DNA. Each exon was PCR amplified using standard conditions. The primers in for exon I were: Forward Exon I (5′-gcg aat tcg gta cca tgg ctg atg ccc aga aca t-3′; SEQ ID NO:13) and Reverse Exon I (5′-cgc ctg tcg aca gat aca gca gc -3′; SEQ ID NO:14). The primers for exon II were: Forward Exon II (5′-tgt atc tgt cga cag gta acc tgg ccg tgc ggc acc c-3′; SEQ ID NO:15) and Reverse Exon II (5′-gcg cgg ccg ctt att ccg gtc ctc ggg c-3′; SEQ ID NO:16). PCR products were subcloned into pCRII and sequenced. For expression in mammalian cells, the putative GALR3 ORF was subcloned into pcDNA-1/amp (Invitrogen) resulting in plasmid GALR3—3; and pcDNA-3 (Invitrogen), resulting in plasmid GALR3-2. 
     EXAMPLE 4 
     RNA Expression Profile 
     Using RNase protection analysis, the relative levels of human GALR3 mRNA was assessed. As shown below GALR3 is expressed in numerous brain regions and peripheral tissues, as observed for GALR1 and GALR2. 
     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Expression 
               
               
                   
                 Tissue 
                 Level 
               
               
                   
                   
               
             
             
               
                   
                 Amygdala 
                 + 
               
               
                   
                 Cerebellum 
                 + 
               
               
                   
                 Frontal Cortex 
                 + 
               
               
                   
                 Hippocampus 
                 + 
               
               
                   
                 Hypothalamus 
                 ++ 
               
               
                   
                 Pituitary 
                 + 
               
               
                   
                 Brain stem 
                 + 
               
               
                   
                 Lung 
                 ++ 
               
               
                   
                 Heart 
                 + 
               
               
                   
                 Spleen 
                 + 
               
               
                   
                 Liver 
                 + 
               
               
                   
                 Pancreas 
                 + 
               
               
                   
                 Duodenum 
                 + 
               
               
                   
                 Colon 
                 + 
               
               
                   
                 Straited muscle 
                 ++ 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 5 
     Radioligand Binding 
     Pharmacology of Human GALR3 
     Mammalian COS-7 cells were transfected by electroporation. COS-7 cells (1×10 7 ) were suspended in 0.85 ml of Ringers&#39; buffer and 15 mg of the GALR3-2 or GALR3—3 expression plasmid was added to a 0.4 mm electroporation cuvette (Bio-Rad, Hercules, Calif.). Current was applied (960 mF, 260 V) using a Bio-Rad Electroporator device and the cells were transferred to a T-180 flask (Corning). Expression was allowed to proceed for 72 hrs. 
     Membranes were prepared from transfected cells following dissociation in enzyme-free dissociation solution (Specialty Media, Lavallette, N.J.) by disruption in a Dounce homogenizer in ice-cold membrane buffer (10 mM Tris, pH 7.4, 10 mM PMSF, 10 mM phosphoramidon, and 40 mg/ml bacitracin). After a low speed (1100×g for 10 min. at 4° C.) and a high speed centrifugation (38,700×g for 15 min. at 4° C.), membranes were resuspended in buffer and protein concentration determined (Bio-Rad assay kit). Binding of  125 I-human or porcine galanin (specific activity of 2200 Ci/mmol, DuPont NEN) was measured in membranes using a buffer of 25 mM Tris pH 7.4, 0.5% BSA, 2 mM MgCl 2 , 40 μg/ml bacitracin, 4 mg/ml phosphoramidon, and 10 μM leupeptin in a total volume of 250 ml. 70 μM  125 I-human or porcine galanin was used. Transfected cells expressing plasmid GALR3—3 were bound with  125 I-human galanin whereas cells expressing plasmid GALR3-2 were bound with  125 I-porcine galanin. Reactions were initiated by the addition of membranes and the incubation was allowed to proceed at room temperature for 1 hour. Non-specific binding was defined as the amount of radioactivity remaining bound in the presence of 1 mM respectively unlabeled galanin and was generally not above 200 cpm (&lt;10% of total radioactivity bound). Titration of membrane protein from 1 to 50 μg was conducted. In competition studies various concentrations of unlabeled human or porcine galanin were included along with  125 I-porcine galanin (70 pM) in cells expressing the GALR3-2 plasmid. Incubations were terminated by rapid filtration through GF/C filters which had been presoaked with 0.1% polyethylamine using a TOMTEC (Orange, Conn.) cell harvester. The results were analyzed using the Prism software package (GraphPad, San Diego, Calif.). The table below illustrates that both clones confer specific binding to COS-7 cells for both human and porcine galanin radioligands as a function of protein concentration. In COS-7 cells mock transfected with expression vector only (no GALR3 gene), no specific binding of either radioligand was observed. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                 Clone GALR3-3 
                 Clone GALR3-2: 
               
