WO1996034877A1 - 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

HUMAN NEUROPEPTIDE RECEPTOR

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptides of the present invention are human 7-transmembrane 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 is the commonest nutritional disorder in Western societies. More than three in ten adult Americans weigh at least 20% in excess of 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 Type II diabetes, hypertension, hyperlipidemia and certain cancers (Grundy, S.M., and Barnett, J.P., Disease-a- Month, 36:645-696 (1990)).

Five single-genε 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 homologue have been reported (Zhang, Y. , et al., Nature, 372:425-431 (1994)). The ob gene encodes a 4.5-kb adipose tissue 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 (id. 425) .

Of the brain regions implicated in the regulation of feeding behavior, the ventromedial nucleus of the hypothalamus (VMH) is 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 feedback loop in which the amount of stored energy is sensed by the hypothalamus, which adjusts food intake and energy expenditure to maintain 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 balance by interacting with the hypothalamus (Kennedy, G.C., Proc. R. Soc.148:578-592 (1953)).

The inability to identify the putative signal from fat has hindered 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 animal with a VMH lesion across a vascular graft to an untreated animal (a parabiosis experiment) resulted in a reduction of food intake in the intact animal. 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 part of a homeostatic mechanism to maintain constancy of the adipose mass (Zhang, Y., et al., Nature, 372:425-431, 431 (1994) ) . 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) ) .

Parabiosis experiments suggest that the ob receptor is encoded by the mouse db (diabetes) gene (Coleman, D.L. , Diabetologia, 14:141-148 (1978)) . Mice having a mutation in the db gene are also obese, with the defect possibly being 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 four types of neuropeptide Y receptors called Y_1# 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 the central nervous system (CNS) and the peripheral nervous system (PNS) . In the PNS, neuropeptide Y is found in the noradrenergic sympathetic innervation of blood vessels and other smooth muscle tissues and in neurons within the enteric nervous system. Neuropeptide Y immunoreactive fibers also occur in the non-vascular smooth muscle, surrounding exocrine glands and surface epithelia. Neuropeptide Y also occurs in subpopulations of neurons and is generally co-localized with other neurotransmitters, particular noradrenaline.

In the CNS, neuropeptide Y is contained in GABAergic interneurons in higher centers and in predominantly catecholaminergic cells that project further caudally. For example, 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 the locus coeruleus (LC) . In the hypothalamus, neuropeptide Y is found predominantly in the arcuate nucleus and lateral hypothalamus.

Within the peripheral nervous system, neuropeptide Y is present in postganglionic sympathetic nerves, and is co- localized as stated above with other neurotransmitters, including catecholamines. 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 of 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 containing 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 demonstration of all durations in neuropeptide Y concentrations in hypertensive animal 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 predominantly 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 Alzheimer'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 regulatory agent and pituitary function as well as potential neuromodulatory function in stress responses and in reproductive function.

In accordance with one aspect of the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.

In accordance with a further aspect of the present invention, there are provided processes for producing such receptor polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences encoding the receptor polypeptides of the present invention, under conditions promoting expression of said polypeptides and subsequent recovery of said polypeptides.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such receptor polypeptides.

In accordance with another aspect of the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation of the receptor polypeptides of the present invention.

In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and activate the receptor polypeptide of the present invention which are useful in the prevention and/or treatment of obesity, hyperlipidemia, certain cancers, to stimulate neuronal growth, to regulate neurotransmission, to enhance activity levels and utilization of ingested foods.

In accordance with another aspect ^"of the present invention there is provided a method of administering 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 inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of Alzheimer's disease, Type II Diabetes Mellitus, epilepsy, stress, anxiety, hypertension, cardiovascular disease, 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. The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of the neuropeptide receptor polypeptide of the present invention. The standard one- letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.) .

Figure 2 shows the cDNA sequence and the corresponding deduced amino acid sequence of the neuropeptide receptor splice variant 1 polypeptide of the present invention. The standard one-letter abbreviation for amino acids is used.

Figure 3 shows the cDNA sequence and the corresponding deduced amino acid sequence of the neuropeptide receptor splice variant 2 polypeptide of the present invention. The standard one-letter abbreviation for amino acids is used.

Figure 4 illustrates the amino acid sequence and seven transmembrane regions of the neuropeptide receptor. The transmembrane regions are underlined and denoted with a TM.

Figure 5 illustrates the amino acid sequence and seven transmembrane regions of the neuropeptide receptor splice variant 1. The transmembrane 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 transmembrane 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 receptors for ligands, both known and unknown, which modulate the activity of cells in both the central nervous system and peripheral tissues regulated by the central nervous system. Examples 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 present 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 for 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 (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone(s) deposited as ATCC Deposit No. on April 27, 1995.

The polynucleotide of this invention was discovered in a cDNA library derived from human adult hypothalamus. It is structurally related to the G protein-coupled receptor family. The neuropeptide receptor polypeptide contains an open reading frame encoding a protein of 402 amino acid residues. The neuropeptide receptor protein exhibits the highest degree of homology to human neuropeptide _λ receptor protein with 52 % similarity 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 includes cDNA, 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 (SEQ ID N0:1) or that of the deposited clone(s) or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 (SEQ ID N0:1) or the deposited cDNA(s) .

The polynucleotides which encode for the mature polypeptide of Figure 2 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA(s) may include: only the coding sequence for the mature polypeptide; the coding sequence 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 mature polypeptide.

Thus, 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 non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced 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 mature polypeptide encoded by the cDNA of the deposited clone(s) as well as variants of such polynucleotide which variants encode for a fragment, derivative 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 include deletion variants, substitution variants and addition or insertion variants. Specific examples of such variants include the polynucleotide sequences as set forth in SEQ ID NOS:3 and 5 which encode for splice 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 l (SEQ ID N0:1) 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 substantially 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 domain 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 marker 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 mammalian 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 hereinabove-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, 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 embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID N0:1) or the deposited cDNA(s) , i.e. function as a soluble neuropeptide receptor by retaining the ability to 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 polynucleotides 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 hereinabove described, and which does not retain 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 maintained 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 TJ.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. The present invention further relates to a polypeptide which has the deduced amino acid sequence of Figure 2 (SEQ ID N0:2) 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" 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 retaining 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 recombinant 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 or (iv) splice variants of the mature polypeptide which may have one or more amino acids deleted from the mature polypeptide yet still retain activity corresponding to the mature polypeptide. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons) .

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also relates to vectors which include 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 may 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 recombinant 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 combinations 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 endonuclease 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: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P_L 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. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably 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 for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Streptomyces. Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruseε; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into 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, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen) , pbs, pDIO, phagescript, psiX174, pbluescript_^ SK, pbskε, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; pTRC99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia) . Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, 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 (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P_R, P_L and trp. Eukaryotic promoters include CMV immediate 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 containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian 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 manner 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 for producing the full- length polypeptides. Fragments of the polynucleotides of the present invention may be used in a similar manner to synthesize the full-length polynucleotides of the present invention.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, 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 enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPi gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , o.-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant 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 tvphimurium and various species within the genera Pseudomonaε, 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" sections are combined with an appropriate promoter and the structural sequence 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. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblaεtε, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necesεary riboεome binding sites, polyadenylation site, splice donor and acceptor siteε, tranεcriptional termination εequences, and 5' flanking nontranscribed sequences. DNA sequenceε derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranεcribed genetic elementε.

