AU764565B2

AU764565B2 - NPY-Y7 receptor gene

Info

Publication number: AU764565B2
Application number: AU45914/99A
Authority: AU
Inventors: Herbert Herzog
Original assignee: Garvan Institute of Medical Research
Current assignee: Garvan Institute of Medical Research
Priority date: 1998-06-29
Filing date: 1999-06-29
Publication date: 2003-08-21
Anticipated expiration: 2019-06-29
Also published as: AU4591499A

Description

WO 00/00606 PCT/AU99/00523 1 NPY-Y7 RECEPTOR GENE Field of Invention: The present invention relates to isolated polynucleotide molecules which encode a novel neuropeptide Y (NPY) receptor designated NPY-Y7. In addition, the present invention relates to the use of these molecules in the production of NPY-Y7 receptors using recombinant DNA technology and to methods of screening and testing compounds for agonist or antagonist activity.

Background of the Invention: Neuropeptide Y (NPY) forms a family (called the pancreatic polypeptide family) together with pancreatic polypeptide (PP) and peptide YY (PYY), which all consist of 36 amino acids and possess a common tertiary structure. NPY receptors, members of the G protein- coupled receptor superfamily, when activated influence a diverse range of important physiological parameters, including effects on psychomotor activity, central endocrine secretion, anxiety, reproduction. vasoactive effects on the cardiovascular system and strongly stimulates food consumption. Specific agonists and antagonists of NPY are therefore likely to be of substantial benefit for therapy of a wide range of clinical disorders. As NPY possess a compact tertiary structure and different parts of the molecule are required for interaction with different subtypes of the receptor, the logical developments of both agonists and antagonists is critically dependent upon the availability and knowledge of specific receptor structure.

It is presently known that NPY binds specifically to at least six receptors; Y1, Y2, Y3, Y4, Y5 (or "atypical Y1") and Y6. While it has been demonstrated that NPY receptors couple to the adenylate cyclase second messenger system, it remains probable that additional NPY receptor subtypes exist since there is evidence that phosphatidylinositol turnover, cations, and arachidonic acid may also function as second messengers for NPY.

Since NPY agonists and antagonists may have commercial value as, for example, potential anti-hypertensive agents, cardiovascular drugs, neuronal growth factors, anti-psychotics, anti-obesity and anti-diabetic agents, the ability to produce NPY receptors by recombinant DNA technology would be WO 00/00606 PCT/AU99/00523 2 advantageous. To this end, DNA molecules encoding Y1, Y2, Y4, Y5 and Y6 have previously been isolated.

The present inventors have now isolated novel DNA molecules encoding the human and murine NPY-Y7 receptors.

Summary of the Invention: Thus, in a first aspect, the present invention provides an isolated polynucleotide molecule encoding an NPY-Y7 receptor or a functionally equivalent fragment thereof.

The encoded NPY-Y7 receptor is characterised by the N-terminal amino acid sequence:

MXIX

2

MX

3

EKWDX

4 NSSE (SEQ ID NO: 1), wherein

X

2

X

3 and X 4 are selected from codable amino acids but, preferably, X, is selected from Phe and Ser, X 2 is selected from Ile and Thr, X, is selected from Asn and Ser, and X, is selected from Thr and Ser.

More preferably, the polynucleotide molecule encodes a human NPY- Y7 receptor of about 408 amino acids or a murine NPY-Y7 receptor of about 405 amino acids.

Most preferably, the polynucleotide molecule encodes a human NPY- Y7 receptor having an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2 or a murine NPY-Y7 receptor having an amino acid sequence subtantially corresponding to that shown as SEQ ID NO: 3.

The polynucleotide molecule may comprise a nucleotide sequence substantially corresponding or, at least, showing at least 90% (more preferably, at least 95%) homology to that shown at nucleotides 1 to 1903 or nucleotides 369 to 1592 of SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent NPY-Y7 receptor fragment.

The 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 NPY-Y7 receptor.