               
                 Membrane Protein 
                   125 I-human galanin 
                   125 I-porcine galanin 
               
               
                 (μg) 
                 (cpm) 
                 (cpm) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ND 
                 ND 
               
               
                 5 
                 211 
                 695 
               
               
                 10 
                 407 
                 1134 
               
               
                 20 
                 886 
                 1763 
               
               
                 50 
                 2061 
                 3728 
               
               
                   
               
             
          
         
       
     
     Competition curves for  125 I-porcine galanin against human and porcine galanin were generated to to determine the IC50 for both unlabeled peptides (clone GALR3-2), as shown in FIG.  7 . The IC50 values for porcine and human galanin were 16 nM and 93 nM, respectively. 
     EXAMPLE 6 
     Rat GALR3 
     Identification and Cloning of Rat GalR3 Gene 
     Primers based on the intronless human GALR3 sequence from TM4 and TM7 were designed and used to PCR amplify with Pfu DNA polymerase the rat GALR3 ortholog from rat genomic DNA. A PCR product of the appropriate size (approximately 400 bp) that hybridized with an Exon 2 probe from the human GALR3 gene was subcloned into pBluescript vector (Stratagene, La Jolla, Calif.). The DNA sequence is shown is FIG.  8  and the deduced amino acid sequence is shown in FIG.  9 . DNA sequence analysis revealed significant homology with human GALR3: approximately 95% protein sequence identity for 129 amino acids spanning TM4 through TM-7. 
     RNA Expression 
     Northern blot analysis using a probe dervived from the rat GALR3 ORF has revealed expression of rat GALR3 mRNA in hypothalamus, mid-brain, pons, and whole fetal brain. 
     
       
         
