NOVEL RECEPTOR

Abstract: The invention provides isolated polynucleotide molecules encoding a novel G-protein-coupled receptor (designated TSR32). These isolated polynucleotide molecules can be used to express the receptor in cells which can then be used to screen compounds for agonist and antagonist activity.

Description: NOVEL RECEPTOR Field of the Invention :- The present invention relates to isolated polynucleotide molecules which encode a novel seven-transmembrane G-protein-coupled receptor designated TSR32. In addition, the present invention relates to the use of these molecules in the production of TSR32 receptors using recombinant technology and to methods of screening compounds for agonists and/or antagonists of the TSR32 receptor. Background of the Invention:- Proteins with seven-transmembrane (7TM) segments define a superfamily of receptors with a common stmcture comprising an extracellular N-terminus, three extramembranous loops on either side of the plasma membrane, and a cytoplasmic C-terminus. The majority of such proteins f inction as cell-surface receptors for a variety of ligands, including small molecules, peptides, hormones, ions, and external sensory stimuli such as odorants (Watson, S. and Arkinstall, S., The G-protein Linked Receptor Facts Book, Academic Press, London, 1994). One family within the 7TM superfamily is known as the secretin receptor family and comprises member receptors with specificity for secretin, calcitonin, glucagon, glucagon-like peptide 1. parathyroid hormone, parathyroid-related peptide, corticotropin- releasing factor (CRF), growth hormone-releasing hormone (GHRH), gastric inhibitory polypeptide, pituitary adenylate-cyclase-activating polypeptide (PACAP), vasoactive intestinal peptides (VIP) and insect diuretic hormone (DHR). The present inventor has now identified a further member receptor of the secretin receptor family. This receptor, designated TSR32, appears to be expressed in a large variety of tissue types including heart, kidney, lung and brain with highest levels in the thyroid gland. Since agonists and/or antagonists for this receptor may have commercial value, for example as regulators of important thyroid functions in growth, development and metabolic activity, the ability to produce TSR32 receptors by recombinant DNA technology would be advantageous. Disclosure of the Invention:- Thus, in a first aspect, the present invention provides an isolated polynucleotide molecule encoding a G-protein-coupled receptor, designated TSR32, which is characterised by the N-terminal amino acid sequence: MTPQSLLQTT (SEQ ID NO: 1), or a functionally equivalent fragment of said receptor. Preferably, the polynucleotide molecule encodes a human TSR32 receptor of about 693 amino acids. More preferably, the isolated polynucleotide molecule encodes a human TSR32 receptor comprising an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2 or a functionally equivalent fragment thereof. The nucleotide sequence of a polynucleotide molecule in accordance with the first aspect, may comprise a nucleotide sequence substantially corresponding to or, showing at least 75% (preferably, at least 90% or. even more preferably, at least 95%) sequence identity to that shown as SEQ ID NO: 3 or SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent TSR32 receptor fragment. The polynucleotide molecvile may be incorporated into plasmids or expression vectors (including viral vectors), which may then be introduced into S iitable host cells (e.g. bacterial, yeast, insect and mammalian host cells). Such host cells may be used to express the TSR32 receptor encoded by the isolated polynucleotide molecule. Accordingly, in a second aspect, the present invention provides a host cell transformed with the polynucleotide molecule