The neuropeptide receptor polypeptide of the present invention can be recovered and purified from recombinant cell cultures by methods including ammonium εulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phoεphocelluloεe chromatography, hydrophobic interaction chromatography, affinity 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 recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycoεylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The polynucleotides and polypeptides of the preεent invention may be employed aε research reagents and materials for discovery of treatments and diagnosticε to human diεease.

The human neuropeptide receptor polypeptides of the present invention may be employed in a proceεε for εcreening 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 thoεe compoundε are εelected which bind to and interact with the receptor. The binding or interaction can be meaεured directly by uεing radioactively labeled compoundε of intereεt or by the εecond meεεenger effect reεulting from the interaction or binding of the candidate compound. Alternatively, the candidate compoundε 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 enhance 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. For example, to screen for compounds which inhibit activation of the receptor polypeptide of the present invention, the compound and a ligand known to bind to the receptor are both contacted with the melanophore cells. Inhibition of the signal generated by the ligand indicates that the compound inhibits activation of the receptor.

The screen may be employed for determining a compound which bindε to and activates the receptor polypeptide of the present invention by contacting such cellε with compoundε to be screened and determining 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, transfected CHO cells) in a system which measureε extra¬ cellular pH changes caused by receptor activation, for example, aε deεcribed in Science, volume 246, pageε 181-296 (October 1989) . For example, compoundε may be contacted with a cell which expreεεeε an neuropeptide receptor polypeptide of the present invention and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential compound is effective as an activator or inhibitor.

Another example involves introducing RNA encoding a neuropeptide receptor of the present invention into Xenopuε 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 inhibition of or an increase in intracellular calcium.

Another example involves expresεing a neuropeptide receptor polypeptide of the preεent invention on the surface of a cell wherein the receptor is linked to a phospholipaεe C or D. As representative examples of such cellε there may be mentioned endothelial cells, smooth muscle cellε, embryonic kidney cells, etc. The screening may be accompliεhed aε hereinabove deεcribed by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second εignal.

Another method involveε determining inhibition of binding 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 preεent invention εuch that the cell expresseε the receptor on itε εurface 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 bindε to the receptor aε 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 expresεing a neuropeptide receptor polypeptide on the surface of a cell wherein the receptor is linked to a εecond meεεenger to increaεe cytoεolic calcium levelε in tranεfected CHO cellε. An example of εuch a method comprises transfecting CHO cells with a nucleic acid sequence encoding a receptor of the present invention such that the receptor iε expreεεed on the εurface thereof. The tranεfected 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 inhibit 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. To measure intracellular cyclic AMP levels, cyclic AMP is asεayed in whole cellε treated for 15 minuteε at 37°C with 100 micromolar isobutylmethylxanthine (IBMX; Sigma) . Transfected cells (1 x 10⁶ / 0.5 ml reaction) are incubated with 10 micromolar forskolin and variouε concentrationε of known or unknown ligands to the receptor. Reactions are terminated with the addition of HC1 to 0.1M, incubation at room temperature for 15 minutes, neutralization and sample dilution in 50 mM sodium acetate, pH 6.2. Cyclic AMP is quantified by using a radioimmunoassay (Dupont/NEN) .

To meaεure levelε of intracellular calcium, tranεfected cellε are suspended in loading medium (modified RPMI 1640 medium/10 mM Hepes/1% newborn calf serum) and incubated in a spinner flaεk at 37°C for 2.5 hour at 1 x 10⁶ cellε per ml. Cellε are then treated with 1 micromolar Fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes) for 30 minuteε at 37°C, washed twice with loading medium, and reεuspended at 5 x 10⁶ cells/ml. Immediately before fluorescence spectroεcopy, cellε are recovered by centrifugation at 1000 rpm and reεuεpended at 1 x 10 cellε/ml in a modified Krebε buffer (135 mM NaCl/4.7 mM KC1/1.2 mM MgS0₄/l.2 mM KH₂P0₄/5 mM NaHC0₃/l mM CaCl₂/2.8 mM glucose/10 mM hepes, pH 7.4) containing sulfinpyrazone. Bombesin is purchased from Sigma and Auspep. Fluorescence recordingε are made on a Hitachi fluoreεcence spectrometer (F4010) at 340 nm (excitation) and 505 nm (emisεion) over 10 minuteε with slit widths of 5 nm and responεe time of 2 εecondε. Intracellular calcium iε quantified by uεing 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 by administering to a host a compound which binds to and activates the receptor polypeptides of the present invention. Such a compound is other than the ob gene product disclosed in Zhang, et al., Nature, 372:425-431 (1994) . The receptor polypeptide of the present invention maps to a human chromosome which correspondε to the poεition of the mouse chromosome which encodeε for the receptor of the ob gene product. The human ob gene encodeε a "εatiety" factor which bindε to and activateε the receptor polypeptide of the preεent invention. Accordingly, a compound which activateε 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, enhance 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 hyperlidimeia, type II diabetes and certain cancers.

Theεe compoundε may alεo be employed to treat and/or prevent other conditionε related to an underexpreεεion of the receptor polypeptide of the preεent invention or ligandε which bind thereto, for example, to εtimulate neuronal growth.

Specific exampleε of compounds which inhibit activation of the receptor polypeptides of the present invention include an antibody, or in some caseε an oligonucleotide, which bindε to the receptor but does not elicit a second meεεenger reεponse such that the activity of the receptor iε prevented.

Another example iε proteinε which are cloεely 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 reεponεe.

Another example includeε an antiεense construct prepared through the use of antisense technology. Antisenεe technology can be uεed to control gene expreεsion 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 sequence, which encodes for the mature polypeptideε of the preεent invention, iε used to deεign 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 tranεcription (triple helix -εee Lee et al., Nucl. Acidε Reε., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing tranεcription and the production of a neuropeptide receptor polypeptide of the present invention. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the receptor (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisenεe Inhibitorε of Gene Expreεsion, CRC Press, Boca Raton, FL (1988) ) . The oligonucleotides deεcribed above can alεo be delivered to cellε such that the antisenεe RNA or DNA may be expreεεed in vivo to inhibit production of the receptorε.

Another example iε a εmall molecule which bindε to a neuropeptide receptor polypeptide of the preεent invention, making it inaccessible to ligands such that normal biological activity iε prevented. Examples of small moleculeε 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 inhibit activation of the receptor polypeptides of the present invention.

