AU718702B2 - Mammalian melanocyte stimulating hormone receptors and uses - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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Name and Address of Applicant: Actual Inventor(s): Address for Service: State of Oregon, acting by and through The Oregon State Board of Higher Education on behalf of The Oregon Health Sciences University 3181 S.W. Sam Jackson Park Road Portland Oregon 97201-3098 UNITED STATES OF AMERICA Roger D. Cone and Kathleen Mountjoy Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Mammalian Melanocyte Stimulating Hormone Receptors and Uses Invention Title-.

The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 MAMMALIAN MELANOCYTE STIMULATING

HORMONE

S- RECEPTORS AND USES BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to melanocyte stimulating hormone receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of a human melanocyte stimulating hormone receptor gene. The invention also relates to the isolation, cloning and sequencing of a mouse melanocyte stimulating hormone receptor gene. The invention relates to the construction of eukaryotic recombinant expression constructs capable of expressing these melanocyte stimulating hormone receptors in cultures of transformed eukaryotic cells, and the production of the melanocyte stimulating hormone receptor in such cultures. The invention relates to the use of such cultures of transformed eukaryotic cells to produce homogeneous compositions of such melanocyte stimulating hormone receptors. The invention also provides cultures of such cells producing melanocyte stimulating hormone receptor for the characterization of novel and 20 useful drugs. Antibodies against and epitopes of these melanocyte stimulating hormone receptor proteins are also provided by the invention.

2. Backaround of the Invention The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, Sa-melanocyte stimulating hormone (aMSH), and adrenocorticotropic hormone (ACTH) have well understood roles in control of melanocyte and adrenocortical function, respectively. Both of these hormones, however, are found in a variety of forms with unknown functions. The melanocortin peptides also have a diverse -2array of biological activities in other tissues, including the brain, and immune system, and bind to specific receptors there with a distinct pharmacology [see, Hanneman et al., in Peptide Honnone as Prohormones, G. Martinez, ed. (Ellis Horwood Ltd.: Chichester, UK) pp. 53-82; DeWied Jolles, 1982, Physiol.

Rev. 62: 976-1059 for reviews].

A complete understanding of these peptides and their diverse biological activities requires the isolation and characterization of their corresponding receptors. Some biochemical studies have been reported on the prior art.

Shimuze, 1985, Yale J. Biol. Med. 58: 561-570 discusses the physiology of melanocyte stimulating hormone.

Tatro Reichlin, 1987, Endocrinology 121: 1900-1907 disclose that MSH receptors are widely distributed in rodent tissues.

Solca et al., 1989, J. Biol. Chem. 264: 14277-14280 disclose the molecular weight characterization of mouse and human MSH receptors linked to radioactively and photoaffinity labeled MSH analogues.

Siegrist et al., 1991, J. Receptor Res. 1: 323-331 disclose the quantification of receptors on mouse melanoma tissue by receptor autoradiography.

i The present invention comprises a human melanocyte stimulating hormone 20 receptor gene, the nucleotide sequence of this gene and the deduced amino acid sequence of its cognate protein, a homogeneous composition of the melanocyte stimulating hormone receptor, nucleic acid hybridization probes and a method for determining the tissue distribution of expression of the gene, a recombinant expression construct capable of expressing the gene in cultures of transformed 25 eukaryotic cells, and such cultures of transformed eukaryotic cells useful in the characterization of novel and useful drugs. The present invention also comprises the homologue of the human melanocyte stimulating hormone receptor gene from the mouse.

-3- DESCRIFIION OF THE DRAWINGS Figure 1 illustrates the nucleotide sequence of the mouse (SEQ ID NO:3) and human (SEQ ID NO:5) melanocyte stimulating hormone receptor.

Figure 2 presents an amino acid sequence comparison between the mouse and human melanocyte stimulating hormone receptor proteins.

Figure 3 illustrates binding of melanocyte stimulating hormone receptor agonists to mouse melanocyte stimulating hormone receptor expressed in human 293 cells.

Figure 4A illustrates the tissue distribution of human melanocyte stimulating hormone receptor gene expression by Northern blot hybridization.

Figure 4B illustrates the tissue distribution of mouse melanocyte stimulating hormone receptor gene expression by Northern blot hybridization.

ea e SUMMARY OF THE INVENTION The present invention relates to the cloning, expression and functional characterization of mammalian melanocyte stimulating hormone receptor (MSH

R

genes. The invention comprises the nucleotide sequence of these genes encoding the mammalian MSH's and the deduced amino acid sequences of the cognate proteins, as well as tissue distribution patterns of expression of these genes.

In particular, the present invention is directed toward the isolation, characterization and pharmacological use of the human MSHR, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the human MSH", a recombinant eukaryotic expression construct capable of expressing the human MSH' in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the human MSHR, a homogeneous composition of the human MSHR, and antibodies against and epitopes of the human MSH

R

The present invention is also directed toward the isolation, characterization and pharmacological use of the mouse MSH', the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the mouse MSH 1 a recombinant eukaryotic expression construct capable of expressing the mouse MSH R in cultures of transformed eukaryotic cells and such 20 cultures of transformed eukaryotic cells that synthesize the mouse MSH', a homogeneous composition of the mouse MSHR, and antibodies against and epitopes of the mouse MSHR.

It is an object of the invention to provide a nucleic acid comprising a nucleotide sequence encoding a mammalian MSHR. In a preferred embodiment of the invention, the nucleotide sequence encodes the human MSHR. In another preferred embodiment, the nucleotide sequence encodes the mouse MSHR.

The present invention includes a nucleic acid comprising a nucleotide sequence encoding a human MSH' receptor derived from a DNA molecule isolated from a human genomic library (SEQ ID NO:5). In this embodiment of the invention, the nucleotide sequence includes 1635 nucleotides of the human MSH' gene comprising 953 nucleotides of coding sequence, 462 nucleotides of untranslated sequence and 220 nucleotides of 3' untranslated sequence.

The present invention also includes a nucleic acid comprising a nucleotide sequence encoding a mouse MSH' derived from a cDNA molecule isolated from a CDNA library constructed with RNA from mouse Cloudman melanoma cells (SEQ ID NO:3). In this embodiment of the invention, the nucleotide sequence includes 1260 nucleotides of the mouse MSH 1 gene comprising 947 nucleotides of coding sequence, 15 nucleotides of 5' untranslated sequence and 298 nucleotides of 3' untranslated sequence.

The invention includes nucleic acids comprising the nucleotide sequences of mammalian MSH's, most preferably mouse and human MSHRs (SEQ ID and includes allelic variations of these nucleotide sequences and the corresponding MSH' molecule, either naturally occurring or the product of in vitro chemical or genetic modification, each such variant having essentially the same nucleotide sequence as the nucleotide sequence of the corresponding MSH

R

disclosed herein, wherein the resulting MSHR molecule has substantially the same biological properties as the MSH' molecule corresponding to the nucleotide sequence described herein. The term "substantially homologous to" as used in this invention encompasses such allelic variability as described in this paragraph.