of the first aspect. In a third aspect, the present invention provides a method of producing TSR32 receptors or functionally equivalent fragments thereof, comprising culturing the host cell of the second aspect under conditions enabling the expression of the polynucleotide molecule and, optionally, recovering the TSR32 receptor or fragments thereof. Preferably, the host cell is mammalian or of insect origin. Where the cell is mammalian, it is presently preferred that it be a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell. Where the cell is of insect origin, it is presently preferred that it be an insect Sf9 cell. In a preferred embodiment, the TSR32 receptor or fragments thereof are expressed onto the surface of the host cells. The polynucleotide molecules of the present invention represent a new subfamily of the secretin receptor family which may be of interest both clinically and commercially as it is expressed in many tissue types and may therefore be involved in a wide variety of systems. By using the polynucleotide molecules of the present invention it is possible to obtain TSR32 receptor protein or functionally equivalent fragments thereof in a substantially pure form. Accordingly, in a fourth aspect, the present invention provides a G- protein-coupled receptor, designated TSR32, which is characterised by the N- terminal amino acid sequence: MTPQSLLQTT (SEQ ID NO: 1), or a functionally equivalent fragment thereof, in a substantially pure form. Preferably, the purified TSR32 receptor has an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2. In a fifth aspect, the present invention provides an antibody or antibody fragment capable of specifically binding to a TSR32 receptor according to the fourth aspect. The antibody may be monoclonal or polyclonal, however, it is presently preferred that the antibody is a monoclonal antibody. Suitable antibody fragments include Fab, (F(ab') 2 and scFv. In a sixth aspect, the present invention provides a non-human animal transformed with a polynucleotide molecule according to the first aspect of the present invention. In a seventh aspect, the present invention provides a method for detecting agonist and/or antagonist compounds of the TSR32 receptor, comprising contacting a TSR32 receptor, functionally equivalent fragment thereof or a host cell transformed with and expressing the polynucleotide molecule of the first aspect, with a test compound under conditions enabling the activation of the TSR32 receptor or functionally equivalent fragment thereof, and detecting an increase or decrease in the activity of the TSR32 receptor or functionally equivalent fragment thereof. An increase or decrease in activity of the receptor or functionally eq iivalent fragment thereof may be detected by measuring changes in CAMP production, CA 2+ levels or IP3 turnover after activating the receptor or fragment thereof with specific agonists or antagonists. In a further aspect, the present invention provides an oligonucleotide or polynucleotide probe comprising a icleotide seqLience of 10 or more nucleotides, the probe comprising a nucleotide sequence such that the probe specifically hybridises to the polynucleotide molecule of the first aspect under high stringency conditions (Sambrook et ah, Molecular Cloning: a laboratory manual, Second Edition, Cold Spring Harbor Laboratory Press). Preferably the probe is labelled. In a still further aspect, the present invention provides an antisense polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridising to an mRNA molecule which encodes a TSR32 receptor so as to prevent translation of the mRNA molecule. Such antisense polynucleotide molecules may include a ribozyme region to catalytically inactivate mRNA to which it is hybridized. The polynucleotide molecule