AU764155B2 - Novel GABA-B receptor - Google Patents

Description

WO 00/00602 PCT/AU99/00524 1 NOVEL GABA-B RECEPTOR Field of the Invention: The present invention relates to isolated polynucleotide molecules which encode a novel transmembrane G-protein coupled receptor designated GABA-B2. The novel receptor appears to be activated by the neurotransmitter y-amino butyric acid (GABA).

Background of the Invention: y-amino butyric acid (GABA) is the principal inhibitory neurotransmitter in the brain, whose action is mediated by two types of receptors, GABA-A and GABA-B. GABAergic inhibitory neurons typically form short pathways from striatum to substantia nigra and from cerebellar cortex to deep cerebellar nuclei), although at least one long pathway projecting from the posterior hypothalamus to the cerebral cortex has also been recognised. This long pathway is believed to provide a direct pathway by which limbic, emotional and visceral information may be transferred to the cortex (Vincent et al., Science 220: 1309-1311, 1993).

GABA-B receptors, such as the GABA-Bla and GABA-Bib receptors, are predominantly present in the brain where they are believed to play a major role in learning and memory. In view of these functions and the known benefit of GABA-B agonists baclofen) in the treatment of spasticity, anxiety and depression, there is considerable interest in isolating genes encoding GABA-B receptor subtypes so as to enable the recombinant production of GABA-B receptors for the development of novel therapeutics.

Summary of the Invention: In a first aspect, the present invention provides an isolated polynucleotide molecule encoding a GABA-B2 receptor or a functionally equivalent fragment thereof.

Preferably, the encoded GABA-B2 receptor is characterised by the Nterminal amino acid sequence: MASPRSSGQP (SEQ ID NO: 1) More preferably, the isolated polynucleotide molecule encodes a human GABA-B2 receptor of about 941 amino acids.

WO 00/00602 PCT/AU99/00524 2 Most preferably, the isolated polynucleotide molecule encodes a GABA-B2 receptor having an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.

The polynucleotide molecule of the first aspect may comprise a nucleotide sequence substantially corresponding to, or showing at least (more preferably, at least 95%) homology to that shown at nucleotides 1 to 3256 or nucleotides 140 to 2962 of SEQ ID NO: 3 or any portion thereof encoding a functionally equivalent GABA-B2 receptor fragment.

The isolated polynucleotide molecule may be incorporated into plasmids or expression vectors (including viral vectors), which may then be introduced into suitable bacterial, yeast, insect and mammalian host cells.

Such host cells may be used to express the GABA-B2 receptor encoded by the isolated polynucleotide molecule.

Accordingly, in a second aspect, the present invention provides a mammalian, insect, yeast or bacterial host cell transformed with the polynucleotide molecule of the first aspect.

In a third aspect, the present invention provides a method of producing GABA-B2 receptors or functionally equivalent fragments thereof, comprising culturing the host cell of the second aspect under conditions enabling the expression of GABA-B2 receptors or functionally equivalent 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, monkey kidney (COS) cell or human embryonic kidney 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 GABA-B2 receptors or fragments thereof are expressed onto the surface of the host cell.

By using the polynucleotide molecule of the present invention it is possible to obtain GABA-B2 receptor protein or fragments thereof in a substantially pure form.

Accordingly, in a fourth aspect, the present invention provides a GABA-B2 receptor or a functionally equivalent fragment of said receptor, in a substantially pure form.

WO 00/00602 PCT/AU99/00524 3 In a fifth aspect, the present invention provides an antibody capable of specifically binding to the GABA-B2 receptor of the fourth aspect. Such antibodies may be produced by any of the methods routine to the art.

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 or antagonist agents of a GABA-B2 receptor, comprising contacting a GABA-B2 receptor, functionally equivalent fragment thereof or a cell transfected with and expressing the polynucleotide molecule of the first aspect, with a test agent under conditions enabling the activation of a GABA- B2 receptor, and detecting an increase or decrease in activity of the GABA-B2 receptor or functionally equivalent fragment thereof.

An increase or decrease in activity of the receptor or functionally equivalent fragment thereof may be detected by measuring changes in cAMP production, Ca 2 levels or IP3 turnover after activating the receptor molecule with specific agonists or antagonists.

In a further aspect, the present invention provides an oligonucleotide or polynucleotide probe comprising a nucleotide sequence 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 al., Molecular cloning: a laboratory manual, Second Edition, Cold Spring Harbor Laboratory Press).

In a still further aspect, the present invention provides an antisense oligonucleotide or polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridising to an mRNA molecule which encodes a GABA-B2 receptor so as to prevent translation of the mRNA molecule.

