CA2220036A1 - Human neuropeptide receptor - Google Patents

Abstract

Human neuropeptide receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods of using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the neuropeptide receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the neuropeptide receptor nucleic acid sequences and an altered level of the soluble form of the receptors.

Description

W 096~34877 PCTrUS95/05616 ~WAN ~:u~Or~sr~-lv K~ .O~

T]his invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides an,d polype~ptides, as well as the production of such polynucleotides and polypeptides. The polypeptides o~ the present invention are human 7-tr~n~m~mhralle G-protein coupled receptors. More particularly, the polypeptides of the present invention are neuropeptide receptor polypeptides, sometimes hereinafter referred to as neuropeptide receptor polypeptides. The invention also relates to inhibiting the action of such polypeptides.
Obesity i8 the comm--n~st nutritional disorder in Western societies. More than three in ten adult Americans weigh at least 2096 in excess o~ their ideal body weight (Burroa, M., The New York Times, 17 July 1994). Increased body weight is an important public health problem because it is associated with '~ype II diabetes, hypertension, hyperlipidemia and certain cancers (Grundy, S.M., and Barnett, J.P., Disease-a-Month, 36:645-696 (1990)).
~ ive single-gene mutations in the mouse obesity gene (ob) which result in an obese phenotype have been described (Friedman, J.M. & Leibel, R. L., Cell, 66:217-220 (1990)).
The cloning and sequencing of the mouse ob gene and its human W O 96/34877 PCTrUS95/05616 homologue have been reported ~Zhany, Y., et al., Nature, 372:425-431 (1994)). The ob gene encodes a 4.5-kb adipose ti~sue mRNA with a highly conserved 167-amino-acid open reading frame. The predicted amino-acid sequence is 84~
identical between human and mouse and has features of a secreted protein. The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot ( d. 425).
Of the brain regions implicated in the regulation of feeding behavior, the ventromedial nucleus of the hypothAlAmll~ (VMH) i8 considered to be the most important satiety center in the central nervous system (CNS). The energy balance in mAmmAls is therefore postulated to be controlled by a feedhack loop in which the amount of stored energy is sensed by the hypothAlAmll~, which adjusts food intake and energy expenditure to mA;ntAin a constant body weight (Ombeck, J.R., Yale J. Biol. Med., 20:545-552 (1948) and Kennedy, G.C., Proc. R. Soc.148:578-592 (1953)). In the lipostasis theory, the size of the body fat depot is regulated by the CNS, with a product of body fat metabolism affecting energy hAlAnce by interacting with the hypothalamus (Kennedy, G.C., Proc. R. Soc.148:578-592 (1953)).
The ;nAh;lity to identify the putative signal from fat has h;n-lered the validation of the lipostasis theory. The possibility that at least one component of the signalling system circulates in the bloodstream was first suggested by Hervey (Dietrich, W., et al., Genetics, 131:423-447 (1992)), who showed that the transfer of blood from an An;mAl with a VMH lesion across a vascular graft to an untreated An;~Al (a parabiosis experiment) resulted in a reduction of food intake in the intact An;mAl It is now significant that there is evidence that the ob gene product is secreted, suggesting that ob may encode this circulating factor.
The ob signal may act directly or indirectly on the CNS
to inhibit food intake and/or regulate energy expenditure as W O 96~34877 PCT/US95/05616 part oi- a homeostatic mechanism to m~n~in constancy of the adipose mass (Zhang, Y., et al., Nature, 372:425-431, 431 ~1994)'l. The ob gene apparently encodes a protein secreted by fat., and mutations apparently prevent translation or expression of the gene (Rink, T., Nature, 372:406-407 (1994)l.
Parabiosis experiments suggest thaLt the ob receptor i8 encoded by the mouse db (diabetes) gene (Coleman, D.L., Diabetologia, 14:141-148 (1978)). Mice having a mutation in the db gene are al~o obese, with the de~ect possibly beiny a receptor defect. (Id. at 406).
Neuropeptide Y is similar to the ob gene product in that it mediates the feeding response. Neuropeptide Y acts on at least ~our types of neuropeptide Y receptors called Y" Y2, Y3 and an atypical Y~ receptor, which mediates the feeding response stimulated by neuropeptide Y.
Neuropeptide Y has a wide range of biological functions.
Neuropeptide Y is found to be widely distributed in th.e central nervous system (CNS) and the peripheral nervou~
system. (PNS). In the PNS, neuropeptide Y is found in thLe noradrenergic sympathetic innervation o:E blood vessels and other smooth muscle tissues and in neurons within the enteric nervous system. Neuropeptide Y ;mmllnoreactive fibers al~o occur in the non-vascular smooth muscle, surrounding exocrinLe glands and surface epithelia. Neuropeptide Y also occurs in subpopulations of neurons and is generally co-localized with other neurotransmitters, particular nora.drenaline.
]:n the CNS, neuropeptide Y is cont~ n~ in GABAergi.c interneurons in higher centers and in pre~om~nAntly catechol~m~nergic cells that project further caudally. For examp]e, neuropeptide Y is contained in interneurons in the cortex, hippocampus, amygdala, basal forebrain and striatum, whereas in the brain stem, neuropeptide Y is ~contained in ~ noradrenergic neurons of the A, and A2 groups in the medulla, and l_he locus coeruleus (LC). In the hypoth~1~mll~, neuropeptide Y is found pre~min~ntly in the arcuate nucleus and lateral hypoth~
Within the peripheral nervous system, neuropeptide Y is present in postganglionic sympathetic nerves, and is co-localized as stated above with other neurotransmitters, including catechol ~mi nPfi, When used pharmacologically, neuropeptide Y has been shown to have a potent vasoconstrictor activity as well as dramatically potentiating the vasoconstriction caused by many other pressor agents.
Particularly high concentrations of neuropeptide Y are found in the sympathetic nerves supplying the coronary, cerebral and renal vasculature and when infused into these vascular beds, neuropeptide Y causes prolonged vasoconstriction that is not reversed by adrenergic blocking agents. These observations have lead to the proposal that neuropeptide Y is the candidate transmitter for pathological vasospasm, a major cause o~ morbidity and mortality when involving the coronary and cerebral vessels.
Neuropeptide Y also appears to be involved in interaction with the renin angiotensin system. Neuropeptide Y contA;n;ng sympathetic nerve terminals are found on the juxta-glomerular apparatus of the renal cortex and neuropeptide Y influences renin release. These data, together with the ~Pmon~tration of all durations in neuropeptide Y concentrations in hypertensive ~n;m~l models and the pressor response to infusion of the peptide, have resulted in implications of this peptide in hypertension.
Within the central nervous system neuropeptide Y is located pre~om;n~ntly within interneurons where it appears to have a regulatory role. It therefore has widespread and diverse effects including effects on memory and a possible role in Al7he;mPr~s disease. Neuropeptide Y is the most potent known substance to cause an increase in feeding and may play a role in the genetic basis of Type II Diabetes Mellitus. Neuropeptide Y may also play a role as a W 096/34877 PCTrUS95/05616 regulat.ory agent and pituitary function as well as potential neuromodulatory function in stress responses and in reprod~lctive function.
In accordance with one aspect of the present invention~
~ there aLre provided novel mature receptor polypeptides as well as bio]ogically active and diagnostically or therapeutically ~ u8eful fragments, analogs and derivatives thereo~. The receptor polypeptides of the present inv~ntion are of human origin.
~ n accordance with another aspect of the presen~
invent:ion, there are provided isolated nucleic acid molecules encoding the receptor polypeptides of the present invention, includ:ing mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs thereof and biologically active and diagno,stically or therapeutically useful fragments thereof..
I:n accordance with a further aspect of the present invention, there are provided processes for producing such receptor polypeptides by recomhin~nt techniq~ues comprising culturing rec~mbin~nt prokaryotic and/or eukaryotic host cells, cont~;n;ng nucleic acid seq[uences encoding the receptor polypeptides of the present invention, under conditions promoting expression of sai.d polypeptides an.d subsequent recovery of said polypeptides.
I.n accordance with yet a further aspect of the presen.t invent.ion, there are provided antibodies against such receptor polypeptides.
In accordance with another aspect of the present invent:ion there are provided methods of screening for compo~mds which bind to and activate or inhibit activation of the receptor polypeptides of the present invention.
:[n accordance with still another embo~-im~nt of the present invention there are provi.ded processes of ~m; nistering compounds to a host which bind to and activat:e the receptor polypeptide of the present invention which axe usefu:L in the prevention and/or treatment of obesity, hyperlipidemia, certain cancers, to stimulate neuronal growth, to regulate neurotransmission, to ~nh~nce activity levels and utilization of ingested foods.
In accordance with another aspect ~of the present invention there is provided a method of ~m;n; stering the receptor polypeptides of the present invention via gene therapy to treat conditions related to underexpression of the polypeptides or underexpression of a ligand to the receptor polypeptide.
In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and ; nh; h~ t activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of Al~he;mer's disease, Type II Diabetes Mellitus, epilepsy, stress, anxiety, hypertension, cardiovascular disea~e, psychotic conditions and obesity caused by neuropeptide Y.
In accordance with yet another aspect of the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polynucleotide sequences of the present invention.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.
In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

