AU772193B2 - Neuropeptide Y-Y5 receptor - Google Patents

Description

AUSTRALIA

Patents Act 1990 Garvan Institute of Medical Research

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT r Invention Title: Neuropeptide Y-Y5 receptor The following statement is a full description of this invention including the best method of performing it known to us:- NEUROPEPTIDE Y-Y5 RECEPTOR The present invention relates to isolated DNA molecules which encode the neuropeptide Y-Y5 receptor. In addition the present invention relates to the use of these molecules in the production of the neuropeptide receptor using recombinant technology and to methods of screening and testing compounds for neuropeptide Y (NPY) agonist or antagonist activity.

In developed affluent countries the prevalence of obesity is alarming and it is now a massive contribution to morbidity and mortality in addition to being socially disadvantageous. Fat deposition in the abdominal area is a particular problem in relation to risk of Type II diabetes and cardiovascular disease. However, until recently, the molecular mechanisms controlling appetite, energy expenditure and adiposity have been surprisingly illunderstood.

Obesity has well-known associations with non-insulin-dependent diabetes (NIDDM), hypertension, dyslipidaemia and coronary heart disease, as well as less obvious links with diseases such as osteoarthritis and various malignancies; it also causes considerable problems through reduced mobility and decreased quality of life. Seven forms of rodent obesities, determined .i by single gene mutations, have been identified: yellow adipose [Ad], diabetes fat [fat], tubby [tub] and obese [ob] in the mouse and fatty [fa] in the rat. The obese phenotypes caused by these mutations differ in their age of onset, severity and the degree of insulin resistance. Similar 25 phenotypes can also be seen in obese humans. Recently the molecular bases for some of these mutations has been elucidated. Of these the [ob] gene product "leptin" has created the most interest. However, many other factors are also involved in regulating energy balance and body fat distribution.

Four factors appear most likely to have an important role: these are 30 neuropeptide Y (NPY), corticotropin releasing factor (CRF)/ACTH/glucocorticoids, insulin and galanin. In particular, NPY and its receptors play an important role in the regulation of appetite and in a related manner, obesity.

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. Neuropeptide Y (NPY) receptors, members of the G protein-coupled receptor superfamily, are activated by one of the most abundant peptides in the mammalian nervous system and subsequently 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 most importantly, potent effects on appetite. A number of neuropeptides and classical neurotransmitters, including noradrenaline and serotonin, modulate ingestive behaviours. However, NPY stands out from the many neurotransmitters with experimental effects on food intake in being able to induce obesity. Injections of NPY into the paraventricular nucleus (PVN), have been shown to increase, in a dose dependent manner, feeding and drinking behaviour in the rat. A single injection of NPY can increase food intake several-fold for several hours and is effective even during the light phase when rats usually eat little, and in animals that have already eaten to satiety. Consequently, NPY peptides are certainly among the most potent orexygenic substances known in either food-deprived or satiated animals. Repeated NPY injections into the PVN result in a massive and persistent feeding response and the rats ultimately develop obesity, with a true increase in body fat content. The importance of NPY as a mediator of appetite/obesity regulation is further enhanced by the very recent report that the obese gene product leptin inhibits NPY synthesis and release.

Injections of NPY into the paraventricular nucleus cause a prompt and robust increase in plasma ACTH levels and there is clear evidence that NPY-induced ACTH secretion is mediated by corticotropin releasing factor (CRF). However, its mode of action as well as its interaction with CRF within the brain is largely unknown, as are 25 its interrelationships with other hormones, such as insulin. Nevertheless an agent Swhich increases appetite and raises glucocorticoid levels might be important in generating central obesity.

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 possesses 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 five receptors; Y1, 35 Y2, Y3, Y4 and Y1-like (or "atypical 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 advantageous. To this end, DNA molecules encoding Y1, Y2, Y3 and Y4 have previously been isolated.

The present inventors have now isolated novel DNA molecules encoding the human, mouse and rat Y1-like (hereinafter referred to as NPY-Y5) receptors. Similar DNA molecules encoding human and rat NPY-Y5 have been described in International (PCT) Patent Specification No. WO 96/16542, however, these encode receptors with, in the case of the human NPY-Y5, an additional 10 N-terminus amino acids, and, in the case of the rat NPY-Y5, an additional 11 N-terminus amino acids. Through analysis of several cDNA clones and RT-PCR using specific primers for intron and exon sequences, the present inventors have confirmed that the human, mouse and rat NPY-Y5 receptor does not include these additional 10/11 amino acids. The DNA molecules described in WO 96/16542 may thus exhibit lower expression rates over those of the present invention. In addition, the receptors encoded by the DNA molecules described in WO 96/16542, may show lower and possibly altered activity.

This specification contains nucleotide and amino acid sequence information prepared using PatentIn Version 3.1, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator 25 <210> followed by the sequence identifier <210>1, <210>2, <210>3, etc).

The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents 35 thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

For the purposes of nomenclature, the nucleotide sequences set forth in the Sequence Listing are as follows: SEQ ID NO: 1 relates to a genomic DNA molecule encoding the human NPYreceptor; SEQ ID NO: 2 relates to the predicted amino acid sequence encoded by SEQ ID NO: 1; SEQ ID NO: 3 relates to a cDNA encoding the human NPY-Y5 receptor; SEQ ID NO: 4 relates to the predicted amino acid sequence encoded by SEQ ID NO: 3; SEQ ID NO: 5 relates to a cDNA encoding the rat NPY-Y5 receptor; SEQ ID NO: 6 relates to the predicted amino acid sequence encoded by SEQ ID NO: SEQ ID NO: 7 relates to a nucleotide sequence of a genomic DNA encoding the mouse NPY-Y5 receptor; and SEQ ID NO: 8 relates to the predicted amino acid sequence encoded by SEQ ID NO: 7.

According to one aspect of the invention there is provided an isolated DNA molecule encoding an NPY-Y5 receptor consisting of 445 amino acids in length.

Preferably, the DNA molecule encodes a human or rat NPY-Y5 receptor. More preferably, the DNA molecule encodes a human NPY-Y5 receptor. Also preferably, 25 the DNA molecule has a nucleotide sequence that is at least 80% homologous to the nucleotide sequence shown: a. at nucleotides 6291 to 7625 in SEQ ID NO: 1; b. at nucleotides 63 to 1397 in SEQ ID NO: 3; or c. at nucleotides 115 to 1449 in SEQ ID NO: Also preferably, the DNA molecule is at least 95% homologous to the nucleotide sequence shown: a. at nucleotides 6291 to 7625 in SEQ ID NO: 1; b. at nucleotides 63 to 1397 in SEQ ID NO: 3; or c. at nucleotides 115 to 1449 in SEQ ID NO: 35 In another aspect of the invention there is provided an isolated DNA molecule encoding an NPY-Y5 receptor consisting of 445 amino acids in length, wherein said DNA molecule comprises a nucleotide sequence selected from the group consisting of nucleotides 6291 to 7625 of SEQ ID NO: 1, nucleotides 63 to 1397 of SEQ ID NO: 3 and nucleotides 115 to 1449 of SEQ ID NO: 5. Preferably, the DNA molecule consists of nucleotides 6291 to 7625 of SEQ ID NO: 1 or substantially corresponds to said nucletotides 6291 to 7625 of SEQ ID NO: 1. Preferably, the DNA molecule consists of nucleotides 63 to 1397 of SEQ ID NO: 3 or substantially corresponds to said nucleotides 63 to 1397 of SEQ ID NO: 3. Preferably, the DNA molecule consists of nucleotides 115 to 1449 of SEQ ID NO: 5 or substantially corresponds to said nucleotides 115 to 1449 of SEQ ID NO: In another aspect of the invention there is provided a plasmid or expression vector including a DNA molecule according to the invention.

In another aspect of the invention there is provided a host cell transformed with the DNA molecule according to the invention. Preferably, the cell is a bacterial, yeast, mammalian or insect cell. More preferably, the cell is a Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) 293 cell or Sf9 cell. More preferably, the cell expresses the NPY-Y5 receptor onto the cell's surface.

In a further aspect, the present invention provides a method of producing receptors comprising culturing the transformed host cell under conditions.

enabling the expression of the DNA molecule and optionally recovering the receptor.

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

By using the nucleic acid molecules of the present invention it is possible to obtain neuropeptide Y-Y5 receptor protein in a substantially pure form.

S••In another aspect of the invention there is provided an isolated receptor polypeptide consisting of 445 amino acids in length. Preferably, the receptor has an amino acid sequence substantially corresponding to any one of the amino acid sequences of SEQ ID Nos 2, 4 or 6.

In another aspect of the invention there is provided an antibody capable of specifically binding to the NPY-Y5 receptor polypeptide according to the invention.

In another aspect there is provided a non-human animal transformed with a DNA molecule according to the invention.

In another aspect of the invention there is provided a method for detecting agonist or antagonist agents of NPY-Y5 receptor, comprising contacting a 6 receptor according to the invention or a cell transformed with and expressing a DNA molecule according to the invention, with a test agent under conditions enabling the activation of the NPY-Y5 receptor, and detecting an increase or decrease in the NPYreceptor activity.

In another aspect of the invention there is provided a nucleic acid probe comprising a nucleotide sequence of 10 or more nucleotides capable of specifically hybridizing to a unique sequence within the DNA molecule according to the invention.

In another aspect of the invention, there is provided an antisense nucleic acid molecule comprising a nucleotide sequence capable of specifically hybridizing to an mRNA molecule which encodes NPY-Y5 receptor so as to prevent translation of the mRNA molecule to which it is hybridised.

The term "substantially corresponding" as used herein in relation to the nucleotide sequences shown in SEQ ID Nos 1, 3, 5 and 7 is intended to encompass minor variations in the nucleotide sequence 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 "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 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 S. 25 acids.

S°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.

The invention is hereinafter described by way of the following non-limiting example and further, with reference to the accompanying figures.

Brief Description of the Figures *o FIG. 1 provides the nucleotide sequence (SEQ ID NO: 1) of a genomic DNA molecule S. encoding the human NPY-Y5 receptor and includes the predicted amino acid S 35 sequence (SEQID NO: 2).

o0 FIG. 2 provides the nucleotide sequence (SEQ ID NO: 3) of a cDNA encoding the human NPY-Y5 receptor and includes the predicted amino acid sequence (SEQ ID NO: 4).