This invention additionally provideε a method of utilizing εuch compounds which inhibit activation for treating abnormal conditions related to an excess of activity of a neuropeptide receptor polypeptide of the present invention for treating obesity εince the neuropeptide receptor polypeptides of the present invention may bind neuropeptide Y which is the most potent known subεtance 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 polypeptideε of the present invention may be employed to treat and/or prevent hypertension εince neuropeptide Y stimulates renin release and neuropeptide Y is known to have potent vasoconstrictor activity when involving the coronary and cerebral vesεelε.

The compoundε may alεo be employed to treat Alzheimer'ε diεeaεe εince neuropeptide Y receptorε are prevalent in the central nervouε εyεtem and are localized predominantly within interneuronε where they appear to have regulatory roleε in memory and Alzheimerε diεeaεe.

The compounds may also be employed to εuppresε excitatory tranεmission 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 nervouε εystem indicates that the compounds which inhibit the neuropeptide receptor polypeptides of the present invention may be used as an antipsychotic drug by regulating neurotransmisεion.

The compoundε which inhibit the receptor polypeptideε of the preεent invention may alεo be employed to treat pathological vasospaεm 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 compriseε contacting the ligand to be identified with a cell compriεing the coding εequence of a neuropeptide receptor and expressing same on its surface under conditions sufficient for binding of ligands previously identified aε binding to such a receptor. In other embodiments cell membrane fractions comprising the receptor or iεolated receptorε free or immobilized on εolid εupportε may be used to measure binding of the ligand to be tested. When recombinant cells are used for purposes of expresεion of the receptor it is preferred to use cells with little or no endogenous receptor activity so that binding, if any, is due to the presence of the expresεed receptor of intereεt. Preferred cellε include human embryonic kidney cellε, monkey kidney (HEK-293 cellε), fibroblaεt (COS) cellε, Chineεe hamster ovary (CHO) cells, Drosophila or murine L-cellε. It is also preferred to employ as a host cell, one in which a receptor responεive second messenger system exists. Well known second mesεenger εyεtems include increaseε or decreaεeε in phoεphoinoεitide hydrolyεis, adenylate cyclase, guanylate cyclaεe, or ion channel activity in reεponεe to ligand binding to extracellular receptor domainε. In a further embodiment a εpecifically deεigned indicator of receptor binding can be conεtructed. For example, a fuεion protein can be made by fuεing the receptor of thiε invention with a protein domain which is sensitive to receptor ligand binding. Such a domain referred to here as an indicator domain iε 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 of a cell by detecting "the presence of mRNA coding for the receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the 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 polypeptideε of the present invention. These related receptors may be identified by homology to a neuropeptide receptor polypeptide of the present invention, by low stringency crosε hybridization, or by identifying receptorε that interact with related natural or εynthetic ligandε and or elicit εimilar 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 geneε which have a high εequence similarity to the genes of the present invention, or which have εimilar biological activity. Probeε of this type preferably have 50 baseε or more. The probe may alεo be uεed to identify a cDNA clone correεponding to a full length tranεcript and a genomic clone or cloneε that contain the complete gene of the preεent invention including regulatory and promoter regionε, exonε and intronε. An example of a screen of this type comprises isolating the coding region of the gene by using the known DNA εequence to εynthesize an oligonucleotide probe. Labeled oligonucleotides having a εequence complementary to that of the geneε of the preεent invention are uεed to εcreen a library of human cDNA, genomic DNA or mRNA to determine which memberε of the library the probe hybridizes to.

The neuropeptide receptor polypeptides and compounds identified above which are polypeptideε, 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 provided 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 containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expresεion of a polypeptide in vivo by, for example, procedureε known in the art. Aε known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the preεent invention may be adminiεtered to a patient for engineering cellε in vivo and expreεεion of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachingε of the present invention. For example, the expresεion vehicle for engineering cellε may be other than a retroviruε, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable 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 immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Viruε, and mammary tumor viruε. In one embodiment, the retroviral plaεmid vector iε derived from Moloney Murine Leukemia Viruε.

The vector includeε one or more promoterε. Suitable promoterε which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniσues. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and jδ-actin promoters) . Other viral promoters which may be employed include, but are not limited to, adenovirus promoterε, thymidine kinaεe (TK) promoterε, and B19 parvovirus promoters. The selection of a εuitable promoter will be apparent to thoεe εkilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention iε under the control of a εuitable promoter. Suitable promoterε which may be employed include, but are not limited to, adenoviral promoters, such as 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 εhock promoters; the albumin promoter; the ApoAI promoter,- human globin promoters,- viral thymidine kinase promoters, such as the Herpeε Simplex thymidine kinase promoter,- retroviral LTRs (including the modified retroviral LTRs hereinabove described) ,- the 0-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controlε the genes encoding the polypeptides.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cellε which may be tranεfected include, but are not limited to, the PE501, PA317, ψ-2 , ψ-ΑM, PA12, T19-14X, VT-19-17-H2, ψCRE , i^CRIP, GP+E-86, GP+envAml2, and DAN cell lineε as described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990) , which is incorporated herein by reference in its entirety. The vector may tranεduce the packaging cellε through any meanε known in the art. Such meanε include, but are not limited to, electroporation, the uεe of lipoεomeε, and CaP0₄ precipitation. In one alternative, the retroviral plaεmid vector may be encapεulated into a lipoεome, or coupled to a lipid, and then adminiεtered to a host. The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(ε) encoding the polypeptideε. Such retroviral vector particleε then may be employed, to tranεduce eukaryotic cells, either in vi tro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(ε) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cellε, aε well aε hematopoietic stem cellε, hepatocyteε, fibroblaεtε, myoblasts, keratinocyteε, endothelial cells, and bronchial epithelial cells.

The soluble neuropeptide receptor polypeptides and compounds which bind to and activate or inhibit activation of a receptor of the preεent 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 includeε but iε not limited to εaline, buffered εaline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation εhould εuit the mode of adminiεtration.

The invention alεo provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositionε of the invention. Aεεociated with εuch container(ε) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or εale of pharmaceuticalε or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the soluble neuropeptide receptor polypeptides or compounds of the present invention may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be adminiεtered in a convenient manner εuch aε by the topical, intravenous, intraperitoneal, intramuscular, subcutaneouε, intranaεal or intradermal routes. The pharmaceutical compositions are adminiεtered in an amount which iε effective for treating and/or prophylaxiε of the specific indication. In general, the pharmaceutical compositionε will be adminiεtered in an amount of at leaεt about 10 μg/kg body weight and in moεt caεeε they will be adminiεtered in an amount not in exceεε of about 8 mg/Kg body weight per day. In moεt caεes, the dosage is from about 10 g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

The present invention alεo contemplateε the use of the genes of the present invention as a diagnostic, for example, some diseases result from inherited defective genes. Theεe geneε can be detected by comparing the εequenceε of the defective gene with that of a normal one. Subεequently, one can verify that a "mutant" gene iε aεsociated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector for expresεion in a functional asεay εystem (e.g., colorimetric asεay, expreεεion on MacConkey plateε, complementation experimentε, in a receptor deficient εtrain of HEK293 cellε) aε yet another meanε to verify or identify mutations. Once "mutant" genes have been identified, one can then screen population for carrierε of the "mutant" receptor gene.