           
             16 
           
           
             1 
             2263 
             DNA 
             human 
           
            1
ccaggtcggg ggagttagat cccggggtca agcaaccaga actgggggct cttgcctgag     60
gattccagct tctcttccca ggtgcccgtc tgatggggag atggctgatg cccagaacat    120
ttcactggac agcccaggga gtgtgggggc cgtggcagtg cctgtggtct ttgccctaat    180
cttcctgctg ggcacagtgg gcaatgggct ggtgctggca gtgctcctgc agcctggccc    240
gagtgcctgg caggagcctc gcagcaccac ggacctgttc atcctcaacc tggcggtggc    300
tgacctctgc ttcatcctgt gctgcgtgcc cttccaggcc accatataca cgctggatgc    360
ctggctcttt ggggccctcg tttgcaaggc cgtgcacctg ttcatctacc tcaccatgaa    420
cgccagcagc tttacgctgg ctgctgtatc tgtggacagg tgcgctgtgc ctggggcctg    480
gctgggcagg gctgtggggg cgggggttgg gggaggagtc ctgaacagat cctcactggc    540
cttaggaagg agagagtggg ggaccagaaa gggaggtggg tgggaggaaa caaaagctcc    600
ctgacccctc gcaagcagcc tctgggcacc tgcagggcgt gcttgagggg actgtcctgc    660
ccttcccctc ctccactgtg aacttccaga ggacgcctct gagtctcaag tggcagcaca    720
gggtctggca catagtaagt gctctgtaag cgcgaaatga atcgcaaaag aagctcacga    780
atgcgttcat cagttttttt gttttgtttt gttttgttgt tttttttttt ttggatcttg    840
gctcactgca acctctgcct cctgggttcc agcgattctc ctgccacagc ctcctgagta    900
gctgggatta caggccacca cacctggcta attttttgta tttttagtag aaacggggtt    960
ttgccatgtt ggacaggctg gtctcgaacc cctgacctca agtgatccgc ccgcctcggc   1020
ctcccaagtg ctgggattac aggcgtgagc caccgcgccc agcccagcta ttttctaact   1080
gcccacacct ggccaagctg tgcacacatc tgcttccaca gcttgaaact tggggtcaaa   1140
tccaggctca ctccagctga tgaccctggg caagtcactt ctctctggac ctcatctgac   1200
gcatccataa aataatccta gaaataacaa gtcaccggga tcgggccctt gctaggtgca   1260
agggcctaag caccttgcgc gttcacaccc ttaatccccg ccacgtcccc cacggttcac   1320
aggaggcgca ctgggccgca gggcccgggc gcgggacgtg gcgcgggccc ctgcgggagg   1380
gcacctgccc gccccgctga ccasgcgccc tccgcaggta cctggccgtg cggcacccgc   1440
tgcgctcgcg cgccctgcgc acgccgcgta acgcccgcgc cgcagtgggg ctggtgtggc   1500
tgctggcggc gctcttctcg gcgccctacc tcagctacta cggcaccgtg cgctacggcg   1560
cgctggagct ctgcgtgccc gcctgggagg acgcgcgccg ccgcgccctg gacgtggcca   1620
ccttcgctgc cggctacctg ctgcccgtgg cygtggtgag cctggcctac gggcgcacgc   1680
tgcgcttcct gtgggccgcc gtgggtcccg cgggcgcggc ggcrgccaar gcgcggcgga   1740
gggcgackgg ccgcgcgggg cgcgccatgc tggcggtggc cgcgctctac gcgctmtgct   1800
ggggtccgca ccacgcgctc atcctgtgct tctggtacgg ccgmttcgcc ttcagcccgg   1860
ccacctacgc mtgccgcctg gcctcacact gcctggccta cgccaactcm tgcctcaacc   1920
cgctcgtmta cgcgctcgcc tcgcgccact tccgcgcgcg cttccgccgc ctgtggccgt   1980
gcggycgccg acgccgccac cgtgcccgcc gcgccttgcg tcgcgtccgc cccgcgtcct   2040
cgggcccacc cggctgcccc ggagacgccc ggcctagcgg gacgctgctg gctggtggcg   2100
gccagggccc sgagcccagg gagggacccg tccacggcgg agaggctgcc cgaggaccgg   2160
aataaaccct gccgcctgga ctccgcctgt gtccgtctgt ctcactcccg ttctccgaag   2220
gcgggacgcc accgggccag ggatggggca atgccacgag ctc                     2263
 