Such antisense oligonucleotide or polynucleotide molecules may include a ribozyme region to catalytically inactivate mRNA to which it is hybridised.

The polynucleotide molecule of the first aspect of the invention may be a dominant negative mutant which encodes a gene product causing an altered phenotype by, for example, reducing or eliminating the activity of endogenous GABA-B2 receptors.

WO 00/00602 PCT/AU99/00524 4 The term "substantially corresponding" as used 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 biological activity of the GABA-B2 receptor. These variations may include conservative amino acid 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, Na-alkalamino acids.

The term "substantially corresponding" as used herein in relation to nucleotide sequences is intended to encompass minor variations in the nucleotide sequences 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 required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded protein.

The term "functionally equivalent fragment/s" as used herein is intended to refer to fragments of the GABA-B2 receptor that exhibit binding specificity and activity that is substantially equivalent to the GABA-B2 receptor from which it/they is/are derived.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Reference to percent homology made in this specification have been calculated using the BLAST program blastn as described by Altschul, S.F. et al., "Capped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Research, Vol. 25, No. 17, pp. 3389-3402 (1997).

Brief description of the accompanying Figures: Figure 1 provides the nucleotide sequence of a cDNA encoding the human GABA-B2 receptor and includes the predicted amino acid sequence.

Figure 2 shows the degree of identity between the predicted amino acid sequence of the human GABA-B2 and GABA-Blb receptors.

WO 00/00602 PCT/AU99/00524 Detailed Disclosure of the Invention: Human GABA-B2 receptor cDNA A human hippocampus cDNA library (Stratagene) was screened under low stringency conditions with a 184 bp 32 P-labelled fragment (corresponding to nucleotides 2051 to 2235 of SEQ ID NO: 3) originated from a human brain EST clone (z43654). A cDNA clone encoding a complete human GABA-B gene was obtained from the screen. The nucleotide sequence of the cDNA clone is shown as SEQ ID NO: 3 and encodes a protein of 941 amino acids (SEQ ID NO: 2) consisting of a large extracellular domain (amino acids 1 to 480) followed by 7 hydrophobic regions (amino acids 481 to 494, 518 to 545, 558 to 578, 597 to 618, 653 to 676, 691 to 713 and 719 to 743) typical of Gprotein coupled receptors.

Sequence comparison with other G-protein coupled receptors identify GABA-Bla, GABA-Bib (Figure 1) and metabotropic glutamate receptors (Kaupman, K. et al., Nature 386: 239-246, 20 March 1997 and Pin, J. et al., Neuropharmacology 34: 1-26, 1995) as the most closely related group.

Northern analysis has identified brain as the tissue with the highest expression of the human GABA-B2 mRNA. In particular, Northern analysis experiments using multiple tissue Northern blots (Clontech) identified high levels of expression of the human GABA-B2 mRNA in the cerebellum, cerebral cortex, occipital pole, frontal lobe and temporal lobe. Lower levels of expression were seen in the thalamus, amygldala, hippocampus, substantia nigra, putamen, subthalamic nucleus, caudate nucleus, and medulla. No apparent expression was seen in the spinal cord and corpus callosum.

Further, in situ hybridisation of rat brain sections has identified discrete areas of expression in the hippocampus, amygdala, the piriform cortex and also the hypothalamus. This mRNA distribution is consistent with the expression of other subtypes of the GABA-B receptor family.

Chromosomal Localisation of Human GABA-B2 receptor gene In order to determine the chromosomal localisation of the human GABA-B2 receptor gene, the complete cDNA clone was nick-translated with biotin-14-dAPT and hybridised in situ at a final concentration of 5 ng/ml to metaphase chromosomal spreads from two normal males. The fluoerescence in situ hybridisation (FISH) method was modified from that previously described (Callen, DF et al., Ann Genet 33: 219-221 1990) in that chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a CCD camera and computer enhancement software.

Twenty metaphases from a first normal male were examined for fluorescent signal. All of these metaphases showed signal on one or both chromatids of chromosome 9 in the region 9q21. There was a total of 7 nonspecific background dots observed in these 20 metaphases. A similar result was obtained from hybridisation of the probe to 20 metaphases from a second normal male.

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.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

0 Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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EDITORIAL NOTE APPLICATION NUMBER 45915/99 The following Sequence Listing pages 1 to 9 are part of the description.

The claims pages follow on pages to "11".

