US20030044973A1

US20030044973A1 - Mammalian adrenocorticotropic hormone receptors and uses

Info

Publication number: US20030044973A1
Application number: US10/151,431
Authority: US
Inventors: Roger Cone; Kathleen Mountjoy
Original assignee: Oregon State
Current assignee: Oregon State
Priority date: 1992-04-10
Filing date: 2002-05-20
Publication date: 2003-03-06
Also published as: WO1993021315A1; US6392027B1; US5554729A; CA2133845A1; US6046011A; CA2133845C; US5280112A; US5773229A

Abstract

The present invention relates to a mammalian adrenocorticotropic hormone receptor. The invention is directed toward the isolation, characterization and pharmacological use of mammalian adrenocorticotropic hormone receptor, the gene corresponding to this receptor, a recombinant eukaryotic expression construct capable of expressing a mammalian adrenocorticotropic hormone receptor in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize mammalian adrenocorticotropic hormone receptor. The invention also provides for screening ACTH^Ragonists and antagonists in vitro using preparations of receptor from such cultures of eukaryotic cells transformed with a recombinant eukaryotic expression construct comprising the ACTH_Rreceptor gene. The invention specifically provides human and bovine ACTH^Rgenes.

Description

[0001] This invention was made with government support under 1R01DK41921-03, 1R01DK43859-01, and 1P01DK44239-10A1 by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to adrenocorticotropic 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 adrenocorticotropic hormone receptor gene. The invention also relates to the isolation, cloning and sequencing of a bovine adrenocorticotropic hormone receptor gene. The invention relates to the construction of eukaryotic recombinant expression construct capable of expressing these adrenocorticotropic hormone receptors in culture of transformed eukaryotic cells, and the production of the adrenocorticotropic hormone receptor in such cultures. The invention relates to the use of such cultures of transformed eukaryotic cells to produce homogeneous compositions of such adrenocorticotropic hormone receptors. The invention also provides culture of such cells producing adrenocorticotropic hormone receptor for the characterization of novel and useful drugs.

2. Background of the Invention

The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, alpha-adrenocorticotropic hormone (αMSH), 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 array 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 Hormone 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 ben reported on the prior art.

Oelofsen & Ramachandran, 1983, Arch. Biochem. Biophys. 225: 414-421 disclose receptor binding studies on ACTH receptors on rat adipocytes.

Mertz & Catt, 1991, Proc. Natl. Acad. Sci. USA 88: 8525-8529 disclose functional expression of ACTH receptors in Xenopus laevis oocytes following injection of total cellular RNA from adrenal tissue.

Moore et al., 1991, Endocrinology 34: 107-114 relates to Allgrove syndrome, an autosomal recessive syndrome characterized by ACTH insensitivity.

The present invention comprises a human adrenocorticotropic hormone receptor gene, the nucleotide sequence of this gene and the deduced amino acid sequence of its cognate protein, a homogeneous composition of the adrenocorticotropic 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 eukryotic cells, and such cultures of transformed eukryotic cells useful in the characterization of novel and useful drugs. The present invention also comprises the bovine adrenocorticotropic hormone receptor gene.

SUMMARY OF THE INVENTION

The present invention relates to the cloning, expression and functional characterization of mammalian adrenocorticotropic hormone receptor (ACTH ^R) gene. The invention comprises the nucleotide sequence of these genes encoding the mammalian ACTH^Rs 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 ACTH ^R, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the human ACTH^R, a recombinant eukaryotic expression construct capable of expressing the human ACTH^Rin cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the human ACTH^R, a homogeneous composition of the human ACTH^R, and antibodies against and epitopes of the human ACTH^R.

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

It is an object of the invention to provide a nucleotide sequence encoding a mammalian ACTH ^R. In a preferred embodiment of the invention, the nucleotide sequence encodes the human ACTH^R. In another preferred embodiment, the nucleotide sequence encodes the bovine ACTH^R.

The present invention includes a nucleotide sequence encoding a human ACTH ^Rreceptor 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 2028 nucleotides of the human ACTH^Rgene comprising 893 nucleotides of coding sequence, 696 nucleotides of 5′ untranslated sequence and 439 nucleotides of 3′ untranslated sequence.