           
             2 
             368 
             PRT 
             human 
           
            2
Met Ala Asp Ala Gln Asn Ile Ser Leu Asp Ser Pro Gly Ser Val Gly
 1               5                  10                  15
Ala Val Ala Val Pro Val Val Phe Ala Leu Ile Phe Leu Leu Gly Thr
            20                  25                  30
Val Gly Asn Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro Ser
        35                  40                  45
Ala Trp Gln Glu Pro Arg Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu
    50                  55                  60
Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe Gln Ala
65                  70                  75                  80
Thr Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala Leu Val Cys Lys
                85                  90                  95
Ala Val His Leu Phe Ile Tyr Leu Thr Met Asn Ala Ser Ser Phe Thr
            100                 105                 110
Leu Ala Ala Val Ser Val Asp Arg Tyr Leu Ala Val Arg His Pro Leu
        115                 120                 125
Arg Ser Arg Ala Leu Arg Thr Pro Arg Asn Ala Arg Ala Ala Val Gly
    130                 135                 140
Leu Val Trp Leu Leu Ala Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr
145                 150                 155                 160
Tyr Gly Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Trp
                165                 170                 175
Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala Thr Phe Ala Ala Gly
            180                 185                 190
Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu
        195                 200                 205
Arg Phe Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala Ala Ala Lys
    210                 215                 220
Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala Met Leu Ala Val
225                 230                 235                 240
Ala Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His Ala Leu Ile Leu
                245                 250                 255
Cys Phe Trp Tyr Gly Arg Phe Ala Phe Ser Pro Ala Thr Tyr Ala Cys
            260                 265                 270
Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro
        275                 280                 285
Leu Val Tyr Ala Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg Arg
    290                 295                 300
Leu Trp Pro Cys Gly Arg Arg Arg Arg His Arg Ala Arg Arg Ala Leu
305                 310                 315                 320
Arg Arg Val Arg Pro Ala Ser Ser Gly Pro Pro Gly Cys Pro Gly Asp
                325                 330                 335
Ala Arg Pro Ser Gly Thr Leu Leu Ala Gly Gly Gly Gln Gly Pro Glu
            340                 345                 350
Pro Arg Glu Gly Pro Val His Gly Gly Glu Ala Ala Arg Gly Pro Glu
        355                 360                 365
 
           
             3 
             1107 
             DNA 
             human 
           
            3
atggctgatg cccagaacat ttcactggac agcccaggga gtgtgggggc cgtggcagtg     60
cctgtggtct ttgccctaat cttcctgctg ggcacagtgg gcaatgggct ggtgctggca    120
gtgctcctgc agcctggccc gagtgcctgg caggagcctg gcagcaccac ggacctgttc    180
atcctcaacc tggcggtggc tgacctctgc ttcatcctgt gctgcgtgcc cttccaggcc    240
accatctaca cgctggatgc ctggctcttt ggggccctcg tctgcaaggc cgtgcacctg    300
ctcatctacc tcaccatgta cgccagcagc tttacgctgg ctgctgtctc cgtggacagg    360
tacctggccg tgcggcaccc gctgcgctcg cgcgccctgc gcacgccgcg taacgcccgc    420
gccgcagtgg ggctggtgtg gctgctggcg gcgctcttct cggcgcccta cctcagctac    480
tacggcaccg tgcgctacgg cgcgctggag ctctgcgtgc ccgcctggga ggacgcgcgc    540
cgccgcgccc tggacgtggc caccttcgct gccggctacc tgctgcccgt ggctgtggtg    600
agcctggcct acgggcgcac gctgcgcttc ctgtgggccg ccgtgggtcc cgcgggcgcg    660
gcggcggccg aggcgcggcg gagggcgacg ggccgcgcgg ggcgcgccat gctggcggtg    720
gccgcgctct acgcgctctg ctggggtccg caccacgcgc tcatcctgtg cttctggtac    780
ggccgcttcg ccttcagccc ggccacctac gcctgccgcc tggcctcaca ctgcctggcc    840
tacgccaact cctgcctcaa cccgctcgtc tacgcgctcg cctcgcgcca cttccgcgcg    900
cgcttccgcc gcctgtggcc gtgcggccgc cgacgccgcc accgtgcccg ccgcgccttg    960
cgtcgcgtcc gccccgcgtc ctcgggccca cccggctgcc ccggagacgc ccggcctagc   1020
gggaggctgc tggctggtgg cggccagggc ccggagccca gggagggacc cgtccacggc   1080
ggagaggctg cccgaggacc ggaataa                                       1107
 