FIG. 3 provides the nucleotide sequence (SEQ ID NO: 5) of a cDNA encoding the rat receptor and includes the predicted amino acid sequence (SEQ ID NO: 6).

FIG. 4 provides the nucleotide sequence (SEQ ID NO: 7) of a genomic DNA encoding the mouse NPY-Y5 receptor and includes the predicted amino acid sequence (SEQ ID NO: 8).

FIG. 5 shows the degree of identity between the predicted amino acid sequence of the human (SEQ ID NOS: 2 and rat (SEQ ID NO: 6) and mouse (SEQ ID NO: 8) receptor proteins.

FIGS. 6a-f provide graphical results of binding assays conducted with CHO cells expressing NPY-Y5, Y5 ligands assayed were NPY, Leu 31 Pro 34 NPY, PP, PYY, NPY 2-36 and PYY 13-36.

FIG. 7 provides graphical results of cAMP assays conducted on CHO cells expressing using the ligands NPY, Leu 31 Pro 34 NPY, PP, PYY and NPY 2-36.

Example 25 EXPERIMENTAL PROCEDURES cDNA and Genomic Library Screening A human genomic P1 DNA library (Genome-Systems), a human foetal brain cDNA library Seeburg, University of Heidelberg) and a rat hypothalamic cDNA library (Stratagene) were screened with a 632 bp 32 P-labelled EcoRI/Pstl fragment flanking exon 1C of the human NPY-Y1 gene. Hybridisation with the probe was **performed in a solution containing 6xSSC, •o solution, 0.1 SDS and 100mg/ml denatured and sheared salmon sperm DNA at 60 °C for 16 h. Filters were washed twice for 15 min in 2xSSC/0.1 %SDS at 60 °C followed by a 15 min wash in 0.1xSSC/0.1% SDS and exposed to X-ray film (Kodak, X-Omat) using an intensifying screen at 70 °C for 16h. P1 DNA from positive clones was isolated according to the manufacturer's protocol. The DNA was digested with EcoRI, HindIII, BamHI and PstI then subcloned into the Bluescript SK vector (Stratagene) generating clones covering all of the human Y1 and Y5 genes.

Nucleotide Sequence Determination.

Supercoiled plasmid DNA was alkaline-denatured and sequenced by the dideoxy chain termination method using T7 polymerase (Promega) (Sambrook et al., 1992). The oligonucleotide primers used initially were complementary to the flanking region of the vector and then based on sequences obtained in order to complete the sequence analysis.

Restriction Map Determination.

P1 DNA was digested with restriction enzymes EcoRI, BamHI, HindIII, alone and in all possible combinations, electrophoresed on a 0.8 agarose gel, alkaline-denatured (0.4 M NaOH), capillary-transferred using 0.4 M NaOH to Hybond N+ membranes and hybridised with several specific oligonucleotides, cDNAs and genomic DNA fragments obtained from the subcloning.

25 In Situ Hybridisation Analyses Sense and antisense riboprobes to the human NPY-Y5 receptor were synthesised using the DIG RNA Labelling Kit (SP6/T7) (Boehringer Mannheim). cDNA corresponding to the coding region of the human receptor was linearised and transcribed with either T7 (for antisense riboprobe) or SP6 (for sense riboprobe) RNA polymerase according to the manufacturers instructions using digoxygenin labelled dUTP.

Postmortem brain tissue was obtained from a young adult male without neurological disease. Specific brain regions were dissected and "fixed by immersion in formalin for 36 hours and then embedded in paraffin.

6 mm serial sections were collected on slides subbed in chrom alum and stored at 40C until used. Sections were dewaxed in Histoclear (National Diagnostics) for 5 min, rehydrated in 100%, 70% and 50% alcohol for 2 min each then washed in phosphate buffered saline (PBS) for 5 min.

Sections were pretreated for 10 min at room temperature with mg/ml proteinase K (Boehringer Mannheim) in 50mM Tris, pH 7.5, 5 mM EDTA. Sections were then washed twice with 0.1M glycine (in PBS) for 2 min, once in PBS then incubated for 1 h at room temperature in hybridisation buffer: 2 x SSPE, 50% formamide, 5% dextran sulfate, 1 x Denhardt's reagent, 100mg/ml tRNA type X-SA (Sigma). Digoxigenin labelled riboprobes to sense and antisense DNA (500ng) in 75ul of hybridisation buffer were added to the sections and hybridised at 420C for 18 h in a humidified environment using a Hybaid Omnislide PCR Thermal Cycler (Integrated Sciences). After hybridisation, sections were washed at room temperature in 2 x saline sodium citrate (SSC) buffer, 0.15M NaCl/0.015 M Na-citrate, pH 7.0 for 10 min, then 0.2 x SSC for 30 min followed by treatment with 20mg/ml RNase [Sigma], in 10mM Tris, pH mM NaCl for 15 min at room temperature. After RNase treatment the slides were washed in 2 x SSC for 5 min at room temperature then 0.2 x SSC at 370C for 30 min.

Tissues were processed for immunological detection by washing for 10 min in buffer A (100mM Tris-HC1, pH 7.5, 150 mM NaC1), then incubated S: for 30 min with a 2% blocking solution (Boehringer Mannheim) with 0.3% Triton X-100 in buffer A. The sections were then incubated for 2 hours with an alkaline phosphatase-conjugated anti-digoxigenin antiserum (Boehringer Mannheim, diluted 1/500 in buffer A plus 0.5% blocking reagent), washed 25 twice for 5 min each in buffer A followed by a wash in 100mM Tris-HC1, pH 100mM NaC1, 50mM MgCl2 for 2 min. The labelled probes were visualised using nitro blue tetrazolium and bromochloro-indoyl phosphate as substrates for 18 hours in the dark. Sections were washed for 10 min in Tris-HC1, pH 8.0, 1 mM EDTA, then 3 quick washes in distilled water, 30 mounted with Aquamount [Gurr] and examined using a Zeiss Axiophot microscope with Nomarsky optics using a blue filter.

Expression of NPY The rat Y5 receptor protein was expressed as follows: the mammalian expression construct rpHzl7 was made by subcloning a 1.9 kb fragment containing the whole coding region and almost the entire 3' untranslated region of the rat NPY-Y5 cDNA into the pPRC/CMV vector (Invitrogen).

The construct is under the control of the CMV promoter and contains the neomycin gene for selection. The expression construct rpHzl7 was transfected into mammalian cell lines CHO-K1 and HEK using a modified calcium phosphate transfection method.

Binding Assay The coding region of the NPY-Y5 receptor was subcloned in the pRC/CMV expression vector and transfected into the chinese hamster ovary (CHO) K1 cell line by using a modified calcium phosphate transfection method. CHO cells were maintained under 5% CO 2 in Dulbecco's modified Eagles medium (DMEM)/Ham's F-12 medium with 2mM glutamine and 10% fetal calf serum. Stably transfected cells were selected with neomycin and tested for the ability to bind NPY/PYY analogues. Transfected cells (1x10 6 were incubated in 0.5ml assay buffer Tris-HC1, pH 7.4, 2mM CaC12, 5mM KC1, 120mM NaC1, 1mM MgC1 2 0.1% bovine serum albumin] in the presence of 0.05nM 1251 labeled NPY and increasing concentrations of human NPY and related peptides. Cells were incubated for 3 hours at 15 0 C then layered onto 0.5ml horse serum before being pelleted in a microcentrifuge for 4 min. Radioactivity was measured for 1 min in a y counter.

Results of binding assays involving CHO cells expressing NPY-Y5 receptor are shown in Table 1, expressed as a percentage of the maximal specifically bound radiolabeled NPY. Results are the pooled data from three separate binding curves with triplicate points.

TABLE 1 Peptide ICso (nM) Mean +/-SE NPY 7. 2 Leu31 Pro34 NPY 7.3+/-0.3 PP 3 PYY 25 NPY 2-36 27+/-3.4 PYY 13-36 1000 o o cAMP Assays CHO cells expressing NPY-Y5 receptor were grown and maintained in Dulbecco's modified Eagles medium: Hams F12 medium (1:1 v/v) supplemented with 2mM L-glutamine and 10% foetal calf serum at 37 0 C under an atmosphere of 100/ CO, in humidified air in 150cm 3 flasks.

Experiments were performed in 24 well cluster dishes when cells had reached confluence.

Inhibition of forskolin-stimulated [3HI-cAMP accumulation Cell monolayers were incubated for 2h at 37 0 C in Iml/well of HEPES buffered Hanks solution (HBH; 20mM, pH 7.4) containing 3 H]-adenine (74kBq/well). Prior to the addition of agonist, cells were incubated in Iml/well HBH containing the phosphodiesterase inhibitor Ro 20-1724 for Agonists (in 10pl HBH) were added to the assay system following the addition of forskolin (10pM) and the incubation continued for The temperature of the incubation medium was maintained at 37°C during these manipulations. Incubations were terminated by the addition of conc. HC1 to each well which lysed the cells. 3 H]-cAMP content of the supernatant buffer from each well was isolated by sequential ion exclusion Dowex-alumina chromatography. After the addition of emulsifier scintillator 20 (15ml), radioactivity was determined by liquid scintillation counting.

Results are provided in Table 2.

TABLE 2 25 Peptide ICso Values (n=3) 9*.

NPY 163.7±70.0nM PYY 45.1±31.4nM **PP 73.4±47.4nM [2-36]NPY 242.5±171.4nM Leu 3 Pro 4 NPY 75.9±38.3nM e$* 11

RESULTS

Identification of NPY-Y5 receptor gene The cloning and characterisation of the 5' upstream region of the human NPY-Y1 receptor gene, while confirming the existence of several alternative 5' exons for the Y1 gene (Ball et al., 1995), also revealed a region of extensive homology with G-protein coupled receptors in exon 1C, involving a partial open reading frame in the opposite orientation. Comparison of this 200 amino acid sequence, which contained parts of the third intracellular loop and transmembrane domains VI and VII, with the Genbank database, identified the human NPY-Y1 receptor as the closest related receptor with 37 identity. Subcloning and sequencing of the entire 7kb area between exon 1C and exon 1B of the Y1 gene confirmed the presence of a gene encoding a novel NPY receptor subtype named Y5 (SEQ ID NO: Screening of human fetal brain and rat hypothalamic cDNA libraries with a 632 bp human genomic Y5 fragment under high stringency identified full length cDNA clones for both species. These sequences encode a 445 amino acid long Y5 receptor (SEQ ID NOs 4 and 6 respectively). The human genomic sequence (SEQ ID NO: 1) shows two candidate initiator ATG codons, however analysis of several cDNA clones and RT-PCR using specific primers for intron and exon sequences has established that one of these ATG codons (located 30 nucleotides upstream of the other ATG) is located within an intron. The overall identity between the human and rat receptors after this correction is 89%. Figure 5 shows that the degree of identity between the predicted amino acid sequence of the human and rat NPY-Y5 receptors.