The invention also includes a protein comprised of a predicted amino acid 20 sequence for the mouse (SEQ ID NO:4) and human (SEQ ID NO:6) MSHR deduced from the nucleotide sequence comprising the complete coding sequence of the mouse (SEQ ID NO:3) and human (SEQ ID NO:5) MSH gene as described herein.

S" In another aspect, the invention comprises a homogeneous composition of a 35.3 kilodalton mouse MSH" or derivative thereof, wherein the amino acid sequence of the MSHI or derivative thereof comprises the mouse MSH-R sequence shown in Figure 2 (SEQ ID NO:4).

In another aspect, the invention comprises a homogeneous composition of a 34.7 kilodalton human MSH' or derivative thereof, wherein the amino acid sequence of the MSH or derivative thereof comprises the human MSH-R sequence shown in Figure 2 (SEQ ID NO:6).

This invention provides both nucleotide and amino acid probes derived from these sequences. The invention includes probes isolated from either cDNA or genomic DNA clones, as well as probes made synthetically with the sequence information derived therefrom. The invention specifically includes but is not limited to oligonucleotide, nick-translated, random primed, or in vitro amplified probes made using cDNA or genomic clone embodying the invention, and oligonucleotide and other synthetic probes synthesized chemically using the nucleotide sequence information of cDNA or genomic clone embodiments of the invention.

It is a further object of this invention to provide sequences of mammalian MSH', preferably the mouse or human MSHR, for use as nucleic acid hybridization probes to determine the pattern, amount and extent of expression of this receptor in various tissues of mammals, including humans. It is also an object of the present invention to provide nucleic acid hybridization probes derived from the sequences of the mouse or human MSH' to be used for the detection and diagnosis of genetic diseases. It is an object of this invention to provide nucleic acid hybridization probes derived from the DNA sequences of the mouse or human MSH' to be used for the detection of novel related receptor genes.

20 The present invention also includes synthetic peptides made using the nucleotide sequence information comprising cDNA or genomic clone embodiments of the invention. The invention includes either naturally occurring or synthetic peptides which may be used as antigens for the production of MSH-specific antibodies, or used for competitors of the MSH' molecule for drug binding, or 25 to be used for the production of inhibitors of the binding of agonists or antagonists or analogues thereof to MSH' molecule.

The present invention also provides antibodies against and epitopes of mammalian MSH's, preferably mouse or human MSH proteins. It is an object of the present invention to provide antibodies that is immunologically reactive to a mammalian MSH' protein. It is a particular object of the invention to provide a monoclonal antibodies to mammalian MSH protein, most preferably mouse or human MSHR protein.

It is also an object of the present invention to provide a hybridoma cell line that produces such an antibody. It is a particular object of the invention to provide a hybridoma cell line that is the result of fusion between a nonimmunoglobulin producing mouse myeloma cell line and spleen cells derived from a mouse immunized with a human cell line which expresses MSH' antigen. The present invention also provides a hybridoma cell line that produces such an antibody, and that can be injected into a living mouse to provide an ascites fluid from the mouse that is comprised of such an antibody.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody that is immunologically reactive to a mammalian MSHR, preferably a mouse or human

MSH

1 and in a pharmaceutically acceptable carrier.

It is a further object of the present invention to provide an epitope of a mammalian MSH R protein wherein the epitope is immunologically reactive to an antibody specific for the mammalian MSH

R

In preferred embodiments, the epitope is derived from mouse of human MSHR protein.

It is another object of the invention to provide a chimeric antibody that is immunologically reactive to a mammalian MSHR protein. In a preferred S 20 embodiment, the chimeric antibody is a monoclonal antibody. In a preferred embodiment, the MSHP is a mouse or human MSH

R

The present invention provides a recombinant expression construct comprising the nucleotide sequence of a mammalian MSH', preferably the mouse or human MSH R and sequences sufficient to direct the synthesis of mouse or human MSH' in cultures of transformed eukaryotic cells. In a preferred embodiment, the recombinant expression construct is comprised of plasmid sequences derived from the plasmid pcDNAI/neo and cDNA or genomic DNA of mouse or human MSH' gene. This invention includes a recombinant expression construct comprising essentially the nucleotide sequences of genomic or cDNA clones of mouse or human MSH' in an embodiment that provides for their expression in cultures of transformed eukaryotic cells.

It is also an object of this invention to provide cultures of transformed eukaryotic cells that have been transformed with such a recombinant expression construct and that synthesize mammalian, preferably mouse or human, MSH

R

protein. In a preferred embodiment, the invention provides human 293 cells that synthesize mouse MSH In an additional preferred embodiment, the invention provides human 293 cells that synthesize human MSHR protein.

The present invention also includes protein preparations of mammalian, preferably mouse or human MSHR, and preparations of membranes containing mammalian MSHR, derived from cultures of transformed eukaryotic cells. In a preferred embodiment, cell membranes containing mouse MSHR protein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of mouse MSH'. In another preferred embodiment, cell membranes containing human MSHR protein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of human MSHR. It also an object of this invention to provide mammalian, preferably mouse or human MSHR for use in the in vtro screening of novel adenosine agonist and antagonist compounds. In a preferred embodiment, membrane preparations containing the mouse MSH

R

derived from **cultures of transformed eukaryotic cells, are used to determine the drug dissociation properties of various novel adenosine agonist and antagonist compounds in vWro. In another preferred embodiment, membrane preparations containing the human MSHR, derived from cultures of transformed eukaryotic cells, are used to determine the drug dissociation properties of various novel "adenosine agonist and antagonist compounds in vitro. These properties are then used to characterize such novel compounds by comparison to the binding properties of known mouse or human MSH' agonists and antagonists.

The present invention will also be useful for the in vivo detection of analogues of agonists or antagonists of MSHR, known or unknown, either naturally occurring or as the embodiments of a drug.

It is an object of the present invention to provide a method for the quantitative detection of agonists or antagonists, or analogues thereof, of MSH known or unknown, either naturally occurring or as the embodiments of a drug. It is an additional object of the invention to provide a method to detect such agonists, antagonists, or analogues thereof in blood, saliva, semen, cerebrospinal fluid, plasma, lymph, or any other bodily fluid.

According to a first embodiment of the invention, there is provided a method of screening a compound for binding to a melanocyte stimulating hormone receptor, the method comprising the following steps: transforming a host cell with a recombinant expression construct encoding a mammalian melanocyte stimulating hormone receptor, wherein the cells of the io transformed cell culture express the receptor; and assaying the transformed cell with the compound to determine whether the compound binds to the melanocyte stimulating hormone receptor.

According to a second embodiment of the invention, there is provided a method of screening a compound for competitive binding to a melanocyte stimulating hormone 15 receptor, the method comprising the following steps: transforming a host cell with a recombinant expression construct encoding a mammalian melanocyte stimulating hormone receptor, wherein the cells of the S transformed cell culture express the receptor; and assaying the transformed cell with the compound in the presence and in the absence of an agonist for the melanocyte stimulating hormone receptor; and determining whether the compound competes with the agonist for binding to the melanocyte stimulating hormone receptor.