           
             4 
             368 
             PRT 
             human 
           
            4
Met Ala Asp Ala Gln Asn Ile Ser Leu Asp Ser Pro Gly Ser Val Gly
 1               5                  10                  15
Ala Val Ala Val Pro Val Val Phe Ala Leu Ile Phe Leu Leu Gly Thr
            20                  25                  30
Val Gly Asn Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro Ser
        35                  40                  45
Ala Trp Gln Glu Pro Gly Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu
    50                  55                  60
Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe Gln Ala
65                  70                  75                  80
Thr Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala Leu Val Cys Lys
                85                  90                  95
Ala Val His Leu Leu Ile Tyr Leu Thr Met Tyr Ala Ser Ser Phe Thr
            100                 105                 110
Leu Ala Ala Val Ser Val Asp Arg Tyr Leu Ala Val Arg His Pro Leu
        115                 120                 125
Arg Ser Arg Ala Leu Arg Thr Pro Arg Asn Ala Arg Ala Ala Val Gly
    130                 135                 140
Leu Val Trp Leu Leu Ala Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr
145                 150                 155                 160
Tyr Gly Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Trp
                165                 170                 175
Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala Thr Phe Ala Ala Gly
            180                 185                 190
Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu
        195                 200                 205
Arg Phe Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala Ala Ala Glu
    210                 215                 220
Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala Met Leu Ala Val
225                 230                 235                 240
Ala Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His Ala Leu Ile Leu
                245                 250                 255
Cys Phe Trp Tyr Gly Arg Phe Ala Phe Ser Pro Ala Thr Tyr Ala Cys
            260                 265                 270
Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro
        275                 280                 285
Leu Val Tyr Ala Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg Arg
    290                 295                 300
Leu Trp Pro Cys Gly Arg Arg Arg Arg His Arg Ala Arg Arg Ala Leu
305                 310                 315                 320
Arg Arg Val Arg Pro Ala Ser Ser Gly Pro Pro Gly Cys Pro Gly Asp
                325                 330                 335
Ala Arg Pro Ser Gly Arg Leu Leu Ala Gly Gly Gly Gln Gly Pro Glu
            340                 345                 350
Pro Arg Glu Gly Pro Val His Gly Gly Glu Ala Ala Arg Gly Pro Glu
        355                 360                 365
 
           
             5 
             348 
             PRT 
             mouse 
           
            5
Met Glu Leu Ala Met Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro
 1               5                  10                  15
Glu Pro Pro Ala Pro Glu Ser Arg Pro Leu Phe Gly Ile Gly Val Glu
            20                  25                  30
Asn Phe Ile Thr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val
        35                  40                  45
Leu Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly
    50                  55                  60
Lys Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala
65                  70                  75                  80
Asp Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr
                85                  90                  95
Ala Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His
            100                 105                 110
Tyr Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala
        115                 120                 125
Met Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser
    130                 135                 140
Ser Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe Ile Trp
145                 150                 155                 160
Ala Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr His Gln Arg Leu
                165                 170                 175
Phe His Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu Gln Trp Pro Asn
            180                 185                 190
Lys Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly Tyr
        195                 200                 205
Leu Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu Asn
    210                 215                 220
His Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala Ser
225                 230                 235                 240
Lys Lys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val Phe Gly
                245                 250                 255
Ile Ser Trp Leu Pro His His Val Val His Leu Trp Ala Glu Phe Gly
            260                 265                 270
Ala Phe Pro Leu Thr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His
        275                 280                 285
Cys Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe
    290                 295                 300
Leu Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys His
305                 310                 315                 320
Val Cys Asp Glu Ser Pro Arg Ser Glu Thr Lys Glu Asn Lys Ser Arg
                325                 330                 335
Met Asp Thr Pro Pro Ser Thr Asn Cys Thr His Val
            340                 345
 