The exon which encodes the 5' untranslated region of the human Y5 gene is separated by a 2.7kb intron from exon 2 and is located about 2.8kb upstream of exon 25 1B of the NPY-Y1 gene. The close proximity of these two 5' exons orientated in opposite directions suggests a possible co-regulation of transcription of both genes through a common promoter region.

An interesting feature of the human Y5 gene, however, is the harbouring of exon 1C of the NPY-Y1 gene within the coding region of the NPY-Y5 gene. The 100 bp long exon 1C encodes, in its opposite strand, a part of the Y5 sequence containing most of the third intracellular loop of the receptor protein. This cytoplasmic loop can vary significantly in size between G-protein coupled receptors and is thought to be involved in determination of the specificity of coupling to different G-protein complexes. In contrast to all other known NPY receptor subtypes, this region in the Y5 receptor is unusually large, consisting of about 150 amino acids. In the corresponding region of the corresponding region of the *0* 12 NPY-Y1 gene, shortly after the fifth transmembrane domain, a small 97 bp intron containing an in frame stop codon interrupts the coding region (SEQ ID NO: 1) suggesting that this noncoding region has gained two additional functions after duplication. One is to encode part of the Y5 protein sequence and the other is to fulfil a regulatory function in tissue specific transcription, as an alternatively spliced exon of the Y1 gene. Transcription activation of exon 1C certainly will have an effect on Y5 expression, most likely inhibiting mRNA production. However, such a mechanism may represent only one aspect of a regulatory interaction between these two receptor genes. The close proximity of exon 1B of the Y1 gene and exon 1 of the Y5 gene suggests an additional control mechanism(s) for the specific transcriptional activation of one or the other gene.

Pharmacological characterisation of the Y5 receptor NPY binding analysis of CHO cell lines stably expressing the rat Y5 receptor subtype show a ligand specificity and rank order of potency (NPY NPY PYY[Leu 3 ,Pro 34 NPY[2-36] PP PYY[13-36] indicative of a NPY receptor with a Y1-like pharmacology, as well as responding strongly to the feeding specific ligand NPY[2-36] (Figure 6a-f). The same profile of selectivity for these different NPY analogues can be seen in the results obtained from experiments measuring the inhibition of adenylate cyclase activity (Figure 7).

In situ hybridisation analysis A comprehensive study was made of the distribution of the Y5 receptor mRNA in hypothalamic regions of the human hypothalamus. Hybridisation with a 25 sense probe to Y5 showed no specific labelling, however, antisense probe showed :extremely high expression of Y5 receptor mRNA is found in large neurons of the paraventricular nucleus. High levels are also found in the dorsomedial nucleus, supraoptic nucleus and in the mamillary body as well as in the midline thalamic nuclei. Within the nuclei the distribution was not always homogenous. For example in the dorsomedial region, clearly unlabelled large pyramidal neurons were found mingled with 0 0 0 oo* labelled neurons, suggesting funtional specialisation. Preliminary results for the Y1 receptor suggest that the human NPY-Y1 receptor has a similar distribution to that of the Y5 receptor, however, with some identifiable differences supporting the theory of a co-regulatory transcription activation of the two genes.

Expression of The expressed Y5 receptor protein appears to have a unique distribution and relative affinities for different NPY/PYY/PP analogues. It is also expected that the Y5 receptor will be functionally unique, relative to other NPY receptors, and may be very important in, for example, the development of drugs for a number of conditions such as appetite/obesity disorders, hypertension, locomotor problems, memory loss, sleeping disorders, migraine and gastrointestinal (GI) and cardiovascular disorders.

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 20 be considered in all respects as illustrative and not restrictive.

go o References:- 1. Ball, Shine, J. Herzog, H. (1995). Multiple promoters regulate tissue-specific expression of the human NPY-Y1 receptor gene. J. Biol. Chem.

270, 27272-27276.

2. Sambrook, Fritsch, E.F. Maniatis, T. (1992). Molecule cloning (A Laboratory Manual) 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

I

SEQUENCE LISTING <110> Garvan Institute of Medical Research <120> Neuropeptide Y-Y5 receptor <130> 93311/SHK <160> 8 <170> Patentln version 3.1 <210> <211> <212> <213> 1 8371