of the first aspect of the invention may be a dominant negative mutant which encodes a gene product ca ising an altered phenotype by, for example, reducing or eliminating the activity of endogenous TSR32 receptors. The term "substantially corresponding" as vised herein in relation to amino acid sequences is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in the biological activity of the TSR32 receptor. These variations may include conservative amino acids substitutions. The substitutions envisaged are:- G, A, V, I, L, M: D, E; N. Q; S, T; K, R, H; F. Y, W, H; and P, Nα-alkalamino acids. The term "substantially corresponding" as used herein in relation to nucleotide sequences is intended to encompass minor variations in the nucleotide seqLience which due to degeneracy in the DNA code do not result in a change in the encoded protein. Further, this term is intended to encompass other minor variations in the sequence which may be reqLiired to enhance expression in a particular system but in which the variations do not result in a decrease and biological activity of the encoded protein. The term "functionally equivalent fragment/s" as used herein is intended to refer to fragments of the TSR32 receptor that exhibit binding specificity and activity that is substantially equivalent to the full length receptor from which it/they is/are derived. The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or without the inclusion of a further step, component or feature or group of steps, components or features. Brief description of the figures:- Figure 1 provides the nucleotide sequence of a cDNA encoding the human TSR32 receptor and includes the predicted amino acid sequence. Figure 2 provides a sequence alignment of the amino acid sequences of the human TSR32 receptor and the human epididymis-specific HE6 receptor (Osterhoff, C, DNA and Cell Biology, Vol. 16 No. 4. 1997). Example :- Degenerative oligonucleotides corresponding to conserved regions within the human Glucagon-like peptide receptor (GLPl) family were vised to amplify specific DNA sequences from a variety of cDNA library DNA's. Library DNA from human hypothalamus and heart yielded fragments of the expected size. These fragments were subcloned and sequenced. One fragment, 456bp long, revealed a novel 7TM receptor sequence distantly related to the secretin receptor family. Screening a human heart cDNA library (Stratagene) using this fragment under high stringency conditions identified two positive hybridising clones, 1 and 2.9kb in size, respectively. The larger clone contains an open reading frame of 2079 nucleotides encoding a 693 amino acid long protein (designated TSR32). The nucleotide and pvitative amino acid seqvience is provided at Figure 1. A search of the EMBL/GenBank database revealed low but significant sequence similarities to the secretin receptor family of TM7 receptors. The seqvience similarity to the above receptor sequences is only about 25% identity and also limited to the transmembrane region. However, the highest overall sequence similarity was found to four other G-protein coupled receptor subtypes, the human epididymis-specific HE6 (31% identity), the human lymphocyte antigen CD97 (33% identity), the orphan human receptor EMR1 (27% identity), the insect DHR (26% identity) and the rat latrophilin- related protein 1 precursor (35% identity) all of which share a large extracellular domain with numerous putative O- and N-glycosylation sites. Except for DHR, in all of the receptors there is a cysteine motif preceding the first transmembrane domain. Further, in contrast to the CD97 and EMR1 receptors, TSR32 does not contain any EGF-domains or calcium binding sites in the extracellular domain. Interestingly, at the gene level the exon/intron organisation of the