Individuals carrying mutations in the gene of the preεent invention may be detected at the DNA level by a variety of techniqueε. Nucleic acids used for diagnosis may be obtained from a patient's cellε, including but not limited to such aε 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 analyεiε. RNA or cDNA may also be used for the εame purpose. As an example, PCR primers complimentary to the nucleic acid of the inεtant invention can be used to identify and analyze mutations in the gene of the preεent invention. For example, deletionε and inεertionε can be detected by a change in εize of the amplified product in compariεon to the normal genotype. Point mutationε can be identified by hybridizing amplified DNA to radio labeled RNA of the invention or alternatively, radio labeled antisense DNA sequenceε of the invention. Perfectly matched εequenceε can be diεtinguiεhed from mismatched duplexes by RNase A digeεtion or by differenceε in melting temperatures. Such a diagnostic would be particularly useful for prenatal or even neonatal testing.

Sequence differences between the reference gene and "mutants" may be revealed by the direct DNA sequencing method. In addition, cloned DNA segmentε may be used aε probeε to detect εpecific DNA εegmentε. The sensitivity of this method iε greatly enhanced when combined with PCR. For example, a sequence primer is used with double stranded PCR product or a εingle εtranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radio labeled nucleotide or by an automatic sequencing procedure with fluorescent-tags.

Genetic testing based on DNA εequence differenceε may be achieved by detection of alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Sequences changes at specific locationε may alεo be revealed by nucleuε protection assayε, such RNase and Si protection or the chemical cleavage method (e.g. Cotton, et al., PNAS. USA. 85:4397-4401 1985) .

In addition, εome diεeases are a result of, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes of the present invention can be uεed aε a reference to identify individuals expressing a decrease of functions asεociated with receptors of thiε type. The present invention also relates to a diagnostic asεay for detecting altered levelε of soluble forms of the neuropeptide receptor polypeptideε of the preεent invention in variouε tissues. Assays used to detect levels of the soluble receptor polypeptideε in a sample derived from a hoεt are well known to those of skill in the art and include radioimmunoasεays, competitive-binding assayε, Western blot analysis and preferably as ELISA assay.

An ELISA assay initially comprises preparing an antibody specific to antigens of the neuropeptide receptor polypeptides, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidaεe enzyme. A sample iε now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any neuropeptide receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter 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. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period iε a measurement of the amount of neuropeptide receptor proteins present in a given volume of patient sample when compared against a standard curve.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromoεome. Moreover, there is a current need for identifying particular siteε on the chromoεome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAε to chromoεomeε according to the present invention iε an important firεt εtep in correlating thoεe sequences with genes aεεociated with diεease.

Briefly, sequences can be mapped to chromosomeε by preparing PCR primerε (preferably 15-25 bp) from the cDNA. Computer analyεiε of the 3' untranslated region is used to rapidly εelect primerε that do not εpan more than one exon in the genomic DNA, thuε complicating the amplification proceεε. Theεe primerε are then used for PCR screening of somatic cell hybrids containing individual human chromosomeε. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybridε iε a rapid procedure for aεεigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primerε, εublocalization can be achieved with panelε of fragmentε from εpecific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategieε that can εimilarly be uεed to map to itε chromosome include in situ hybridization, prescreening with labeled flow-εorted chromoεomeε and preεelection by hybridization to conεtruct chromoεome εpecific-cDNA librarieε.

Fluoreεcence in si tu hybridization (FISH) of a cDNA clone to a metaphaεe chromoεomal spread can be used to provide a preciεe chromoεomal location in one step. This technique can be used with cDNA as short as 50 or 60 baseε. For a review of thiε technique, εee Verma et al., Human Chromoεomes: a Manual of Basic Techniques, Pergamon Presε, New York (1988) . 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, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) . The relationship between geneε and diseases that have been mapped to the same chromosomal region are then identified through linkage analysiε (coinheritance of phyεically adjacent genes) .

Next, it is necesεary to determine the differenceε 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 iε 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 asεociated with the diεeaεe 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 expresεing them can be uεed as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodieε. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedureε known in the art may be used for the production of such antibodieε and fragmentε.

Antibodieε generated againεt the polypeptideε corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptideε to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a εequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodieε can then be uεed to iεolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuouε cell line cultureε can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodieε (Cole, et al., 1985, in Monoclonal Antibodieε and Cancer Therapy, Alan R. Liεs, Inc., pp. 77-96).

Techniques described for the production of εingle chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce εingle chain antibodieε to immunogenic polypeptide productε of thiε invention. Alεo, tranεgenic mice may be uεed to expreεε humanized antibodies to immunogenic polypeptide products of this invention.

The present invention will be further deεcribed with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following exampleε certain frequently occurring methodε and/or termε will be deεcribed.

"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmidε in accord with publiεhed procedureε. In addition, equivalent plaεmidε to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA referε to catalytic cleavage of the DNA with a reεtriction enzyme that actε only at certain sequences in the DNA. The various reεtriction enzymeε used herein are commercially available and their reaction conditions, cofactors and other requirements were uεed aε would be known to the ordinarily εkilled artiεan. For analytical purpoεes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragmentε for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate bufferε and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation timeε of about 1 hour at 37"C are ordinarily uεed, but may vary in accordance with the εupplier'ε inεtructionε. After digeεtion the reaction iε electrophoreεed directly on a polyacrylamide gel to iεolate 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 strandε which may be chemically εyntheεized. Such εynthetic oligonucleotideε have no 5' phoεphate and thuε 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 proceεs of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T. , et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known bufferε and conditions with 10 units to T4 DNA ligase ("ligaεe") per 0.5 μg of approximately equimolar amountε of the DNA fragmentε to be ligated.