According to a third embodiment of the invention, there is provided a method of screening a compound to determine if the compound is an inhibitor of a melanocyte 25 stimulating hormone receptor, the method comprising the following steps: o o* transforming a host cell with a recombinant expression construct encoding a mammalian melanocyte stimulating hormone receptor, wherein the cells of the transformed cell culture express the receptor; and assaying the transformed cell culture with the compound to determine whether the compound is capable of inhibiting binding to the melanocyte stimulating hormone receptor by a melanocyte stimulating hormone receptor agonist.

Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

[R:\LIBFF]08635.doc:gcc DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The term "melanocyte stimulating hormone receptor" as used herein refers to proteins substantially homologous to, and having substantially the same biological activity as, the protein coded for by the nucleotide sequence depicted in Figure 1 (SEQ ID NO:3). This definition is intended to encompass natural allelic variations in the melanocyte stimulating hormone receptor sequence.

Cloned genes of the present invention may code for MSHs of any species of origin, including, for example, mouse, rat, rabbit, cat, and human, but preferably code for receptors of mammalian, most preferably mouse and human, origin.

Nucleic acid hybridization probes provided by the invention comprise DNA sequences that are substantially homologous to the DNA sequences in Figure 1A (SEQ ID NO:3) and IB (SEQ ID NO:5). Nucleic acid probes are useful for detecting MSH 1 gene expression in cells and tissues using techniques well-known in the art, including but not limited to Northern blot hybridization, in situ hybridization and Southern hybridization to reverse transcriptase polymerase chain reaction product DNAs. The probes provided by the present invention, including oligonucleotides probes derived therefrom, are useful are also useful for Southern hybridization of mammalian, preferably human, genomic DNA for screening for restriction fragment length polymorphism (RFLP) 20 associated with certain genetic disorders.

The production of proteins such as the MSH' from cloned genes by genetic engineering is well known. See, U.S. Patent No. 4,761,371 to Bell et al.

at Col. 6 line 3 to Col. 9 line 65. (The disclosure of all U.S. patent references cited herein is to be incorporated herein by reference.) The discussion which o 25 follows is accordingly intended as an overview of this field, and is not intended to reflect the full state of the art.

DNA which encodes the MSH' may be obtained, in view of the instant disclosure, by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by combinations of these procedures, as illustrated below.

Screening of mRNA or genomic DNA may be carried out with oligonucleotide -11probes generated from the MSH' gene sequence information provided herein.

Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with know procedures and used in conventional hybridization assays, as described in greater detail in the Examples below. In the alternative, MSH' gene sequences may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the MSH gene sequence provided herein. See U.S. Patent Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis.

The MSHR may be synthesized in host cells transformed with a recombinant expression construct comprising a DNA sequence encoding the MSH. Such a recombinant expression construct can also be comprised of a vector that is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding the MSH' and/or to express DNA which encodes the MSH'. For the purposes of this invention, a recombinant expression construct is a replicable DNA construct in which a DNA sequence encoding the MSH t is operably linked to suitable control sequences capable of effecting the expression of the MSH' in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen.

Generally, control sequences include a transcriptional promoter, an optional 20 operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of trnsformants.

Vectors useful for practicing the present invention include plasmids, viruses (including phage), retroviruses, and integratable DNA fragments fragments integratable into the host genome by homologous recombination). The vector replicates and functions independently of the host genome, or may, in some 30 instances, integrate into the genome itself. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the -12intended expression host. A preferred vector is the plasmid pcDNAI/neo.

Transformed host cells are cells which have been transformed or transfected with recombinant expression constructs made using recombinant DNA techniques and comprising a mammalian MSH'. Transformed host cells may ordinarily express the mammalian MSH but host cells transformed for purposes of cloning or amplifying nucleic acid hybridization probe DNA need not express the receptor.

When expressed, the mammalian MSH' will typically be located in the host cell membrane.

DNA regions are operably linked when they are functionally related to each other. For example: a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

Generally, operably linked means contiguous and, in the case of leaders sequences, contiguous and in the same translational reading frame.

Cultures of cells derived from multicellular organisms are a desirable host for recombinant MSH' synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. However, mammalian cells are preferred, as illustrated in the Examples. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture, 20 Academic Press, Kruse Patterson, editors (1973). Examples of useful host cell lines are human 293 cells, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Human 293 cells are preferred. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to S: 25 be expressed, along with a ribosome binding site, RNA splice sites (if introncontaining genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source polyoma, adenovirus, VSV, or MPV), or may be provided by the host cell chromosomal replication mechanism. If the vector is -13integrated into the host cell chromosome, the latter may be sufficient.

The invention provides homogeneous compositions of mammalian MSHR protein produced by transformed eukaryotic cells as provided herein. Such homogeneous compositions are intended to be comprised of mammalian MSH

R

protein that comprises 90% of the protein in such homogenous composition.

Mammalian MSH' protein made from cloned genes in accordance with the present invention may be used for screening agonist compounds for MSH

R

activity, or for determining the amount of a MSHR agonist or antagonist drug in a solution blood plasma or serum). For example, host cells may be transformed with a recombinant expression construct of the present invention, MSHR expressed in that host, the cells lysed, and the membranes from those cells used to screen compounds for MSH" binding activity. Competitive binding assays in which such procedures may be carried out are well known in the art. By selection of host cells which do not ordinarily express MSHRs, pure preparations of membranes containing MSH's can be obtained. Further, MSH R agonists and antagonists can be identified by transforming host cells with vectors of the present invention. Membranes obtained from such cells can be used in binding studies wherein the drug dissociation activity is monitored.

The recombinant expression constructs of the present invention are useful 20 in molecular biology to transform cells which do not ordinarily express the MSHR to thereafter express this receptor. Such cells are useful as intermediates for S' making cell membrane preparations useful for receptor binding assays, which are in turn useful for drug screening. Further, genes and vectors comprising the recombinant expression construct of the present invention are useful in gene 25 therapy. For such purposes, retroviral vectors as described in U.S. Patent No.

4,650,764 to Temin Watanabe or U.S. Patent No. 4,861,719 to Miller may be employed. Cloned genes of the present invention, or fragments thereof, may also be used in gene therapy carried out homologous recombination or site-directed mutagenesis. See generally Thomas Capecchi, 1987, Cell 51: 503-512; 30 Bertling, 1987, Bioscience Reports 2:107-112; Smithies et 1985, Nature 312: 230-234.

-14- Oligonucleotides of the present invention are useful as diagnostic tools for probing MSH receptor gene expression in tissues. For example, tissues can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques, as explained in greater detail in the Examples below, to investigate native expression of this receptor or pathological conditions relating thereto. Further, chromosomes can be probed to investigate the presence or absence of the MSH' gene, and potential pathological conditions related thereto, as also illustrated by the Examples below.