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 NPY-Y7 receptors or functionally equivalent fragments thereof, comprising WO 00/00606 PCT/AU99/00523 3 culturing the host cell of the second aspect under conditions enabling the expression of NPY-Y7 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 NPY-Y7 receptors or functionally equivalent fragments thereof are expressed onto the surface of the host cell.

The polynucleotide molecule of the present invention encodes an NPY receptor which may be of interest both clinically and commercially as it is expressed in many regions of the body and neuropeptides of the NPY family affect a wide number of systems.

By using the polynucleotide molecule of the present invention it is possible to obtain NPY-Y7 receptor protein or fragments thereof in a substantially pure form.

Accordingly, in a fourth aspect, the present invention provides a NPY- Y7 receptor or a functionally equivalent fragment of said receptor, in a substantially pure form.

In a fifth aspect, the present invention provides an antibody or fragment thereof capable of specifically binding to the NPY-Y7 receptor or functionally equivalent fragment of the fourth aspect.

In a sixth aspect, the present invention provides a non-human animal transformed with the polynucleotide molecule of the first aspect of the present invention.

In a seventh aspect, the present invention provides a method for detecting agonist or antagonist agents of an NPY-Y7 receptor, comprising contacting an NPY-Y7 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 an NPY- Y7 receptor, and detecting an increase or decrease in activity of the NPY-Y7 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 or fragment with specific agonist or antagonist agents.

WO 00/00606 PCT/AU99/00523 4 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 laboratoly 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 an NPY-Y7 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 NPY-Y7 receptors.

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 NPY-Y7 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 NPY-Y7 receptor that exhibit binding specificity and activity that is substantially equivalent to the NPY-Y7 receptor from which it/they is/are derived.

WO 00/00606 PCT/AU99/00523 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.

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 shows the degree of identity between the predicted amino acid sequence of the human NPY-Y1, NPY-Y2 and NPY-Y7 receptors.

Figure 2 provides a graph showing the inhibition of human 2 5

I]PYY

binding with various NPY-related peptides on human NPY-Y7 membranes.

The results were obtained through competitive displacement of [1 25 I]pYY on membranes of COSm6 cells transiently expressing human NPY-Y7 receptors.

Membranes were incubated with 2 5 I]PYY (50pM) and increasing concentrations of peptide competitors. Data are representative of a single experiment with each point measured in triplicate.

Figure 3 provides a schematic diagram of the murine NPY-Y7 receptor gene. The gene covers approximately 12 kb and consists of three exons.

Figure 4 shows the degree of identity between the predicted amino acid sequence of the human and murine NPY-Y7 receptors.

Detailed Disclosure of the Invention: Human NPY-Y7 cDNA Human amygdala and testis cDNA libraries (Stratagene) were screened under low strigency conditions with a 401 bp 32P-labelled fragment (corresponding to nucleotides 507 to 908 of SEQ ID NO: 4) originated from a human fetal brain EST clone (GenBank AA449919). Two overlapping cDNA clones were obtained from the screen. The combined nucleotide sequence (hy7) of the clones is shown as SEQ ID NO: 4 and encodes a protein of 408 amino acids (SEQ ID NO: 2).

WO 00/00606 PCT/AU99/00523 6 Sequence comparison with other G protein coupled receptors identified neuropeptide Y receptors as the most closely related group with approximately 32% amino acid sequence identity to the Y1 receptor subtype (Figure Further, in situ hybridisation studies of rat brain sections has identified a NPY-Y7 mRNA distribution (expression was found to occur in the amygdala, the CA3 region of the hippocampus and the piriform cortex) which is consistent with the expression of other NPY-receptor subtypes (Blomquist, and Herzog, TINS 20(7), 1997) and is in agreement with the suggestions of the existence of further Y-receptor family members. This mRNA distribution suggests important functions for the NPY-Y7 receptor in the regulation of the circadian rhythm, anxiety and metabolic status.