Unleεε otherwiεe εtated, tranεformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Example 1 Bacterial Expression and Purification of the Neuropeptide Receptor

The DNA sequence encoding for neuropeptide receptor,

ATCC # is initially amplified uεing PCR oligonucleotide primerε correεponding to the 5' and 3' end εequenceε of the proceεεed neuropeptide receptor gene (minus the signal peptide εequence) and the vector sequences 3' to the gene. Additional nucleotides correεponding to neuropeptide receptor nucleotide sequence are added to the 5' and 3' sequenceε respectively. The 5' oligonucleotide primer has the sequence 5' CACTAAAGCTTAATGGAGCCCTCAGCCACC 3' (SEQ ID NO:7) contains a Hind III restriction enzyme site followed by 18 nucleotides of neuropeptide receptor coding sequence starting from the preεumed terminal amino acid of the processed protein codon. The 3' sequence 5' ACAAGTCCTTGTCC.TTCTAGAGGGC 3' (SEQ ID NO:8) and contains an Xbal site. The restriction enzyme sites correspond to the restriction enzyme siteε on the bacterial expreεεion vector pQE-9 (Qiagen, Inc. Chatεworth, CA) . pQE-9 encodeε antibiotic resistance (Amp^r) , 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 and Xbal. The amplified εequenceε are ligated into pQE-9 and are inεerted in frame with the sequence encoding for the hiεtidine tag and the RBS. The ligation mixture iε then uεed to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure deεcribed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989) . M15/rep4 containε multiple copieε of the plaεmid pREP4, which expreεses the lad repressor and also conferε kanamycin reεiεtance (Kan^r) . Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resiεtant colonieε are εelected. Plaεmid DNA iε iεolated and confirmed by reεtriction analyεiε. Clones containing the desired conεtructε are grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The O/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.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Iεopropyl-B-D-thiogalacto pyranoεide") iε then added to a final concentration of l mM. IPTG induceε by inactivating the la repressor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hourε. Cellε are then harveεted by centrifugation. The cell pellet iε εolubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, εolubilized neuropeptide receptor iε purified from thiε εolution by chromatography on a Nickel-Chelate column under conditionε that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984). The protein iε eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpoεe of renaturation adjusted to 3 molar guanidine HC1, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

Example 2 Expresεion of Recombinant Neuropeptide Receptor in COS cells The expreεεion of plasmid, neuropeptide receptor HA is derived from a vector pcDNA3/Amp (Invitrogen) containing: l) SV40 origin of replication, 2) ampicillin reεiεtance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation εite. A DNA fragment encoding the entire neuropeptide receptor precurεor and a HA tag fuεed in frame to itε 3' end iε cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspondε to an epitope derived from the influenza hemagglutinin protein as previouεly deεcribed (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767) . The infusion of HA tag to the target protein allows eaεy detection of the recombinant protein with an antibody that recognizeε the HA epitope.

The plaεmid conεtruction εtrategy iε deεcribed aε followε:

The DNA sequence encoding for neuropeptide receptor,

ATCC # , is conεtructed by PCR uεing two primers: the

5' primer 5' CCTAGGATGCCCCTCTGCTGCAGCGG 3' (SEQ ID NO:9) contains a BamHI site; the 3' sequence 5' ACAAGTCCTTGT CCTTCTAGAGGGC 3' (SEQ ID NO:10) containε complementary εequenceε to an Xbal εite, tranεlation stop codon, and the last 17 nucleotides of the neuropeptide receptor coding εequence (not including the εtop codon) . Therefore, the PCR product contains a BamHI site, coding sequence, a tranεlation termination εtop codon and an Xbal site. The PCR amplified DNA fragment and the vector, pcDNA3/Amp, are digested with BamHI and Xbal restriction enzymeε and ligated. The ligation mixture iε tranεformed into E. coli εtrain SURE (Stratagene Cloning Syεtems, La Jolla, CA) the transformed culture iε plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA iε iεolated from tranεformantε and examined by reεtriction analyεiε for the preεence of the correct fragment. For expreεεion of the recombinant neuropeptide receptor, COS cellε are tranεfected with the expreεsion vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A^"Laboratory Manual, Cold Spring Laboratory Preεε, (1989)) . The expression of the neuropeptide receptor HA protein is detected by radio- labelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Presε, (1988)) . Cellε are labelled for 8 hourε with ³⁵S-cyεteine two dayε post transfection. Culture media are then collected and cells are lyεed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Triε, pH 7.5) . (Wilεon, I. et al., Id. 37:767 (1984)) . Both cell lyεate and culture media are precipitated with a HA εpecific monoclonal antibody. Proteinε precipitated are analyzed on 15% SDS-PAGE gelε.

Example 3 Cloning and expreεεion of Neuropeptide Receptor uεincr the baculoviruε expreεsion svεtem

The DNA sequence encoding the full length neuropeptide receptor protein, ATCC # , iε amplified uεing PCR oligonucleotide primerε correεponding to the 5' and 3' εequenceε of the gene:

The 5' primer has the εequence 5' CGGGATCCGCCATCATGGAG CCCTCAGCCACC 3' (SEQ ID NO:11) and containε a BamHI restriction enzyme site (in bold) followed by 6 nucleotideε reεembling an efficient εignal for the initiation of tranεlation in eukaryotic cells (J. Mol. Biol. 1987, 196. 947-950, Kozak, M.) . The initiation codon for translation "ATG" iε underlined) .

The 3' primer haε the εequence 5' ACAAGTCCTTGTC I r AGAGGGC 3' (SEQ ID NO:12) and containε the cleavage εite for the restriction endonuclease Xbal and 5 nucleotides complementary to the 3' non-translated sequence of the neuropeptide receptor gene. The amplified εequenceε are iεolated from a 1% agaroεe gel uεing a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) . The fragment iε then digeεted with the endonucleaεes BamHI and Xbal and then purified as described in Example 1. Thiε fragment iε deεignated F2.

The vector pA2 (modification of pVL941 vector, diεcussed below) is used for the expresεion of the neuropeptide receptor protein uεing the baculoviruε expreεεion system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methodε for baculoviruε vectorε and inεect cell culture procedureε, Texaε Agricultural Experimental Station Bulletin NO:l, 3 and 5555) . Thiε expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhidrosiε virus (AcMNPV) followed by the recognition sites for the restriction endonucleaεes BamHI and Xbal. The polyadenylation εite of the εimian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant viruses the beta-galactosidaεe gene from E.coli iε inεerted in the εame orientation aε the polyhedrin promoter followed by the polyadenylation εignal of the polyhedrin gene. The polyhedrin εequenceε are flanked at both εideε by viral εequenceε for the cell-mediated homologouε recombination of co-tranεfected wild-type viral DNA. Many other baculoviruε vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39) .

The plasmid is digested with the restriction enzymes BamHI and Xbal and then dephosphorylated uεing calf intestinal phosphatase by procedures known in the art. The DNA iε then iεolated from a 1% agaroεe gel aε deεcribed in Example 1. This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. DH5α are then tranεformed and bacteria identified that contained the plaεmid (pBac neuropeptide receptor) with the neuropeptide receptor gene uεing the enzymeε BamHI and Xbal . The εequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of the plasmid pBac neuropeptide receptor are co- tranεfected with 1.0 μg of a commercially available linearized baculoviruε ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987) ) . lμg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac neuropeptide receptor are mixed in a sterile well of a icrotiter plate containing 50 μl of serum free Grace's medium (Life Technologieε Inc., Gaitherεburg, MD) . Afterwardε 10 μl Lipofectin pluε 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the tranεfection mixture iε added drop wiεe to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tisεue culture plate with 1 ml Grace' medium without εerum. The plate iε rocked back and forth to mix the newly added εolution. The plate iε then incubated for 5 hourε at 27°C. After 5 hourε the tranεfection εolution iε removed from the plate and 1 ml of Grace'ε inεect medium εupplemented with 10% fetal calf serum is added. The plate iε put back into an incubator and cultivation continued at 27°C for four dayε.