W 096/:34877 PCTrUS95/0~616 The following drawings are illustrative o~ embodiments of the invention and are not meant to limit the scope of the invention as encomr~sed by the cl ~ms .
F:igure 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of the neuropeptide receptor polypeptide of the present invention. The st~n~rd one-~ letter abbreviation for amino acids is u~ed. Se~l~nc~ng was performed using a 373 Automated DNA sequencer ~Applied Biosystems, Inc.).
Figure 2 shows the cDNA sequence and the correspondingdeduced amino acid sequence of the neuropeptide receptor splice variant 1 polypeptide o~ the pre~ent invention. The st~n~rd one-letter abbreviation for amino acids is used.
P~igure 3 shows the cDNA sequence and the corresponding deduced amino acid sequence o~ the neuropeptide receptor splice! variant 2 polypeptide of the pre~ent invention. The st~n~l~d one-letter abbreviation for amino acids is used.
Figure 4 illustrates the amino acid sequence and seven tr~nsmemhrane regions of the neuropeptide receptor. The tr~nfim~mh~ane regions are underlined and denoted with a TM.
~ igure 5 illustrates the amino aci~ sequence and seven tr~n~m~mhrane regions of the neuropeptide receptor splice variant 1. The tr~nsm~mhrane regions are underlined and denoted with a TM.
]Figure 6 illustrates the amino acid sequence and seven transmembrane regions of the neuropeptide receptor splice variant 2. The transmemhrane regions are underlined and denoted with a TM.
Figure 7 shows the amino acid homology between the human neuropeptide receptor polypeptide of the present invention (and the human neuropeptide Y~ receptor).
The receptor polypeptides of the present invention are recepltors for ligands, both known and unl~nown, which modulate the activity of cells in both the central nervous system and peripheral tissues regulated by the central nervous system.
, ~ CA 02220036 1997-10-31 5 ~ 16 Exam~les of such ligands are neuropeptide Y, substance P, the human ob gene product and neurokinin B. Accordingly, modulation of the activity of receptor polypeptides of the pre~ent invention will have a broad range of therapeutic and diagnostic applications, particularly with respect to the treatment of obesity.
The present inventors have isolated a full-length cDNA
clone encoding a human neuropeptide receptor polypeptide.
The present full-length cDNA has been mapped to a location on human chromosome 1 position p31-34 which corresponds to a location on the mouse chromosome 4 where the db gene is found. The mouse db gene is thought to encode the receptor ~or the obesity gene product In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptide having the deduced amino acid sequence of Figures 2 (S~Q ID NO:2) or ~or the mature polypeptide encoded by the cDNA of the clone~s) depasited as ATCC Deposit No. 97128 on April 28, 1995.
The polynucleotide of this invention was discovered in a cDNA library derived from human adult hypoth~l~m~ It is structurally related to the G protein-coupled receptor family. The neuropeptide receptor polypeptide cont~in~ an open reading frame encoding a protein of 402 amino acid residues. The neuropeptide receptor protein exhibits the highe~t degree of homology to human neuropeptide Yl receptor protein with 52 % ~imilarity and 26 % identity over the entire amino acid sequence.
The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA incl~des cDN~, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequences which encode the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SFQ ID

,~lOED S~

W 096/34877 PCTrUS95/05616 NO:1) or that of the deposited clone(s) or may be a di~feren~
coding se~uence which coding sequence, aLs a result of the re~lln~ncy or degeneracy of the genetic code, encodes the same n~lture polypeptide as the DNA of Figure 1 (SEQ ID NO:1) or the deposited cDNA(s).
The polynLucleotides which encode for the mature ~ polypeotide of Figure 2 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited c~NA(s) mi3Ly include:
only the coding sequence for the mature polypeptide; the coding se~uence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3~ of the coding sequence for the mi3Lture polypeptide.
T]lus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which :includes additional coding and/or n.on-coding sequence.
T]he present invention further relates to variants of the herPin.~bove described polynucleotides which encode for fragme:nts, analogs and derivatives of the polypeptides having the delduced amino acid sequence of Figure 2 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone(s). The variants of the polynucleotide may be naturally occurring allelic variants of the polynucleotides or non-naturally occurring variants of the polynucleotides.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 2 (SEQ ID NO:2) or the same maLture polypeptide encoded by the cDNA of the deposited clone(s) as well as variants of such polYnucleotide which variants encode ~or a fragment, deriva.tive or analog of the polypeptide of Figure 2 (SEQ ID
NO:2) or the polypeptide encoded by the cDNA of the deposited clone~s). Such nucleotide variants inclu,de deletion variants, substi~ution variants and addition or insertion variants. Specific examples of such variants include the _g_ polynucleotide sequences as set forth in SEQ ID NOS:3 and 5 which encode for ~plice variant 1 and 2, respectively, of the polypeptide of the present invention.
As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID
NO:1) or of the coding sequence of the deposited clone(s).
As known in the art, an allelic variant is an alternate form of polynucleotide sequences which may have a substitution, deletion or addition of one or more nucleotides, which does not sub~tantially alter the function of the encoded polypeptides.
The polynucleotides may also encode for a soluble form of the neuropeptide receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the TM and intracellular ~m~i n of the full-length polypeptide of the present invention.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a m~rker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a m~mm~l ian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to the her~;nAhove-described sequences if there is at least 70~, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, W 096/.34877 PCTrUS95/0~616 the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred ~mho~m~nt encode polypeptides which either retain substantially the same biological $unction or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SBQ I]D NO:1) or the deposited cDNA(s), i.e. function as a soluble neuropeptide receptor by retA~n~ng the ability t~
bind the ligands for the receptor even though the polypeptide does not function as a membrane bound neuropeptide receptor, for example, by eliciting a second messenger response.
Alternatively, the polynucleotides may be polynurleotides which have at least 20 bases, preferably 30 bases ,and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which have an identity thereto, as her~nAhove described, and which does not rel_ain activity. Such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO: 1, or for variants thereof, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer The deposit~s) referred to herein will be ~AtntA~n~
under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. 112.
The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein.. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

W 096134877 PCT~US95/05616 The present invention ~urther relates to a polypeptide which has the deduced amino acid sequence of Figure 2 (SEQ ID
NO:2) or which has the amino acid sequence encoded by the deposited cDNA(s), as well as fragments, analogs and derivatives of such polypeptide.
The terms ~fragment," "derivative'l and "analog" when referring to the polypeptide of Figure 2 (SEQ ID NO:2) or that encoded by the deposited cDNA(s), means polypeptides which either retain substantially the same biological function or activity as such polypeptides, i.e., function as a soluble neuropeptide receptor by retA;n~ng the ability to bind the ligands of the receptors even though the polypeptides do not function as membrane bound neuropeptide receptors. An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. Specific examples are splice variant 1 and 2 of Figures 2 and 3 (SEQ ID NO:4 and 6), respectively.
The polypeptides of the present invention may be reComh;n~nt polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.
A fragment, derivative or analog of the polypeptide of Figure 2 (SEQ ID NO:2) or that encoded by the deposited cDNA(s) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which one or more of the amino acid residues includes a substituent group, (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), (iv) one in which the additional amino acids are fused to the mature polypeptide, such as sequence which is employed for purification of the mature polypeptide sequence W 096/34877 PCTrUS95/05616 or (iV'I splice variants of the mature polypeptide whieh may have one or more amino acids deleted from the mature polypeptide yet still retain activity c~rresponding to the mature polypeptide. Such fragments, derivatives and analogs are dee~med to be within the scope of those skilled in the art from the t~h~ngs herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and pre~erably are purified to homogeneity.
The tenm "gene" means the segment of DNA involved in producing a polypeptide chain; it includes reyions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons).
I~he term "isolated" means that the material is removed from its original environment (e.g., the natural envi~unllle~t if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living ~ni~l iS not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materi.als in the natural system, is isolated. Such polymlcleotides could be part of a vector and/or such polymlcleotides or polypeptides could be part of a compol,ition, and still be isolated in that such vector or composition is not part of its natural environm~nt.
The present invention also relates to vectors which inclu~e polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered ~transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector m~y be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the human neuropeptide receptor genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recom.binant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from comh;nAtions of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction ~n~onllclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: hTR or SV40 promoter, the E. coli. lac or trP, the phage 1 Amh~lA PL
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.