Radio-ligand binding experiments has shown that the protein encoded by the hy7 cDNA shows highest affinity for human PYY (Figure These experiments were conducted using COS-6 or HEK (293) cells transiently expressing recombinant Y7 receptor protein. The radio-ligand binding (Herzog, H. et al., Proc. Natl. Acad. Sci. USA 89:5794-5798, 1992) suggests that the NPY-Y7 receptor has a pharmacology similar to the Y2 receptor (Rose, J. Biol. Chem. 270:22661-22664, 1995). The rank of potency for the Y7 receptor is: PYY>NPY>[2-36]PYY>[3-36]NPY>[13-36]NPY>>(Leu 3 1, Pro34)NPY>PP.

Chronmosomal Localisation of the Human Y7 gene Screening of a medium resolution Stanford G3 panel of 83 clones was performed to further refine the map position of the hy7 gene. PCR amplification was carried out on this panel using primers hy7-A (5'GGATGGCCATTTGGAAAC3') and hy7-B (5'CCAATCCTTCCATACATG3'), corresponding to nucleotides 507-524 and 890-907 of the hy7 cDNA (SEQ ID NO: respectively. The analysis indicated that the hy7 gene is most closely associated with the marker SHGC-418 on the long arm of chromosome 4.

This map location is defined by markers AFM191xh2 and AFM347ZH1.

Assessment of the flanking markers using the Whitehead/MIT STS-Based Map of the Human Genome )(http://www-genome.wi.mit.edu/cgibin/contig/phys_map) in conjunction with The Genome Directory (Adams, et al. Nature 377 Suppl. (1995)) identifies 4q21.3 as the most likely position of the hy7 gene.

WO 00/00606 PCT/AU99/00523 7 Mouse Y7 ienomic DNA Using a 32 P-labelled fragment of the hy7 cDNA a mouse genomic

BAC

library (Genome Systems) was screened. A clone encoding the entire gene of the mouse equivalent to hy7 was isolated (SEQ ID NO: The gene covers approximately 12 kb and is divided by two introns into three exons (Figure Figure 4 shows the degree of identity between the predicted amino acid sequence of the human and murine NPY-Y7 receptors.

Pharmacological characterisation pcDNA3.1-hy7 cDNA was transiently transfected into the COSm6 cell line using FUGENE and 5mg of DNA/106 cells. The COSm6 cells were grown in Dulbecco's modified Eagles medium supplemented with 2mM glutamine and 10% fetal calf serum, in 5% CO 2 at 37 0 C. Membranes were harvested with COSm6 cells 72hr post-transfection. Adherent cells were washed twice in ice-cold phosphate buffered saline and lysed using a glass homogeniser in ice-cold hypotonic buffer (50mM Tris-HCI, pH 7.4, 0.1% bacitracin).

Membranes were pelleted by high speed centrifugation (30,000 x g, homogenised again in ice-cold hypotonic buffer and collected again by high speed centrifugation (30,000 x g, 15min, The final membrane pellet was resuspended into lml of ice-cold binding buffer (50mM Tris-HCI, pH7.4, 10mM NaC1, 5mM MgCl2, 2.5mM CaC12, 0.1% bacitracin, 0.1% bovine serum albumin. Membrane suspensions were diluted in binding buffer to yield membrane protein concentrations of 0.05mg/ml. Under these conditions non-specific binding of [1 25 1]PYY to membranes was less than 2 5 I]PYY and unlabelled peptide competitors were also diluted to the required concentrations in binding buffer. Samples were prepared by mixing binding buffer, unlabelled peptide or binding buffer (50ml), [1 2 5

I]PYY

50ml) and membrane suspension (100ml). Samples were incubated at room temperature for 2hr. Incubations were terminated by centrifugation (4min) and pellets collected. Radioactivity was measured for Imin in a g counter.

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.

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.

*000 0*

I

EDITORIAL NOTE APPLICATION NUMBER 45914/99 The following Sequence Listing pages 1/6 to 6/6 are part of the description. The claims pages follow on pages to "13".