WO 00/00602 PCT/AU99/00524 1 Sequence Listings: Applicant: Garvan Institute of Medical Research Title of Invention: Novel GABA-B Receptor Prior Application Number: PP4384 Prior Application Filing Date: 1998-06-29 Number of SEQ ID NOs: 4 Software: PatentIn Ver. 2.1 SEQ ID NO: 1 Length: Type: PRT Organism: Homo sapiens Sequence: 1 Met Ala Ser Pro Arg Ser Ser Gly Gln Pro 1 5 SEQ ID NO: 2 Length: 941 Type: PRT Organism: Homo sapiens Sequence: 2 Met Ala Ser Pro Arg Ser Ser Gly Gln Pro Gly Pro Pro Pro Pro Pro 1 5 10 Pro Pro Pro Pro Ala Arg Leu Leu Leu Leu Leu Leu Leu Pro Leu Leu 25 Leu Pro Leu Ala Pro Gly Ala Trp Gly Trp Ala Arg Gly Ala Pro Arg 40 Pro Pro Pro Ser Ser Pro Pro Leu Ser Ile Met Gly Leu Met Pro Leu 55 Thr Lys Glu Val Ala Lys Gly Ser Ile Gly Arg Gly Val Leu Pro Ala 70 75 WO 00/00602 WO 0000602PCT/AU99/00524 Val1 Tyr Gly Met Ser 145 Pro Pro Tyr Ser Giu 225 Lys Gin Tyr Trp Lys 305 Pro Gin Lys Glu Phe Leu Val1 130 Leu Val1 Ser Gin Giu 210 Ile Lys As n Gly T rp 290 As n Leu Tyr Phe Leu Leu Lys 115 Phe Gin Leu Asp T rp 195 Val Ser Leu Met Ser 275 Glu Leu Ser Giu His Ala Asp 100 Al a Gly Gly Al a As n 180 Lys Arg Asp Lys Al a 260 L-ys Gin Leu Ser Arg 340 Gi y Ile Leu Phe Gly Trp Asp 165 Al a Arg As n Thr Gi y 245 Al a Tyr Val1 Al a Lys 325 Giu Tyr Giu Arg Tyr Val As n 150 Lys Val1 Val1 Asp Giu 230 As n Lys Gin His Al a 310 Gin Tyr Al a Gin Leu Asp Cys 135 Leu Lys As n Gly Leu 215 Ser Asp Val Trp Thr 295 Met Ile As n Tyr Thr 375 Ile Tyr Al a 120 Pro Val1 Lys Pro Thr 200 Thr Phe Val Phe Ile 280 Giu Giu Lys As n Asp 360 Ar g Asp 105 Ile Ser Gin Tyr Al a 185 Leu Gly Ser Arg Cys 265 Ile Al a Gi y Thr Lys 345 Gly As n 90 Th r Lys Val1 Leu Pro 170 Ile Thr Val1 As n Ile 250 Cys Pro As n Tyr Ile 330 Arg li 1e G1 u Giu Tyr Thr Ser Tyr Leu Gin Leu Asp 235 Ile Al a G liy Ser Ile 315 Ser Ser T rp Ser Cys Gi y Ser 140 Phe Phe Lys Asp Tyr 220 Pro Leu Tyr T rp Ser 300 Gi y Gi y Gi y Vali Ser 380 Leu Asp Pro 125 Ile Al a Phe Leu Va I 205 Gi y Cys Gi y Giu Tyr 285 Arg Val Lys Val Ile 365 Leu As n 110 As n Ile Al a Arg Leu 190 Gin Giu Th r Gin Giu 270 Gi u Cys Asp Thr Gly 350 Al a Arg Al a His Al a Th r Thr 175 Lys Ar g Asp Ser Phe 255 As n Pro Leu Phe Pro 335 Pro Lys Pro Lys Leu Glu Thr 16 0 Val1 His Phe Ile Val1 240 Asp Met Ser Arg Giu 320 Gin Ser Thr 355 Arg Ala Met Glu Leu Gin 370 Leu His Ala Ser Arg His Gin Arg WO 00/00602 WO 0000602PCT/AU99/00524 Ile 385 As n Phe Asp Leu Lys 465 Leu Ser Lys Leu Ser 545 Thr Arg Lys Leu Vai 625 Ile Leu Phe Gin Al a Arg Ser Giu 450 Asp Tyr Al a Met S er 530 Giu Val1 Val1 Asp Cys 610 Glu Arg Gly Leu Asp Met As n Arg 435 Ile Lys Ser Phe Ser 515 Tyr Lys Gi y His Gin 595 Ile Lys Pro Ile Al a 675 Phe As n Gi y 420 Giu Ile Thr Ile Leu 500 Ser Al a Thr Tyr Al a 580 Lys Leu Tyr Leu Val 660 Trp Tyr 390 Thr Arg Lys Asp Ile 470 Ser Phe Tyr Ile Glu 550 Thr Phe Leu Cys Met 630 Gi u Al a Thr Thr As n Met