-W 096/.S4877 PCTfUS95/05616 The v~ector may also include appropriate sequences for ampli~ying expression.
In addition, the expre8sion vectors pre~erably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance ~or eukaryoti.c cell culture, or such as tetracycline or ampicilli.n resistance in E. coli.
I'he vector cont~ ni n~ the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or contrcll sequence, may be employed to transform an appropriate host to permit the host to express the protein.
~ -s representative examples of a~.o~,iate hosts, there may be mentioned: bacterial cells, such as E. coli, strePt:omyces~ S~lm~nella tYphimurium; fungal cells, such as yeast; insect cells such as Droso~hila S2 and S~odo~tera Sf9;
~n~l cells such as CHO, COS or Bowes m~l ~no~ i adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of tho~e skilled in the art from the teachings herein.
r~Ore particularly, the present invention also includes reco~inant constructs comprising one or more of the sequences as broadly described above. The construc~s compr:ise a vector, such as a plasmid or viral vector, in1o which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, $or example, a promoter, operably linked to the sequence. Large numbers o~ suitable vectors and promoters are known to those of skill in the art, and are co~m~rcially available. The following vectors are provid,ed by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDlO, phagescript, psiXl74, pbluescript~ SK, pbsks, pNH8A, p~l6a, pNHl8A, pNH46A (Stratagene!; pTRC99a, pKK223-3, p~K233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, .

W 096/34877 PCTrUS95/05616 pSV2CAT, pO&44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chlor~mph~n;col transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR~ PL and trp.
Eukaryotic promoters include CMV ~mm~ te early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
In a further embodiment, the present invention relates to host cells cont~;n;ng the above-described constructs. The host cell can be a higher eukaryotic cell, such as a m~mm~lian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, ~1986)).
The constructs in host cells can be used in a conventional m~nner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Fragments of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates ~or producing the full-length polypeptides. Fragments of the polynucleotides of the present invention may be used in a similar m~nner to W 096/.34877 PCT~US95tO5616 synthe~size the full-length polynucleotides of the present invention.
Mature proteins can be expressed in mAmm~1~An cells, yeast, bacteria, or other cells under the control of a~l~Liate promoters. Cell-free translation systems can also b~e employed to produce such protein~ using RNAs derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are de~cribed by Sambrook, et al., Molecular Cloning: A Laboratory ~nll~l, Second ~dition, Cold Spring Harbor, N.Y., ~1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an ~nh~ncer sequence into the vector. Rnh~ncers are cis-acting el~m~nts of DNA, usually about from 10 to 3~0 bp that act on a promoter to increase its transcription.
Examples including the SV40 ~nh~ncer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter ~nh~ncer, the polyoma ~nh~ncer on the late side of the replication origin, and adenovirus enhancers.
Generally, reco~h; n~nt expression vectors will include origins of replication and selectable markers permitting transf-ormation of the host cell, e.g., the aml?icillin resist:ance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promol-ers can be derived from operons PnCo~ng glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ~-factor, acid ]?hosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in a~o~riate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion o~ translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence ~ , .

W O 96/34877 PCTrUS9S/05616 can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recomh~n~nt product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella tY~himurium and various species within the genera Psell~omon~s~ Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone'l sections are combined with an appropriate promoter and the structural se~uence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

W 096/.34877 PCTAUS95/0~616 Microbial cells employed in expression o~ proteins cc~n be disrupted by any convenient method, including freeze-thi~w cycling, sonication, mechanical disruption, or use o~ ce].l lysing agents, such methods are well know to those skilled i.n the art.
~ arious mAmm~lian cell culture systems can also be employed to express recom-h~nAnt protein. ~xamples of m~ l ian expression Cystems include ~he COS-7 lines of monkey kidney fibroblasts, described by ~luzman, Cell, 23:175 (1981), and other cell lines cApAbl~ of expressing a comlpat.ible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. ~A~'l ian eXprecsion vectors will comprise an origin of replication, a suitable promoter and PnhAncer, and also any necessary ribosome binding sites, polyaclenylation site, splice donor and acceptor sites, transc:riptional termination sequences) and 5' flankillg nontricmscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites m~y be used to provide the required nontranscribed genetic ele~ents.
The neuropeptide receptor polypeptide of the present invenl_ion can be recovered and purified from recombinant cell cultu:res by methods including Ammon~um sulfate or ethanol precipitation, acid extraction, anion or cation ~xrh~nge chrom~tography, phosphocellulose chromatography, hydrophobic interiaction chromatography, affinil:y chromatography, hydroxylapatite chromatography and lectin chromatography.
Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The neuropeptide receptor polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recomh~nAnt techniques from a prokaryotic or eukaryotic host (for exam.F~le, by bacterial, yeast, higher plant, insect and W 096/34877 PCTrUS9510~616 m~mmAl~An cells in culture). Depending upon the host employed in a reco~h~ n~nt production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.
The human neuropeptide receptor polypeptides of the present invention may be employed in a process for screening compounds which bind to and activate the receptor polypeptide and for compounds which bind to and inhibit activation of the receptor polypeptides of the present invention.
In general, the neuropeptide receptor in isolated, immobilized or cell bound form is contacted with a plurality of compounds and those compounds are selected which bind to and interact with the receptor. The h; nA; ng or interaction can be measured directly by using radioactively labeled compounds of interest or by the second messenger effect resulting from the interaction or binding of the candidate compound. Alternatively, the candidate compounds can be subjected to competition screening assays, in which a known ligand, preferably labeled with an analytically detectable reagent, most preferably radioactivity, is introduced with the compound to be tested and the compound~s capacity to inhibit or ~nhAnce the binding of the labeled ligand is measured. Compounds are screened for their increased afffinity and selectivity to the receptor polypeptide of the present invention.
One such screening procedure involves the use of melanophores which are transfected to express the neuropeptide receptor of the present invention. Such a screening technique is described in PCT WO 92/01810 published February 6, 1992.

W 096/34877 PCTrUS95/05616 For example, to screen for compounds which i nhi bit activation of the receptor polypeptide of the present invention, the compound and a ligand ~lown to bind to the receptor are both contacted with the melanophore cell~.
Tnh; hi tion of the signal generated by the ligand indicates that t:he compound inhibits activation of the receptor.
~ he screen rnay be employed for determining a compo~ld which binds to and activate~ the receptor polypeptide of the present invention by contacting such cells with com~ounds ~o be screened and deter~ining whether such compound generates a signal, i.e., activates the receptor.
Other examples include the use of cells which express a neuropeptide receptor of the present invention (for example, transiected CHO cells) in a system which nnea~ures extra-cellu:Lar pH changes caused by receptor activation, for exarnp:Le, as described in Science, volume 246, pages 181-296 (October 1989). ~or example, compounds may be contacted with a celL which expresses an neuropeptide receptor polypepti~e of the present invention and a second messenger response, e.g. signal transduction or pH changes, may be measured to deten~ine whether the potential compound is effective as ~n activator or inhibitor.
~ nother example involves introducing RNA encoding a neuropeptide receptor of the present invention into Xenopus oocytes to transiently express the receptor. The oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of ~ nh- hi tion of or an increase in intracellular calcium.
Another exan~ple involves expres~3ing a neuropeptide receptor polypeptide of the present invention on the surface of a cell wherein the receptor i~ linked to a phospholipase C or D. As representative examples of such cells there n~y be rnentioned endothelial cells, smooth mu~scle cells, embr~onic kidney cells, etc. The screening may be accomplished as hereinabove described by detecting activation W 096t34877 PCT/US95/05616 of the receptor or ~nh~ h; tion of activation of the receptor from the phospholipase second signal.
Another method involves determining ;nh; h; tion of h;n~ing of labeled ligand to cells which have a neuropeptide receptor on the surface thereof. Such a method involves transfecting a eukaryotic cell with DNA encoding an neuropeptide receptor polypeptide of the present invention such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
Another screening technique involves expre~sing a neuropeptide receptor polypeptide on the surface of a cell wherein the receptor is linked to a second messenger to increase cytosolic calcium levels in transfected CHO cells.
An example of such a method comprises transfecting CHO cells with a nucleic acid sequence encoding a receptor of the present invention such that the receptor is expressed on the surface thereof. The transfected cell is then incubated in a reaction mixture with labeled calcium in the presence of a compound to be screened. The ability of the compound to increase calcium up-take or ;nh;h;t calcium up~take can then be determined by measuring the amount of labeled calcium transported into the cells by taking advantage of the label, e.g., radioactivity.
Compounds may also be identified by the above methods which bind to specific subregions within the CNS that are important for specific behaviors through indirect interactions with a neuropeptide receptor polypeptide of the present invention.