After four dayε the εupernatant iε collected and a plaque assay performed εimilar aε deεcribed by Summerε and Smith (εupra) . As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is uεed which allowε an eaεy iεolation of blue εtained plaqueε. (A detailed deεcription of a "plaque aεsay" can also be found in the uεer'ε guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) . Four dayε after the εerial dilution the viruε iε added to the cellε and blue stained plaqueε are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruεeε is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the εupernatant containing the recombinant baculoviruses is used to infect Sf9 cells seeded in 35 mm disheε. Four dayε later the εupernatantε of theεe culture diεhes are harvested and then stored at 4°C.

Sf9 cellε are grown in Grace' ε medium εupplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-neuropeptide receptor at a multiplicity of infection (MOD of 2. Six hours later the medium is removed and replaced with SF900 II medium minuε methionine and cyεteine (Life Technologieε Inc., Gaithersburg) . 42 hourε later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cyεteine (Amerεham) are added. The cellε are further incubated for 16 hourε before they are harveεted by centrifugation and the labelled proteinε viεualized by SDS- PAGE and autoradiography.

Example 4 Expression via Gene Therapy

Fibroblastε are obtained from a εubject by εkin biopεy. The reεulting tiεεue iε placed in tiεεue-culture medium and εeparated into small pieces. Small chunks of the tissue are placed on a wet surface of a tisεue culture flaεk, approximately ten pieceε are placed in each flaεk. The flaεk iε turned upεide down, cloεed tight and left at room temperature over night. After 24 hourε at room temperature, the flaεk iε inverted and the chunkε of tiεεue remain fixed to the bottom of the flaεk and freεh media (e.g., Ham'ε F12 media, with 10% FBS, penicillin and streptomycin, is added. Thiε iε then incubated at 37°C for approximately one week. At thiε time, freεh media iε added and εubsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer iε trypsinized and scaled into larger flaεkε. pMV-7 (Kirεchmeier, P.T. et al, DNA^", 7:219-25 (1988) flanked by the long terminal repeatε of the Moloney murine εarcoma viruε, is digested with EcoRI and Hindlll and εubεequently treated with calf inteεtinal phoεphatase. The linear vector is fractionated on agarose gel and purified, uεing glaεs beadε.

The cDNA encoding a polypeptide of the present invention is amplified using PCR primerε which correεpond to the 5' and 3' end εequences respectively. The 5' primer containing an EcoRI εite and the 3' primer having containε a Hindlll εite. Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained 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 tiεεue 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 iε then added to the media and the packaging cellε are transduced with the vector. The packaging cells now produce infectiouε viral particleε containing the gene (the packaging cellε are now referred to aε producer cellε) .

Freεh media iε added to the tranεduced producer cellε, and subεequently, the media iε harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectiouε viral particleε, iε filtered through a millipore filter to remove detached producer cellε and thiε media iε then uεed to infect fibroblast cells. Media is removed from a εub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. Thiε media iε removed and replaced with freεh media. If the titer of viruε iε high, then virtually all fibroblasts will be infected and no selection is required. If the titer iε very low, then it iε necessary to use a retroviral vector that haε a εelectable marker, εuch as neo or his.

The engineered fibroblaεtε are then injected into the hoεt, either alone or after having been grown to confluence on cytodex 3 microcarrier beadε. The fibroblaεtε now produce the protein product.

Numerouε modificationε and variationε of the preεent invention are possible in light of the above teachings and, therefore, within the εcope of the appended claimε, the invention may be practiced otherwiεe than aε particularly deεcribed.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: LI, ET AL.

(ii) TITLE OF INVENTION: Human Neuropeptide Receptor

(iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART & OLSTEIN

(B) STREET: 6 BECKER FARM ROAD

(C) CITY: ROSELAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: 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) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1209 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS : SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGGAGCCCT CAGCCACCCC AGGGGCCCAG ATGGGGGTCC CCCCTGGCAG CAGAGAGCCG 60 TCCCCTGTGC CTCCAGACTA TGAAGATGAG TTTCTCCGCT ATCTGTGGCG TGATTATCTG 120 TACCCAAAAC AGTATGAGTG GGTCCTCATC CCAGCCTATG TGGCTGTGTT CGTCGTGGCC 180

CTGGTGGGCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240

ACCAACTACT TCATTGTCAA CCTGTCCCTG GCTGACGTTC TGGTGACTGC TATCTGCCTG 300

CCGGCCAGCC TGCTGGTGGA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360

GTCATCCCCT ATCTACAGGC TGTGTCCGTG TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420

GCCCTGGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480

GCCCGTGGCT CCATCCTGGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540

GCAGTCATGG AATGCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG GCTCTTCTCA 600

GTCTGTCATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTGCTTCTTT 660

ATTGTCACCT ACCTGGCCCC ACTGGGCCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720

AACCTCTGGG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCGGAA CTGGAAGCGC 780

CCCTCAGACC AGCTGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCGGGGC 840

CGCGCCTTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900

ATGGTGGTGC TGCTGGTCTT CGCCCTCTGC TACCTGCCCA TCAGCGTCCT CAATGTCCTT 960

AAGAGGGTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAGCTGTCTA CGCCTGCTTC 1020

ACCTTCTCCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080

CTCAGTGGCA AATTCCGGGA GCAGTTTAAG GCTGCCTTCT CCTGCTGCCT GCCTGGCCTG 1140

GGTCCCTGCG GCTCTCTGAA GGCCCCTAGT CCCCGCTCCT CTGCCAGCCA CAAGTCCTTG 1200

TCCTTGTAG 1209

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 402 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS :

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Glu Pro Ser Ala Thr Pro Gly Ala Gin Met Gly Val Pro Pro