Vai Thr 455 Leu Al a As n Met Phe 535 Thr Al a Lys Val1 T rp 615 Giu His Tyr Arg Asp Phe Glu Gi y 440 Ile Glu Leu Ile As n 520 Leu Leu Phe As n Ile 600 Gin Pro Cys Lys As n 680 His Phe Thr 425 Glu Arg Gin Thr Lys 505 As n Phe Cys Gi y Vali 585 Vai Aila Asp Glu Gly 665 Val Thr Leu 395 Gly Vai 410 Ile Lys Tyr Asn Phe Gin Leu Arg 475 IIe Leu 490 Asn Arg Leu Ile Gly Leu Thr Vai 555 Ala Met 570 Lys Met Giy Gly Val Asp Pro Ala 635 Asn Thr 650 Leu Leu Ser Ile Gly Arg Ile lie Leu 400 Thr Phe Al a Gly 460 Lys Gi y Asn Ile Asp 540 Ar g Phe Lys Met Pro 620 Gi y His Met Pro Gi y Thr Val Ser Ile Met Gin Leu 525 Gly Thr Al a Lys Leu 605 Leu Arg Met Leu Ala 685 Gin Gin 430 Al a Glu Ser Le TLys 510 Giy S er T rp Lys Lys 590 Leu Arg Asp Thr Phe 670 Leu Val 415 Phe Asp Pro Leu Met 495 Leu Gly Phe Ile Thr 575 Ile Ile Ari Ile Ile 655 Gly As n Val Gin Thr Pro Pro 480 Al a Ile Met Val Leu 560 Trp Ile Asp Thr Ser 640 Trp Cys Asp Ser Lys Tyr Ile Gly Met Ser Val Tyr Asn Vai Gly Ile Met Cys Ile 690 695 700 WO 00/00602 PTA9/02 PCT/AU99/00524 Ile 705 The Cys Al a Giu Thr 785 Lys Gin Giu Gi y Gin 865 Asp Pro Ser Gly Cys Leu Al a Asp 770 Ser Ile Asp Leu Gi y 850 T rp Ile Ile Cys Al a Ile Vai Thr 755 Set Arg Thr Thr As n 835 Lys As n As n Leu Val 915 Al a Val1 Phe 740 Gin Lys Leu Gi u Pro 820 Asp Ala Thr Ser His 900 Ser Val Al a 725 Val1 As n Thr Giu Leu 805 Giu Ile Ile Th r Pro 885 His Pro Ser 710 Leu Pro Arg Ser Gi y 790 Asp Lys Leu Leu Giu 870 Gi u Ala Cys Phe Val1 Lys Arg Thr 775 Leu Lys Thr As n Lys 855 Pro His Tyr Val1 Arg 935 Leu Ile Leu Phe 760 Set Gin Asp Th r Leu 840 As n Set Ile TLeu Set 920 Thr Ile Ile 745 Gin Val1 Ser Leu Tyr 825 Gly His Atg Gin Pro 905 Pro Arg Phe 730 Thr Phe Thr Giu Giu 810 Ile As n Leu Thr Arg 890 Ser Thr Asp 715 Cys Leu Thr Ser As n 795 Giu Lys Phe Asp Cys 875 Ar g T-1e Al a Gin Ser Arg Gin Val1 780 His Vali Gin Thr Gin 860 Lys Leu Gi y Set Gi y 940 Pro Thr Thr As n 765 As n Arg Thr As n Giu 845 Asn Asp Set Gi y Pro 925 As n Ile As n 750 Gin Gin Leu Met His 830 Set Pro Pro Leu Val 910 Arg Val Thr 735 Pro Lys Al a Arg Gin 815 T yr Th r Gin Ile Gin 895 Asp His Gin 720 Leu Asp Lys Set Met 800 Leu Gin Asp Leu Giu 880 Leu Al a Arg His Val 930 Pro Pro Set Phe Val Met Val Set SEQ ID NO: 3 Length: 3256 Type: DNA Organism: Homno sapiens WO 00/00602 WO 0000602PCT/AU99/00524 Sequence: gaattccgac gcactccttg ccgcagcccg gccgccgcca tctggcgccc gccgctctcC gcgcggtgtg gcgcccctac gaaagccttc ctqtccatcc ttcttttgct gaccgtccca gtggaagcgc cctgactgga cgatccctgt gtttgaccag tagtaaatat cacggaagcc ca ttggcgtg tccacagcag ccacgggtac gacactgcat gctgggcagg agttgtattc cagggaggtg caccatcagg gcggaagatc catggccagt gtcgagtcca atttctcttt cgtcaggacc gacctggaga ccagaaactg ctggcaggct gggcggtgtg gcgcggggcg cggcgcggca ccgccgcccg ggggcctggg atcatgggcc ctccccgccg ttcctcgacc tacgatgcga gtcacatcca gcaaccacgc tcagacaatg gtgqgcacgc gttctgtatq accagtgtca