W O 96/34877 PCTrUS95/05616 To mea~ure intracellular cyclic AMP levels, cyclic A~P
is assayed in whole cells treated for 15 minutes at 37~C wi~.h 100 micromolar isobutylmethyl~Anth~ne (IBMX; Sigma).
TransfeCted cells (1 x 106 / 0.5 ml reaction) are incubated with ~0 micromolar forskolin and various concentrations of known or unknown ligands to the receptor. Reactions are terminated with the addition of HCl to O.lM, incubation at room t:emperature for 15 minutes, neutralization and sample dilution in 50 mM sodium acetate, pH 6.2. Cyclic AMP i.s quantified by using a radioimmlln~as8ay (Dupont/NEN~.
~ o measure levels of intracellular calcium, transfected cells are suspended in loading medium (modified RPMI 1640 medium/10 mM Hepes/1~ newborn calf serum) and incubated in a spinner ~lask at 37~C ~or 2.5 hour at 1 x 106 cells per ml.
Cells are then treated with 1 micromolar Fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes) for 30 minutes at 37~C, washed twice with loading medium, and resuspended at 5 x 106 cells~ml. Tmm~iAtely be~ore fluorescence ~;pectroscop~,r, cells are recovered by centrifugatioIl at 1000 rpm aIld resuspended at 1 x 10 cells/ml in a modified Krebs buffer (135 mM NaCl/4.7 mM KCl/1.2 mM MgSO4/1.2 mM KH2PO4/5 mM
NaHCO3/l mM CaCl2/2.8 mM glucose/10 mM hepes, pH 7.~) cont~tn;ng sulfinpyrazone. Bombesin is purchased ~rom Si~na and Auspep. Fluorescence recordings are made on a Hitachi fluorescence spectrometer (F4010) at 340 nm (excitation) and 505 m~ (emission) over 10 minutes with slit widths of 5 ~m and response time of 2 seconds. Intracellular calcium is quantified by using equations described by Grynkiewicz, ,et al., ,J. Bio. Chem. 260:3440-3450, 1985.
The invention also provides a method of treating and/or preventing obesity ~y A~m; ni stering to a host a compound which binds to and activates the receptor polypeptides of the present invention. Such a compound is o~her than the ob gene product disclosed in Zhang, et al., Nature, 372:425-431 (1994). The receptor polypeptide of the present invention W 096/34877 PCTrUS95/05616 maps to a human chromosome which corresponds to the position of the mouse chromosome which encodes for the receptor of the ob gene product. The human ob gene encodes a "satiety"
factor which binds to and activates the receptor polypeptide of the present invention. Accordingly, a compound which activates the receptor of the present invention will decrease appetite and prevent obesity.
The compounds described above may also be employed to enhance activity level, modify eating behavior, PnhAnce utilization of ingested foods and regulate deposition of fat stores. Conditions related to obesity may also be treated by the compounds which bind to and activate the receptor polypeptides of the present invention including hyperli~;m~, type II diabetes and certain cancers.
These compounds may also be employed to treat and/or prevent other conditions related to an underexpression of the receptor polypeptide of the present invention or ligands which bind thereto, for example, to stimulate neuronal growth.
Specific examples of compounds which inhibit activation of the receptor polypeptides of the present invention include an antibody, or in some cases an oligonucleotide, which binds to the receptor but does not elicit a second messenger response such that the activity of the receptor i8 prevented.
Another example is proteins which are closely related to the ligands of the receptor, i.e. a fragment of the ligand, which have lost biological function and when binding to the receptor, elicit no response.
Another example includes an antisense construct prepared through the u~e of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide se~uence, which encodes for the mature , polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see hee et al., Nucl. Aci~ds Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988);
and r)ervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of a neuropeptide recepltor polypeptide of the present invention. The antisense RNA o:Ligonucleotide hybridizes to the m~NA in vivo and blocks translation of the mRNA molecule into the receptor (antisen~e - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Bxpression, CRC Pres~, Boca Raton, FL (1988)). The oligonucleotides de8cribed above can al80 ]~e delivered to cells such that the antisense RNA or DNA
m~Ly be expressed in vivo to t nh~ h~ t production of the receptors.
~ nother example is a small molecule which binds to a neuropeptide receptor polypeptide of the present invention, making it inaccessible to ligands such that normal biological activity is prevented. Examples of small molecules include but ,are not limited to small peptides or peptide-like molecules and neuropeptide Y fragments and/or derivatives.
Soluble forms of a neuropeptide receptor polypeptide of the present invention, e.g., a fragment of the receptor, which binds to the ligand and prevents the ligand from interacting with membrane bound receptors may also t nh; h; t activation of the receptor polypeptides of the present invention.
This invention additionally provides a method of utilizing such compounds which inhibit activation for treat:ing abnorm~Ll conditions related to an excess of activity of ~ neuropeptide receptor polypeptide of the present invention for treating obesity since the neuropeptide receptor polypeptides of the present invention may bind -W 096/34877 PCTrUS95/05616 neuropeptide Y which is the most potent known substance to cause an increase in feeding behavior and type II Diabetes Mellitus since neuropeptide Y may play a role in the genetic basis of this disease.
The compounds which inhibit activation of the receptor polypeptides of the present invention may be employed to treat and/or prevent hypertension since neuropeptide Y
stimulates renin release and neuropeptide Y is known to have potent vasoconstrictor activity when involving the coronary and cerebral vessels.
The compounds may also be employed to treat Al~h~im~r~s disease since neuropeptide Y receptors are prevalent in the central nervous system and are localized pre~ominAntly within interneurons where they appear to have regulatory roles in memory and Al~heim~rs disease.
The compounds may also be employed to suppress excitatory transmission by neuropeptide Y in the hippocampus and therefore may be employed to treat epileptic seizure, stress and anxiety.
The prevalence of neuropeptide Y receptors in the central nervous system indicates that the compounds which inhibit the neuropeptide receptor polypeptides of the present invention may be used as an antipsychotic drug by regulating neurotransmission.
The compounds which inhibit the receptor polypeptides of the present invention may also be employed to treat pathological vasospasm involving coronary and cerebral vessels.
This invention also provides a method for determining whether a ligand not known to be capable of binding to a neuropeptide receptor of the present invention can bind thereto which comprises contacting the ligand to be identified with a cell comprising the coding se~uence of a neuropeptide receptor and expressing same on its ~urface under conditions sufficient for binding of ligands previously W 096/34877 PCT/US95/0~616 identified as htn~tng to such a receptor. In other emboAtm~nts cell membrane fractions comprising the receptor or isolated receptors free or tmmnh;lized on solid suppor~s may be used to measure binding of the ligand to be tested When r~ecombinant cells are used for purposes of expression of the receptor it is preferred to use cells with little or no endogenous receptor activity so that htnAtn~, if any, is d~e to the presence of the expressed receptor of interest.
Preferred cells include human embryonic kidney cells, monkey kidney (HEK-293 cells), fibroblast (COS) cells, ~htne~e hamster ovary (CH0) cells, Droso~hila or murine L-cells. It is al!~o preferred to employ as a host cell, one in which a recep1:0r responsive second messenger system exists. Well known second messenger systems include increases or decreas~s in phosphoinositide hydrolysis, adenylate cyclase, guanyla~e cyclase, or ion ch~nnel activity in response to ligand binding to extracellular receptor A~; n~ . In a further embodiment a specifically designed indicator of receptor hi nAi1ng can be constructed. For example, a fusion protein can b~e made by fusing the receptor of this invention with a protein Anm~tn which is sensitive to receptor ligand h;nAtng Such a ~om~in referred to here as an indicator ~om~in is capable, itself, or in association with accessory molecules, of generating an analytically detectable signal which is indicative or receptor ligand binding.
This invention also provides a method of detecting expression of a neuropeptide receptor polypeptide of the present invention on the surface o~ a cell by detecting the presence of mRNA coding for the receptor which comprises obt~intng total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least lO nucleotides capable of specifically hybridizing with a sequence included within th~e sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA
-hybridized to the probe, and thereby detecting the expression of the receptor by the cell.
The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. These related receptors may be identified by homology to a neuropeptide receptor polypeptide of the present invention, by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the neuropeptide receptor polypeptides of the present invention.
Fragments of the genes may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the genes of the present invention, or which have similar biological activity. Probes of this type preferably have 50 bases or more. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene of the present invention including regulatory and promoter regions, exons and introns. An example of a screen of this type comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the genes of the present invention are used to ~creen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The neuropeptide receptor polypeptides and compounds identified above which are polypeptide~, may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.~
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being W 096/.34877 PCTrUS9510~616 provicled to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be~ engineered by procedures known in the art by use of a retroviral particle contA~nin~ RNA encoding a polypeptide o$
the present invention.
C;~m; l Arly~ cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, proceclures known in the art. As known in the art, a producer cell ~or producing a retroviral particle cont~ntng RNA
encodiLng the polypeptide of the present invention may ~e n-l stered to a patient for engineering cells in vivo alld expression of the polypeptide in vivo. These and other metho~s for ~min; stering a polypeptide of the prese~t inven1ion by such method should be apparent to those skilled in the art from the teachings of the present invention. For examp:Le, the expression vehicle for engineering cells may ~e other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after co~h~n~tion with a suita]ble delivery vehicle.
Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human ~mml~nodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and ~mm~ry tumor virus.
In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, 1_he retroviral LTR; the SV40 promoter; and the hum~n cytomegalovirus (CMV) promoter described in Miller, et al., ~iotechnic~ues, Vol. 7, No. 9, 980-990 (1989), or any other ~ promc>ter (e.g., cellular promoters such as eukaryotic cellular promoters including, but n~t limited to, the W 096/34877 PCTrUS95105616 histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings cont~;ned herein.
The nucleic acid sequence encoding the polypeptide o~
the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such a the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs here;n~hove described); the ~-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the genes encoding the polypeptides.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by re~erence in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