           
             6 
             346 
             PRT 
             rat 
           
            6
Met Glu Leu Ala Pro Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro
 1               5                  10                  15
Glu Pro Pro Ala Glu Pro Arg Pro Leu Phe Gly Ile Gly Val Glu Asn
            20                  25                  30
Phe Ile Thr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val Leu
        35                  40                  45
Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly Lys
    50                  55                  60
Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala Asp
65                  70                  75                  80
Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr Ala
                85                  90                  95
Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His Tyr
            100                 105                 110
Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala Met
        115                 120                 125
Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser Ser
    130                 135                 140
Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe Ile Trp Ala
145                 150                 155                 160
Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr Tyr Gln Arg Leu Phe
                165                 170                 175
His Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu His Trp Pro Asn Gln
            180                 185                 190
Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly Tyr Leu
        195                 200                 205
Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu Asn His
    210                 215                 220
Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala Ser Lys
225                 230                 235                 240
Lys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val Phe Gly Ile
                245                 250                 255
Ser Trp Leu Pro His His Val Ile His Leu Trp Ala Glu Phe Gly Ala
            260                 265                 270
Phe Pro Leu Thr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His Cys
        275                 280                 285
Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe Leu
    290                 295                 300
Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys Arg Val
305                 310                 315                 320
Cys Asn Glu Ser Pro His Gly Asp Ala Lys Glu Lys Asn Arg Ile Asp
                325                 330                 335
Thr Pro Pro Ser Thr Asn Cys Thr His Val
            340                 345
 
           
             7 
             349 
             PRT 
             human 
           
            7
Met Glu Leu Ala Val Gly Asn Leu Ser Glu Gly Asn Ala Ser Cys Pro
 1               5                  10                  15
Glu Pro Pro Ala Pro Glu Pro Gly Pro Leu Phe Gly Ile Gly Val Glu
            20                  25                  30
Asn Phe Val Thr Leu Val Val Phe Gly Leu Ile Phe Ala Leu Gly Val
        35                  40                  45
Leu Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly
    50                  55                  60
Lys Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala
65                  70                  75                  80
Asp Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr
                85                  90                  95
Ala Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His
            100                 105                 110
Tyr Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala
        115                 120                 125
Met Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser
    130                 135                 140
Ser Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Cys Ile Trp
145                 150                 155                 160
Ala Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr His Gln Gly Leu
                165                 170                 175
Phe His Pro Arg Ala Ser Asn Gln Thr Phe Cys Trp Glu Gln Trp Pro
            180                 185                 190
Asp Pro Arg His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly
        195                 200                 205
Tyr Leu Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu
    210                 215                 220
Asn His Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala
225                 230                 235                 240
Ser Lys Lys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val Phe
                245                 250                 255
Gly Ile Ser Trp Leu Pro His His Ile Ile His Leu Trp Ala Glu Phe
            260                 265                 270
Gly Val Phe Pro Leu Thr Pro Ala Ser Phe Leu Phe Arg Ile Thr Ala
        275                 280                 285
His Cys Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala
    290                 295                 300
Phe Leu Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys
305                 310                 315                 320
His Ile Arg Lys Asp Ser His Leu Ser Asp Thr Lys Glu Asn Lys Ser
                325                 330                 335
Arg Ile Asp Thr Pro Pro Ser Thr Asn Cys Thr His Val
            340                 345
 
           
             8 
             371 
             PRT 
             mouse 
           
            8
Met Asn Gly Ser Asp Ser Gln Gly Ala Glu Asp Ser Ser Gln Glu Gly
 1               5                  10                  15
Gly Gly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe Ala
            20                  25                  30
Leu Ile Phe Leu Val Gly Ala Val Gly Asn Ala Leu Val Leu Ala Val
        35                  40                  45
Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile Leu
    50                  55                  60
Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe
65                  70                  75                  80
Gln Ala Thr Ile Tyr Thr Leu Asp Asp Trp Val Phe Gly Ser Leu Leu
                85                  90                  95
Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser Ser
            100                 105                 110
Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg Tyr
        115                 120                 125
Pro Met His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala Ala
    130                 135                 140
Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr Leu
145                 150                 155                 160
Ser Tyr Tyr Ser Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His Pro
                165                 170                 175
Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp Leu Cys Thr Phe Val
            180                 185                 190
Phe Ser Tyr Leu Leu Pro Val Leu Val Leu Ser Leu Thr Tyr Ala Arg
        195                 200                 205
Thr Leu His Tyr Leu Trp Arg Thr Val Asp Pro Val Ala Ala Gly Ser
    210                 215                 220
Gly Ser Gln Arg Ala Lys Arg Lys Val Thr Arg Met Ile Val Ile Val
225                 230                 235                 240
Ala Val Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu Ile Leu
                245                 250                 255
Cys Val Trp Phe Gly Arg Phe Pro Leu Thr Arg Ala Thr Tyr Ala Leu
            260                 265                 270
Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn Pro
        275                 280                 285
Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg Lys
    290                 295                 300
Ile Cys Ala Gly Leu Leu Arg Arg Ala Pro Arg Arg Ala Ser Gly Arg
305                 310                 315                 320
Val Cys Ile Leu Ala Pro Gly Asn His Ser Gly Gly Met Leu Glu Pro
                325                 330                 335
Glu Ser Thr Asp Leu Thr Gln Val Ser Glu Ala Ala Gly Pro Leu Val
            340                 345                 350
Pro Ala Pro Ala Leu Pro Asn Cys Thr Thr Leu Ser Arg Thr Leu Asp
        355                 360                 365
Pro Ala Cys
    370
 