aatatggcag cagtggatca aactcatccc gatttcgagc tatgagagag gcctacgatg gccagcagcc atcatcctca cggaatgggg aaggtgggag ttccaagggt tccctacctc gcttttctct tacatgaaca ggccttgatg tqgattctca gtccacgcca cttgtgatcg gtggaccccc tacaaagggc gcgggccggg tggcttcccc cgcgcctgct gctgggcgcg tcatgccgct tqgaactggc tgcggctcta taaaatacgg tcattgcaga ctgttctagc cggtgaatcc tgacgcaaga gcgaggacat aaaagctgaa caaaagtgtt ttccgggctg gctgcctccg ccctgagctc agtacaacaa gcatctgggt ggcaccagcg atgccatgaa agagaatgga agtacaacgc ccgaaccacc tctacagcat tcttcaacat accttatcat gatcctttgt ccgtgggcta tcttcaaaaa tggggggcat tgcgaaggac agggacttaa ccaggccatg gcggagctcc actgctactg gggcgccccc caccaaggag catcgagcag tgacacggag gccgaaccac gtccctccaa cgataagaaa agccattcta cgtt cagagg tgagatttca ggggaatgat ctgttgtgca gtacgagcct gaagaatctg caagcagatc caagcggtca catcgccaag gatccaggac cgaqaccaac gaccattaaa tgtggccgac aaaagacaag cctctctgcc caagaaccgg ccttggaggg ctctgaaaag cacgaccgct tgtgaaaatg gctgctgatc agtggagaag tcaacgcaag cgqgccgagt gggcagcccg ctgctgccqc cggccgccgc gtggccaagg atccgcaacg tgcgacaacg ttgatggtgt ggctggaatc aaataccctt aagttgctca t-tctctgagg gacaccgaga gtgcggatca tacgaggaga tcttggtggg cttgctgcca aagaccatct ggcgtggggc acactgcaga ttcaactaca ttcttcgqgg tttactcaat acactggaga accatcatcc ctcaccatcc aatcagaagc atgctctcct acctttgaaa tttggggcca aagaagaaga gacctgtgta tacagcatgg cttatgaccc gagccggcgc ggccgccgcc tgctgct-gcc ccagcagccc gcagcatcgg agtcactcct caaaagggtt ttggaggcgt tggtgcagct atttctttcg agcactacca tgcggaatga gcttctccaa tccttggcca acatgtatgg agcaggtgca tggagggcta caggaaagac ccagcaagtt gggccatgga cggaccacac izcacgggtca tLtcaagacag tcatcaatga tggagcagct tcgggatgat tcataaagat atgcttccat cactttgcac tgtttgcaaa tcatcaagga tcctgatctg agccggaccc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 WO 00/00602 WO 0000602PCT/AU99/00524 agcaggacgg catctggctt agcttgggag gagtgtdtac ggaccagccc caccctctgc aacgcagaac cacctcggtc aaaccatcgc gcagctgcag caatgacatc aaaaaatcac atgcaaagat ccagctcccc tgtcagcccc ccgagtcatg gaaccacact ctgggcacca gcagggggaq aagaagagga tatcaaactg SEQ ID NO: gatatctcca gqcatcgtct acccgcaacg aacgtgggga aatgtgcagt ctggtattcg aggcgattcc accagtqtga ctgcgaatga gacacaccag ctcaacctgg ctcgatcaaa cctatagaag atcctccacc tgcgtcagcc gtctcgggCC gggcagaggg tggctggcct acttggcacc acggaaatgg gaattc tccgccctct atgcctacaa tcagcatccc tcatgtgcat tctgcatcqt tgccgaagct agttcactca accaagccag agatcacaga aaaagaccac gaaacttcac atccccagct atataaactc acgcctacct ccaccgccag tqtaagggtg gtctgctgca ctcaggacca tgacctcgag gacgtcttcc cctggagcac gggacttctc cgcactcaac catcggggcc ggctctggtc catcaccctg gaatcagaag cacatcccgc gctqgataaa ctacattaaa tgagagcaca acagtggaac tccagaacac cccatccatc cccccgccac gqaggcctgg gaaacactgt ctcggatggc ccttatttgt ttaacatctq tgtgagaaca atgttgttcg gacagcaagt gctgtctcct atcatcttct agaacaaacc aaagaagatt ctggagggcc gacttggaag caqaaccact gatggaggaa acaacagagc atccagcgtc ggaggcgtgg