WO 00/00606 PCT/AU99/00523 1/6 Sequence Listings: Applicant: Garvan Institute of Medical Research Title of Invention: NPY-Y7 Receptor Gene Prior Application Number: PP 4385 Prior Application Filing Date: 1998-06-29 Number of SEQ ID NOs: Software: PatentIn Ver. 2.1 SEQ ID NO: 1 Length: 14 Type: PRT Organism: Artificial Sequence Feature: Other Information: Description of Artificial Sequence: N-terminal consensus sequence Sequence: 1 Met Xaa Xaa Met Xaa Glu Lys Trp Asp Xaa Asn Ser Ser Giu 1 5 SEQ ID NO: 2 Length: 408 Type: PRT Organism: Homo sapiens Sequence: 2 Met Phe Ile Met Asn Glu Lys Trp Asp Thr Asn Ser Ser Glu Asn Trp 1 5 10 His Pro Ile Trp Asn Val Asn Asp Thr Lys His His Leu Tyr Ser Asp 25 Ile Asn Ile Thr Tyr Val Asn Tyr Tyr Leu His Gln Pro Gln Val Ala 40 Ala Ile Phe Ile Ile Ser Tyr Phe Leu Ile Phe Phe Leu Cys Met Met WO 00/00606 PTA9/02 PCT/AU99/00523 Gly Asn Thr Vai Vai Cys Phe Ile Val Met Arg Asn Lys His Met His Thr Val1 Gly Gi y Asp 145 Lys Ile Tyr Cys Val 225 Met Thr Gin Set Leu 305 Al a Gi y Vali G1 y T rp Ile 130 Arg Thr Met Arg Arg 210 Leu Tyr Gly Lys T rp 290 Set His Phe Thr Ile Pro 115 Ser Phe Al a Set Vai 195 Giu Phe Giy Arg Ile 275 Leu Pro Trp Phe As n Phe 100 Phe Val1 Gin Phe Pro 180 Arg Asp Al a Arg Lys 260 Ile Pro As n Leu As n 340 Leu Cys Gly Al a Cys Val1 165 Ser Leu Trp As n le 245 As n Lys Leu Glu Al a 325 Gi u

P

M

A

V

A

P.

I1 2;

ME

Ti 3: P1 70 he Ile et Pro sn Thr la Ser 135 al Val 50 ie Ile la Vai sn Ser ro Asn 215 le Tyr 30 'ly Ile ln Giu et Leu rp Thr 295 u Gin 10 ie Giy ~n Phe Leu Asn Leu 90 Ile Thr Leu 105 Met Cys Lys 120 Val Phe Thr Tyr Pro Phe Met Ile Ile 170 Met Leu His 185 Gin Asn Lys 200 Gin Giu Met Leu Ala Pro Set Leu Phe 250 Gin Trp His 265 Leu Ile Val 280 Leu Met Met Ile Ilie Asn Asn Ser Ser 330 Arg Arg Gly 75 Al a Leu Ile Leu Lys 155 T rp Val Thr Arg Leu 235 Arg Vai Al a Leu Ile 315 Val Phe Ile Asp Ser Val 140 Pro Val1 Gin Set Lys 220 Set Al a Val Leu Set 300 Tyr As n Gin Set As n Gly 125 Al a Lys Leu di u Pro 205 Ile Leu Al a Set Leu 285 Asp Ile Pro di u Al a 365 Asp le 110 Leu Ile Leu Al a Giu 190 Val1 Tyr Ile Va I Arg 270 Phe Tyr Tyr Ile Al a 350 Leu Leu Ile Ala Vai Gin Ala Val Thr Ile 160 Ile Tht 175 Lys Tyt Tyr Trp Thr Thr Va1 Ile 240 Pro His 255 Lys Lys Ile Leu Ala Asp Pro Phe 320 Ile Tyr 335 Phe Gin Leu Gin Leu Cys Gin Lys Arg Ala Lys Pro Met Giu 355 360 Tyr Thr Leu WO 00/00606 PCT/AU99/00523 3/6 Lys Ala Lys Ser His Val Leu Ile Asn Thr Ser Asn Gin Leu. Val Gln .370 375 380 Giu Ser Thr Phe Gin Asn Pro His Gly Glu Thr Leu Leu Tyr Arg Lys 385 390 395 400 Ser Ala Glu Asn Pro Asn Arg Asn 405 SEQ ID NO: 3 Length: 405 Type: PRT Organism: Mus Inusculus Sequence: Met As n Ile Al a Gi y Thr Val Gi y Gi y Asp 145 Lys Ile Ser His As n Val As n Val Gi y T rp Ile 130 Arg Thr Met *Thr Ile Ile Phe Thr Thr Ile Pro 115 Ser Phe Al a Thr Met Trp Thr Ile Val As n Phe 100 Phe Val Ar g Phe Pro Ser Ser Tyr Ser Val1 Phe Cys Gi y Al a Cys Val 165 Ser Glu Gi y Val1 Ser Cys 70 Leu Met Ser Al a Val1 150 Thr Alia Ly's Trp Asp Ser Asn Ser Ser Giu, Ser Trp As n As n Tyr 55 Phe Ile Pro Ser Ser 135 Val Ile Ile Asp Tyr 40 Leu.