TSR32-encoding cDNA. at least within the region encoding the transmembrane domain, is most similar to the gene structure of the PACAP receptor family, with fewer bvit identical exon/intron borders. Northern analysis indicates a low level of expression of the receptor mRNA in a large variety of tissues including kidney, lung, cerebellum and heart. The highest level of mRNA however, was found in the thyroid gland. This expression pattern of the receptor mRNA indicates that TSR32 has important functions in metabolic regulations throughout the body via the thyroid gland. The gene for TSR32 has been mapped to human chromosome 16q31 by in situ hybridisation and this localisation was confirmed by radiation hybrid analysis. An avitosomal recessive disorder (Bardet Biedl Syndrome; Kwitek- Black, A.E. et al, Nature Genetics 5 : 392-396, 1993) has also been linked to this region. Features of this syndrome include obesity, retinal degeneration, hypogonadism and mental retardation. The expression of TSR32 examined by in situ hybridisation and Northern analysis overlaps strongly with the tissues affected in the Bardet Biedl Syndrome making TSR32 a good candidate for this locus. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Sequence listings:- Applicant: Garvan Institute of Medical Research Title of Invention: Novel receptor Number of SEQ ID Nos : 4 SEQ ID NO: 1 Length: 10 Type PRT Organism: Homo sapiens Sequence: 1 Met Thr Pro Gin Ser Leu Leu Gin Thr Thr 1 5 10 SEQ ID NO: 2 Length 693 Type PRT Organism: Homo sapiens Sequence: 2 Met Thr Pro Gin Ser Leu Leu Gin Thr Thr Leu Phe Leu Leu Ser Leu 1 5 10 15 Leu Phe Leu Val Gin Gly Ala His Gly Arg Gly His Arg Glu Asp Phe 20 25 30 Arg Phe Cys Ser Gin Arg Asn Gin Thr His Arg Ser Ser Leu His Tyr 35 40 45 Lys Pro Thr Pro Asp Leu Arg lie Ser lie Glu Asn Ser Glu Glu Ala 50 55 60 Leu Thr Val His Ala Pro Phe Pro Ala Ala His Pro Ala Ser Arg Ser 65 70 75 80 Phe Pro Asp Pro Arg Gly Leu Tyr His Phe Cys Leu Tyr Trp Asn Arg 85 90 95 His Ala Gly Arg Leu His Leu Leu Tyr Gly Lys Arg Asp Phe Leu Leu 100 105 110 Ser Asp Lys Ala Ser Ser Leu Leu Cys Phe Gin His Gin Glu Glu Ser 115 120 125 Leu Ala Gin Gly Pro Pro Leu Leu Ala Thr Ser Val Thr Ser Trp Trp 130 135 140 Ser Pro Gin Asn lie Ser Leu Pro Ser Ala Ala Ser Phe Thr Phe Ser 145 150 155 160 Phe His Ser Pro Pro His Thr Ala Ala His Asn Ala Ser Val Asp Met 165 170 175 Cys Glu Leu Lys Arg Asp Leu Gin Leu Leu Ser Gin Phe Leu Lys His 180 185 190 Pro Gin Lys Ala Ser Arg Arg Pro Ser Ala Ala Pro Ala Ser Gin Gin 195 200 205 Leu Gin Ser Leu Glu Ser Lys Leu Thr Ser Val Arg Phe Met Gly Asp 210 215 220 Met Val Ser Phe Glu Glu Asp Arg lie Asn Ala Thr Val Trp Lys Leu 225 230 235 240 Gin Pro Thr Ala Gly Leu Gin Asp Leu His lie His Ser Arg Gin Glu 245 250 255 Glu Glu Gin Ser Glu lie Met Glu Tyr Ser Val Leu Leu Pro Arg Thr 260 265 270 Leu Phe Gin Arg Thr Lys Gly Arg Ser Gly Glu Ala Glu Lys Arg Leu 275 280 285 Leu Leu Val Asp Phe Ser Ser Gin Ala Leu Phe Gin Asp Lys Asn Ser 290 295 300 Ser Gin Val Leu Gly Glu Lys Val Leu Gly lie Val Val Gin Asn Thr 305 310 315 320 Lys Val Ala Asn Leu Thr Glu Pro Val Val Leu Thr Phe Gin His Gin 325 330 335 Leu Gin Pro Lys Asn Val Thr Leu Gin Cys Val Phe Trp Val Glu Asp 340 345 350 Pro Thr Leu Ser Ser Pro Gly His Trp Ser Ser Ala Gly Cys Glu Thr 355 360 365 Val Arg Arg Glu Thr Gin Thr Ser Cys Phe Cys Asn His Leu Thr Tyr 370 375 380 Phe Ala Val Leu Met Val Ser Ser Val Glu Val Asp Ala Val His Lys 385 390 395 400 His Tyr Leu Ser Leu Leu Ser Tyr Val Gly Cys Val Val Ser Ala Leu 405 410 415 Ala Cys Leu Val Thr lie Ala Ala Tyr Leu Cys Ser Arg Val Pro Leu 420 425 430 Pro Cys Arg Arg Lys Pro Arg Asp Tyr Thr lie Lys Val His Met Asn 435 440 445 Leu Leu Leu Ala Val Phe Leu Leu Asp Thr Ser Phe Leu Leu Ser Glu 450 455 460 Pro Val Ala Leu Thr Gly Ser Glu Ala Gly Cys Arg Ala Ser Ala