agacatgtgc cccgggcctc cggctctggc actcaggtgg gaagtcctta caaacaagga cccatatgac gttgtttctt acatcgggat tcctgacccg qcagcaccat cagatgcagc ctaaaacgtc tacagtcaga aggtcaccat accaagagct aggccattt cctctcgaac ggctgtccct acgccagctg caccctcctt ccccgtgaca tgcggagaag acaggacggg tttcttcaca ggcgctggga 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3256 Length: 844 Type: PRT Organism: Homa sapiens Sequence: 4 Met Gly Pro Gly Gly Pro Cys 1 5 Leu Leu Val Met Ala Ala Gly Pro His Leu Pro Arg Pro His Glu Arg Arg Ala Val Tyr Ile 55 Thr Pro Val Ala 25 Pro Arg Gly Trp Pro Leu Pro Leu Val Trp Ala Gly Val Pro Pro Leu Phe Pro Ser His Ser Pro Ser Ser Ser Gly Gly Al a WO 00/00602 PCT/AU99/00524 7 Trp Pro Gly Gly Gin Ala Cys Gin Pro Ala Val Glu Met Ala Leu Glu 70 75 Asp Val Asn Ser Arg Arg Asp Ile Leu Pro Asp Tyr Glu Leu Lys Leu 90 Ile His His Asp Ser Lys Cys Asp Pro Gly Gin Ala Thr Lys Tyr Leu 100 105 110 Tyr Glu Leu Leu Tyr Asn Asp Pro Ile Lys Ile Ile Leu Met Pro Gly 115 120 125 Cys Ser Ser Val Ser Thr Leu Val Ala Glu Ala Ala Arg Met Trp Asn 130 135 140 Leu Ile Val Leu Ser Tyr Gly Ser Ser Ser Pro Ala Leu Ser Asn Arg 145 150 155 160 Gin Arg Phe Pro Thr Phe Phe Arg Thr His Pro Ser Ala Thr Leu His 165 170 175 Asn Pro Thr Arg Val Lys Leu Phe Glu Lys Trp Gly Trp Lys Lys Ile 180 185 190 Ala Thr Ile Gin Gin Thr Thr Glu Val Phe Thr Ser Thr Leu Asp Asp 195 200 205 Leu Glu Glu Arg Val Lys Glu Ala Gly Ile Glu Ile Thr Phe Arg Gin 210 215 220 Ser Phe Phe Ser Asp Pro Ala Val Pro Val Lys Asn Leu Lys Arg Gin 225 230 235 240 Asp Ala Arg Ile Ile Val Gly Leu Phe Tyr Glu Thr Glu Ala Arg Lys 245 250 255 Val Phe Cys Glu Val Tyr Lys Glu Arg Leu Phe Gly Lys Lys Tyr Val 260 265 270 Trp Phe Leu Ile Gly Trp Tyr Ala Asp Asn Trp Phe Lys Ile Tyr Asp 275 280 285 Pro Ser Ile Asn Cys Thr Val Asp Glu Met Thr Glu Ala Val Glu Gly 290 295 300 His Ile Thr Thr Glu Ile Val Met Leu Asn Pro Ala Asn Thr Arg Ser 305 310 315 320 Ile Ser Asn Met Thr Ser Gin Glu Phe Val Glu Lys Leu Thr Lys Arg 325 330 335 Leu Lys Arg His Pro Glu Glu Thr Gly Gly Phe Gin Glu Ala Pro Leu 340 345 350 Ala Tyr Asp Ala Ile Trp Ala Leu Ala Leu Ala Leu Asn Lys Thr Ser 355 360 365 WO 00/00602 WO 0000602PCT/AU99/00524 Gly As n 385 Phe Met Ile Asp Lys 465 Leu Ile As n Pro Val 545 Tyr Lys Leu Al a Ala 625 Leu Tyr Glu Gly Thr Gly Trp Tyr 435 T rp Phe Ser As n Leu 515 Gly Gin Ser Giu Al a 595 Trp Glu His Tyr Lys 675 Gi y Ile Val1 Thr 420 Tyr Ile Arg Leu Ser 500 Thr Leu Al a Met Giu 580 Thr Gin Glu Cys Lys 660 Ser Arg Th r Ser 405 Leu Asp Gi y Phe Gi y 485 His Al a Asp Ar g Phe 565 Lys Vali Ile Pro Ser 645 Gly Val Ser Asp 390 Gly Ile Ser Gly Leu 470 -11e Val1 Val Gi y Leu 550 Thr Lys Gly Val1 Lys 630 Ser Leu Ser Gly 375 Gin His Gi u Thr Ser 455 Ser Val Arg Gi y Tyr 535 Trp Lys Giu Leu Asp 615 Glu Arg Leu Thr Vai Ile Val1 Gin Lys 440 Pro Gin Leu Tyr Cys 520 His Leu Ile Trp Leu 600 Pro Asp Lys Leu Giu 680 8.