Ile Leu.

Ile Met 120 Val Tyr Vai Met Th r 25 Tyr Leu Val As n Thr 105 Cx's Phe Pro Ile Leu Gin Leu Ile Ile Leu 90 Leu.

Lys Thr Phe Ile 170 His His His Gin Phe Val Arg Asn 75 Ala Ile Leu Asp Ile Ser Leu. Val 140 Ly's Pro 155 Trp Gly T rI Pro Leu Arg Ser As n Gi y 125 Al a Lys Leu T vr Gin Cys His Asp Ile 110 Leu.

Ile Leu.

Ala Ser Val1 Met Met Leu Ile Val Al a Thr Ile 175 Asp Al a Val His Leu Al a Gn Vai Val1 160 Aila 180 His Val Gin Glu Glu Lys Tyr Tyr Arg Val Arg Leu Ser Ser His Asn Lys Thr Ser Thr Val Tyr Trp WO 00/00606 PCT/AU99/00523 4/6 195 200 205 Cys Arq Glu Asp Trp Pro Arg His Giu Met Arg Arg Ile Tyr Thr Thr 210 215 220 Val Leu Phe Ala Ile Ile Tyr Leu Ala Pro Leu Ser Leu Ile Val Ile 225 230 235 240 Met Tyr Ala Arg Ile Gly Ala Ser Leu Phe Lys Thr Ala Ala His Cys 245 250 255 Thr Gly Lys Gin Arg Pro Val Gin Cys Met Tyr Gin Glu Lys Gin Lys 260 265 270 Val Ile Lys Met Leu Leu Thr Val Ala Leu Leu Phe Ile Leu Ser Trp 275 280 285 Leu Pro Leu Trp Thr Leu Met Met Leu Ser Asp Tyr Thr Asp Leu Ser 290 295 300 Pro Asn Lys Leu Arg Ile Ile Asn Ile Tyr Ile Tyr Pro Phe Ala His 305 310 315 320 Trp Leu Ala Plie Cys Asn Ser Ser Vai Asn Pro Ile Ile Tyr Gly Phe 325 330 335 Phe Asn Giu Asn Phe Arg Asn Gly Phe Gin Asp Ala Phe Gin Ile Cys 340 345 350 Gin Lys Lys Ala Lys Pro Gin Glu Ala Tyr Set Leu Arg Ala Lys Arg 355 360 365 Asn Ile Vai Ile Asn Thr Set Gly Leu Leu Vai Gin Giu Pro Vai Ser 370 375 380 Gin Asn Pro Gly Gly Giu Asn Leu Gly Cys Gly Lys Ser Ala Asp Asn 385 390 395 400 Pro His Arg Asn Pro 405 SEQ ID NO: 4 Lenqth: 1903 Type: DNA Organism: Homo sapiens Sequence: 4 ctcgaqatcc attgtgctct aaaggcctcc tgagtagctg ggactacagg cgcccgccac cacgcctggc taattttttt gtatttttag tagggacggc gtttcactgt gttagccaga 120 tggtctccat ctcccgacct cgtgatccac ccacctcggc ctcccaaagt gctgggatta 180 WO 00/00606 WO 0000606PCT/AU99/00523 caggcgtgag ttctgcttcc gacatacaag tgactgctat Ccatctggaa tgaactacta tcttcttttt atatgcacac gcatattctg acacgatgtg cgttagttgc tcactatcaa tgtctccatc actcccagaa tgaggaagat ttgtcatcat gcaggaagaa tgctcctgat tgctctcaga acccttttgc tcttcaacga aaagagcaaa catctaatca ataggaaaag taacagcagt tatttaaatc taaaacattt aagatcataa tgaataaata *accgcgcccg atattacagg aaacatcaaa gttcatcatg tgtcaatgac tcttcaccag gtgcatgatg agtcactaat catgcctata caagatcagt aattqctgta gacagcgttt tgCagtaatg taaaaccagt ctacaccact gtatggaagg ccaggagcag tgtggCcctg ctacgctgac acactggctg gaatttccgc gcctatggaa gcttgtccag tgctgaaaac gagatttaaa cattgctttt actgaaagcc acaatcttat tatttctaga gccaatttcc tttcctcagt aagattgaat aatgagaaat acaaagcatc cctcaagtgg ggaaatactg Ctcttcatct acactgctgg ggattggtcc gataggttcc gtcattatta ttacatgtgc Ccagtctact gtgctgtttg attggaattt tggca cgtgg ctttttattc ctttctccaa gcattcggca