The invention also provides antibodies that are immunologically reactive to a mammalian MSH'. The antibodies provided by the invention can be raised in animals by inoculation with cells that express a mammalian MSHR or epitopes of a mammalian MSH' using methods well known in the art. Animals that can be used for such inoculations include individuals from species comprising cows, sheep, pigs, mice, rats, rabbits, hamsters, goats and primates. Preferred animals for inoculation are rodents (including mice, rats, hamsters) and rabbits. The most preferred animal is the mouse.

Cells that can be used for such inoculations, or for any of the other means used in the invention, include any cell line which naturally expresses a O mammalian MSH', or any cell or cell line that expresses a mammalian MSH n or 20 any epitope therein as a result of molecular or genetic engineering, or that has been treated to increase the expression of a mammalian MSH" by physical, biochemical or genetic means. Preferred cells are human cells, most preferably human 293 cells that have been transformed with a recombinant expression S construct comprising DNA sequences encoding a mammalian MSHR and that express the mammalian MSH' gene product.

The present invention provides monoclonal antibodies that are immunologically reactive with an epitope that is a mammalian MSH' present on the surface of mammalian cells, preferably human or mouse cells. These antibodies are made using methods and techniques well known to those of skill in the art.

Monoclonal antibodies provided by the present invention are produced by nybridoma cell lines, that are also provided by the invention and that are made by methods well known in the art. Hybridoma cell lines are made by fusing individual cells of a myeloma cell line with spleen cells derived from animals immunized with cells expressing a mammalian MSH", including human cells, as described above. The myeloma cell lines used in the invention include lines derived from myelomas of mice, rats, hamsters, primates and humans. Preferred myeloma cell lines are from mouse, and the most preferred mouse myeloma cell line is P3X63-Ag8.653. The animals from whom spleens are obtained after immunization are rats, mice and hamsters, preferably mice, most preferably Balb/c mice. Spleen cells and myeloma cells are fused using a number of methods well known in the art, including but not limited to incubation with inactivated Sendai virus and incubation in the presence of polyethylene glycol (PEG). The most preferred method for cell fusion is incubation in the presence of a solution of 45% PEG-1450. Monoclonal antibodies produced by hybridoma cell lines can be harvested from cell culture supernatant fluids from in vitro cell growth; alternatively, hybridoma cells can be injected subcutaneously and/or into the peritoneal cavity of an animal, most preferably a mouse, and the monoclonal antibodies obtained from blood and/or ascites fluid.

Monoclonal antibodies provided by the present invention can also be 20 produced by recombinant genetic methods well known to those of skill in the art, 0 S. and the present invention encompasses antibodies made by such methods that are immunologically reactive with an epitope of a mammalian MSHR.

The present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a mammalian MSHR. Such fragments 25 can be produced by any number of methods, including but not limited to proteolytic cleavage, chemical synthesis or preparation of such fragments by SO. means of genetic engineering technology. The present invention also encompasses single-chain antibodies that are immunologically reactive with an epitope of a mammalian MSHR made by methods known to those of skill in the art.

o* 0 30 The present invention also encompasses an epitope of a mammalian MSH that is comprised of sequences and/or a conformation of sequences present in the -16mammalian MSH' molecule. This epitope may be naturally occurring, or may be the result of proteolytic cleavage of the mammalian MSH R molecule and isolation of an epitope-containing peptide or may be obtained by synthesis of an epitope-containing peptide using methods well known to those skilled in the art.

The present invention also encompasses epitope peptides produced as a result of genetic engineering technology and synthesized by genetically engineered prokaryotic or eukaryotic cells.

The invention also includes chimeric antibodies, comprised of immunologically reactive light chain and heavy chain peptides to an epitope that is a mammalian MSH'. The chimeric antibodies embodied in the present invention include those that are derived from naturally occurring antibodies as well as chimeric antibodies made by means of genetic engineering technology well known to those of skill in the art.

The Examples which follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.

EXAMPLE 1 Isolation of an aMSH Receptor Probe by Random PCR Amplification of Human Melanoma cDNA Using Degenerate Olionucleotide Primers In order to clone novel G-protein coupled receptors, human melanoma 25 cDNA was used as template for a polymerase chain reaction (PCR)-based random cloning experiment PCR was performed using a pair of degenerate oligonucleotide primers corresponding to the putative third and sixth transmembrane regions of G-protein coupled receptors (Libert et al., 1989, Science 244: 569-72; Zhou et al., 1990, Nature M4: 76-80). The PCR products 30 obtained in this experiment were characterized by nucleotide sequencing. Two novel sequences representing novel G-protein-coupled receptors were identified.

PCR amplification was performed as follows. Total RNA was isolated from a human melanoma tumor sample by the guanidinium thiocyanate method -17- (Chirgwin et al., 1979, Biochemistry 18: 5294-5299). Double-stranded cDNA was synthesized from total RNA with murine reverse transcriptase (BRL, Gaithersburg, MD) by oligo-dT priming [Maniatis et al., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), 1990]. The melanoma cDNA mixture was then subjected to 45 cycles of PCR amplification using 500 picomoles of degenerate oligonucleotide primers having the following sequence: Primer III (sense):

GAGTCGACCTGTG(C/T)G(CT)(C/G)AT(C/T)(A/G)CIIT(GT)GAC(C/A)G(C/G)T

AC

(SEQ ID NO: 1) and Primer VI (antisense):

CAGAATTCAG(T/A)AGGGCAICCAGCAGAI(GIC)(G/A)(T/C)GAA

(SEQ ID NO:2) in 100 tl of a solution containing 50 mM Tris-HCl (pH 2.5 mM MgCi 2 0.01% gelatin, 200 gM each dNTP, and 2.5 units of Taq polymerase (Saiki et al., 1988, Science 239: 487-491). These primers were commercially synthesized by Research Genetics Inc. (Huntsville, AL). Each PCR amplification cycle consisted of incubations at 94°C for 1 min (denaturation), 45 0 C for 2 min (annealing), and 72"C for 2 min (extension).

Amplified products of the PCR reaction were extracted with phenol/chloroform and precipitated with ethanol. After digestion with EcoRI and S SalI, the PCR products were separated on a 1.2% agarose gel. A slice of this 30 gel, corresponding to PCR products of 300 basepairs (bp) in size, was cut out and z purified using glass beads and sodium iodide, and the insert was then cloned into a pBKS cloning vector (Stratagene, LaJolla, CA).

A total of 172 of such pBKS clones containing inserts were sequenced using Sequenase S. Biochemical Corp., Cleveland, OH) by the dideoxynucleotide chain termination method (Sanger et al., 1977, Proc. Natl.

Acad. Sci. USA 74: 5463-5467). Two types of sequences homologous to other -18- G-protein coupled receptors were identified.

EXAMPLE 2 Isolation and Seauence Analysis of Mouse aMSH Recentor cDNA Probes isolated in Example 1 was used to screen a Cloudman melanoma cDNA library in order to isolate a full-length cDNA corresponding to the cloned probe. One clone was isolated from a library of 5 x 106 clones screened as described below. This clone contained an insert of 2.6 kilobases The nucleotide sequence of the complete coding region was determined, as shown in Figure IA (SEQ ID NO:3).