W 096/34877 PCT~US95/05616 The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence (8) encod:ing the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence~s) encoding the polypeptide. Eukaryotic cells which may be transduced inclu~e, but are not limited to, em~ryonic stem cells, embry~nic carC~n~m~ cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cell~, and bro~ch;~l epithelial cells.
'rhe soluble neuropeptide receptor polypeptides and com~pounds which bind to and activate or inhibit activation of a receptor of the present invention may also be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the soluble neuropeptide receptor polypeptide or compounds, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The invention also provides a pharmaceutical pack or kit com~prising one or more cont~;ners filled with one or more of the iLngredients of the pharmaceutical compositions of the inven,tion. Associated with such cont~;ner(s) can be a notice in t~le form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human ~m; n; stration. In addit:ion, the soluble neuropeptide receptor polypeptides or compounds of the present invention may be employed in conjtmction with other therapeutic compounds.
The pharmaceutical compositions may be administered in a convenient m~nner such as by the topical, intravenous, W 096/34877 PCTrUS95105616 intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are ~m; n~ stered in an amount which is effective for treating and/or prophylaxis of the speCific indication. In general, the pharmaceutical compositions will be ~m; n; stered in an amount of at least about 10 ~g/kg body weight and in most cases they will be ~m; n; stered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ~g/kg to about 1 mg/kg body weight daily, taking into account the routes of ~m~ n; stration, s~mptoms, etc.
The present invention also contemplates the use of the genes of the present invention as a diagnostic, for example, some diseases result from inherited defective genes. These genes can be detected by comparing the sequences of the defective gene with that of a normal one. Subsequently, one can verify that a "mutant" gene is associated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector for expression in a functional assay sy6tem (e.g., colorimetric assay, expression on MacConkey plates, complementation experiments, in a receptor deficient strain of HEK293 cells) as yet another means to verify or identify mutations. Once "mutant" genes have been identified, one can then screen population for carriers of the "mutant" receptor gene.
Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR
(Saiki, et al., Nature, 324:163-166 1986) prior to analysis.
RNA or cDNA may also be used for the same purpose. As an example, PCR primers complimentary to the nucleic acid of the W 096/.34877 PCTrUS95/0~616 instant invention can be used to identi~y and analyze mutat:ions in the gene o~ the present invention. For example, delet:ions and insertions can be detected by a change in size of the amplified product in co~r~ri.son to the normal genotype. Point mutations can be identified by hybridizing ampli:Eied DNA to radio labeled RNA of the invention or alternatively, radio labeled antisense DNA sequences of the invenltion. Perfectly matched sequences can be distingll;sh~A
from mismatched duplexes by RNase ~ digestion or by differences in melting temperatu~es. Su~h a diagno8tic would be pa~ticularly useful for prenatal or even neonatal testing.
Sequence differences between the re~erence gene and ~mutants" may be revealed by the direct DNA sequencing methold. In addition, cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly ~nh~nced when combined with PCR. For example, a secluence primer is used with double stranded PCR
product or a single stranded template molecule generated by a modified PCR. The secluence determination is performed by conventional procedures with radio labeled nucleotide or by an automatic sequencing procedure with fluorescent-tags.
Genetic testing based on DNA secluence differences may be achieved by detection o~ alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Secluences changes at specific locations may also be revealed by nucleus protection assays, such RNase and S1 protection or the chemical cleavage method (e.g. Cotton, et al., PNAS, USA, 85:4397-4401 1985).
In addition, some diseases are a result of, or are characterized by changes in gene expression which can be detec:ted by changes in the mRNA. Alternatively, the genes of the present invention can be used as a reference to identify individuals expressing a decrease of functio~s associated with receptors of this type.

W 096/34877 PCTrUS95/05616 The present invention al~o relates to a diagnostic assay for detecting altered levels of soluble forms of the neuropeptide receptor polypeptides of the present invention in various tissues. Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a ho~t are well known to those of skill in the art and include radioi ~ ~noA~says, competitive-h;n~ing assays, Western blot analysis and preferably as 8LISA assay.
An BLISA assay initially comprises preparing an antibody ~pecific to antigens of the neuropeptide receptor polypeptides, preferably a monoclonal antibody. In addition a ~e~o-Ler antibody is prepared against the monoclonal Ant;hoAy To the reporter antibody is attAch~ a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a solid su~oLL, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein h;n~ing sites on the dish are then covered by incubating with a non-specific protein such as bovine serum A lhl-m; n Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antiho~es attach to any neuropeptide receptor proteins attAche~ to the poly~tyrene dish. All unbound monoclonal antibody is ~ ~e~
out with buffer. The ~~-Ler antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to neuropeptide receptor proteins. Unattached reporter antibody is then washed out. Peroxida~e substrates are then added to the dish and the amount of color developed in a given time period is a mea~urement of the ~-.~,~ of neuropeptide receptor proteins present in a given volume of patient sample ~ when compared against a stAn~Ard curve.
The se~lences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on SI~BSTlllrrE SHEET (RULE 2~) W 096/.34877 PCTrUS95/05616 an individual human chromosome. Moreover, there is a current need ior identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available ~or marking chrom~somal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those seq[uences with genes associated with disea~3e.
]3riefly, seq~uences can be mapped to chromosomes by prepa:ring PCR primers (pre~erably 15-25 bp) ~rom the cDN~.
Compu~er analysis o~ the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the ampli$ication process.
These primers are then used for PCR screening of somatic cell hybril~s cont~n~ng individual human chromosomes. Only those hybri~s cont~n;ng the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure ~or assigning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous m~nner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) o~ a cDNA
clone to a met~ph~e chromosomal spread can be used to provide a precise chromosomal location in one step. This techniq[ue can be used with cDNA as short as 50 or 60 bases.
For a review o~ this technique, ee ~erma et al., Human Chromosomes: a ~nll~l o~ Basic Techniq[ues, Pely~oll Press, New York (1988).

W 096/34877 PCTrUS95/0~616 The above techniques were utilized to map the gene corresponding to the neuropeptide receptor of the present invention to chromosome 1 position 31-34.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, MPn~plian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immllnogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chim~ric, single chain, and hllm~nized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be W O 96/34877 PCTrUS95/0~616 obtained by direct injection of the polypeptides into an ~ntm;~ l or by ~r~m~n~fitering the polypeptides to an ~n~m~l, prefe:rably a nonhllm~n. The antibody so obt~; n~rl will then bind the polypeptides itself. In this m~nnPr, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypleptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any techniclue which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma techniclue (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma techniclue, the human B-cell hybridoma technique (Kozbor et al., 1983, Tmmllnology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniclues described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to ;mmllnogenic polypeptide products of this invention. Also, transgenic mice may be used to express hllm~n~zed antibodies to ~nnogenic polypeptide products of this invention.
The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to suLch examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate underst~n-~ng of the ~ollowing exam;c)les certain frecluently occurring methods and/or terms will be described.
"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The start:ing plasmids herein are either co~rcially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain se~uences in the DNA. The various restriction enzymes used herein are c~m~rcially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid CA 02220036 1997-10-31 PC~/US9 5 / O
1PEA~VS 3 ~ MAR ~97 frag~ents (Maniatis, T , et al., Id., p. 146). Unl~ess otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA liga8e ("ligase") per 0.5 ~g of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transform~tion was performed as described in the method o~ Graham, F. and van der Eb, A., Virology, 52:456-457 (1973).

Example 1 Bac~rial Expression and Purification of the Neuropeptide Recel~tor The DNA sequence encoding for neuropeptide receptor, ATCC # 97128 is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the proc,-ssed neuropeptide receptor gen~ (minus the si~lal peptide sequence) and the vector sequences 3~ to the gene.
Addiltional nucleotides corresponding to neuropeptide recep~or nucleotide sequence are added to the 5~ and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' C~CTAAAGG-l-lAATGGAGCCCTCAGCCACC 3' (SEQ ID NO:7) cont~;n~
a Hind III restriction enzyme site followed by 18 nucleotides of neuropeptide receptor coding sequence starting from the preslumed terminal amino acid of the processed protein codon.
The :3~ sequence 5' ACAAGTCCTTGTC~-l-L~-lAGAGGGC 3' (SEQ ID N0:8) and contains an Xbal site. The restriction enzyme sites corrlespond to the restriction enzyme sites on the bacterial exprlession vector pQE-9 (Qiagen, Inc. Chatsworth, CA). pQE-9 enco,des antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is then digested with Hind III ~d Xbal. The amplified sequences are ligated into pQ~-9 and are inserted in ~rame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used AMENDE~ S~t~

W 096/:34877 PCTAUS95/05616 to trans~onm E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory ~n~ , Cold Spring Laboratory Press, (1989). M15/rep4 cnnt~'n~ multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kanr). Trans~ormants are identi~ied by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis. Clones cont~;n~ng the desired constructs are grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The 0/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.~) of between 0.4 an.d 0.6. IPTG (~IIsopropyl-B-D-thiogalacto pyranosidell) is then added to a ~inal concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet: is solubilized in the chaotropic agent 6 Molar Guanicline HCl. After clarification, solubilized neuropeptide recept:or is purified from this solution ~y chromatography on a NicX:el-Chelate column under conditions that allow for tight binding by proteins cont~n~ng the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984). The protein is elutec~ from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incu~ation i.n this solution for 12 hours the protein is dialyzed to 10 mmola:r sodium phosphate.