cgtggtttcc gcttataccc gaatctacat cccaacagga aagagctagt tgtqgctttg ctctctggca gttgtataaa gaacagttaa tttcttagtt gcctctgccc acctcttctc 240 tgcgaaatta gtcttaataa gggacacaaa atctgtactc caqcaatctt tggtttgctt taaacctggc acaatattat agggaatatc agtgtgtggt tgatcatctg aagaagaaaa ggtgccgqga ccaacatcta cactcttcag tgtccaggaa tctcatggct atgaactgca acagcagtgt aagaagcttt taaaagctaa ttcaaaaccc attagtgatg gtgataatcc cacttcaaat aaaaaattaa aatacgtaga aaaaaaaaaa ggatgttaat gagtgaagca Ctcttcagaa agatattaat cattatttcc tattgtaatg cataagtgat agcaggatgg tgtcgcagct cta ccctttt ggtcctagcc atattaccga agactqgcca cctggCtccc ggctgcagtt gaagcagaag gcccctgtgg gatcatcaac caatcccatc ccagctccag aagccatgtg tcatggggaa gaagaattaa taactctact ttttcaaaga aaataaacaa gtgacttaga aaa tatagctttt 300 tgtagatcag 360 aactggcatc 420 attacctatg 480 tactttctga 540 aggaacaaac 600 ttactaqttg 660 ccatttggaa 720 tcagtcttta 780 aaaccaaagc 840 atcaccatta 900 gtgagactca 960 aatcaggaaa 1020 ctctccctca 1080 cctcacacag 1140 atcattaaga 1200 actctaatga 1260 atctacatct 1320 atttatggtt 1380 ctctgccaaa 1440 ctcataaaca 1500 accttgcttt 1560 aagaaactac 1620 acgcattata 1680 atgttctaaa 1740 aaatggtcat 1800 catgtttgca 1860 1903 SEQ ID NO: Length: 1228 Type: DNA Organism: Mus musculus WO 00/00606 WO 0000606PCTIAU99/00523 Sequence: atgtccacca agtggcaatg tatctccacc ttgtgcatgq acagtcacta tgtatgccta tgcaagatca gcaatagctg aagacagcct tctgcaataa aataaaacca atctatacca atgtatgcaa cgtccagtgc gccctccttt actgacctgt tggctcgcct tttcgcaatg gcctattccc gaaccggtgt ccacacagga tgagcgagaa atacacagca agccccaagt tgggaaatac atttcttgat tcacattgct gtgggctggt tggacagatt ttgtcacgat tgttacatgt gcacagtcta cggtgctatt ggattggggc agtgcatgta tcatcctttc ctcctaacaa tctgcaacag gtttccaaga tgagagcgaa ctcaaaaccc atccttgata atqggactca tcactggtat ggcagctgtc tgtcgtttgC cttaaacctt ggacaacatc gcaagggata ccgctgtgtg tgtgatcatc acaagaagaa ctqgtgtcgg tgccatcatc ttccctcttC tcaagagaaa ctggcttccc actgcgtatc cagtgtcaac tgctttccag acgcaacata aggtggggaa gaggaatg aactcttcag tcagatatca ttcatcagct tttattgtga gccataagtg atagcaggat tcagttgcgg gtctacccct tggggcctgg aaatactacc gaggactggc tat cttgctc aagacggcag cagaaggtca ctgtggaccc atcaacatct cctattattt atctgccaaa gtcataaaca aatttgggat aaagctggaa acattaccta cctacctcct taaggaatag atttactqgt ggccattcgq Cttccgtctt ttaagccaaa ccatcgccat gtqtgagact caagacacga Ctctctcact cacactgcac tcaagatgct tgatgatgct acatctaccc atggattctt agaaagccaa catcggqcct gtggaaaaag tcacatctgg tgtgaactac gatctttgtc acacatgcac tggaatattc aagcagcatg caccttggtt gctcactgtc tatgactcca cagctcccac aatgaggagg cattgttatc aggcaagcag gctgactgtg ctcagactat tttcgcccac taatgaaaat gccccaggaa gctggtgcag tgcagacaat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1228