DNA

Homo sapiens <220> <221> ODS <222> (6291) .(7625) <223> <400> 1 ctgcagcggc cgccgggtag caggtccctg agcggggtgg aggcacttgt gagcaggtcc ggagagtgtc gctgcccccg caccccgcgg gcagcccgcg cccgggaggc aaatgcgagc ccaagttcac gccaaaacga ttggcagtcc ttagaatttg tttatctttc ttgagaacat ttgtatcatt cctttgtgaa cttatttaaa ttattcttga gtctaggtct ttatttttta agagattaaa aaaagagaat gatttcttgt ttatatagca tgcataaaaa ctgctcattg gcttttttgt cttattgtac cggggcgccc gcctgcaccg ggagcccgca ggaaggtgaa caggagtgaa ctccggttcc ggggatccgc gctggacacg ggtgcccggg gggcgccact agctgcgctc aataacaaac agacctttag ttttacctta gaatgcaatt gaagggaggg caattcaaat ggaatcatgc gcaggaggaa aagaatgatg tgtacaagtc gaactatcta cagatataga taagttgcca gttctgtgtt gagtaaatat caaataatat atttcagaag tgacagcact tgttttcagg ttgttttttc aaaattttcc cgaggtacgg aggggccgtg cccgtctctg gggagcgcgg gagacagcgg caactcaccc gaaggagcct ctctggcgct tcagggctca caggccgtcc taaggtttgc atccatagaa ttaatctttt caccatggaa tttagatttc tggtgcatgc tgtgccagat tgctgttcca aaagaattac tgtattttca agacttttaa cttggttaaa agagtttata tagtttaata tcagcctgga agtaattagg tccatgttaa attcacatat cgtaataaca aaaaaggaag atttaagtgc taatgtttaa gctcccgccc gcgggtcccc gtgccagggc ctggaaaaat agagggtact gggtggagca cctggggatg agcccggctg acctagcggg agctgccgcg gctcctgttt ctcgaattcc aaaggaactg aatagcctaa agatttctca ctgtgtgttt tctggcttta cgatcacagc atatatttta taaagcaaaa tatcctttga atacttaaat agaaaattcc aagaactttt aattctgatg tatgacaaaa aatatttttt atcattactt cttgttgaaa ggaaagggta gttcctgtac actaggccct ctccctgcca gcgcgggctg gttgtcgggg ggggattagg gggctgaatt ggcgcgggcc gggcggggga gcatccggag tctctggcga cggtccagcg gcgaggtgtc agaaacggga aggcgttgtg ggctcttttc agggaagaga gtcagctgag agaaaaaacc aaaacagaca ttcttttgtg aattattcaa attccctgca ctattcagaa agtaaacctt atttttcaca gtgggagata gtctcatgct ctctggctat ttataataga tttggatttc agtttaatgg cttgagtttt ggctaccagg acccctttcg cgagtctgcg gtcccaagag gtggcggaac ctttcgtgcc gaacccggga tggacaaagc ctgggaacag ggCCgggggC ctgacccgag ttcatataac attctttttt ttggaccaaa agcagaattt aactctgctg cagagctgta atgggaatat atagttgata tgattgtcat acaaagaaac gttcctccta ggtttcattt taaaaagata ctttttactc caactaatac gtcaatatca atttcataat taaaatatgt cattgtaggt aaaaaatcct caagttaaat aggcactttt 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 aaaaaaacta gcaatattca gaatgttcat gatggtgtgg taaaatgaat accaatctat gctatagaga tcatcatcca cttgaattaa ttagagcaga aaagaaggaa aattatatat atcctcagtt tactaaggag aataaaagtt tacatttttt agcctttatc ggtatctcct ctataaatgt tcatactcta caagggaaat tatgtgtaat tccattcgaa actcaaaact aattttggac gcacaaagcc aaggaaaggc gaaagtaagt attataaata aggaaattag agagtaggtg atgcgagtca tggctagggt aaaggatgta gtgaaaaact tgctacacat cacgtccacc ttatttagtt tctgtgcaga ggtggggcta aaatgagtgt cagtagctac aataatatat atttgggcct tctatataga gcagagcacc caactaatca aaattatctc ttttctccag atgtggtttt ttaataacag aaatgttgta aatgaagtta cctcattgca atatataaag tcctgaattt taaactgctt atgctttatt tataatagag acattgtttc agcaatttta gttgtagaag tatcggtaac caagagaaga taaataggat gcaaaagcac ctgagaggcc cttttgtacc ctaagaatga gttagcttcc tgtaactttt agaactgaag accacccctc gtaagaagaa ctatatatta gagggagagc tgaatgaatc ataagtattc tactatcagt ttttatatct atattcttgg tagcagaata ttcttagctg tttcttaaaa tgtccaatga acaatttaat actgcacagt aatatatctt tccaaattta ttttggggcc atacatatac tcccacattg ttctaaaacg aaacatcatt aggtctaacc tattaccaca cactaagtaa gggccatcta aactgacctg acaactaagc catgaataag cagtcaaagc attgactggg aggtgttctt atgaatacaa agaaaataat ctcatagcac ttaaacttat ccccacccgc attcttcctt gatattacat aggagaaagt aagtgctaag attatattca caggagaatg cagcaatcta catagcacct tatgttcaat ttcattaata tactcaaatg atactcaaga gcattcaata cgcggaaatg acagttttag aagtgctaaa atttgaaaca acacatatat ccagaatgat attggtaaga tctagaatga acatggaatg aattgtgctg gtatcatcca atttctctct ccacaaagtt aggattgcag ctgaattgag tgttttgatt aatatgatga tgtcattgag tcctaagtct ttggtcaaca aatgatgact ttgtaatgaa cctccctgcc cattggagca gtgtgtatgt tgactacagt agagaatttt gcagtaatgc ccattcattt cagttactca tgcattagtc aaatactttt gaaggtgcct ttttaaggaa tggcatttat ttttatgtgc gatatacttc ttgcatgttg ctatctttat tctataattt atgtacacac tcatttctgt aatatttact aaataattaa gagaaaaatc catcttctct cagttacatg tgttcttgct agaagaaagg ttacaagcag ccaggggatc agaagcttgc ataatatgat gcaatatcaa ttgaattaac tagaatcact ctgtcatcct tgtatgtaca tccagcattt aagattcaca ttatgtggta cacaccaaaa taaattgctt atgtgtccat attaattcat gggtgtagag gtctttatgc tgaatgaata acccctttaa tgaaagaagc ttcatcttct atatatttaa ttttttcatt cttgtgtgat tgtctgtcta caatggttct aaaaatatag tattttaaag tagcatccac gagttttatc tgaattttag ttcttcaaaa ttccaaaaag taggtgttac attgattcaa cctgtacaca atcagaactc tgacctatcc tcagtaggtc tgtaaattgt ttatccttta tgtagaagtt gtttgaaact caatagtatt 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 tgccatttgg tgtttactat tagatctaga atatatatca gttttctgta cctctcatgc aggagtttga attagccaga aggctgagat cactactgca gtaaaaaaaa attggtatgt tagaggtaaa cttattaccg ccttgaacaa taatcaacag tcagtggttg agaaagatgt atttaatgag gcataaattt ttttattctc ttgggatgga acaaggtaga acaactgtaa ccaggacaag gataagatta agtgattgca gttatttact ttagaagtaa cttggaacga atcccagcac gtgaaacccg tagtcccagc tgcagtgagc tcaaaaaaaa aaatttattg taatttaaat aataagtaga caatctctac ggaaattctt ctataatccc gaccagcctg tgtagtggca gagaggattg ccccagcctg aaaaaagtcc tataggcaat tacgtaatgc ggagttatat tgaattcatt gtgaaatttg tcctaatcag catcatccag tgcctactat gaacattgta tcataaacat ttccaaagtg tgggtaggca acagaatgta tgacttttaa tgtgactata aagcatgtat acagaaattt tgataaaatt gttaaaggta tttgggaggc gtctctacta tactcgggag cgagatggcg aaaaaaaaaa aacgaagacc aaacatgtat taaacatata gacaatgtat gttaaaaaaa aacagtttgg ggcaacatag cgtgcctgta cttgagtcca ggcaacagaa tttttttttt ttagtgcttc cattgatgta taatatcact gactcgttca attgtttgtt agataatctg cattgcgtcc atgcatctgg cgaactaggt ttatttaata agtaagagat ttataatgca acaggagttc gtttggccta ctacatacca cttgaagttc taaaaatgca aaatttacta tcataaactt cgaggcgggc aaaatacaaa gctgaggcag ccacagcact aagatatcat tgcaggtccc ttttatagtt ttgataggta ttattggaat cattaaagtg gaggccaagg ccagacccca gtccacgtgc ggcgttcaag tgagaccctt aaacgagagg atcaggcagt tgacattaat gctacattta taagccaaaa tttttaaaac gcacatctca acacagtcaa gtcatgagat gatatttgtt cctgcagtga agtttctgct atgaaagcag tgaagaggag gattgagata ggtatattga ttgtctacat atctactacc taatcattat tctgggctgg ggatcacgag aaattagctg gagaatggcg ccagcctggg aaacttcctt tcataaatta ttagtataat acaaaagtgg taatttcttt gctgggcaca tgggaggttt tctccacaaa ctgtagtcca gttacaatga tttctaagaa agggagtcct aggatcaaaa ttaatttgaa cgtttaaggt tctatacaca gccaacagcc aaccattgag cagtagagtt agtgatccta actagagttt tgaagttact tttccattgc attatgatat atcatgtcca gaaataaatg cttggagaat ttgccttttt ttaacataaa tggctgatac gcacggtgct gtcaggagat ggcgcagtgg tgaacccggg cgacagagcg aggagattaa aagataacag tattcaatta ttttttaact gttggtttgt gtggttcatg acttgaggcc aaatagaaag gctgcttggg gctgtggtca aaataaaaag tttgcctctt gtctaatatg atgaagaaaa ataatgtttt gtttttaaat tgctagtctg gattggtcac tgataaatat ttctcaagga tgtttgaacg ctgccatgta