Arg Tyr Vai Pro 425 Asp) Pro Lys Al a Ile 505 Ser Ile Leu TrD Arg 585 Vai Leu Ile Met Leu 665 Lys Leu Arg Phe 410 Gin Asp Al a Leu Val2 490 Gin Leu Gly Gi y Tro 570 Lys Gi y His Asp As n 650 Leu Ile Giu Al a 395 Asp Gly Leu Asp Phe 475 Val As n Al a Arg Leu 555 Val1 Thr Met Arg Val 635 Thr Gly As n Asp 380 Met Al a Gi y Ser Gin 460 l1 e Cys Ser Leu As n 540 Gi y Hi-4s Leu Asp Th r 620 Ser T rp Ile Asp Phe As n Ser Ser Trp 445 Thr Ser Leu Gin Al a 525 Gin Phe Thr Glu Val 605 le Ile Leu Phe His 685 As n Ser Gi y Tyr 430 Ser Leu Val Ser Pro 510 Al a Phe Ser Giv Pro 590 Leu Giu Leu Gi y Leu 670 Arg Tyr Ser Ser 415 Lys Lys Val1 Ser Ohe 495 As n Val1 Pro iLeu Phe 575 Trp Thr Thr Pro Ile 655 Ala Al a As n Ser 400 Arg Lys Thr Ile Va 11 480 As r; Leu Phe Phe Gi y 560 Thr Lys Leu Phe Gin 640 Phe Tyr Val WO 00/00602 WO 0000602PCT/AU99/00524 Gly Val1 705 Ser Val1 Al a Glu Ile 785 Ser Pro Ser Met 690 Th r Leu Pro Gin Lys 770 Al a Arg Ser Cys Al a Met Al a Lys Asp 755 Ser Gi u Gin Gly Asp 835 Tyr Leu Val 725 Arg Met Leu Giu Leu 805 Leu Ser Al a Gin Ser Ile Gi y 760 Lys Vai Arg Gi y His 840 Val Gin Tyr Th r 745 Ser Giu Ser Arg Pro 825 Leu Leu Asp Ile 730 Arg Ser As n Giu His 810 Pro Leu Cys Al a 715 Thr Giy Thr Ar g Leu 795 Pro Giu Tyr Leu 700 Al a Leu Giu As n Giu 780 Ar g Pro Pro Lys Ile Phe Val T rp As n 765 Leu H Is Thr Pro Al a Phe Leu 735 Ser Giu Lys Leu Pro 815 Arg