lie 465 470 475 480 Phe Leu His Phe Ser Leu Leu Thr Cys Leu Ser Trp Met Gly Leu Glu 485 490 495 Gly Tyr Asn Leu Tyr Arg Leu Val Val Glu Val Phe Gly Thr Tyr Val 500 505 510 Pro Gly Tyr Leu Leu Lys Leu Ser Ala Met Gly Trp Gly Phe Pro lie 515 520 525 Phe Leu Val Thr Leu Val Ala Leu Val Asp Val Asp Asn Tyr Gly Pro 530 535 540 lie lie Leu Ala Val His Arg Thr Pro Glu Gly Val lie Tyr Pro Ser 545 550 555 560 Met Cys Trp lie Arg Asp Ser Leu Val Ser Tyr lie Thr Asn Leu Gly 565 570 575 Leu Phe Ser Leu Val Phe Leu Phe Asn Met Ala Met Leu Ala Thr Met 580 585 590 Val Val Gin lie Leu Arg Leu Arg Pro His Thr Gin Lys Trp Ser His 595 600 605 Val Leu Thr Leu Leu Gly Leu Ser Leu Val Leu Gly Leu Pro Trp Ala 610 615 620 Leu lie Phe Phe Ser Phe Ala Ser Gly Thr Phe Gin Leu Val Val Leu 625 630 635 640 Tyr Leu Phe Ser lie lie Thr Ser Phe Gin Gly Phe Leu lie Phe lie 645 650 655 Trp Tyr Trp Ser Met Arg Leu Gin Ala Arg Gly Gly Pro Ser Pro Leu 660 665 670 Lys Ser Asn Ser Asp Cys Ala Arg Leu Pro lie Ser Ser Gly Ser Thr 675 680 685 Ser Ser Ser Arg lie SEQ ID NO: 3 Length 2082 Type DNA Organism: Homo sapiens Sequence : 3 atgactcccc agtcgctgct gcagacgaca ctgttcctgc tgagtctgct cttcctggtc 60 caaggtgccc acggcagggg ccacagggaa gactttcgct tctgcagcca gcggaaccag 120 acacacagga gcagcctcca ctacaaaccc acaccagacc tgcgcatctc catcgagaac 180 tccgaagagg ccctcacagt ccatgcccct ttccctgcag cccaccctgc ttcccgatcc 240 ttccctgacc ccaggggcct ctaccacttc tgcctctact ggaaccgaca tgctgggaga 300 ttacatcttc tctatggcaa gcgtgacttc ttgctgagtg acaaagcctc tagcctcctc 360 tgcttccagc accaggagga gagcctggct cagggccccc cgctgttagc cacttctgtc 420 acctcctggt ggagccctca gaacatcagc ctgcccagtg ccgccagctt caccttctcc 480 ttccacagtc ctccccacac ggccgctcac aatgcctcgg tggacatgtg cgagctcaaa 540 agggacctcc agctgctcag ccagttcctg aagcatcccc agaaggcctc aaggaggccc 600 tcggctgccc ccgccagcca gcagttgcag agcctggagt cgaaactgac ctctgtgaga 660 ttcatggggg acatggtgtc cttcgaggag gaccggatca acgccacggt atggaagctc 720 cagcccacag ccggcctcca ggacctgcac atccactccc ggcaggagga ggagcagagc 780 gagatcatgg agtactcggt gctgctgcct cgaacactct tccagaggac gaaaggccgg 840 agcggggagg ctgagaagag actcctcctg gtggacttca gcagccaagc cctgttccag 900 gacaagaatt ccagccaagt cctgggtgag aaggtcttgg ggattgtggt acagaacacc 960 aaagtagcca acctcacgga gcccgtggtg ctcactttcc agcaccagct acagccgaag 1020 aatgtgactc tgcaatgtgt gttctgggtt gaagacccca cattgagcag cccggggcat 1080 tggagcagtg ctgggtgtga gaccgtcagg agagaaaccc aaacatcctg cttctgcaac 1140 cacttgacct actttgcagt gctgatggtc tcctcggtgg aggtggacgc cgtgcacaag 1200 cactacctga gcctcctctc ctacgtgggc tgtgtcgtct ctgccctggc ctgccttgtc 1260 accattgccg cctacctctg ctccagggtg cccctgccgt gcaggaggaa acctcgggac 1320 tacaccatca aggtgcacat gaacctgctg ctggccgtct tcctgctgga cacgagcttc 1380 ctgctcagcg agccggtggc cctgacaggc tctgaggctg gctgccgagc cagtgccatc 1440 ttcctgcact tctccctgct cacctgcctt tcctggatgg gcctcgaggg gtacaacctc 1500 taccgactcg tggtggaggt ctttggcacc tatgtccctg gctacctact caagctgagc 1560 gccatgggct ggggcttccc catctttctg gtgacgctgg tggccctggt ggatgtggac 1620 aactatggcc ccatcatctt ggctgtgcat aggactccag agggcgtcat ctacccttcc 1680 atgtgctgga tccgggactc cctggtcagc tacatcacca acctgggcct cttcagcctg 1740 gtgtttctgt tcaacatggc catgctagcc accatggtgg tgcagatcct gcggctgcgc 1800 ccccacaccc aaaagtggtc acatgtgctg acactgctgg gcctcagcct ggtccttggc 1860 ctgccctggg ccttgatctt cttctccttt gcttctggca ccttccagct tgtcgtcctc 1920 taccttttca gcatcatcac ctccttccaa ggcttcctca tcttcatctg gtactggtcc 1980 atgcggctgc aggcccgggg tggcccctcc cctctgaaga gcaactcaga ctgcgccagg 2040 ctccccatca gctcgggcag cacctcgtcc agccgcatct ag 2082 SEQ ID NO: 4 Length 2834 Type DNA Organism: Homo sapiens Sequence : 4 gaattccggc agcagggtct cgctctgtca cacaggctgg agtgcagtgg tgtgatcttg 60 gctcatcgta acctccacct cccgggttca agtgattctc atgcctcagc ctcccgagta 120 gctgggatta caggtggtga cttccaagag tgactccgtc