ttgtaaataa gtagcatcag gccgagttga gaatttttat aatattaatg ctttccttac ttaatacatc ttgaattgcc cacgcctgta cgagaccacg cgggcgcctg aggcggagct agactccgtc taaggtcacg 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 ggagctgatt gtaatattta gtttccctct gaatagatta atttaaagta gtcatgtaat gtttttttgg ttgcttacaa atgtcttttt attccaagca ggactataat atg gat Met Asp 6296 tta gag ctc Leu Giu Leu act gct gcc Thr Ala Ala gac gag tat tat Asp Giu Tyr Tyr aac aag aca ctt gcc aca gag aat aat Asn Lys Thr Leu Ala Thr Giu Asn Asn 10 gat ttc cca gtc tgg gat gac tat aaa Asp Phe Pro Val Trp Asp Asp Tyr Lys 6344 act cgg aat Thr Arg Asn 6392 agc Ser 35 agt gta gat gac tta Ser Val Asp Asp Leu 40 cag tat ttt ctg att Gin Tyr Phe Leu Ile 45 atg ggg aat cta ctt Met Gly Asn Leu Leu 60 ggg ctc tat aca ttt Gly Leu Tyr Thr Phe att tta atg gct ctc Ile Leu Met Ala Leu 6440 6488 gta agt ctt ctt ggc Val Ser Leu Leu Gly atg aaa aag cgt aat Met Lys Lys Arg Asn cag aag act Gin Lys Thr gta aac ttc ctc ata ggc aat Val Asn Phe Leu Ile Giy Asn 6536 ctg gcc ttt Leu Aia Phe ctg acg tct Leu Thr Ser 100 tct gat atc ttg gtt gtg ctg ttt tgc tca cct ttc aca Ser Asp Ile Leu Val Vai Leu Phe Cys Ser Pro Phe Thr 90 gtc ttg ctg gat cag tgg atg ttt ggc aaa gtc atg tgc Val Leu Leu Asp Gin Trp Met Phe Gly Lys Val Met Cys 105 110 cct ttt ctt caa tgt gtg tca gtt ttg gtt tca act tta Pro Phe Leu Gin Cys Val Ser Val Leu Val Ser Thr Leu 6584 6632 cat His 115 att atg Ile Met 6680 att tta Ile Leu ata tca att Ile Ser Ile 135 gcc att gtc agg tat Ala Ile Val Arg Tyr 140 cat atg ata aaa His Met Ile Lys cat ccc His Pro 145 6728 ata tct aat aat tta aca gca aac Ile Ser Asn Asn Leu Thr Ala Asn 150 ggc tac ttt ctg ata gct act Gly Tyr Phe Leu Ile Ala Thr 160 6776 gtc tgg Val Trp agt ctt Ser Leu 180 aca cta ggt ttt gcc Thr Leu Giy Phe Ala 165 gtg gaa ctt caa gaa Val Giu Leu Gin Glu 185 atc tgt tct ccc ctt cca gtg ttt cac Ile Cys Ser Pro Leu Pro Val Phe His 170 175 aca ttt ggt tca gca ttg ctg agc agc Thr Phe Gly Ser Ala Leu Leu Ser Ser 190 6824 6872 agg tat tta Arg Tyr Leu 195 tgt gtt gag Cys Val Glu 200 tca tgg cca tct Ser Trp Pro Ser ttt act atc tct Phe-Thr Ile Ser ctt act gta agt Leu Thr Val Ser 230 tta ttg cta gtt cag tat Leu Leu Leu Val Gin Tyr 215 220 cat aca agt gtc tgc aga His Thr Ser Val Cys Arg 235 gat tca tac aga att gcc Asp Ser Tyr Arg Ile Ala 205 210 att ctg ccc tta gtt tgt Ile Leu Pro Leu Val Cys 225 agt ata agc tgt gga ttg Ser Ile Ser Cys Gly Leu 240 6920 6968 7016 tcc aac aaa gaa aac aga ctt Ser Asn Lys Giu Asn Arg Leu 245 gaa gaa aat gag atg atc aac tta act Glu Glu Asn Giu Met Ile Asn Leu Thr 250 255 ggg cct cag gtg aaa ctc tct ggc agc Gly Pro Gin Val Lys Leu Ser Gly Ser 270 7064 ctt cat Leu His 260 cca tcc aaa aag agt Pro Ser Lys Lys Ser 265 7112 aaa tgg agt tat tca Lys Trp Ser Tyr Ser 280 ttc atc aaa aaa cac Phe Ile Lys Lys His 285 aga aga aga tat ago Arg Arg Arg Tyr Ser .290 aag aag aca gca tgt Lys Lys Thr Ala Cys 295 tta oct got Leu Pro Ala oca gaa aga cot tot caa gag Pro Giu Arg Pro Ser Gin Glu 300 305 ttt ggo tot gta aga agt cag Phe Gly Ser Vai Arg Ser Gin 320 7160 7208 7256 7304 aac cac too Asn His Ser ata ott cca gaa Ile Leu Pro Glu otc tot toa Leu Ser Ser 325 too agt aag tto ata Ser Ser Lys Phe Ile 330 cca ggg gtc ccc act tgo ttt gag Pro Giy Val Pro Thr Cys Phe Glu 335 ata aaa Ile Lys 340 tct gtt Ser Val 355 cct gaa gaa aat toa Pro Giu Giu Asn Ser 345 gat gtt oat gaa ttg aga gta aaa ogt Asp Val His Giu Leu Arg Vai Lys Arg 350 7352 aca aga ata Thr Arg Ile aag aga tot oga agt Lys Arg Ser Arg Ser 365 gtt tto tao aga otg Val Phe Tyr Arg Leu 370 aco ata otg ata tta Thr Ile Leu Ile Leu 375 ttc cat gtg gta act Phe His Val Vai Thr 390 gta ttt got gtt agt Vai Phe Aia Vai Ser 380 tgg atg oca ota Trp Met Pro Leu cac ott His Leu 385 7400 7448 7496 7544 tto aag ttg Phe Lys Leu 405 gtg tat Vai Tyr gat ttt aat gao Asp Phe Asn Asp 395 tgo att tgt oat Cys Ile Cys His 410 aat ott att tca aat agg oat Asn Leu Ile Ser Asn Arg His 400 ttg ttg ggo atg atg too tgt Leu Leu Giy Met Met Ser Cys 415 tgt ctt aat cca att cta tat ggg ttt ctt aat Cys Leu Asn Pro Ile Leu Tyr Gly Phe Leu Asn 420 425 gat tta gtg tcc ctt ata cac tgt ctt cat atg Asp Leu Val Ser Leu Ile His Cys Leu His Met aat ggg att aaa gct Asn Gly Ile Lys Ala 430 7592 taataattct cactgtttac 7645 caaggaaaga ataaatagaa gtcagttcat tgtgcagtca ttccatgttg aaccaactca taatttcaaa ttaatgttta tgacagatgt cctgtaggaa tcctagtaat atacaacatt tgtgaa acaaatgctg attttgttaa aatatatgga gtgtcaatcc tcttataaac gtacttttgt cttgaagaat gtttgtgaag atgcccaaat aatgctattg acacaagtaa aaacactacc gggtcatata catggaattt agataatttt aatctgtaat aaatgaattg ccaaaaatat atctaccagc tacagaaaaa tatgattata cctattgaga tttaccaaat aataaaagtt aaatatattt aatttatgtg atgtgttata ttcactttag tattttttgt aataagaaaa tatctatatc atttaatatg atcttcaaca attggtcaaa aaagaatttt atgataacta aaagagttct gtaggattaa aaggttgtat tgaaagtaaa aatttttctc atttctactc cctggaaaat ttaactacag ttgtcaattt aaatcctttc tttacatata ggattcaaat tttatttagt taccttccac agttatatct gaggaacttt cataggcttc cacaactaaa ttttggaagt aactccactg cagactcatt tattctaaaa 7705 7765 7825 7885 7945 8005 8065 8125 8185 8245 8305 8365 8371 gttttcatat acatcaaaac <210> 2 <211> 445 <212> PRT <213> Homo sapiens <400> 2 Met Asp Leu Glu Leu Asp Glu Tyr Tyr Asn Lys Thr Leu Ala Thr Glu 1 5 10 Asn Asn Thr Tyr Lys Ser Thr Phe Val Ala Thr Arg Asn Ser Asp Phe Pro Val Val Asp Asp Leu Gin Tyr Phe Leu Ile Trp Asp Asp Gly Leu Tyr Ser Leu Leu Phe Met Gly Asn Leu Ile Leu Met Ala Leu Met Lys Lys Gin Lys Thr Asn Phe Leu Asn Leu Ala Asp Ile Leu Leu Phe Cys Ser Pro Phe Thr Leu Met Cys His 115 Thr Leu Ile Vai Leu Leu Trp Met Phe Met Pro Phe Cys Vai Ser Giy Lys Vai 110 Leu Vai Ser Met Ile Lys Leu Ile Ser Ile Vai Arg 130 His Pro Ile Ser Asn Thr Aia Asn Tyr Phe Leu Thr Vai Trp Giy Phe Aia Ser Pro Leu Pro Vai 175 Leu Leu Phe His Ser Giu Leu Gin Phe Giy Ser Ser Ser Arg Tyr Leu Cys Vai Giu Ser Trp Pro Ser Asp Ser Tyr Arg Ile Ala 210 Phe Thr Ile Ser Leu Leu Leu Val Gin 215 Ile Leu Pro Leu Vai 225 Cys Leu Thr Val His Thr Ser Val Arg Ser Ile Ser Giy Leu Ser Asn Giu Asn Arg Leu Giu Asn Giu Met Ile Asn 255 Leu Thr Leu Giy Ser His 275 Pro Ser Lys Lys Giy Pro Gin Val Lys Leu Ser 270 Arg Arg Arg Lys Trp Ser Tyr Phe Ile Lys Lys Tyr Ser 290 Lys Lys Thr Ala Val Leu Pro Ala Giu Arg Pro Ser Gin 305 Giu Asn His Ser Ile Leu Pro Giu Asn Phe Gly Ser Vai 315 Ser Gin Leu Ser Ser Ser Lys Phe Pro Giy Vai Pro Thr Cys 335 Phe Giu Ile Lys Arg Ser 355 Pro Giu Glu Asn Asp Vai His Giu Leu Arg Val 350 Val Phe Tyr Vai Thr Arg Ile Lys Arg Ser Arg Arg Leu 370 Thr Ile Leu Ile Val Phe Ala Val Trp Met Pro Leu His 385 Leu Phe His Val Thr Asp Phe Asn Asn Leu Ile Ser Arg His Phe Lys Leu Val Tyr Cys Ile Cys 405 410 Ser Cys Cys Leu Asn Pro Ile Leu Tyr Gly 420 425 Lys Ala Asp Leu Val Ser Leu Ile His Cys 435 440 His Leu Leu Gly Met Met 415 Phe Leu Asn Asn Gly Ile 430 Leu His Met 445 <210> <211> <212> <213> 3 2143