ExamPle 2 Expression of Recombinant Neuro~e~tide Rece~tor in COS cells IPEA/US 3 ~ 1997 The expression of plasmid, neuropeptide receptor HA is derived from a vector pcDNA3/Amp (Invitrogen) cont~;n;ng~
SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a Sv40 intron and polyadenylation site.
A DNA fragment encoding the entire neuropeptide receptor precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV
promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as pre~iously described (I. Wilson, H. ~iman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to the target protein allows ea~y detection of the recombinant protein with an antibody that recognizes the HA
epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding for neuropeptide receptor, ATCC # 97128, is constructed by PCR using two primers: the 5' primer 5' CCTAGGATGCCCCTCTGCTGCAGCGG 3' (S~Q ID NO:9) contains a BamHI site; the 3' sequence 5~ ACAA~l~-l-l~l CCTTCTAGAGGGC 3' (SEQ ID NO:10) contains co~plementary sequences to an XbaI site, translation stop codon, and the last 17 nucleotides of the neuropeptide receptor coding sequence (not including the stop codon). Therefore, the PCR
prod.uct contains a BamHI site, coding sequence, a translation term~ination stop codon and an XbaI site. The PCR amplified DNA fragment and the vector, pcDNA3/Amp, are digested with BamH:I and XbaI restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain SUR~ (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and re~istant colonies are selected. Plasmid DNA is isolated from transformants and exam.ined by restriction analysis for the presence of the AMENDED S~T

CA 02220036 1997-10-31 PCT/lJS9 5 / O 5 6 t6 1P~IS 3 1 MAR 1997 correct fragment. For expression of the recombinant neuropeptide receptor, COS cells are transfected with the expression vector by DEAE-DEXTRAN me~hod (J. Sambrook, E.
Frit;sch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the neuropeptide receptor HA protein is detected by radio-labelling and immllnoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with 35S-cysteine two days post transf~ction. Culture media are then collected and cells are lysed with detergent (RIPA
buffer (150 mM NaCl, 1% NP-40, 0.1~ SDS, 1% NP-40, 0.5% DOC, 50m~[ Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)).
sotklcell lysate and culture media are precipitated with a HA
specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.

Example 3 Clor.Linq and exPression of Neuropeptide Rece~tor usinq the baculovirus expression s~stem The DNA sequence encoding the fu]l length neuropeptide receptor protein, ATCC # 97128, is amplified using PCR
olicronucleotide primers corresponding to the 5' and 3' seqllences of the gene:
The 5~ primer has the sequence 5~ CGGGATCCGCCATCATGGAG
CCCTCAGCCACC 3~ (SEQ ID NO:11) and contains a BamHI
rest:riction enzyme site (in bold) followed by 6 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (J. Mol. Biol. 1987, 196, 947-950, Xozak, M.). The initiation codon for translation "ATG" is underlined).
The 3~ primer has the sequence 5~ ACAAGT~ ~lCCTTCT
AGA~GGC 3' (SEQ ID NO:12) and contains the cleavage site for the restriction endonuclease XbaI and 5 nucleotides comE~lementary to the 3~ non-translated sequence of the .q.~
AMENDE~

W 096i34877 PCTrUS95/05616 neuropeptide receptor gene. The amplified sequences are isolated from a 1% agarose gel using a romm~cially available kit ("Geneclean," BI0 101 Inc., La Jolla, Ca.). The fragment is then digested with the en~onllcleases BamHI and XbaI and then purified as described in ~xample 1. This fragment is designated F2.
The vector pA2 (modification of pVL941 vector, discussed below) is used for the expression of the neuropeptide receptor protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A m~nll~l of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experim~ntAl Station Bulletin N0:1, 3 and 5555). This e~?ression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhidrosis virus (AcMNPV) followed by the recognition sites for the restriction ~n~nllcleases BamHI and XbaI. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of reco~lbinant viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homo]ogous recQm~in~tion of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
The plasmid is digested with the restriction enzymes BamH:[ and XbaI and then dephosphorylated using calf intestinal phosphatase by procedures ~own in the art. The DNA is then isolated from a 1% agarose gel as described in Exam]?le 1. This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 are liga~ed with T4 DNA ligase. DH5~ are then transformed and bactleria identified that cont~in~ the plasmid (pBac W 096/34877 PCTrUS95/05616 neuropeptide receptor) with the neuropeptide receptor gene using the enzyme~; BaTnHI and XbaI. The sequence o~ the cloned fragment is confirmed by DNA sequencing.
5 llg of the plasmid pBac neuropeptide receptor are co-transfected with 1.0 ~g of a commercially available linearized baculovirus ("BaculoGoldn' baculovirus DNA", Pharmingen, San Diego, CA.) using the lipo~ection method (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the plasmid pBac neuropeptide receptor are mixed in a sterile well o~ a microtiter plate cont~;n;ng 50 ~1 of serum free Grace's medium (Life Technologies Inc., Gaithersburg, ~).
Afterwards 10 ~Ll Lipofectin plus 90 ~l Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace' medium without serum. The plate is rocked back and ~orth to mix the newly added solution. The plate is then incubated ~or 5 hours at 27~C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation continued at 27~C i~or four days.
A~ter four days the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. (A
detailed description of a "plaque assay" can also be ~ound in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .

W 096/34877 PCTrUS95/05616 Four days after the serial dilution the virus is added to the cells and blue sti~n~ plac~ues are picked with the tip of an Eppendorf pipette. The agar conti~;ntng the reComhtni~nt viruses is then resuspended in an Eppendorf tube contAtntng 200 ~1 of Grace~s medium. The agar is removed by a brief centrifugation and the supernatant cont~tntng the recnmhtni~nt baculoviruses is used to infect Sf9 cells seeded in 35 mm ~t hPs Four days later the supernatants of these culture dishes are harvested and then stored at 4~C.
Sf9 cells are grown in Grace's medium supplemented with 10% ~eat-inactivated FBS. The cells are infected with the recombinant baculovirus V-neuropeptide receptor at a multiplicity of infection (MOI) of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gait~ersburg). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 3-'S cysteine (Amersham) are added. The cells are $urther incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Exam~le 4 Expression via Gene TherapY
Fibroblasts are obt~nP~ from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is t:urned upside down, closed tight; and left at room temperature over night. After 24 hours at room temperature, the ~Elask is inverted and the chunks oE tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 medii~, with 10~ FBS, penicillin and streptomyc~n, is added.
This is then incubated at 37~C for approximately one week.
At this time, fresh media is added and subsequently changed , every ~everal days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer contAining an EcoRI site and the 3' primer having contains a HindIII site.
Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintAin~ under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10~ calf serum (CS), penicillin and streptomycin. The MSV vector contAining the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles cont~intng the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, contA;n~ng the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and ~uickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marke~r, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the ~rotein product.
Numerous modifications and variations o~ the present inven.tion are possible in light of the above teachings and, there!fore, within the scope of the appended claims, the inven.tion may be practiced otherwise than as particularly described.