           
             9 
             372 
             PRT 
             rat 
           
            9
Met Asn Gly Ser Gly Ser Gln Gly Ala Glu Asn Thr Ser Gln Glu Gly
 1               5                  10                  15
Gly Ser Gly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe
            20                  25                  30
Ala Leu Ile Phe Leu Val Gly Thr Val Gly Asn Ala Leu Val Leu Ala
        35                  40                  45
Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile
    50                  55                  60
Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro
65                  70                  75                  80
Phe Gln Ala Thr Ile Tyr Thr Leu Asp Asp Trp Val Phe Gly Ser Leu
                85                  90                  95
Leu Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser
            100                 105                 110
Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg
        115                 120                 125
Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala
    130                 135                 140
Ala Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr
145                 150                 155                 160
Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His
                165                 170                 175
Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp Leu Cys Thr Phe
            180                 185                 190
Val Phe Ser Tyr Leu Leu Pro Val Leu Val Leu Ser Leu Thr Tyr Ala
        195                 200                 205
Arg Thr Leu Arg Tyr Leu Trp Arg Thr Val Asp Pro Val Thr Ala Gly
    210                 215                 220
Ser Gly Ser Gln Arg Ala Lys Arg Lys Val Thr Arg Met Ile Ile Ile
225                 230                 235                 240
Val Ala Val Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu Ile
                245                 250                 255
Leu Cys Val Trp Phe Gly Arg Phe Pro Leu Thr Arg Ala Thr Tyr Ala
            260                 265                 270
Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn
        275                 280                 285
Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg
    290                 295                 300
Lys Ile Cys Ala Gly Leu Leu Arg Pro Ala Pro Arg Arg Ala Ser Gly
305                 310                 315                 320
Arg Val Ser Ile Leu Ala Pro Gly Asn His Ser Gly Ser Met Leu Glu
                325                 330                 335
Gln Glu Ser Thr Asp Leu Thr Gln Val Ser Glu Ala Ala Gly Pro Leu
            340                 345                 350
Val Pro Pro Pro Ala Leu Pro Asn Cys Thr Ala Ser Ser Arg Thr Leu
        355                 360                 365
Asp Pro Ala Cys
    370
 