DNA

Homo sapiens <220> <221> CDS <222> (63)..(1397) <223> <400> 3 agctcgtcga cctgacctgc cacaaagtta gaagaaagga ttgattcaag aaagactata at atg gat tta gag ctc gac gag tat tat aac aag aca ctt gcc aca Met Asp Leu Giu Leu Asp Glu Tyr Tyr Asn Lys Thr Leu Ala Thr 1 5 10 gag aat aat act Glu Asn Asn Thr gcc act ogg aat Ala Thr Arg Asn gat ttc cca gto Asp Phe Pro Val tgg gat Trp Asp gac tat aaa agc agt gta gat gac tta cag tat ttt ctg att ggg gtc Asp Tyr Lys Ser Ser Val Asp Asp Leu Gin Tyr Phe Leu Ile Gly Val 40 tat aca Tyr Thr atg got Met Ala ttt gta agt ott ott Phe Val Ser Leu Leu otc atg aaa aag cgt Leu Met Lys Lys Arg 70 ggc ttt atg ggg aat cta ott att tta Gly Phe Met Gly Asn Leu Leu Ile Leu 55 aat oag aag act aog gta aao ttc otc Asn Gin Lys Thr Thr Val Asn Phe Leu ata Ile 80 ggo aat ctg gc ttt tot gat atc ttg gtt Gly Asn Leu Ala Phe Ser Asp Ile Leu Val 85 90 gtg otg ttt tgo toa Val Leu Phe Cys Ser cct ttc aoa otg acg Pro Phe Thr Leu Thr 100 tct gtc ttg otg gat cag tgg atg ttt ggo aaa Ser Val Leu Leu Asp Gin Trp Met Phe Gly Lys 105 110 gto atg tgo cat att atg oct ttt ott Val Met Cys His Ile Met Pro Phe Leu 115 120 oaa tgt gtg Gin Cys Val toa aot tta Ser Thr Leu 130 att tta ata toa att gc att gto agg Ile Leu Ile Ser Ile Ala Ile Val Arg 135 toa gtt ttg gtt Ser Val Leu Val 125 tat oat atg ata Tyr His Met Ile 140 ggo tao ttt otg Gly Tyr Phe Leu aaa cat Lys His ccc ata tot aat Pro Ile Ser Asn tta aca goa aac Leu Thr Ala Asn gct act gtc tgg aca Ala Thr Val Trp Thr 165 cta ggt ttt gcc atc Leu Gly Phe Ala Ile 170 tgt tct ccc ctt cca Cys Ser Pro Leu Pro 175 gtg ttt cac agt Val Phe His Ser gtg gaa ctt caa Val Glu Leu Gln aca ttt ggt tca Thr Phe Gly Ser gca ttg Ala Leu 190 ctg agc agc agg Leu Ser Ser Arg 195 aga att gcc ttt Arg Ile Ala Phe 210 tta gtt tgt ctt Leu Val Cys Leu 225 tat tta tgt Tyr Leu Cys act atc tct Thr Ile Ser act gta agt Thr Val Ser 230 gtt gag tca tgg cca tct gat tca tac Val Glu Ser Trp Pro Ser Asp Ser Tyr 200 205 tta ttg cta gtt cag tat att ctg ccc Leu Leu Leu Val Gln Tyr Ile Leu Pro 215 220 cat aca agt gtc tgc aga agt ata agc His Thr Ser Val Cys Arg Ser Ile Ser 235 tgt gga ttg tcc aac aaa Cys Gly Leu Ser Asn Lys 240 245 aac tta act ctt cat cca Asn Leu Thr Leu His Pro 260 gaa aac aga ctt gaa Glu Asn Arg Leu Glu 250 gaa aat gag atg atc Glu Asn Glu Met Ile 255 tcc aaa aag Ser Lys Lys ggg cct cag gtg Gly Pro Gln Val aaa ctc Lys Leu 270 tct ggc agc cat Ser Gly Ser His 275 aaa tgg agt tat tca Lys Trp Ser Tyr Ser 280 ttc atc aaa aaa cac aga aga Phe Ile Lys Lys His Arg Arg 285 aga tat Arg Tyr aag aag aca gca Lys Lys Thr Ala gtg tta cct gct Val Leu Pro Ala gaa aga cct Glu Arg Pro tct caa Ser Gin 305 aga agt Arg Ser 320 gag aac cac tcc Glu Asn His Ser aga ata ctt cca gaa aac ttt ggc tct gta Arg Ile Leu Pro Giu Asn Phe Giy Ser Vai 310 315 1019 cag ctc tct Gin Leu Ser tec agt aag ttc ata Ser Ser Lys Phe Ile 330 cca ggg gtc ccc Pro Gly Vai Pro 1067 1115 tgc ttt gag ata aaa Cys Phe Giu Ile Lys 340 cct gaa gaa aat tca Pro Giu Giu Asn Ser 345 gat gtt cat gaa ttg aga Asp Vai His Giu Leu Arg 350 gta aaa cgt tct gtt aca aga ata aaa Val Lys Arg Ser Val Thr Arg Ile Lys 355 360 aag aga tct cga agt gtt ttc Lys Arg Ser Arg Ser Vai Phe 365 1163 tac aga ctg Tyr Arg Leu 370 acc ata ctg ata Thr Ile Leu Ile gta ttt gct gtt agt tgg atg cca Val Phe Ala Vai Ser Trp Met Pro 380 1211 cta cac Leu His 385 ctt ttc cat gtg gta act gat ttt aat gac aat Leu Phe His Val Vai Thr Asp Phe Asn Asp Asn 390 395 ctt att tca Leu Ile Ser 1259 agg cat ttc aag ttg Arg His Phe Lys Leu 405 gtg tat tgc att tgt cat ttg ttg ggc atg Val Tyr Cys Ile Cys His Leu Leu Gly Met 410 415 1307 atg tcc tgt tgt Met Ser Cys Cys ctt aat cca att Leu Asn Pro Ile 420 tta gtg tcc ctt Leu Vai Ser Leu ggg ttt ctt aat aat ggg Gly Phe Leu Asn Asn Gly 430 1355 att Ile aaa gct gat Lys Ala Asp 435 ata cac tgt ctt cat Ile His Cys Leu His 1397 taataattct atgataacta aaagagttct gtaggattaa cactgtttac tttacatata ggattcaaat tttatttagt caaggaaaga ataaatagaa gtcagttcat tgtgcagtca acaaatgctg at-tttgttaa aatatatgga gtgtcaatcc gggtcatata catggaattt agataatttt aatctgtaat aaatatattt aatttatgtg atgtgttata ttcactttag 1457 1517 1577 1637 aaggttgtat taccttccac tgaaagtaaa agttatatct aatttttctc gaggaacttt atttctactc cataggcttc cctggaaaat cacaactaaa ttaactacag ttttggaagt ttgtcaattt aactccactg aaatcctttc cagactcatt acatcaaaac tattctaaaa <210> 4 <211> 445 <212> PRT <213> Homo sapiens ttccatgttg aaccaactca taatttcaaa ttaatgttta tgacagatgt cctgtaggaa tcctagtaat atacaacatt tgtgaa tcttataaac gtacttttgt cttgaagaat gtttgtgaag atgcccaaat aatgctattg acacaagtaa aaacactacc aaatgaattg ccaaaaatat atctaccagc tacagaaaaa tatgattata cctattgaga tttaccaaat aataaaagtt tattttttgt aataagaaaa tatctatatc atttaatatg atcttcaaca attggtcaaa aaagaatttt gttttcatat 1697 1757 1817 1877 1937 1997 2057 2117 2143 16 <400> 4 Met Asp Leu Giu Leu Asp Giu Tyr Tyr Asn Lys Thr Leu Ala Thr Giu Asn Asn Thr Tyr Lys Ser Ala Thr Arg Asn Asp Phe Pro Val Trp Asp Asp Gly Val Tyr Ser Vai Asp Asp Gin Tyr Phe Leu Thr Phe Val Ser Leu Leu Phe Met Gly Asn Leu Ile Leu Met Leu Met Lys Lys Asn Gin Lys Thr Vai Asn Phe Leu Gly Asn Leu Ala Ser Asp Ile Leu Val Leu Phe Cys Ser Pro Phe Thr Leu Met Cys His 115 Ser Val Leu Leu Gin Trp Met Phe Giy Lys Val 110 Leu Val Ser Ile Met Pro Phe Gin Cys Val Ser Thr Leu 130 Ile Leu Ile Ser Aia Ile Val Arg His Met Ile Lys Pro Ile Ser Asn Asn Leu Thr Aia Asn 150 Giy Tyr Phe Leu Ala Thr Val Trp Thr Leu Giy Phe Ala Ile Cys Ser Pro Leu Pro Val 175 165 170 Phe His Ser Val Giu Leu Gin Thr Phe Gly Ser Ala Leu Leu 190 Ser Tyr Arg Ser Ser Arg Tyr Leu Cys Val 195 Ser Trp Pro Ser Ile Ala 210 Phe Thr Ile Ser Leu Leu Leu Val Gin 215 Ile Leu Pro Leu Cys Leu Thr Val His Thr Ser Val Arg Ser Ile Ser Giy Leu Ser Asn Giu Asn Arg Leu Giu Asn Giu Met Ile Asn 255 Leu Thr Leu Gly Ser His 275 Pro Ser Lys Lys Giy Pro Gin Val Lys Leu Ser 270 His Arg Arg Arg 285 Giu Arg Pro Ser Lys Trp Ser Tyr Ser 280 Cys Val 295 Phe Ile Lys Lys Tyr Ser 290 Lys Lys Thr Ala Leu Pro Ala Gin Giu Asn His Ser 305 Ser Gin Leu Ser Ser 325 Ile Leu Pro Giu Phe Gly Ser Val Ser Ser Lys Phe Ile 330 Pro Gly Val Pro Thr Cys 335 Val His Giu Leu Arg Val 350 Phe Giu Ile Lys Pro Giu Giu Asn Ser Asp 340 345 Lys Arg Ser 355 Arg Leu Thr Vai Thr Arg Ile 18 Lys Arg Ser Phe Ala Val Arg Ser 365 Ser Trp Vai Phe Tyr Met Pro Leu Ile Leu Ile 370 His Leu Phe His Val Asp Phe Asn Leu Ile Ser 385 Arg His Phe Lys Tyr Cys Ile Cys 410 Gly Leu Leu Gly Met Met 415 Giy Ile Ser Cys Cys Pro Ile Leu Phe Leu Asn Lys Ala Asp 435 Val Ser Leu Cys Leu His <210> <211> <212> <213> 2286