W 096/34877 PCTrUS95/05616 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: LI, ET AL.
(ii) TITLE OF lNv~NllON: Hum~n Neuropeptide Receptor (iii) NUMBER OF SEQUENCES: 12 (iv) rORR~:~P~)NL~N~:~; ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) ~:OIJN1~Y: USA
(F) ZIP: 07068 (v) COM~ul~ READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COM~ul~: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: concurrently (C) CLASSIFICATION:
(vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134 (C) REFERENCE/DOCKET NUMBER: 325800-268 (viii) TELECOMMnNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUEN OE CHARACTERISTICS
(A) LENGTH: 1209 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRA-NL~ Nlt:SS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATGGAGCCCT CAGCCACCCC AGGGGCCCAG AlGGGG~lCC CC'C-~-lGGCAG CAGAGAGCCG 60 CCL-lL~lGC CTCCAGACTA TGAAGATGAG ~ L'lCLGCT Al~-l~l~GCG TGATTATCTG 120 CA 02220036 l997-lO-3l TACCCA;~AAC AGTATGAGTG G~1-~-1'~ATC CCAGCCTATG ~LGG~ C~lC~LGGCC 180 L-1~L~a~GCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240 ACCAAC~rACT TCATTGTCAA C~1'~L~C-1~ GCTGACGTTC TGGTGACTGC TATCTGCCTG 300 CCGGCC~GCC TG~1~aLW A CATCACTGAG TCCTGGCTGT TCGGCCATGC C~-l.-lG~AAG 360 GTCA'LCI_C~1' ATCTACAGGC '1'~'1~a'LCC~L~ TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420 GCCCTG~;ACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480 GCC~Ia'L~aGCT CCA'1'~-L~GG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540 GCAGTC~TGG AATGCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG G~-1~-11'~-1'~A 600 ~1~-L~L~ATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTG~-l-L~-l-l-L 660 ATTGTC~CCT ACCTGGCCCC ACT WW CCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720 AAC~1~L~GG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCW AA CTGGAAGCGC 780 CCCTCAraACC AG~-1GGGG~A CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCGGGGC 840 CGCGCCrTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900 ATGW TGaTGC TG-LW 1~11 CGCC~L~L~aC TACCTGCCCA TCA.GCGTCCT CAA1~'1'~L1-1' 960 AAGAGG~aTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAG~L~'1~1'A CGCCTGCTTC 1020 A~-11~-1CCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTAcAAcTTc 10 80 CTCAGT~GCA AA'l lCCGGGA GCAGTTTAAG GCTGCCTTCT CCTGCTGCCT GCCTGGCCTG 1140 G~L~C~-'L~CG G~-1-~-L~-L~AA GGCCCCTAGT CCCCGCTCCT CTGCCAGCCA CAA~-lC~-l-L~ 1200~LC~-1-L~'1'AG 1209 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 402 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STR~NDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQ~ DESCRIPTION: SEQ ID NO:2:
Met Glu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro ~5 Gly Ser Arg Glu Pro Ser Pro Val Pro Pro Asp Tyr Glu Asp Glu Phe Leu Arg Tyr Leu Trp Arg Asp Tyr Leu Tyr Pro Lys Gln Tyr Glu Trp Val Leu Ile Ala Ala Tyr Val Ala Val Phe Val Val Ala l60 Leu Val Gly Asn Thr Leu Val Cys Leu Ala Val Trp Arg Asn His His Met Arg Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ser L~eu Ala ~,sp Val Leu Val Thr Ala Ile Cys Leu. Pro Ala Ser Leu Leu Val Asp Ile Thr Glu Ser Trp Leu Phe Gly His Ala Leu Cys Lys Val Ile Pro Tyr Leu Gln Ala Val Ser Val Ser Val Ala Val Leu Thr Leu Ser Phe Ile Ala Leu Asp Arg Trp Tyr Ala Ile Cys His Pro I,eu Leu Phe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser Ile Leu C;ly Ile Trp Ala Val Ser Leu Ala Ile Met Val Pro Gln Ala ~la Val Met Glu Cys Ser Ser Val Leu Pro Glu Leu Ala Asn Arg 185 190 195~hr Arg Leu Phe Ser Val Cys Asp Glu Arg Trp Ala Asp Asp Leu 200 205 210~yr Pro Lys Ile Tyr His Ser Cys Phe Phe I~e Val Thr Tyr Leu 215 220 225~la Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gln Ile Phe Arg 230 235 - 240~ys Leu Trp Gly Arg Gln Ile Pro Gly Thr Thr Ser Ala Leu Val 245 250 255~rg Asn Trp Lys Arg Pro Ser Asp Gln Leu Gly Asp Leu Glu Gln 260 265 270~ly Leu Ser Gly Glu Pro Gln Pro Arg Gly Arg Ala Phe Leu Ala 275 280 285~lu Val Lys Gln Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu 290 295 300~et Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro Ile Ser 305 310 315~al Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gln Ala 320 325 330~er Asp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp 335 340 345~eu Val Tyr Ala Asn Ser Ala Ala Asn Pro Ile Ile Tyr Asn Phe 350 355 360~eu Ser Gly Lys Phe Arg Glu Gln Phe Lys Ala Ala Phe Ser Cys 365 370 375~ys Leu Pro Gly Leu Gly Pro Cys Gly Ser Leu Lys Ala Pro Ser Pro Arg Ser Ser Ala Ser His Lys Ser Leu Ser Leu (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1110 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATGGAGCCCT CAGCCACCCC AGGGGCCCAG Al~GGG~lLC' C'C'C~-L~GCAG CAGAGAGCCG 60 lL-CCL-l~lGC CTCCAGACTA TGAAGATGAG l-l-lL-lLL~CT AlL-lLl-LGCG TGATTATCTG 120 TACCCA~AAC AGTATGAGTG GGl~-~-lLATC GCAGCCTATG TGG~-lL;lL-l-l C-~lL-L-LLiGCC 180 ACCAACTACT TCATTGTCAA C~-1L;1CC~-1L; GCTGACGTTC TGGTGACTGC TATCTGCCTG 300 CCGGCCAGCC TG~-l-Li~GA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360 GTCATCCCCT ATCTACAGGC lL;lLil~C'~lL TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420 CCC~-lGGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480 GCC'~lGGCT CCAlC~-lGGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540 GCAGTCATGC AATCCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG G~-1L-1-1L-1LA 600 L-lL-lL-lLATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTG~-l-lL-l-l-l 660 CCCTCAGACC AGCTGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCC'GGGGC 840 Al w 1~71~C TG~-l~l~-l-l ~C~-l~-l~C TAC~lCCC~A TCAGCGTCCT CAAl~lC~-l-l 960 AAGAGGC;TGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAG~-L~l~-lA CGCCTGCTTC 1020 AC~-l-l~-lCCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080 CTCAGTGGCC llcc~-l~AG TCTGCTCTAA 1110 (2) INFORMATION FOR SBQ ID NO:4:
~i) SE~ CHARACTERISTICS
(A) LENGTH: 369 BASE PAIRS
(B) TYPE: AMINO ACID
(C) STRAhv~vN~SS: SINGLE
(D) TOPOLOGY: LINEAR
(iiL) MOLECULE TYPE: cDNA
(x.L) SEQ~ DESCRIPTION: SEQ ID NO:4:
Met G:Lu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro Gly Ser Arg Glu Pro Ser Pro Val Pro Pro Asp Tyr Glu Asp Glu Phe Leu Arg Tyr Leu Trp Arg Asp Tyr Leu Tyr Pro Lys Gln Tyr Glu T:rp Val Leu Ile Ala Ala Tyr Val Ala Val Phe Val Val A].a Leu V;~l Gly Asn Thr Leu Val Cys Leu Ala Val Trp Arg Asn Eis His M~et Arg Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ser Leu ~0 Ala A,sp Val Leu Val Thr Ala Ile Cys Leu Pro Ala Ser Leu Leu 10.0 105 Val Asp Ile Thr Glu Ser Trp Leu Phe Gly His Ala Leu Cys Lys Val Ile Pro Tyr Leu Gln Ala Val Ser Val Ser Val Ala Val Leu Thr Leu Ser Phe Ile Ala Leu Asp Arg Trp Tyr Ala Ile Cys Hi.s Pro Leu Leu Phe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser Ile Leu Gly Ile Trp Ala Val Ser Leu Ala Ile Met Val Pro Gln Ala 170 175 1~0 Ala Val Met Glu Cys Ser Ser Val Leu Pro Glu Leu Ala Asn Arg Thr Arg Leu Phe Ser Val Cys Asp Glu Arg Trp Ala Asp Asp Leu Tyr Pro Lys Ile Tyr His Ser Cys Phe Phe Ile Val Thr Tyr Leu Ala Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gln Ile Phe Arg 230 235 2~0 Lys L.eu Trp Gly Arg Gln Ile Pro Gly Thr Thr Ser Ala Leu Val Arg A.sn Trp Lys Arg Pro Ser Asp Gln Leu Gly Asp Leu Glu Gln ~ly Leu Ser Gly Glu Pro Gln Pro Arg Gly Arg Ala Phe Leu Ala 275 280 285~lu Val Lys Gln Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu 290 295 300~et Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro Ile Ser 305 310 315~al Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gln Ala 320 325 330~er Asp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp 335 340 345~eu Val Tyr Ala Asn Ser Ala Ala Asn Pro Ile Ile Tyr Asn Phe Leu Ser Gly Leu Pro Trp Ser Leu Leu (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1133 BASE PAIRS
~B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO:5:
ATGGAGCCCT CAGCCACCCC AGGGGCC~-AG ATGGGGGTCC CCC~-LGGCAG CAGAGACCCC 60 1-CCC.-1~1GC CTCCAGACTA TGAAGATGAG 'l-l-l'~'lC~-l Al~-L~LGGCG TGATTATCTG 120 TACCCAAAAC AGTATGAGTG G~1CL-1~ATC GCAGCCTATG TGG.-1~1~1-1 C~L~1GGCC 180 ACCAACTACT TCATTGTCAA C~-1~LCC~-LG GCTGACGTTC TGGTGACTGC TATCTGCCTG 300 CCGGCCAGCC TG.-1~G1~GA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360 GTCATCCCCT ATCTACAGGC 1~L~1C~1G TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420 GCC~-1GGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480 GCCC~1GGCT CCAL~-1GGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540 GCAGTCATGG AATGCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG G~-1~L1~-1~A 600 ~1~-L~1~ATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTG~-l~ Ll 660 CCCTCAGACC AG~-LGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCG&GGC 840 A1G~L~1GC TGCTGGTCTT CGCC~-L~-1GC TACCTGCCCA TCAGCGTCCT CAA1~1C-11 960 AAGAGGGTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAG~-L~1~1A CGCCTGCTTC 1020 AC~-1-1~-1CCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080 CTCAGTGGAT GTAAAGAGAA GAGTCTAGTT ~-L~1C~-1GAC CATCGTGCCC CGG 1133 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 377 BASE PAIRS
(B) TYPE: AMINO ACID
(C) STR~NDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