The PCR probe was labeled by the random-priming method (Stratagene Primelt, #300387, LaJolla, CA) and used to screen a Cloudman melanoma line cDNA library constructed in the XZAP vector (Stratagene). Library screening was performed using techniques well-known in the art as described in Bunzow et al. (1988, Nature 336: 783-787) at moderate stringency (40% formamide, 1M NaCI, 50mM Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 100g/ml salmon sperm DNA, 10X Denhardt's solution). One cDNA clone was identified (termed mmelA) and its 2.6 kb cDNA insert was isolated and subcloned into pBKS (Stratagene); the resulting plasmid was called pmmelA. Nucleotide sequence analysis and homology comparisons were done on the OHSU computer system with software provided by Intelligenetics Inc.

(Mountain View, CA).

25 The nucleotide sequence of pmmelA (the cDNA clone isolated as described above) is shown in Figure IA (SEQ ID NO:3). The longest open reading frame of this cDNA encodes a predicted protein product of 315 amino acids with a calculated molecular weight of 35.3 kilodaltons The deduced amino acid sequence is shown in Figure 2 (SEQ ID NO:4) as mouse MSH-R. Single letter amino acid codes are used [see, G. Zubay, Biochemistry (2d 1988 (MacMillen Publishing: New York) p.33]. Uppercase lettering indicates amino acid residues in common between the receptor proteins shown; lowercase lettering indicates divergent residues.

-19- Hydrophobicity analysis (Kyte Doolittle, 1982, J. Mol. Biol. 157: 105-132) of the deduced amino acid sequence showed that the protein contains seven hydrophobic stretches of 21 to 26 amino acids apiece. Putative transmembrane domains are overlined and designated with Roman numerals.

EXAMPLE 3 Construction of Mouse aMSHR Expression Plasmids, DNA Transfection and Functional Exnression of the aMSHa Gene Product In order to biochemically characterize the putative mouse aMSHR cDNA isolated as in Example 2, and to confirm that it encodes an aMSH receptor, mmelA was cloned into a mammalian expression vector, this vector transfected into human 293 cells, and cell lines generated that expressed the putative aMSHR receptor at the cell surface. Such cells and membranes isolated from such cells were used for biochemical characterization experiments described below.

The entire coding region of the aMSH cDNA insert from mmelA contained in a 2.1kb fragment was excised from pBSK and subcloned into the BamHI/XhoI sites of pcDNAI/neo expression vector (Invitrogen, San Diego, CA).

The resulting plasmid was called pcDNA-mmelA. pcDNA-mmelA plasmid DNA was prepared in large-scale through one cycle of CsCl gradient ultracentrifugation and 20 Ag pcDNA-mmelA DNA were transfected into each 100mm dish of 293 cells using the calcium phosphate method (see Chen Okayama, 1987, Mol.

Cell. Biol. 7: 2745-2752). After transfection, cells were cultured in DMEM 25 media supplemented with 10% calf serum in a 3% CO, atmosphere at 37*C.

Selection was performed with neomycin (G418; GIBCO) at a concentration of 1000 ug/ml; selection was started 72 hr after transfection and continued for 3 weeks.

The aMSH' is known to couple to G-proteins and thereby activate adenyl 30 cyclase, increasing intracellular levels of cAMP (see Buckley Ramachandran, 1981, Proc. Natl. Acad. Sci. USA 78: 7431-7435; Grahame-Smith et al., 1967, J. Biol. Chem 242: 5535-5541; Mertz Catt, 1991, Proc. Natl. Acad. Sci. USA 8: 8525-8529; Pawalek et al., 1976, Invest. Dermatol. 66: 200-209). This property of cells expressing the aMSH receptor was used analyze expression of the aMSH receptor in cell colonies transfected with the expression vectors described herein as follows. Cells 1xl0 6 were plated in 6-well dishes, washed once with DMEM containing 1 bovine serum albumin (BSA) and 0.5mM IBMX (a phosphodiesterase inhibitor), then incubated for 45 minutes at 37C with varying concentrations of the melanotropic peptides aMSH, 3MSH, yMSH, the MSH peptide analogues Nle, D-Phe'-aMSH (NDP-MSH), and ACTH.

Following hormone treatment, the cells were washed twice with phosphate buffered saline and intracellular cAMP extracted by lysing the cells with lml of 60% ethanol. Intracellular cAMP concentrations were determined using an assay (Amersham) which measures the ability of cAMP to displace cAMP from a high affinity cAMP binding protein (see Gilman, 1970, Proc. Natl. Acad. Sci.

USA 27: 305-312).

The results of these experiments are shown in Figure 3. The abscissa indicates the concentration of each hormone and the ordinate indicates the percentage of basal intracellular cAMP concentration achieved by each treatment.

Points indicate the mean of duplicate incubations; the standard error did not exceed 15% for any data point. None of the peptides tested induced any change in intracellular cAMP in cells containing the vector alone. Cells expressing the murine aMSH receptor responded to melanotropic peptides with a 2-3 fold elevation of intracellular cAMP, similar to levels of cAMP induced by these peptides in the Cloudman cell line (see Pawalek, 1985, Yale J. Biol. Med. 58: 571-578). The ECs values determined for aMSH (2.0x10- 9

ACTH

(8.0xl0M) and the superpotent MSH analogue NDP-MSH (2.8x10-M) correspond closely to reported values (see Tatro et al., 1990, Cancer Res. 1237-1242). As expected, the OMSH peptide had an EC, value comparable to aMSH while -yMSH had little or no activity (see Slominski et al., 1992, Life Sci. 5Q: 1103-1108), confirming the identity of this receptor as a melanocyte aMSH receptor.

a -21- EXAMPLE 4 Isolation and Characterization of a Human pMSHR Genomic Clone In order to isolate a human counterpart of the murine melanocyte aMSH receptor gene, a human genomic library was screened at high stringency formamide, 42*C) using the human PCR fragments isolated as described in Example 1. Two different types of sequences were isolated, corresponding to the two PCR fragments, and were found to encode highly related G protein-coupled receptors. These genomic clones were sequenced as described in Example 2.

One of these genomic clones was determined to encode an human MSH receptor (SEQ ID NO:5). The human MSH receptor has a predicted amino acid sequence (SEQ ID NO:6) that is 75% identical and colinear with the mouse aMSH receptor cDNA sequence (Figure represented as human MSH-R. The predicted molecular weight of the human MSH is 34.7kD.

The predicted amino acid sequences of the mouse aMSHR (SEQ ID NO:4) and human MSH' (SEQ ID NO:6) are aligned in Figure 2. These sequences define the melanocortin receptors as a novel subfamily of the G protein-coupled receptors with a number of unusual features. The melanocortin receptors are the smallest G protein-coupled receptors identified to date (297-317aa) resulting from a short amino terminal extracellular domain, a short carboxy-terminal intracellular domain, and a very small third intracellular loop. The melanocortin receptors are lack several amino acid residues present in most G protein coupled receptors (see Probst et al., 1992, DNA Cell Biol. U1: 1-20), including the proline residues 25 in the 4th and 5th transmembrane domains, likely to introduce a bend in the alpha helical structure of the transmembrane domains and thought to be involved in the formation of the binding pocket (see Applebury Hargrave, 1986, Vision Res.