DNA

Rattus rattus <220> <221> CDS <222> (115)..(1449) <223> <400> gaattoggca ogaggggttt goaaggtggc ttggaagtoa actgcoagta ggaaatagoo atcoacaoao otgagttooa agggggaaga aagagattot tatctgatto tagt atg Met 1 gag ttt aag ctt gag gag oat ttt aac aag aca ttt gto aca gag aac Glu Phe Lys Leu Glu Glu His Phe Asn Lys.Thr Phe Val Thr Glu Asn aat aca got Asn Thr Ala got got cgg aat gca goc ttc Oct gcc Ala Ala Arg Asn Ala Ala Phe Pro Ala gag gac tao Glu Asp Tyr aga ggc Arg Gly ago gta gao gat tta oaa tao ttt otg att ggg oto tat aca Ser Val Asp Asp Leu Gln Tyr Phe Leu Ile Gly Leu Tyr Thr gta agt ott ott ggo ttt Val Ser Leu Leu Gly Phe atg ggo aat ota Met Gly Asn Leu 60 ott att tta atg got Leu Ile Leu Met Ala 309 gtt atg aaa aag ogo Val Met Lys Lys Arg aat cag aag act aoa Asn Gln Lys Thr Thr 75 gao ato ttg gto gto Asp Ile Leu Val Val gtg aao ttt oto ata ggo Val Asn Phe Leu Ile Gly otg ttt tgo too oot tto Leu Phe Cys Ser Pro Phe aao otg gc Asn Leu Ala aoo otg Thr Leu tot gto ttg ttg gat Ser Val Leu Leu Asp 105 cag tgg atg ttt Gln Trp Met Phe aaa ago atg Lys Ser Met tgc cat Cys His 115 atc atg ccg ttc ctt Ile Met Pro Phe Leu 120 caa tgt gtg tca gtt Gin Cys Vai Ser Vai 125 ctg gtt Leu Val tca act Ser Thr att tta ata tca att Ile Leu Ile Ser Ile 135 gcc att gtc agg Ala Ile Val Arg cat atg ata His Met Ile aag cac Lys His 145 cct att tct aac Pro Ile Ser Asn act gtc tgg aca Thr Vai Trp Thr 165 aat tta acg gca aac cat Asn Leu Thr Ala Asn His 150 155 ctg ggc ttt gcc atc tgt Leu Gly Phe Ala Ile Cys 170 ggc tac ttc ctg ata gct Gly Tyr Phe Leu Ile Ala 160 tct ccc ctc cca gtg ttt Ser Pro Leu Pro Val Phe 175 cac agt His Ser gtg gaa ctt aag Val Giu Leu Lys acc ttt ggc tca Thr Phe Gly Ser ctg ctg agt Leu Leu Ser agc caa Ser Gin 195 tat ctc tgt gtt gag Tyr Leu Cys Val Glu 200 tca tgg ccc tct gat tca tac aga att Ser Trp Pro Ser Asp Ser Tyr Arg Ile 205 ttc aca atc tct tta ttg cta gtg cag tat Phe Thr Ile Ser Leu Leu Leu Val Gin Tyr 215 .220 atc ctg cct cta gta Ile Leu Pro Leu Vai 225 tgt tta acg gta Cys Leu Thr Val cat acc agc gtc His Thr Ser Val cga agc ata agc Arg Ser Ile Ser tgt gga Cys Gly 240 ttg tcc cac Leu Ser His gaa aac aga ctc Glu Asn Arg Leu gaa gaa aat gag atg Glu Giu Asn Giu Met atc aac tta Ile Asn Leu 255 acc cta Thr Leu cca tcc aaa aag Pro Ser Lys Lys agg aac cag gca Arg Asn Gin Ala acc ccc agc Thr Pro Ser act caa Thr Gin 275 aag tgg agc tac tca-ttc Lys Trp Ser Tyr Ser Phe 280 *atc aga aag cac aga agg agg tac 981 Ile Arg Lys His Arg Arg Arg Tyr 285 agc Ser 290 aag aag acg gcc tgt Lys Lys Thr Ala Cys 295 gtc tta ccc gcc cca Val Leu Pro Ala Pro 300 gca gga cct tcc cag Ala Gly Pro Ser Gin 305 ggg aag cac cta Gly Lys His Leu ctg tcg cca tcc Leu Ser Pro Ser 325 gtt cca gaa aat Val Pro Giu Asn gcc tcc gtc cgt Ala Ser Val Arg agc cag Ser Gin 320 1029 1077 1125 agt aag gtc att cca Ser Lys Val Ile Pro 330 ggg gtc cca atc tgc ttt gag Gly Val Pro Ile Cys Phe Giu 335 gtg aaa cct Val Lys Pro 340 gaa gaa agc tca gat Giu Giu Ser Ser Asp 345 gct cat gag atg Ala His Giu Met aga gtc aag cgt Arg Val Lys Arg 350 1173 tcc atc Ser Ile 355 act aga ata aaa aag Thr Arg Ile Lys Lys 360 aga tct cga agt Arg Ser Arg Ser gcc gtt agc tgg Ala Val Ser Trp 380 gtt ttc tac aga ctg Val Phe Tyr Arg Leu 365 atg cca ctc cac gtc Met Pro Leu His Val 385 acc Thr 370 ata ctg ata ctc gtg Ile Leu Ile Leu Val 375 1221 1269 1317 ttc cac gtg gtg Phe His Val Val gac ttc aat gat Asp Phe Asn Asp ttg att tcc aat agg Leu Ile Ser Asn Arg ttc aag ctg gta tac tgc atc tgt Phe Lys Leu Val Tyr Cys Ile Cys 405 tgt cta aat ccg atc cta tat ggt Cys Leu Asn Pro Ile Leu Tyr Gly 420 425 cac ttg tta ggc atg atg tcc tgt His Leu Leu Gly Met Met Ser Cys 410 415 ttc ctt aat aat ggt atc aaa gca Phe Leu Asn Asn Gly Ile Lys Ala 430 1365 1413 gac ttg aga gcc ctt atc cac tgc eta cac atg tca Asp Leu Arg Ala Leu Ile His Cys Leu His Met Ser tgattctctc 1459 tgtgcaccaa gatetgaatg caatcagttg attattttcc aatgtatgaa ttatctgggt aaaatattea gtaggagaaa gtcctactac atatcacatt atgtgaagtc aaaagtetgt ctgatttatg ctctatcgtg agagagaaga ccagttcgta tgcagagtca attttatgtc gtgacaagtt atcttcattt aaagtcagaa gatctgctcc taatatttaa aaacactgtc taaatggtgt agatggtatg ttgtacctat ttgtetcaet aacgtggtaa atctacgtaa atgtccatct attggtaata gtcccaaaga etatttcaca ctctattaca tattagtgaa tttttcaaat aaataaaggc ctgtatttec gatagctagt gtcgtatgta aaagtgaaag ttgacacata gateatetto aatacaattt agttgagtga gcatttaact ggcttcttaa gatgtatgca gattggtaaa atgaaaaggt tgtttttata aattattaaa tgtttgttaa tattaggage atttatacag atgttataat catgtgttga tactctgtgg acagatttaa catttttttg taaaagatga attgtcagtt ttcagatttt tgcatcgttg taaettctaa tataaagtaa agtttcagec aagtattctg atggttaatt agtagtttac tttagtgtaa ggaatttcta taaaagtaca ttataatttt taacccgtct gtttagattt atgtteeaaa gatcattttt aagtagatag ccacagaaca 1519 1579 1639 1699 1759 1819 1879 1939 1999 2059 2119 2179 2239 2286 caaacgaaaa aaaaaaa <210> 6 <211> 445 <212> PRT <213> Rattus rattus <400> 6 Met Glu Phe Lys Leu Glu Glu His Phe Asn Lys Thr Phe Val Thr Glu Asn Asn Thr Ala Ala Arg Asn Ala Phe Pro Ala Trp Glu Asp Tyr Arg Gly Ser Val Asp Asp Thr Phe Val Ser Leu Leu Gly 50 Gln Tyr Phe Leu Ile Gly Leu Tyr Phe Met Gly Asn Leu Ile Leu Met Val Met Lys Lys Arg Asn Gln Lys Thr Val Asn Phe Leu Gly Asn Leu Ala Ser Asp Ile Leu Val Val Leu Phe Cys Ser Pro Phe Thr Leu Thr Ser Val Leu Leu 100 Gln Trp Met Phe Gly Lys Ser 110 Met Cys His 115 Ile Met Pro Phe Gln Cys Val Ser Val Leu Val Ser 125 Thr Leu 130 Ile Leu Ile Ser Ile Ala Ile Val Arg Tyr His Met Ile Lys His Pro Ile Ser Asn Asn Leu Thr Ala Asn His Gly Tyr Phe Leu Ala Thr Val Trp Leu Gly Phe Ala Cys Ser Pro Leu Pro Val 175 Phe His Ser Leu Val Giu Leu Lys Giu Thr Phe Gly Ser Ala Leu Leu 190 Ser Tyr Arg Ser Ser Gin 195 Tyr Leu Cys Val Ser Trp Pro Ser Ile Ala 210 Phe Thr Ile Ser Leu Leu Vai Gin Ile Leu Pro Leu Cys Leu Thr Val His Thr Ser Val Arg Ser Ile Ser Giy Leu Ser His Giu Asn Arg Leu Giu Asn Glu Met Ile Asn 255 Leu Thr Leu Ser Thr Gin 275 Pro Ser Lys Lys Arg Asn Gin Ala Lys Thr Pro 270 Arg Arg Arg Lys Trp Ser Tyr Phe Ile Arg Lys Tyr Ser 290 Lys Lys Thr Aia Val Leu Pro Ala Aia Giy Pro Ser Giy Lys His Leu Val Pro Giu Asn Aia Ser Val Arg Gin Leu Ser Pro Ser Ser Lys Vai Ile 325 Giy Vai Pro Ile Cys Phe 335 Giu Vai Lys Giu Giu Ser Ser Ala His Giu Met Arg Val Lys 350 Arg Ser Ile 355 Thr Arg Ile Lys Arg Ser Arg Ser Val Phe Tyr Arg Leu Thr 370 Ile Leu Ile LeuMe PrLuHiPeAl Va Sr Phe Ala Val Ser Met Pro Leu His 385 Phe His Val Val Asp Phe Asn Asp Leu Ile Ser Asn Arg 400 Met Ser 415 His Phe Lys Leu Tyr Cys Ile Cys Leu Leu Gly Met Cys Cys Leu Pro Ile Leu Tyr Phe Leu Asn Asn Gly Ile Lys 430 Ala Asp Leu 435 Arg Ala Leu Ile Cys Leu His Met <210> 7 <211> 1585 <212> DNA <213> Mus musculus <220> <221> CDS <222> (73)..(1470) <223> <400> 7 gttattgtca tagcgtgcta ttgttcttca agctgctaat ggtcactgtc ttcttccaag caggactcta gt atg gag gtt aaa ctt gaa gag cat ttt aac aag aca ttt Met Glu Val Lys Leu Glu Glu His Phe Asn Lys Thr Phe gtc acg Val Thr 15 gag aac aat act gct Glu Asn Asn Thr Ala 20 gco agt cag Ala Ser Gin aac acg gcc tcc cct gcc Asn Thr Ala Ser Pro Ala gag gac tac aga ggc aca gag aac aat act tot gct got cgg Glu Asp Tyr Arg Gly Thr Giu Asn Asn Thr Ser Ala Ala Arg act cog ttt cca Thr Pro Phe Pro oaa tao tto ctg Gin Tyr Phe Leu atg gga aat ota Met Gly Asn Leu aag act aca gtg Lys Thr Thr Val 95 gtc tgg gag gao tat aga Val Trp Giu Asp Tyr Arg 55 att ggg otc tat aca ttt Ile Gly Leu Tyr Thr Phe 70 ggc ago gta gao gao tta Gly Ser Val Asp Asp Leu gta agt ott ott ggt ttt Val Ser Leu Leu Gly Phe ott ato tta Leu Ile Leu aao ttt otc Asn Phe Leu 100 atg got gtt atg aaa aag cgc aat oag Met Ala Val Met Lys Lys Arg Asn Gin 85 ata ggc aac ctg gc tto too gao att Ile Gly Asn Leu Ala Phe Ser Asp Ile 105 gtt gtc ctg ttt tgo Val Val LeuPhe Cys 115 too cot tto aco ctg Ser Pro Phe Thr Leu 120 aco tot gto ttg Thr Ser Val Leu gat cag tgg atg ttc ggo aaa gc atg tgo cat ato atg oca ttc Ott Asp Gin Trp Met Gly Lys Ala Met His Ile Met Pro Phe Leu 140 cag tgt gta tca Gin Cys Vai Ser 145 gtt ctg gtt tca act ctg att tta ata tcg att gcc Vai Leu Vai Ser Thr Leu Ile Leu Ile Ser Ile Ala 150 155 att gtc agg Ile Val Arg 160 tat cat atg ata aag Tyr His Met Ile Lys 165 cac cct ata tct His Pro Ile Ser aat tta aca Asn Leu Thr gca aac cat ggc tac ttc Ala Asn His Gly Tyr Phe 175 gcc atc tgt tct ccc ctc Ala Ile Cys Ser Pro Leu 190 195 ctg ata gca tct gtc Leu Ile Ala Ser Val 180 cca gtg ttt cac agc Pro Val Phe His Ser 200 aca ctg ggc ttt Thr Leu Gly Phe ctt gtg gaa ctt aag Leu Val Giu Leu Lys 205 gaa acc ttt ggc Glu Thr Phe Gly tca tgg ccc tct Ser Trp Pro Ser 225 tta gtt cag tat Leu Val Gin Tyr 240 agt gtc tgc agg Ser Val Cys Arg 255 gca ttg cta agc Ala Leu Leu Ser agc aag tat ttg tgt gtt gag Ser Lys Tyr Leu Cys Val Glu 215 220 gct ttc aca atc tct tta ttg Ala Phe Thr Ile Ser Leu Leu 235 gat tca tac Asp Ser Tyr atc ctg cct Ile Leu Pro agt ata agc Ser Ile Ser 260 aga att Arg Ile 230 cta gta tgt tta aca gta agt cat act Leu Val Cys Leu Thr Val Ser His Thr 245 250 tgt gga ttg tcc cac aaa gaa aac aga Cys Gly Leu Ser His Lys Giu Asn Arg 265 ctc gaa gaa aat gag atg atc aac tta act cta cat cca tcc aaa aag Glu Glu Asn Glu Aie Asn Leu Thr His Pro Ser Lys agt egg gac eag gca Ser Arg Asp Gin Ala 290 tte ato aga aag cac Phe Ile Arg Lys His 305 aaa etc ccc agc act Lys Leu Pro Ser Thr 295 cga aga agg tac age Arg Arg Arg Tyr Ser 310 eaa aag tgg agc Gin Lys Trp Ser tac tca Tyr Ser 300 975 1023 aag aag acg gca tge gtg Lys Lys Thr Ala Cys Vai 315 tta ccc gcc Leu Pro Ala 320 cca gca gga ect tee cag gag.aag cac eta acc gtt eca Pro Aia Gly Pro Ser Gin Giu Lys His Leu Thr Vai Pro 325 330 1071 gaa aac Glu Asn 335 eca ggc tcg gte Pro Giy Ser Vai age cag etg tca Ser Gin Leu Ser cca tee agt aag gtt Pro Ser Ser Lys Vai 345 ect gaa gaa agc tea Pro Giu Giu Ser Ser 365 att Ile 350 cca ggg gte ccg atc Pro Giy Vai Pro Ile 355 tge ttt gag gtg aaa Cys Phe Giu Vai Lys 360 1119 1167 1215 1263 gat get cag gag Asp Ala Gin Glu aga tct cgc agt Arg Ser Arg Ser 385 atg aga gte aag cgt tee Met Arg Val Lys Arg Ser 370 375 gtt ttc tac aga etg act Vai Phe Tyr Arg Leu Thr 390 ctc acg aga ata aag aag Leu Thr Arg Ile Lys Lys 380 ata ttg ata tta gtg ttc Ile Leu Ile Leu Vai Phe 395 get gtt age Ala Val Ser 400 tgg atg cca etc cac Trp Met Pro Leu His 405 gte ttc cac gtg gtg Val Phe His Vai Vai 410 ace gat tte Thr Asp Phe 1311 aat gat aac ctg att tee aat agg eat ttc aag etg gtg tae tge ate 1359 Asn Asp 415 Asn Leu Ile Ser Asn Arg His Phe Lys Leu Val Tyr Cys Ile tgt Cys 430 cac ttg tta ggo atg atg too tgt tgt His Leu Leu Gly Met Met Ser Cys Cys 435 ott aat ccg ato tta tat Leu Asn Pro Ile Leu Tyr 440 445 ttg aga gco ctt atc cac Leu Arg Ala Leu Ile His 460 1407 gga ttc ott aat aat Gly Phe Leu Asn Asn 450 tgc ota cac atg tca Cys Leu His Met Ser 465 ggt atc aaa gca gac Gly Ile Lys Ala Asp 455 1455 tgattototo tgtgoaooga ggagagaaga aatgtggaga 1510 otgoooaoaa taoatotgtg otaattgatg oataatttao ataaaogtgt otggatotga atgooagttt gtaat <210> 8 <211> 466 <212> PRT <213> Mus musoulus <400> 8 Met Giu Val Lys Leu Giu Giu His Phe Asn Lys Thr Phe Val Thr Giu 1 5 10 Asn Asn Thr Ala Ala Ser Gin Asn Thr Ala Ser Pro Ala Trp Giu Asp 25 1570 1585 Tyr Arg Gly Thr Giu Asn Asn Thr Ser Aia.Aia Arg Asn Thr Pro Phe Pro Vai Trp Glu Asp Tyr Gly Ser Val Asp Leu Gin Tyr Phe Ile Gly Leu Tyr Phe Val Ser Leu Gly Phe Met Gly Leu Leu Ile Leu Met Ala Val Met Lys Arg Asn Gin Lys Thr Thr Val Asn Phe Ile Gly Asn Leu Ala Phe Ser Asp Ile 105 Leu Val Val 110 Asp Gin Trp Leu Phe Cys Ser Pro Phe Thr 115 Thr Ser Vai Leu Met Phe 130 Gly Lys Ala Met His Ile Met Pro Phe 140 Leu Gin Cys Val Val Leu Val Ser Thr Leu Ile Leu Ile 150 Ile Ala Ile Val Tyr His Met Ile His Pro Ile Ser Asn Asn Leu Thr Ala 170 Asn His 175 Gly Tyr Phe Ile Ala Ser Val Thr Leu Giy Phe Ala Ile Cys 190 Ser Pro Leu 195 Pro Val Phe His Leu Vai Giu Leu Lys Glu Thr Phe 205 Gly Ser Ala Leu Leu Ser Ser Lys Tyr Leu Cys Val Giu Ser Trp Pro 215 220 Ser Asp Ser Tyr Arg Ile Ala Phe Thr Ile Le LuLuVa Leu Leu Leu Val Tyr Ile Leu Pro Val Cys Leu Thr Ser His Thr Ser Val Cys 255 Arg Ser Ile Asn Giu Met 275 Cys Gly Leu Ser Lys Giu Asn Arg Leu Giu Giu 270 Ser Arg Asp Ile Asn Leu Thr His Pro Ser Lys Gin Ala 290 Lys Leu Pro Ser Gin Lys Trp Ser Ser Phe Ile Arg His Arg Arg Arg Ser Lys Lys Thr Cys Val Leu Pro Pro Ala Giy Pro Gin Giu Lys His Thr Val Pro Giu Asn Pro 335 Gly Ser Val Ser Gin Leu Ser Pro Ser Ser Lys Val 345 Ile Pro Gly 350 Asp Ala Gin Val Pro Ile 355 Cys Phe Giu Val Pro Giu Giu Ser Giu Met 370 Arg Val Lys Arg Ser Leu Thr Arg Ile 375 Lys Arg Ser Arg Val Phe Tyr Arg Thr Ile Leu Ile Val Phe Ala Val Trp Met Pro Leu Val Phe His Val Thr Asp Phe Asn Asp Asn 415 Leu Ile Ser Asn Arg His Phe Lys Leu Val Tyr Cys Ile Cys His Leu 420 425 430 Leu Gly Met Met Ser Cys Cys Leu Asn Pro Ile Leu Tyr Gly Phe Leu 435 440 445 Asn Asn Gly Ile Lys Ala Asp Leu Arg Ala Leu Ile His Cys Leu His 450 455 460 Met Ser 465