W O961'34877 PCT~US95/05616 Met G:Lu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro 15~ly Ser Arg Glu Pro Ser Pro Val Pro Pro Asp Tyr Glu Asp Glu 30~he Ll~u Arg Tyr Leu Trp Arg Asp Tyr Leu Tyr Pro Lys Gln Tyr 45~lu T:~p Val Leu Ile Ala Ala Tyr Val Ala Val Phe Val Val Ala 60~eu Val Gly Asn Thr Leu Val Cys Leu Ala Val Trp Ary Asn His 75~is Met Arg Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ser Leu gO~la A~sp Val Leu Val Thr Ala Ile Cys Leu Pro Ala Ser Leu Leu 100 1()5~al Al~p Ile Thr Glu Ser Trp Leu Phe Gly Hi~ Ala Leu Cys Lys 110 115 120~al Ile Pro Tyr Leu Gln Ala Val Ser Val Ser Val Ala Val Leu 125 130 135~hr L,eu Ser Phe Ile Ala Leu Asp Arg Trp Tyr Ala Ile Cy~ Hi~
140 145 150~ro Lleu Leu Phe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser Ile 155 160 165~eu Gly Ile Trp Ala Val Ser Leu Ala Ile Met Val Pro Gln A~La 170 175 1~30~la V,al Met Glu Cys Ser Ser Val Leu Pro Glu Leu Ala Asn Arg 185 190 195~hr Arg Leu Phe Ser Val Cys Asp Glu Arg Trp Ala Asp Asp Leu 200 205 210~yr Pro Lys Ile Tyr His Ser Cys Phe Phe Ile Val Thr Tyr Leu 215 220 225~la Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gln Ile Phe Arg 230 235 2gO~ys Leu Trp Gly Arg Gln Ile Pro Gly Thr Thr Ser Ala Leu Val 245 250 255~rg Asn Trp Lys Arg Pro Ser Asp Gln Leu Gly Asp Leu Glu Gln 260 265 270~ly Leu Ser Gly Glu Pro Gln Pro Arg Gly Arg Ala Phe Leu Ala 275 280 2~35~lu Val Lys Gln Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu 290 295 300~et Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro Ile Ser 305 310 315~al Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gln Ala 320 325 330~er A.sp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp 335 340 345~eu Val Tyr Ala Asn Ser Ala Ala Asn Pro Ile Ile Tyr Asn Phe 350 355 360~eu Ser Gly Cys Lys Glu Lys Ser Leu Val Leu Ser Pro Ser Cy~;
365 370 375~ro G,ly W 096/34877 PCTrUS95/05616 ~2) INFORMATION FOR SEQ ID NO:7:
(i) SEQU~N~h CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANv~N~SS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CACTA~AGCT TAATGGAGCC CTCAGCCACC 30 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 25 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQU~N~ DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 26 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CCTAGGATGC C~-l~-lGCTG CAGCGG 26 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUEN OE CHARACTERISTICS
(A) LENGTH: 25 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

(2) INFORMATION FOR SEQ ID No~
(i) SEQUENOE CHARACTERISTICS
(A) LENGTH: 32 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR~NDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CGGGATCCGC CATCATGGAG ~C~-L~AGCCA CC 32 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 25 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOhOGY: LINEAR
(i.i) MOLECULE TYPE Oligonucleotide (xi) SEQU~N~ DESCRIPTION: SEQ ID NO:12:
ACAAGTCCTT GT~-l-L~-lAG AGGGC 25

Claims (34)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide as set forth in SEQ ID NO:2;
(b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or (b).

2. The polynucleotide of Claim 1 encoding the polypeptide as set forth in SEQ ID NO:2.

3. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

4. The polynucleotide of Claim 1 wherein said polynucleotide is RNA.

5. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.

6. The polynucleotide of Claim 1 comprising from nucleotide 1 to nucleotide 1209 as set forth in SEQ ID
NO:1.

7. The polynucleotide of Claim 1 encoding a soluble form of the polypeptide of SEQ ID NO:2.

8. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding a mature polypeptide encoded by the DNA contained in ATCC Deposit No. 97128;

(b) a polynucleotide encoding the polypeptide expressed by the DNA contained in ATCC Deposit No. 97128;
(c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).

9. The polynucleotide of Claim 8 wherein said polynucleotide encodes a polypeptide expressed by the DNA
contained in ATCC Deposit No. 97128.

10. The polynucleotide of Claim 8 wherein said polynucleotide encodes a polypeptide expressed by the DNA
contained in ATCC Deposit No. 97128.

11. A vector containing the DNA of Claim 2.

12. A host cell transformed or transfected with the vector of Claim 11.

13. A process for producing a polypeptide comprising: expressing from the host cell of Claim 12 the polypeptide encoded by said DNA.

14. A process for producing cells capable of expressing a polypeptide comprising transforming or transfecting the cells with the vector of Claim 11.

15. A receptor polypeptide selected from the group consisting of:
(i) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof; and (ii) a polypeptide encoded by the cDNA of ATCC
Deposit No. 97128 and fragments, analogs and derivatives of said polypeptide.

16. The polypeptide of Claim 15 wherein the polypeptide has the deduced amino acid sequence of SEQ ID
NO:2.

17. An antibody against the polypeptide of claim 15.

18. A compound which activates the polypeptide of claim 15.

19. A compound which inhibits activation the polypeptide of claim 15.

20. A method for the treatment of a patient having need to activate a neuropeptide receptor comprising:
administering to the patient a therapeutically effective amount of the compound of claim 18.

21. A method for the treatment of a patient having need to inhibit a neuropeptide receptor comprising:
administering to the patient a therapeutically effective amount of the compound of claim 19.

22. The method of claim 20 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA encoding said agonist and expressing said agonist in vivo.

23. The method of claim 21 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA

encoding said antagonist and expressing said antagonist in vivo.

24. A method for identifying compounds which bind to and activate the receptor polypeptide of claim 15 comprising:
providing a recombinant host cell expressing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide;
contacting a plurality of compounds with said host cell under conditions sufficient to permit binding of compounds to the receptor polypeptide; and identifying those compounds capable of receptor binding by detecting the signal produced by said second component.

25. An agonist compound identified by the method of claim 24.

26. A method for identifying compounds which bind to and inhibit activation of the polypeptide of claim 15 comprising:
providing a recombinant host cell expressing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide contacting an analytically detectable ligand known to bind to the receptor polypeptide and a plurality of compounds with said host cell under conditions to permit binding to the receptor polypeptide; and determining whether the ligand binds to the polypeptide by detecting the absence of a signal generated from the interaction of the ligand with the polypeptide.

27. An antagonist compound identified by the method of claim 26.

28. A process for determining whether a ligand not known to be capable of binding to the polypeptide of claim 15 can bind thereto comprising:
contacting a recombinant host cell expressing on the surface thereof the polypeptide with a ligand to be identified under conditions permitting binding and detecting the presence of any ligand-bound receptor.

29. The method of claim 28 wherein the receptor polypeptide or a membrane fraction containing the receptor is isolated from said cell prior to contacting with the ligand to be identified.

30. A method of screening compounds to identify those compounds which bind to the receptor polypeptide of claim 15 comprising:
contacting a recombinant host cell expressing the receptor on the surface thereof with a plurality of candidate compounds and an analytically detectable ligand known to bind to the receptor, under conditions permitting binding to the receptor; and identifying those candidate compounds capable of enhancing or inhibiting the binding of the ligand to the receptor.

31. An antagonist or agonist compound identified by the method of claim 30.

32. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 15 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.

33. The polypeptide of Claim 15 wherein the polypeptide is a soluble fragment of the polypeptide and is capable of binding a ligand for the receptor.

34. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 33 in a sample derived from a host.