ggaggaaaat gactccccag 180 tcgctgctgc agacgacact gttcctgctg agtctgctct tcctggtcca aggtgcccac 240 ggcaggggcc acagggaaga ctttcgcttc tgcagccagc ggaaccagac acacaggagc 300 agcctccact acaaacccac accagacctg cgcatctcca tcgagaactc cgaagaggcc 360 ctcacagtcc atgccccttt ccctgcagcc caccctgctt cccgatcctt ccctgacccc 420 aggggcctct accacttctg cctctactgg aaccgacatg ctgggagatt acatcttctc 480 tatggcaagc gtgacttctt gctgagtgac aaagcctcta gcctcctctg cttccagcac 540 caggaggaga gcctggctca gggccccccg ctgttagcca cttctgtcac ctcctggtgg 600 agccctcaga acatcagcct gcccagtgcc gccagcttca ccttctcctt ccacagtcct 660 ccccacacgg ccgctcacaa tgcctcggtg gacatgtgcg agctcaaaag ggacctccag 720 ctgctcagcc agttcctgaa gcatccccag aaggcctcaa ggaggccctc ggctgccccc 780 gccagccagc agttgcagag cctggagtcg aaactgacct ctgtgagatt catgggggac 840 atggtgtcct tcgaggagga ccggatcaac gccacggtat ggaagctcca gcccacagcc 900 ggcctccagg acctgcacat ccactcccgg caggaggagg agcagagcga gatcatggag 960 tactcggtgc tgctgcctcg aacactcttc cagaggacga aaggccggag cggggaggct 1020 gagaagagac tcctcctggt ggacttcagc agccaagccc tgttccagga caagaattcc 1080 agccaagtcc tgggtgagaa ggtcttgggg attgtggtac agaacaccaa agtagccaac 1140 ctcacggagc ccgtggtgct cactttccag caccagctac agccgaagaa tgtgactctg 1200 caatgtgtgt tctgggttga agaccccaca ttgagcagcc cggggcattg gagcagtgct 1260 gggtgtgaga ccgtcaggag agaaacccaa acatcctgct tctgcaacca cttgacctac 1320 tttgcagtgc tgatggtctc ctcggtggag gtggacgccg tgcacaagca ctacctgagc 1380 ctcctctcct acgtgggctg tgtcgtctct gccctggcct gccttgtcac cattgccgcc 1440 tacctctgct ccagggtgcc cctgccgtgc aggaggaaac ctcgggacta caccatcaag 1500 gtgcacatga acctgctgct ggccgtcttc ctgctggaca cgagcttcct gctcagcgag 1560 ccggtggccc tgacaggctc tgaggctggc tgccgagcca gtgccatctt cctgcacttc 1620 tccctgctca cctgcctttc ctggatgggc ctcgaggggt acaacctcta ccgactcgtg 1680 gtggaggtct ttggcaccta tgtccctggc tacctactca agctgagcgc catgggctgg 1740 ggcttcccca tctttctggt gacgctggtg gccctggtgg atgtggacaa ctatggcccc 1800 atcatcttgg ctgtgcatag gactccagag ggcgtcatct acccttccat gtgctggatc 1860 cgggactccc tggtcagcta catcaccaac ctgggcctct tcagcctggt gtttctgttc 1920 aacatggcca tgctagccac catggtggtg cagatcctgc ggctgcgccc ccacacccaa 1980 aagtggtcac atgtgctgac actgctgggc ctcagcctgg tccttggcct gccctgggcc 2040 ttgatcttct tctcctttgc ttctggcacc ttccagcttg tcgtcctcta ccttttcagc 2100 atcatcacct ccttccaagg cttcctcatc ttcatctggt actggtccat gcggctgcag 2160 gcccggggtg gcccctcccc tctgaagagc aactcagact gcgccaggct ccccatcagc 2220 tcgggcagca cctcgtccag ccgcatctag gcctccagcc cacctgccca tgtgatgaag 2280 cagagatgcg gcctcgtcgc acactgcctg tggcccccga gccaggccca gccccaggcc 2340 agtcagccgc agactttgga aagcccaacg accatggaga gatgggccgt tgccatggtg 2400 gacggactcc cggggctggg gcttttgaat tggccttggg gactactcgg ctctcactca 2460 gctcccacgg gactcagaag tgcgccgcca tgctgcctag ggtactgtcc ccacatctgt 2520 cccaacccag ctggaggcct ggtctctcct tacaacccct gggcccagcc tcattgctgg 2580 gggccaggcc ttggatcttg agggtctggc acatccttaa tcctgtgccc ctgcctggga 2640 cagaaatgtg gctccagttg ctctgtctct cgtggtcacc ctgagggcac tctgcatcct 2700 ctgtcatttt aacctcaggt ggcacccagg gcgaatgggg cccagggcag accttcaggg 2760 ccagagccct ggcggaggag aggccctttg ccaggagcac agcagcagct cgcctacctc 2820 tgagcccgga attc 2834

Claims: Claims: 1. An isolated polynucleotide molecule encoding a G-protein-coupled receptor which is characterised by the N-terminal amino acid seqvience: MTPQSLLQTT (SEQ ID NO: 1), or a functionally equivalent fragment of said receptor. 2. A polynucleotide molecvile according to claim 1, wherein said polynucleotide molecule encodes a G-protein-coupled receptor of human origin of about 693 amino acids in length. 3. A polynucleotide molecule according to claim 2, wherein the polynucleotide molecule encodes a G-protein-coupled receptor comprising an amino acid seqvience substantially corresponding to that shown as SEQ ID NO: 2. 