Claims

1. An isolated polynucleotide molecule encoding an NPY-Y7 receptor or a functionally equivalent fragment thereof, wherein the encoded NPY-Y7 receptor is characterised by the N-terminal amino acid sequence: MXiX 2 MX 3 EKWDX 4 NSSE (SEQ ID NO: 1), wherein X 1 X 2 X 3 and X 4 are selected from codable amino acids.

2. A polynucleotide molecule according to claim 1, wherein Xi is selected from Phe and Ser, X 2 is selected from Ile and Thr, X 3 is selected from Asn and Ser and X 4 is selected from Thr and Ser.

3. A polynucleotide molecule according to claim 1 or 2, wherein the polynucleotide molecule encodes an NPY-Y7 receptor of human origin of about 408 amino acids in length.

4. A polynucleotide molecule according to claim 3, wherein the polynucleotide molecule encodes a human NPY-Y7 receptor having an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.

5. An isolated polynucleotide molecule that encodes an NPY-Y7 receptor polypeptide of human origin comprising the amino acid sequence set forth in SEQ ID NO: 2.

6. A polynucleotide molecule according to claim 1 or 2, wherein the 25 polynucleotide molecule encodes an NPY-Y7 receptor of murine origin of about 405 amino acids in length.

7. A polynucleotide molecule according to claim 6, wherein the polynucleotide molecule encodes a murine NPY-Y7 receptor having an amino acid sequence S 30 substantially corresponding to that shown as SEQ ID NO: 3. o0••

8. An isolated polynucleotide molecule that encodes an NPY-Y7 receptor polypeptide of murine origin comprising the amino acid sequence set forth in SEQ ID NO: 3.

9. A polynucleotide molecule encoding an NPY-Y7 receptor, wherein the polynucleotide molecule comprises a nucleotide sequence showing at least homology to that shown at nucleotides 1 to 1903 or nucleotides 369 to 1592 of SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent NPY-Y7 receptor fragment. A polynucleotide molecule according to claim 9, wherein the polynucleotide molecule comprises a nucleotide sequence showing at least 95% homology to that shown at nucleotides 1 to 1903 or nucleotides 369 to 1592 of SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent NPY-Y7 receptor fragment.

11. A polynucleotide molecule according to claim 8 or 9, wherein the polynucleotide molecule comprises a nucleotide sequence substantially corresponding to that shown at nucleotides 1 to 1903 or nucleotides 369 to 1592 of SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent NPY-Y7 receptor fragment.