5 10 15

Gly Ser Arg Glu Pro Ser Pro Val Pro Pro Aεp Tyr Glu Aεp Glu

20 25 30

Phe Leu Arg Tyr Leu Trp Arg Aεp Tyr Leu Tyr Pro Lys Gin Tyr

35 40 45

Glu Trp Val Leu He Ala Ala Tyr Val Ala Val Phe Val Val Ala

50 55 60

Leu Val Gly Asn Thr Leu Val Cyε Leu Ala Val Trp Arg Asn His

65 70 75

Hiε Met Arg Thr Val Thr Aεn Tyr Phe He Val Aεn Leu Ser Leu

80 85 90

Ala Aεp Val Leu Val Thr Ala He Cyε Leu Pro Ala Ser Leu Leu

95 100 105

Val Aεp He Thr Glu Ser Trp Leu Phe Gly Hiε Ala Leu Cyε Lyε

110 115 120

Val He Pro Tyr Leu Gin Ala Val Ser Val Ser Val Ala Val Leu

125 130 135

Thr Leu Ser Phe He Ala Leu Aεp Arg Trp Tyr Ala He Cyε His

140 145 150

Pro Leu Leu Phe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser He

155 160 165

Leu Gly He Trp Ala Val Ser Leu Ala He Met Val Pro Gin Ala

170 175 180 Ala Val Met Glu Cys Ser Ser Val Leu Pro Glu Leu Ala Asn Arg

185 190 195 Thr Arg Leu Phe Ser Val Cys Aεp Glu Arg Trp Ala Aεp Asp Leu

200 205 210 Tyr Pro Lys He Tyr His Ser Cyε Phe Phe He Val Thr Tyr Leu

215 220 225

Ala Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gin He Phe Arg

230 235 240 Lyε Leu Trp Gly Arg Gin He Pro Gly Thr Thr Ser Ala Leu Val

245 250 255

Arg Aεn Trp Lys Arg Pro Ser Asp Gin Leu Gly Asp Leu Glu Gin

260 265 270

Gly Leu Ser Gly Glu Pro Gin Pro Arg Gly Arg Ala Phe Leu Ala

275 280 285

Glu Val Lys Gin Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu

290 295 300

Met Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro He Ser

305 310 315

Val Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gin Ala

320 325 330

Ser Asp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp

335 340 345

Leu Val Tyr Ala Asn Ser Ala Ala Asn Pro He He Tyr Asn Phe

350 355 360

Leu Ser Gly Lyε Phe Arg Glu Gin Phe Lyε Ala Ala Phe Ser Cyε

365 370 375

Cyε Leu Pro Gly Leu Gly Pro Cyε Gly Ser Leu Lyε Ala Pro Ser

380 385 390 Pro Arg Ser Ser Ala Ser Hiε Lyε Ser Leu Ser Leu

395 400

(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 ATGGGGGTCC CCCCTGGCAG CAGAGAGCCG 60

TCCCCTGTGC CTCCAGACTA TGAAGATGAG TTTCTCCGCT ATCTGTGGCG TGATTATCTG 120

TACCCAAAAC AGTATGAGTG GGTCCTCATC GCAGCCTATG TGGCTGTGTT CGTCGTGGCC 180

CTGGTGGGCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240

ACCAACTACT TCATTGTCAA CCTGTCCCTG GCTGACGTTC TGGTGACTGC TATCTGCCTG 300

CCGGCCAGCC TGCTGGTGGA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360

GTCATCCCCT ATCTACAGGC TGTGTCCGTG TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420

CCCCTGGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480

GCCCGTGGCT CCATCCTGGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540

GCAGTCATGC AATCCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG GCTCTTCTCA 600

CTCTGTCATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTGCTTCTTT 660

ATTGTCACCT ACCTGGCCCC ACTGGGCCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720

AAGCTCTGGG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCGGAA CTGGAAGCGC 780 CCCTCAGACC AGCTGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCGGGGC 840

CGCGCCTTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900

ATGGTGGTGC TGCTGGTCTT CGCCCTCTGC TACCTCCCCA TCAGCGTCCT CAATGTCCTT 960

AAGAGGGTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAGCTGTCTA CGCCTGCTTC 1020

ACCTTCTCCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080

CTCAGTGGCC TTCCCTGGAG TCTGCTCTAA 1110

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 369 BASE PAIRS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Glu Pro Ser Ala Thr Pro Gly Ala Gin Met Gly Val Pro Pro

5 10 15

Gly Ser Arg Glu Pro Ser Pro Val Pro Pro Aεp Tyr Glu Aεp Glu

20 25 30

Phe Leu Arg Tyr Leu Trp Arg Aεp Tyr Leu Tyr Pro Lys Gin Tyr

35 40 45

Glu Trp Val Leu He Ala Ala Tyr Val Ala Val Phe Val Val Ala

50 55 60

Leu Val Gly Asn Thr Leu Val Cys Leu Ala Val Trp Arg Asn His

65 70 75

His Met Arg Thr Val Thr Aεn Tyr Phe He Val Aεn Leu Ser Leu

80 85 90

Ala Aεp Val Leu Val Thr Ala He Cys Leu Pro Ala Ser Leu Leu

95 100 105

Val Asp He Thr Glu Ser Trp Leu Phe Gly Hiε Ala Leu Cyε Lyε

110 115 120

Val He Pro Tyr Leu Gin Ala Val Ser Val Ser Val Ala Val Leu

125 130 135

Thr Leu Ser Phe He Ala Leu Aεp Arg Trp Tyr Ala He Cyε Hiε

140 145 150

Pro Leu Leu Phe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser He

155 160 165

Leu Gly He Trp Ala Val Ser Leu Ala He Met Val Pro Gin Ala

170 175 180

Ala Val Met Glu Cys Ser Ser Val Leu Pro Glu Leu Ala Aεn Arg

185 190 195

Thr Arg Leu Phe Ser Val Cyε Aεp Glu Arg Trp Ala Aεp Aεp Leu

200 205 210

Tyr Pro Lyε He Tyr Hiε Ser Cyε Phe Phe He Val Thr Tyr Leu

215 220 225

Ala Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gin He Phe Arg

230 235 _^ 240

Lys Leu Trp Gly Arg Gin He Pro Gly Thr Thr Ser^" Ala Leu Val

245 250 255

Arg Asn Trp Lys Arg Pro Ser Asp Gin Leu Gly Aεp Leu Glu Gin

260 265 270 Gly Leu Ser Gly Glu Pro Gin Pro Arg Gly Arg Ala Phe Leu Ala

275 280 285

Glu Val Lys Gin Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu

290 295 300

Met Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro He Ser

305 310 315

Val Leu Aεn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gin Ala

320 325 330

Ser Asp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp

335 340 345

Leu Val Tyr Ala Aεn Ser Ala Ala Aεn Pro He He Tyr Aεn Phe

350 355 360

Leu Ser Gly Leu Pro Trp Ser Leu Leu

365

(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) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ATGGAGCCCT CAGCCACCCC AGGGGCCCAG ATGGGGGTCC CCCCTGGCAG CAGAGACCCC 60

TCCCCTGTGC CTCCAGACTA TGAAGATGAG TTTCTCCGCT ATCTGTGGCG TGATTATCTG 120

TACCCAAAAC AGTATGAGTG GGTCCTCATC GCAGCCTATG TGGCTGTGTT CGTCGTGGCC 180

CTGGTGGGCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240

ACCAACTACT TCATTGTCAA CCTGTCCCTG GCTGACGTTC TGGTGACTGC TATCTGCCTG 300

CCGGCCAGCC TGCTGGTGGA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360

GTCATCCCCT ATCTACAGGC TGTGTCCGTG TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420

GCCCTGGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480

GCCCGTGGCT CCATCCTGGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540

GCAGTCATGG AATGCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG GCTCTTCTCA 600

GTCTGTGATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTGCTTCTTT 660

ATTGTCACCT ACCTGGCCCC ACTGGGCCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720

AAGCTCTGGG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCGGAA CTGGAAGCGC 780