Claims (22)

1. An isolated polynucleotide molecule encoding a GABA-B receptor or a functionally equivalent fragment thereof, wherein the encoded GABA-B receptor is characterised by the N-terminal amino acid sequence: MASPRSSGQP (SEQ ID NO: 1).

2. A polynucleotide molecule according to claim 1, wherein the polynucleotide molecule encodes a GABA-B receptor of human origin of about 941 amino acids in length.

3. An isolated polynucleotide molecule that encodes a GABA-B receptor polypeptide wherein said polynucleotide molecule comprises a nucleotide sequence selected from the group consisting of: the sequence set forth in SEQ ID NO: 3; (ii) the sequence consisting of nucleotides 140 to 2962 of SEQ ID NO: 3; and (iii) a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 2.

4. A polynucleotide molecule according to any one of claims 1 to 3, wherein the polynucleotide molecule encodes a human GABA-B receptor having an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2. A polynucleotide molecule encoding a GABA-B receptor, wherein the polynucleotide molecule comprises a nucleotide sequence showing at least 20 90% homology to that shown at nucleotides 1 to 3256 or nucleotides 140 to 2962 of SEQ ID NO: 3 or any portion thereof encoding a functionally equivalent GABA-B receptor fragment.

6. A polynucleotide molecule according to claim 5, wherein the polynucleotide molecule comprises a nucleotide sequence showing at least homology to that shown at nucleotides 1 to 3256 or nucleotides 140 to 8 2962 of SEQ ID NO: 3 or any portion thereof encoding a functionally equivalent GABA-B receptor fragment.

7. A polynucleotide molecule according to claim 5, wherein the polynucleotide molecule comprises a nucleotide sequence substantially corresponding to that shown at nucleotides 1 to 3256 or nucleotides 140 to 2962 of SEQ ID NO: 3 or any portion thereof encoding a functionally equivalent GABA-B receptor fragment.

8. A plasmid or expression vector including a polynucleotide molecule according to any one of claims 1 to 7.

9. A host cell transformed with a polynucleotide molecule according to any one of claims 1 to 7 or a plasmid or expression vector according to claim 8. A host cell according to claim 9, wherein the cell is a mammalian or insect cell.

11. A host cell according to claim 10, wherein the cell is a Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) 293 cell or an insect Sf9 cell.

12. A host cell according to any one of claims 9 to 11, wherein the cell 25 expresses the GABA-B receptor or functionally equivalent fragment thereof encoded by the transformed polynucleotide on the cell surface.

13. An isolated or recombinant GABA-B receptor polypeptide comprising an amino acid sequence which is characterised by the N-terminal amino acid sequence: MASPRSSGQP (SEQ ID NO:1), in a substantially pure form. *o *o *ee •*oo• 9

14. An isolated or recombinant GABA-B receptor polypeptide according to claim 13, wherein said receptor polypeptide is a human receptor polypeptide of about 941 amino acids.

15. An isolated or recombinant receptor polypeptide according to claim 14, wherein said receptor has an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.

16. An antibody or fragment thereof which specifically binds to a GABA-B receptor polypeptide comprising an amino acid sequence that is at least identical to the sequence set forth in SEQ ID NO: 2.

17. The antibody or fragment thereof according to claim 16, wherein said antibody or fragment binds to a GABA-B receptor polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.

18. An antibody or fragment thereof which specifically binds to the isolated or recombinant GABA-B receptor polypeptide according to claim 13 or 14.

19. A non-human animal transformed with a polynucleotide molecule according to any one of claims 1 to 7 or a plasmid or expression vector according to claim 8. 25 20. A method for detecting an agonist or antagonist agents of a GABA-B receptor, comprising contacting a GABA-B receptor according to any one of claims 13 to 15 or a host cell according to any one of claims 9 to 12, with a test agent under conditions enabling the activation of said receptor, and detecting an increase or decrease in the receptor activity.

21. An oligonucleotide or polynucleotide probe comprising a nucleotide sequence of 10 or more nucleotides, the probe comprising a nucleotide sequence such that the probe specifically hybridises to the polynucleotide molecule according to any one of claims 1 to 7 under high stringency conditions. S35 conditions. *o o *oo *eo

22. An antisense oligonucleotide or polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridising to an mRNA molecule which encodes a GABA-B receptor encoded by the polynucleotide molecule according to any one of claims 1 to 7, so as to prevent translation of the mRNA molecule.

23. A method of producing GABA-B receptors or functionally equivalent fragments thereof according to any one of claims 13 to 15, comprising culturing a host cell according to any one of claims 9 to 12 under conditions enabling the expression of GABA-B receptors or functionally equivalent fragments thereof, and optionally recovering the receptors or functionally equivalent fragments thereof.

24. A method of producing a GABA-B receptor polypeptide, said method comprising culturing a host cell transformed with the polynucleotide according to any one of claims 1 to 7 under conditions sufficient for expression of the GABA-B receptor polypeptide encoded by the polynucleotide to occur. A method of producing a GABA-B receptor polypeptide, said method comprising introducing the polynucleotide according to any one of claims 1 to 7 encoding a GABA-B receptor polypeptide into a cell thereby producing a transformed cell and then growing the transformed cell under conditions sufficient for expression of the encoded GABA-B receptor polypeptide to occur. *oooo o *o* o o 11

26. The method of claim 24 or 25 further comprising recovering the GABA- B receptor polypeptide from the host cell substantially free of other proteins. DATED this ninth day of June 2003 Garvan Institute of Medical Research Patent Attorneys for the Applicant: F.B. RICE CO. *0 0 0 0 0 *•go o*oQ