Claims (23)

1. An isolated DNA molecule encoding an NPY-Y5 receptor consisting of 445 amino acids in length.

2. The isolated DNA molecule according to claim 1, wherein said DNA molecule encodes a human or rat NPY-Y5 receptor.

3. The isolated DNA molecule according to either claim 1 or claim 2, wherein said DNA molecule encodes a human NPY-Y5 receptor.

4. The isolated DNA molecule according to any one of claims 1 to 3, wherein the DNA molecule is at least 80% homologous to the nucleotide sequence shown: a. at nucleotides 6291 to 7625 in SEQ ID NO: 1; b. at nucleotides 63 to 1397 in SEQ ID NO: 3; c. at nucleotides 115 to 1449 in SEQ ID NO: The isolated DNA molecule according to any one of claims 1 to 3, wherein the DNA molecule is at least 95% homologous to the nucleotide sequence shown: a. at nucleotides 6291 to 7625 in SEQ ID NO: 1; b. at nucleotides 63 to 1397 in SEQ ID NO: 3; c. at nucleotides 115 to 1449 in SEQ ID NO:

6. An isolated DNA molecule encoding an NPY-Y5 receptor consisting of 445 25 amino acids in length, wherein said DNA molecule comprises a nucleotide sequence selected from the group consisting of nucleotides 6291 to 7625 of SEQ ID NO: 1, nucleotides 63 to 1397 of SEQ ID NO: 3 and nucleotides 115 to 1449 of SEQ ID NO:

7. The isolated DNA molecule encoding an NPY-Y5 receptor according to claim 6, wherein said DNA molecule consists of nucleotides 6291 to 7625 of SEQ 0 ID NO: 1 or substantially corresponds to said nucletotides 6291 to 7625 of SEQ ID NO: 1.

8. The isolated DNA molecule encoding a NPY-Y5 receptor according to claim 6, wherein the DNA molecule consists of nucleotides 63 to 1397 of SEQ ID 16 NO: 3 or substantially corresponds to said nucleotides 63 to 1397 of SEQ ID NO: 3.

9. The isolated DNA molecule encoding a NPY-Y5 receptor according to claim 6, wherein the DNA molecule consists of nucleotides 115 to 1449 of SEQ ID NO: 5 or substantially corresponds to said nucleotides 115 to 1449 of SEQ ID NO: A plasmid or expression vector including a DNA molecule according to any one of the preceding claims.

11. A host cell transformed with the DNA molecule according to any one of claims 1 to 9.

12. The host cell according to claim 11, wherein the cell is a mammalian, insect or bacterial cell.

13. The host cell according to either claim 11 or claim 12, wherein the cell is a Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) 293 cell or Sf9 cell.

14. The host cell according to any one of claims 11 to 13, wherein the cell expresses NPY-Y5 receptor onto the cell's surface. 25 15. An isolated NPY-Y5 receptor polypeptide consisting of 445 amino acids in length.

16. The NPY-Y5 receptor polypeptide according to claim 15, wherein the NPY- receptor has an amino acid sequence substantially corresponding to any one of the amino acid sequences of SEQ ID NOs 4 or 6.

17. An antibody capable of specifically binding to the NPY-Y5 receptor polypeptide according to either claim 15 or claim 16.

18. A non-human animal transformed with a DNA molecule according to any one claims 1 to 9. *o 17

19. A method for detecting agonist or antagonist agents of NPY-Y5 receptor, comprising contacting a NPY-Y5 receptor according to either claim 15 or claim 16 or a cell transformed with and expressing a DNA molecule according to any one of claims 1 to 9, with a test agent under conditions enabling the activation of the NPY-Y5 receptor, and detecting an increase or decrease in the NPY-Y5 receptor activity. A nucleic acid probe comprising a nucleotide sequence of 10 or more nucleotides capable of specifically hybridizing to a unique sequence within the DNA molecule according to any one of claims 1 to 9.

21. An antisense nucleic acid molecule comprising a nucleotide sequence capable of specifically hybridizing to an mRNA molecule which encodes receptor so as to prevent translation of the mRNA molecule.

22. An isolated DNA molecule according to any one of claims 1 to 9, substantially as herein described with reference to the Example and/or accompanying Figures.

23. A plasmid or expression vector according to claim 10, substantially as herein described with reference to the Example and/or accompanying Figures. S: 24. A host cell according to any one of claims 11 to 14, substantially as herein S 25 described with reference to the Example and/or accompanying Figures.

25. An isolated NPY-Y5 receptor polypeptide according to either claim 15 or •I claim 16, substantially as herein described with reference to the Example and/or accompanying Figures. S26. An antibody according to claim 17, substantially as herein described with reference to the Example and/or accompanying Figures.

27. A non-human animal according to claim 18, substantially as herein described with reference to the Example and/or accompanying Figures. 18

28. A method for detecting agonist or antagonist agents of NPY-Y5 receptor according to claim 19, substantially as herein described with reference to the Example and/or accompanying Figures.

29. A nucleic acid probe according to claim 20, substantially as herein described with reference to Example and/or accompanying Figures. An antisense nucleic acid molecule according to claim 21, substantially as herein described with reference to the Example and/or accompanying Figures. Dated this twenty-fourth day of December 2003 Garvan Institute of Medical Research Patent Attorneys for the Applicant: F B RICE CO S. *S