26: 1881-1895), and one or both of the cysteine residues thought to form a disulfide bond between the first and second extracellular loops (see Dixon et al., 30 1987, EMBO J. 6: 3269-3275 and Karnik et al., 1988, Proc. Natl. Acad. Sci.

USA 85: 8459-8463). Remarkably, the melanocortin receptors do not appearhighly related to the other G protein-coupled receptors which recognize peptide ligands, such as the receptors for bombesin (see Spindel et al., 1990, Mol.

o* -22- Endocrinol. 4: 1956-1963) or substance K (see Masu et al., 1987, Nature M2.: 836-838) but rather, are more closely related to the receptor for A'-tetradhydrocannabinol (see Matsuda et 1990, Nature 346: 561-564). The cannabinoid receptor also lacks the conserved proline in transmembrane 5 and the cysteine in the first extracellular loop necessary for disulfide bond formation.

Least parsimony analysis with the receptor sequences shown in Figure 2 suggests the cannabinoid and melanocortin receptors may be evolutionarily related and form a subfamily distinct from the peptide receptors and the amine receptors.

Regardless of whether the similarities are the result of evolutionary conservation or convergence, the sequence and putative structural similarities between the melanocortin and cannabinoid receptors may be informative in the search for the endogenous cannabinoid-like ligand.

EXAMPLE S Tissue Distribution of cMSH Receptors To further gain insight into these receptors, we have examined the tissue distribution of their corresponding mRNAs from various tissues by performing Northern hybridization experiments on RNA isolated from various tissues (see Maniatis et al., ibid.). The results of these experiments are shown in Figures 4A and 4B.

A panel of tissue samples was examined by Northern hybridization analysis performed under high stringency conditions. The same nitrocellulose filter was hybridized successively with a human MSH receptor probe and a mouse MSH receptor probe to determine the distribution of each receptor mRNA. The murine MSH receptor is encoded predominantly by a single mRNA species of 3.9kb, while the human MSH receptor is encoded, in two melanoma samples, *predominantly by a 3.0kb species. High levels of receptor mRNA are seen in both primary mouse melanocytes and mouse melanoma cell lines. In contrast, 3 extremely low levels of receptor mRNA were detected in primary human melanocytes, and many human melanoma samples (see melanoma 1, Fig. 4A).

Most intriguing is the dramatic elevation of MSH-R mRNA seen thus far in 3 of S11 samples tested, such as is seen in melanoma sample #2 (Fig. 4A).

-23- Additionally, we have been unable to detect expression in the brain of any of the receptors described here, despite extensive documentation of MSH binding sites there as well as in other tissues. These finding suggest the existence of alternate forms of these or related receptors that may be specifically expressed in brain tissue.

It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

e

S*

-24- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Cone, Roger D Mountjoy, Kathleen G (ii) TITLE OF INVENTION: Melanocyte Stimulating Hormone Receptor and Uses (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Allegretti Witcoff, Ltd.

STREET: 10 South Wacker Drive, Suite 3000 CITY: Chicago STATE: Illinois COUNTRY: USA ZIP: 60606 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: PCT/US93/03247 FILING DATE: 07-APR-1993

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Noonan, Kevin E REGISTRATION NUMBER: 35.303 REFERENCE/DOCKET NUMBER: 92,154-A (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 312-715-1000 TELEFAX: 312-715-1234 TELEX: 910-221-5317 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..33 OTHER INFORMATION: /function= "Degenerate oligonucleotide primer (sense)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAGTCGACCT GTGYGYSATY RCTKGACMGS TAC 33 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1.31 OTHER INFORMATION: /f unction= "Degenerate oliqonucleotide primer (antisense)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CAGAATTCAO WAGGOCACCA GCAGASRYGA A 31 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1260 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 15..959 I. FEATURE: NAME/KEY: 5'1UTR LOCATION: L..14

FEATURE:

NAME/KEY: 3' UTE LOCATION: 960.31260 -26- Aen Ala Thr Ser His Lau Gly Leu Ala Thr Asn Gin Ser Glu Pro Trp 25 Cy. Leu Tyr Val 5cr Ile Pro Asp Gly Leu Ph. Lou Ser Lou Gly Leu 40 Val Ser Lou Val Glu Asn Val. Lau Val Val Ile Ala Ile Thr Lys Asn 55 Arg Aen Leu His Ser Pro Met Tyr Tyr Ph Ile Cys Cys Lau Ala Leu 70 75 Ser Asp Leu Met Val Ser Val Ser Ile Val Leu Glu Thr Thr Ile Ile 90 Lou Lou Lou Giu Val Gly Ile Lau Val Ala Arg Val Ala Lou Val Gin -100 105 110 Gin Lou Asp Aen Leu Ile Asp Val Leu Ile Cys Gly Ser Met Val Scr 115 120 125 Ser Leu Cy. Phe Lou Gly Ile Ile Ala Ile Asp Ar; Tyr Ile Ser Ile 130 135 140 Ph. Tyr Ala Leu Ar; Tyr His Ser Ile Val Thr Lou Pro Ar; Ala Ar; 145 150 155 160 Arg Ala Val Val Gly Ile Trp, Met Val 5cr Ile Val Sor Ser Thr Lou 165 170 175 Phe 1ie Thr Tyr Tyr Lys His Thr Ala Val Lou Lou Cys Lou Val Thr 180 185 190 Phe Phe Lou Ala Met Lou Ala Lou Met Ala Ile Leu Tyr Ala His Met 195 200 205 *Pho Thr Ar; Ala Cys Gin His Val Gin Gly Ile Ala Gin Lou His Lys 210 215 220 Ar; Ar; Arg Ser Ile Ar; Gin Gly Ph. Cys Lou Lys Gly Ala Ala Thr 225 230 235 240 Lou Thr Ile Lou Lou Giy Ile Ph. Ph. Lou Cys Trp Gly Pro Phe Pho 245 250 255 Lou His Leu Lou Lou Ile Val Lou Cys Pro Gln His Pro Thr Cys Sor 260 265 270 Cyu Ile Phe Lye Asn Ph. Asn Lou Pho Lou Lau Leu Ile Val Lou 8cr 275 280 285 Sor Thr Val Asp Pro Lou Ile Tyr Ala Ph. Ar; Sor Gin Glu Lou Arg 290 295 300 Met 305 (2) -27- Thr Leu Lys Glu Val Leu Leu Cys Sor Trp 310 315 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1633 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 462..1415 (ix) FEATURE: NAME/KEY: 5' UTR LOCATION: L.461 (ix) FEATURE: NAHE/KEY: 3 *UTR LOCATION: 1416.. 1633 (xi) SEQUENCE DESCRIPTION: SEQ ID 110:5: CCCGCATGTG GCCGCCCTCA ATGGAGGGCT CTGAGAACGA AGCTCCATTC TTCCCAGACC TCAGCGCAGC CCTGGCCCAG AGGACGGTCC AGAGGTGTCG AAATGTCCTG GGAACCTGAG GCAGGGAGGG, ACCTGAGGAC CAGGCTTGGT TGTGAGAATC GCCAGGAGGT GTCTGGACTG GCTGGGCCAT GCCTGGGCTG GGTGTGAGGG CAGATCTGGG GGTGCCCAGA TGGAAGGAGG GCCCCCTGGC ACCACCATGA ACTAAGCAGG ACACCTGGAG GGAGGCCTCC AhCGACTCCT TCCTGCTTCC TGGACAGGAC GGA TCC CAG AGA AGA CTT CTG GGC TCC CTC AAC Gly Ser Gln Arg Arg Lou Lou Gly Sor Lou An 10 15 ATC CCC CAG CTG 000 CTG OCT GCC AAC CAG ACA Ile Pro Gln Lou Gly Lou Ala Ala Ann Gln Thr 30 CTTTTAAAAC GCAGAGAAAA GAAGGGAGGA GACAGAGGCC CAGCAGCCAC CAGGGAAGAG CCTGAGCCCA GGCGGTTGAT ACCTGTCCAG CCAGGGAGAG CAGGCATGGG; GACACCCAAG GGGAAGAACT GTGGGGACCT T ATG GCT GTG CAG Met Ala Val Gln TCC ACC CCC ACA GCC Ser Thr Pro Thr Ala GGA GCC CGG TGC CTG Gly Ala Arg Cys Lou -28- GAG GTG TCC ATC TCT GAC GGG CTC TTC CTC AGC CTG GGG CTG GTG AGC 617 Glu Val Ser Ile Ser Asp Gly Lou Ph. Leu Ser Lau Gly Lau Val Ser 45 TTG GTG GAG AAC GCG CTG GTG GTG GCC ACC ATC GCC AAG, AAC CGG AAC 665 Leu Val Glu Agn Ala Leu Val Val Ala Thr Ile Ala.Lye Asn Arg Asn 60 CTG CAC TCA CCC ATG TAC TGC TTC ATC TOC TGC CTG GCC TTG TCG, GAC 713 Lau His Ser Pro Mot Tyr Cys Phe Ile Cys Cys Lau Ala Lou Ser Asp 75 CTG CTG CTG AGC GGG, ACO AAC GTG CTG GAG ACG GCC GTC ATC CTC CTG 761 Lau Lou Val Ser Giy Thr Asn Val Leu Glu Thr Ala Val Ile Leu Leu 90 95 100 CTG GAG CCC COT OCA CTG GTC GCC COG GCT C GTG; CTG CAG, CAG CTG 809 Lou Olu Ala Gly Ala Lou Val Ala Arg Ala Ala Val Lou Gin Gin Lou 105 110 115 GAC AAT CTC ATT GAC GTO ATC ACC TGC ACC TCC ATG CTG TCC AOC CTC 857 Asp Asn Vai Ile Asp Val Ile Thr Cys Ser Sor Met Lou Sor Ser Lou 120 125 130 TGC TTC CTG GGC CCC ATC GCC GTG GAC CGC TAC ATC TCC ATC TTC TAC 905 Cys Ph. Lou Gly Ala Ile Ala Val Asp Arg Tyr Ile Ser Ile Phe Tyr 135 140 145 GCA CTO CGC TAC CAC AGC ATC OTG ACC CTG CCG CGC; GCG CCG CGA GCC 953 Ala Lou Arg Tyr His Sr Ile Val Thr Lou Pro Arg Ala Pro Arg Ala 150 155 160 OTT GCG CCC ATC TGG CTG GCC AGT GTC GTC TTC AGC ACG CTC TTC ATC 1001 Val Ala Ala Ile Trp, Val Ala Ser Val Val Ph. Ser Thr Lou Ph. Ile 165 170 175 180 GC TAr. TC GAC CA iaTj GC GTC CiT CTG TGC CTC GTG GTC TTC TTC 1049 Ala Tyr Tyr Asp His Val Ala Val Lou Lou Cys Leu Val Val Ph. Phe 185 190 195 CTG GCT ATG CTG GTG CTC ATG GCC OTO CTG TAC GTC CAC ATO CTG GCC 1097 Lau Ala Met Lou Val Leu Met Ala Val Lou Tyr Val His Mot Lou Ala 200 205 210 CGG GCC TGC CAG CAC CCC CAG GGC ATC CCC COG CTC CAC AAG AGO CAG 1145 Arg Ala Cys Gin His Ala Gin Cly Ile Ala Arg Lau His Lys Arg Gin *215 220 225 CCC CCC CTC CAC CAG GGC TTT GGC CTT AAA CCC GCT GTC ACC CTC ACC 1193 Arg Pro Val His Gin Gly Phe Gly Lau Lye Gly Ala Val Thr Lou Thr 230 235 240 ATC CTG CTG GGC Ile Lau Lau Gly 245 CTC ACA CTC ATC Leu Thr Leu Ile TTC AAG AAC TTC Ph. Lys Asn Ph.

280 AT? TTC Ile Phe 250 GTC CTC Val Lau TTC CTC TGC Ph. Lou Cya TGC CCC GAG Cys Pro Giu -29- TGG GGC CCC TTC TTC CTG Trp Gly Pro Phe Phe Leu 255

CAT

His 260 265

AAC

Ann CTC TTT CTC Lou Ph. Leu

CC

Ala 285 CAC CCC ACG TGC GGC TGC ATC His Pro Thr Cys Gly CyS Ile 270 275 CTC ATC ATC TGC AAT GCC ATC Lou Ile Ile Cys Amn Ala Ile 290 AGC CAG GAG CTC CGC AGG ACG Ser Gin Giu Lau Arg Arg Thr 305 TGAGCGCGGT GCACGCGCTT 1241 1289 1337 1385 1432 ATC GAC CCC CTC ATC TAC GCC Ile Asp Pro Lou Ile Tyr Ala 295 CTC AAG GAG GTG CTG ACA TGC Lou Lys Giu Val Lau Thr Cys 310 315 TTC CAC Ph. His 300 TCC TGG Ser Trp TAAGTGTGCT GGGCAGAGGG AGGTGGTGAT ATTGTGGTCT GGTTCCTGTG

TGACCCTGGG

CAGTTCCTTA CCTCCCTGGT CCCCGTTTGT CAAAGAGGAT GGACTAAATG

ATCTCTGAAA

GTGTTGAAGC GCGGACCCTT CTGGGCAGGG, AGGGGTCCTG CAAA&CTCCA

GGCAGGACTT

CTCACCAGCA GTCGTGGGAA C 1492 1552 1612 1633 q ce i *a.q 0 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 317 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTIONs SEQ ID Met Ala Val Gin Gly Ser Gin Arg Arg Lou 1 5 10 Thr Pro Thr Ala Ile Pro Gin Lou Gly Lou 20 25 NO :6: Lou Gly Ala Ala Ser Lou Asn Ser Asn Ala Arg Cys Lou GlU Gly Lou Val Ser Lou Val Ser Ile Ser Asp Gly Lou Phe 40 Val Glu Aen Ala Lou Val Val Ala 55 AGin Thr Gly Lou Ser Lou Thr Ile Ala Lys Asn Arg Aen Lou His Sor Pro Met Tyr Cys Ph.