CCCTCAGACC AGCTGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCGGGGC 840

CGCGCCTTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900

ATGGTGGTGC TGCTGGTCTT CGCCCTCTGC TACCTGCCCA TCAGCGTCCT CAATGTCCTT 960

AAGAGGGTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAGCTGTCTA CGCCTGCTTC 1020

ACCTTCTCCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080

CTCAGTGGAT GTAAAGAGAA GAGTCTAGTT CTGTCCTGAC CATCGTGCCC CGG 1133

(2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 377 BASE PAIRS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Glu Pro Ser Ala Thr Pro Gly Ala Gin Met Gly Val Pro Pro

5 10 15

Gly Ser Arg Glu Pro Ser Pro Val Pro Pro Asp Tyr Glu Asp Glu

20 25 30

Phe Leu Arg Tyr Leu Trp Arg Aεp Tyr Leu Tyr Pro Lyε Gin Tyr

35 40 45

Glu Trp Val Leu He Ala Ala Tyr Val Ala Val Phe Val Val Ala

50 55 60

Leu Val Gly Asn Thr Leu Val Cys Leu Ala Val Trp Arg Asn His

65 70 75

His Met Arg Thr Val Thr Asn Tyr Phe He Val Asn Leu Ser Leu

80 85 90

Ala Asp Val Leu Val Thr Ala He Cys Leu Pro Ala Ser Leu Leu

95 100 105

Val Asp He Thr Glu Ser Trp Leu Phe Gly His Ala Leu Cys Lys

110 115 120

Val He Pro Tyr Leu Gin Ala Val Ser Val Ser Val Ala Val Leu

125 130 135

Thr Leu Ser Phe He Ala Leu Asp Arg Trp Tyr Ala He Cyε Hiε

140 145 150

Pro Leu Leu Phe Lyε Ser Thr Ala Arg Arg Ala Arg Gly Ser He

155 160 165

Leu Gly He Trp Ala Val Ser Leu Ala He Met Val Pro Gin Ala

170 175 180

Ala Val Met Glu Cyε Ser Ser Val Leu Pro Glu Leu Ala Asn Arg

185 190 195

Thr Arg Leu Phe Ser Val Cys Asp Glu Arg Trp Ala Aεp Aεp Leu

200 205 210

Tyr Pro Lyε He Tyr Hiε Ser Cyε Phe Phe He Val Thr Tyr Leu

215 220 225

Ala Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gin He Phe Arg

230 235 240

Lyε Leu Trp Gly Arg Gin He Pro Gly Thr Thr Ser Ala Leu Val

245 250 255

Arg Aεn Trp Lyε Arg Pro Ser Aεp Gin Leu Gly Aεp Leu Glu Gin

260 265 270

Gly Leu Ser Gly Glu Pro Gin Pro Arg Gly Arg Ala Phe Leu Ala

275 280 285

Glu Val Lys Gin Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu

290 295 300

Met Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro He Ser

305 310 315

Val Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gin Ala

320 325 330

Ser Asp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp

335 340 345

Leu Val Tyr Ala Aεn Ser Ala Ala Aεn Pro He He Tyr Aεn Phe

350 355 360

Leu Ser Gly Cyε Lyε Glu Lyε Ser Leu Val Leu Ser Pro Ser Cyε

365 370 375 Pro Gly (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 30 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CACTAAAGCT 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) SEQUENCE DESCRIPTION: SEQ ID NO:8: ACAAGTCCTT GTCCTTCTAG AGGGC 25

(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 CCCTCTGCTG CAGCGG 26

(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE 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: ACAAGTCCTT GTCCTTCTAG AGGGC 25

(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 32 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CGGGATCCGC CATCATGGAG CCCTCAGCCA 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) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ACAAGTCCTT GTCCTTCTAG AGGGC 25

Claims

WHAT IS CLAIMED IS:

1. An iεolated 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 l wherein the polynucleotide is genomic DNA.

6. The polynucleotide of Claim 1 compriεing from nucleotide l to nucleotide 1209 aε εet forth in SEQ ID NO:l.

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

8. An isolated polynucleotide comprising a member εelected from the group consisting of:

(a) a polynucleotide encoding a mature polypeptide encoded by the DNA contained in ATCC Deposit No. (b) a polynucleotide encoding the polypeptide expresεed by the DNA contained in ATCC Depoεit No. ,-

(c) a polynucleotide capable of hybridizing to and which iε at leaεt 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 εaid polynucleotide encodeε a polypeptide expreεεed by the DNA contained in ATCC Depoεit No. .

10. The polynucleotide of Claim 8 wherein εaid polynucleotide encodeε a polypeptide expressed by the DNA contained in ATCC Deposit No. .

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: expreεεing from the hoεt cell of Claim 12 the polypeptide encoded by εaid DNA.

14. A proceεs for producing cells capable of expressing a polypeptide comprising tranεforming or tranεfecting the cellε with the vector of Claim 11.

15. A receptor polypeptide εelected from the group conεiεting of:

(i) a polypeptide having the deduced amino acid εequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof; and (ii) a polypeptide encoded by the cDNA of ATCC

Depoεit No. and fragmentε, analogε and derivativeε of said polypeptide.

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

17. An antibody againεt the polypeptide of claim 15.

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

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

20. A method for the treatment of a patient having need to activate a neuropeptide receptor compriεing: adminiεtering 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 compriεing: adminiεtering to the patient a therapeutically effective amount of the compound of claim 19.

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

23. The method of claim 21 wherein εaid compound iε 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 hoεt cell expressing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable εignal in response to the binding of a compound to said receptor polypeptide; contacting a plurality of compounds with said hoεt cell under conditionε εufficient to permit binding of compounds to the receptor polypeptide; and identifying those compounds capable of receptor binding by detecting the εignal produced by εaid εecond component.

25. An agoniεt 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 expresεing on the εurface thereof the receptor polypeptide, εaid receptor being associated with a second component capable of providing a detectable signal in reεponεe to the binding of a compound to εaid receptor polypeptide contacting an analytically detectable ligand known to bind to the receptor polypeptide and a plurality of compoundε with εaid host cell under conditionε to permit binding to the receptor polypeptide; and determining whether the ligand bindε to the polypeptide by detecting the abεence of a εignal generated from the interaction of the ligand with the polypeptide.

27. An antagoniεt 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 conditionε permitting binding and detecting the preεence 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 compoundε to identify thoεe compoundε which bind to the receptor polypeptide of claim 15 compriεing: contacting a recombinant hoεt cell expreεεing the receptor on the εurface thereof with a plurality of candidate compoundε and an analytically detectable ligand known to bind to the receptor, under conditionε permitting binding to the receptor; and identifying thoεe 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 procesε for diagnoεing a diεeaεe or a susceptibility to a diseaεe related to an under-expreεεion of the polypeptide of claim 15 compriεing: determining a mutation in the nucleic acid sequence encoding said polypeptide.

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

34. A diagnostic procesε compriεing: analyzing for the preεence of the polypeptide of claim 33 in a εample derived from a hoεt.