           
             10 
             387 
             PRT 
             human 
           
            10
Met Asn Val Ser Gly Cys Pro Gly Ala Gly Asn Ala Ser Gln Ala Gly
 1               5                  10                  15
Gly Gly Gly Gly Trp His Pro Glu Ala Val Ile Val Pro Leu Leu Phe
            20                  25                  30
Ala Leu Ile Phe Leu Val Gly Thr Val Gly Asn Thr Leu Val Leu Ala
        35                  40                  45
Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile
    50                  55                  60
Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro
65                  70                  75                  80
Phe Gln Ala Thr Ile Tyr Thr Leu Asp Gly Trp Val Phe Gly Ser Leu
                85                  90                  95
Leu Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser
            100                 105                 110
Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg
        115                 120                 125
Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala
    130                 135                 140
Ala Ile Gly Leu Ile Trp Gly Leu Ser Leu Leu Phe Ser Gly Pro Tyr
145                 150                 155                 160
Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His
                165                 170                 175
Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp Ile Cys Thr Phe
            180                 185                 190
Val Phe Ser Tyr Leu Leu Pro Val Leu Val Leu Gly Leu Thr Tyr Ala
        195                 200                 205
Arg Thr Leu Arg Tyr Leu Trp Arg Ala Val Asp Pro Val Ala Ala Gly
    210                 215                 220
Ser Gly Ala Arg Arg Ala Lys Arg Lys Val Thr Arg Met Ile Leu Ile
225                 230                 235                 240
Val Ala Ala Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu Ile
                245                 250                 255
Leu Cys Val Trp Phe Gly Gln Phe Pro Leu Thr Arg Ala Thr Tyr Ala
            260                 265                 270
Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn
        275                 280                 285
Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg
    290                 295                 300
Thr Ile Cys Ala Gly Leu Leu Gly Arg Ala Pro Gly Arg Ala Ser Gly
305                 310                 315                 320
Arg Val Cys Ala Ala Ala Arg Gly Thr His Ser Gly Ser Val Leu Glu
                325                 330                 335
Arg Glu Ser Ser Asp Leu Leu His Met Ser Glu Ala Ala Gly Ala Leu
            340                 345                 350
Arg Pro Cys Pro Gly Ala Ser Gln Pro Cys Ile Leu Glu Pro Cys Pro
        355                 360                 365
Gly Pro Ser Trp Gln Gly Pro Lys Ala Gly Asp Ser Ile Leu Thr Val
    370                 375                 380
Asp Val Ala
385
 
           
             11 
             390 
             DNA 
             rat 
           
            11
ccctacctca gctactacgg cacggtgcgc tacggccggc tcgagctctg cgtgcccgct     60
tgggaggagg acgcgcggcg gcgcgcgctg gacgtggcca ccttcgccgc gggctacctg    120
ctgccggtgg ccgtggtgag cctggcctac ggacgcacgc tatgtttcct atgggccgcc    180
gtgggtcccg cgggcagcgc ggcagcagag gcgcgcagac gggcgaccgg ccgggcggga    240
cgccgcatgc tggcagtggc gctctacgcg ctttgctggg gcccgcacca cgcgctcatc    300
ctctgcttct ggtacggtcc gttcgccttc agcccggcca cctacgcctg tcgcctggcc    360
tcacactgcc tcgcctacgc caactcctgc                                     390
 
           
             12 
             129 
             PRT 
             rat 
           
            12
Pro Tyr Leu Ser Tyr Tyr Gly Thr Val Arg Tyr Gly Arg Leu Glu Leu
 1               5                  10                  15
Cys Val Pro Ala Trp Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala
            20                  25                  30
Thr Phe Ala Ala Gly Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala
        35                  40                  45
Tyr Gly Arg Thr Leu Cys Phe Leu Trp Ala Ala Val Gly Pro Ala Gly
    50                  55                  60
Ser Ala Ala Ala Glu Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg
65                  70                  75                  80
Arg Met Leu Ala Val Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His
                85                  90                  95
Ala Leu Ile Leu Cys Phe Trp Tyr Gly Pro Phe Ala Phe Ser Pro Ala
            100                 105                 110
Thr Tyr Ala Cys Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser
        115                 120                 125
Cys
 
           
             13 
             34 
             DNA 
             Artificial Sequence 
             
               PCR primer 
             
           
            13
gcgaattcgg taccatggct gatgcccaga acat                                 34
 
           
             14 
             23 
             DNA 
             Artificial Sequence 
             
               PCR primer 
             
           
            14
cgcctgtcga cagatacagc agc                                             23
 
           
             15 
             37 
             DNA 
             Artificial Sequence 
             
               PCR primer 
             
           
            15
tgtatctgtc gacaggtaac ctggccgtgc ggcaccc                              37
 
           
             16 
             28 
             DNA 
             Artificial Sequence 
             
               PCR primer 
             
           
            16
gcgcggccgc ttattccggt cctcgggc                                        28