70 75 Ile Cys Cys Lou Ala Val Lau Lou Ser 145 Ala Thr Val His His 225 Val Phe Re Cu 305 Sor Lau Gin 115 8cr Ph.

Arg Pho Ph.

195 Lou Arg Lou Lou Cl's 275 Ala Arg Asp Lou 100 Lau Lou Tyr Ala Ile 180 Ph.

Ala Gin Thr His 260 Ile I le Thr Lou Leu Asp Cyu Ala Val 165 Ala Lou Arg Arg Ile 245 Leu Ph.

Ile Lou Lou Giu Asn Ph.

Lou 150 Ala Tyr Ala Ala Pro 230 Lou Thr Lys Asp Lys 310 Val Ala Val Lou 135 Arg Ala Tyr Met cys 215 Val Lou Lou Asn Pro 295 Giu Sor Giy Ile 120 Gly Tyr Ile Asp Lou 200 Gin His Gly Ile Ph.

280 Lou Val Gly Ala 105 Asp Ala His Trp His 185 Val His Gin Ile Val 265 Asn Ile Lou Thr 90 Lou Val Ilie Sor Val 170 Val Lou Ala Gly Ph.

250 Lou Lou Tyr Thr Aun Val Ile Ala Ile 155 Ala Ala Met Gin Ph.

235 Ph.

Cys Ph.

Ala Cl's 315 Val Ala Thr Val 140 Val Sor Val Ala Gly 220 Gly Lou Pro Lou Pho 300 Ser Lou Glu Arg Ala 110 Cym Ser 125 Asp Arg Thr Lou Val Val Lou Lou 190 Val Lou 205 Ile Ala Lou Lys Cym Trp Giu His 270 Ala Lou 285 His Ser Trp Thr Ala Ala Val Ser Met Tyr Ile Pro Arg 160 Phe 175 Cys Lou Tyr Val Arg Lou Gly Ala 240 Gly Pro 255 Pro Thr Ile Ile Gin Glu

Claims (19)

1. A method of screening a compound for binding to a melanocyte stimulating hormone receptor, the method comprising the following steps: transforming a host cell with a recombinant expression construct encoding a mammalian melanocyte stimulating hormone receptor, wherein the cells of the transformed cell culture express the receptor; and assaying the transformed cell with the compound to determine whether the compound binds to the melanocyte stimulating hormone receptor.

2. The method of claim 1, wherein the mammalian melanocyte stimulating 0o hormone receptor is a human melanocyte stimulating hormone receptor having an amino acid sequence identified as SEQ ID No. 6.

3. The method of claim 2, wherein the mammalian melanocyte stimulating hormone receptor is a mouse melanocyte stimulating hormone receptor having an amino acid sequence identified in Figure 2. i

4. A method of screening a compound for competitive binding to a melanocyte stimulating hormone receptor, the method comprising the following steps: transforming a host cell with a recombinant expression construct encoding a mammalian melanocyte stimulating hormone receptor, wherein the cells of the transformed cell culture express the receptor; and assaying the transformed cell with the compound in the presence and in the absence of an agonist for the melanocyte stimulating hormone receptor; and determining whether the compound competes with the agonist for binding to the melanocyte stimulating hormone receptor.

5. The method of claim 4, wherein the mammalian melanocyte stimulating 2 hormone receptor is a human melanocyte stimulating hormone receptor having an amino acid sequence identified as SEQ ID No. 6.

6. The method of claim 4, wherein the mammalian melanocyte stimulating hormone receptor is a mouse melanocyte stimulating hormone receptor having an amino acid sequence identified in Figure 2. o0

7. The method of any one of claims 4-6, wherein the compound is detectably- labeled.

8. The method of any one of claims 4-6, wherein the melanocyte stimulating hormone receptor agonist is detectably-labeled.

9. The method of claim 8, wherein the melanocyte stimulating hormone receptor i tompetitor is quantitatively characterized by assaying the transformed cell culture with S[R:LIBFF]08635.doc:gcc OJI" R:\LBFF]08635.doc:gcc 32 stimulating hormone receptor agonist and measuring the extent of competition with binding t6 the melanocyte stimulating hormone receptor thereby.

The method of claim 9, wherein the mammalian melanocyte stimulating hormone receptor is a human melanocyte stimulating hormone receptor having an amino acid sequence identified as SEQ ID No. 6.

11. The method of claim 9, wherein the mammalian melanocyte stimulating hormone receptor is a mouse melanocyte stimulating hormone receptor having an amino acid sequence identified in Figure 2.

12. A method of screening a compound to determine if the compound is an inhibitor in of a melanocyte stimulating hormone receptor, the method comprising the following steps: transforming a host cell with a recombinant expression construct encoding a mammalian melanocyte stimulating hormone receptor, wherein the cells of the transformed cell culture express the receptor; and assaying the transformed cell culture with the compound to determine i whether the compound is capable of inhibiting binding to the melanocyte stimulating hormone receptor by a melanocyte stimulating hormone receptor agonist.

13. The method of claim 12, wherein the mammalian melanocyte stimulating hormone receptor is a human melanocyte stimulating hormone receptor having an amino acid sequence identified as SEQ ID No. 6.

14. The method of claim 12, wherein the mammalian melanocyte stimulating hormone receptor is a mouse melanocyte stimulating hormone receptor having an amino acid sequence identified in Figure 2.

15. The method of claim 12, wherein the melanocyte stimulating hormone receptor inhibitor is quantitatively characterized by assaying the transformed cell culture with 25 varying amounts of the inhibitor in the presence of a detectably-labeled melanocyte stimulating hormone receptor agonist and measuring the extent of inhibition of binding of the agonist to the melanocyte stimulating hormone receptor thereby.

16. The method of claim 15, wherein the mammalian melanocyte stimulating hormone receptor is a human melanocyte stimulating hormone receptor having an amino 3 acid sequence identified as SEQ ID No. 6.

17. The method of claim 15, wherein the mammalian melanocyte stimulating hormone receptor is a mouse melanocyte stimulating hormone receptor having an amino acid sequence identified in Figure 2. [R:\LIBFF]08635.doc:gcc 33

18. A method of screening a compound for binding to a melanocyte stimulating Hormone receptor, substantially as hereinbefore described with reference to any one of the Examples.

19. A method of screening a compound for competitive binding to a melanocyte S stimulating hormone receptor, substantially as hereinbefore described with reference to any one of the Examples. A method of screening a compound to determine if the compound is an inhibitor of a melanocyte stimulating hormone receptor, substantially as hereinbefore described with reference to any one of the Examples. Dated 14 February, 2000 State of Oregon, acting by and through The Oregon State Board of Higher Education on behalf of The Oregon Health Sciences University Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON S S 5*555 1 °•o *oo o*o* [R:\LIBFF08635 .doc:gcc