12. An isolated polynucleotide molecule encoding an NPY-Y7 receptor polypeptide, i..i twherein the polynucleotide molecule comprises a nucleotide sequence selected from the group consisting of: the sequence set forth in SEQ ID NO: 4; 25 (ii) a sequence comprising nucleotides 369 to 1592 of SEQ ID NO: 4; and (iii) a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 2. -13. The isolated polynucleotide molecule according to claim 12, wherein the polynucleotide molecule comprises the nucleotide sequence set forth in SEQ ID NO: 4 30 or a fragment thereof comprising nucleotides 369 to 1592 of SEQ ID NO: 4. •go•* *o 11

14. A plasmid or expression vector including a polynucleotide molecule according to any one of claims 1 to 13. A host cell transformed with the isolated polynucleotide molecule according to any one of claims 1 to 13 or a plasmid or expression vector according to claim 14.

16. A host cell according to claim 15, wherein the cell is a mammalian or insect cell.

17. A host cell according to claim 16, wherein the cell is a Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) 293 cell or an insect Sf9 cell.

18. A host cell according to any one of claims 15 to 17, wherein the cell expresses the NPY-Y7 receptor or functionally equivalent fragment thereof on its surface.

19. An NPY-Y7 receptor which is characterised by the N-terminal amino acid sequence: MXIX 2 MX 3 EKWDX 4 NSSE (SEQ ID NO: 1), wherein X 1 X 2 X 3 and X 4 are selected from codable amino acids, or a functionally equivalent fragment of said receptor, in a substantially pure form. A receptor according to claim 19, wherein said receptor is a human receptor of about 408 amino acids.

21. A receptor according to claim 20, wherein said receptor has an amino acid 25 sequence substantially corresponding to that shown as SEQ ID NO: 2.

22. A receptor according to claim 20, wherein said receptor is a murine receptor of about 405 amino acids.

23. A receptor according to claim 22, wherein the receptor has an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 3. ft S 12

24. An isolated NPY-Y7 receptor in a substantially pure form, wherein said polypeptide is encoded by the isolated polynucleotide according to any one of claims 8, 12, or 13. The isolated NPY-Y7 receptor polypeptide according to claim 24, wherein said NPY-Y7 receptor polypeptide is a human receptor polypeptide consisting of about 408 amino acids in length.

26. The isolated NPY-Y7 receptor polypeptide according to claim 24 or 25, wherein said NPY-Y7 receptor polypeptide has the amino acid sequence set forth in SEQ ID NO: 2.

27. The isolated NPY-Y7 receptor polypeptide according to claim 24, wherein said NPY-Y7 receptor polypeptide is a murine receptor polypeptide consisting of about 405 amino acids in length.

28. The isolated NPY-Y7 receptor polypeptide according to claim 24 or 25, wherein said NPY-Y7 receptor polypeptide has the amino acid sequence set forth in SEQ ID NO: 3. i 29. An antibody or fragment thereof which specifically binds to an NPY-Y7 receptor according to any one of claims 19 to 28. 25 30. A non-human animal transformed with a polynucleotide molecule according to any one of claims 1 to 13 or a plasmid or expression vector according to claim 14.

31. A method for detecting an agonist or antagonist of an NPY-Y7 receptor, comprising contacting an NPY-Y7 receptor according to any one of claims 19 to 28 or a host cell according to any one of claims 15 to 18, with a test agent under conditions *go •go•* *o 13 enabling the activation of said receptor, and detecting an increase or decrease in the receptor activity.

32. A method of producing an NPY-Y7 receptor polypeptide comprising culturing the host cell according to any one of claims 15 to 18 under conditions enabling the expression of an NPY-Y7 receptor polypeptide encoded by the polynucleotide molecule according to any one of claims 1 to 13 or the plasmid or expression vector according to claim 14, and optionally recovering the expressed receptor polypeptide.

33. An antisense polynucleotide comprising a nucleotide sequence selected from the group consisting of: a sequence that is complementary to the sequence set forth in SEQ ID NO: 4; (ii) a sequence that is complementary to a sequence comprising nucleotides 369 to 1592 of SEQ ID NO: 4; and (iii) a sequence that is complementary to a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 2.

34. Use of the antisense polynucleotide of claim 33 in the preparation of a reagent to prevent translation of mRNA encoding an NPY-Y7 polypeptide in a cell. DATED this ninth day of June 2003 Garvan Institute of Medical Research Patent Attorneys for the Applicant: F.B. RICE CO.