4. An isolated polynucleotide molecvile encoding a G-protein-coupled receptor, wherein the polynucleotide molecule comprises a nucleotide seqvience showing at least 75% seqvience identity to that shown as SEQ ID NO: 3 or SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent receptor fragment. 5. A polynucleotide molecvile according to claim 4, wherein the polynucleotide molecule comprises a nucleotide seqvience showing at least 90% seqvience identity to that shown as SEQ ID NO: 3 or SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent receptor fragment. 6. A polynucleotide molecule according to claim 5, wherein the polynucleotide molecule comprises a nucleotide seqvience showing at least 95% seqvience identity to that shown as SEQ ID NO: 3 or SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent receptor fragment. 7. A plasmid or expression vector including a polynucleotide molecvile according to any one of the preceding claims. 8. A host cell transformed with a polynucleotide molecule according to any one of claims 1 to 6. 9. A host cell according to claim 8, wherein the cell is a mammalian or insect cell. 10. A host cell according to claim 9, wherein the cell is a Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) 293 cell or an insect Sf9 cell. 11. A host cell according to any one of claims 8 to 10 wherein the cell expresses the G-protein-coupled receptor or functionally equivalent fragment thereof onto the cell's surface. 12. A G-protein-coupled receptor which is characterised by the N-terminal amino acid seqvience: MTPQSLLQTT (SEQ ID NO: 1), or a functionally equivalent fragment of said receptor, in a substantially pure form. 13. A receptor according to claim 12. wherein said receptor is a human receptor of about 693 amino acids. 14. A receptor according to claim 13, wherein the receptor has an amino acid seqvience substantially corresponding to that shown as SEQ ID NO: 2. 15. An antibody or fragment thereof which specifically binds to a G- protein-coupled receptor according to any one of claims 12 to 14. 16. A non-human animal transformed with a polynucleotide molecule according to any one of claims 1 to 6. 17. A method for detecting agonist or antagonist agents of a G-protein- coupled receptor comprising, contacting a G-protein-coupled receptor according to any one of claims 12 to 14 or a host cell transformed with and expressing a DNA molecvile according to any one of claims 1 to 6, with a test agent under conditions enabling the activation of the receptor, and detecting an increase or decrease in the receptor activity. 18. An oligonucleotide or polynvicleotide probe comprising a nucleotide seqvience of 10 or more nucleotides, the probe comprising a nucleotide seqvience such that the probe specifically hybridises to the polynucleotide molecule of any one of claims 1 to 6 under high stringency conditions. 19. An antisense polynucleotide molecule comprising a nucleotide seqvience capable of specifically hybridising to an mRNA molecule which encodes a G-protein-coupled receptor encoded by the polynucleotide molecvile of any one of claims 1 to 6, so as to prevent translation of the mRNA molecule. 20. A method of producing G-protein-coupled receptors or functionally equivalent fragments thereof according to any one of claims 12 to 14, comprising cviltviring a host cell according to any one of claims 8 to 11 under conditions enabling the expression of the polynvicleotide molecule and optionally recovering the receptors or functionally equivalent fragments thereof.

Inventors: HERZOG HERBERT, HERZOG, HERBERT
Applicants: GARVAN INST MED RES, HERZOG HERBERT, GARVAN INSTITUTE OF MEDICAL RESEARCH, HERZOG, HERBERT
Application year: 1998
Main classification: C07K7
IPC codes: A01K67, C07K7, C07K14, C12N5, C12N15, C12P21, C12Q1, C12R1