The present invention also includes a nucleotide sequence encoding a bovine ACTH ^RR derived from a cDNA molecule isolated from a cDNA library construct with bovine RNA (SEQ ID NO:3). In this embodiment of the invention, the nucleotide sequence includes 1106 nucleotides of the bovine ACTH^Rgene comprising 893 nucleotides of coding sequence, 133 nucleotides of 5′ untranslated sequence and 82 nucleotides of 3′ untranslated sequence.

The invention includes nucleotide sequences of mammalian ACTH ^Rs, most preferably bovine and human ACTH^Rs (SEQ ID NOS:3&5), and includes allelic variations of these nucleotide sequences and the corresponding ACTH^Rmolecule, 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 ACTH^Rdisclosed herein, wherein the resulting ACTH^Rmolecule has substantially the same biological properties as the ACTH^Rmolecule 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 predicted amino acid sequence for the bovine (SEQ ID NO:4) and human (SEQ ID NO:6) ACTH ^Rdeduced from the nucleotide sequence comprising the complete coding sequence of the bovine (SEQ ID NO:3) and human (SEQ ID NO:5) ACTH^RR gene as described herein.

In another aspect, the invention comprises a homogeneous composition of a 34 kilodalton bovine ACTH ^Ror derivative thereof, wherein the amino acid sequence of the ACTH^Ror derivative thereof comprise a sequence shown in FIG. 3 (SEQ ID NO:4).

In another aspect, the invention comprises a homogeneous composition of a 34 kilodalton human ACTH ^Ror derivative thereof, wherein the amino acid sequence of the ACTH^Ror derivative thereof comprises a sequence shown in FIG. 3 (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 ACTH ^R, preferably the bovine or human ACTH^R, 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 bovine or human ACTH^Rto 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 bovine or human ACTH^Rto be used for the detection of novel related receptor genes.

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 ACTH ^R-specific antibodies, or used for competitors of the ACTH^Rmolecule for drug binding, or to be used for the production of inhibitors of the binding of agonists or antagonist or analogues thereof to ACTH^Rmolecule.

The present invention also provides antibodies against and epitopes of mammalian ACTH ^Rs, preferably bovine or human ACTH^Rproteins. It is an object of the present invention to provide antibodies that is immunologically reactive to a mammalian ACTH^Rprotein. It is a particular object of the invention to provide a monoclonal antibodies to mammalian ACTH^Rprotein, most preferably bovine or human ACTH^Rprotein.

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 non-immunoglobulin producing bovine myeloma cell line and spleen cells derived from a bovine immunized with a human cell line which expresses ACTH ^Rantigen. The present invention also provides a hybridoma cell line that produces such an antibody, and that can be injected into a living bovine to provide an ascites fluid from the bovine 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 ACTH ^R, preferably a bovine or human ACTH^RR, and in a pharmaceutically acceptable carrier.

It is a further object of the present invention to provide an epitope of a mammalian ACTH ^Rprotein wherein the epitope is immunologically reactive to an antibody specific for the mammalian ACTH^R. In preferred embodiments, the epitope is derived from bovine of human ACTH^Rprotein.

It is another object of the invention to provide a chimeric antibody that is immunologically reactive to a mammalian ACTH ^Rprotein. In a preferred embodiment, the chimeric antibody is a monoclonal antibody. In a preferred embodiment, the ACTH^Ris a bovine or human ACTH^R.

The present invention provides a recombinant expression construct comprising the nucleotide sequence of a mammalian ACTH ^R, preferably the bovine or human ACTH^Rand sequences sufficient to direct the synthesis of bovine or human ACTH^Rin 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 bovine or human ACTH^Rgene. This invention includes a recombinant expression construct comprising essentially the nucleotide sequences of genomic or cDNA clones of bovine or human ACTH^Rin 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 bovine or human, ACTH ^Rprotein. In a preferred embodiment, the invention provides human 293 cells that synthesize bovine ACTH^R. In an additional preferred embodiment, the invention provides human 293 cells that synthesize human ACTH^Rprotein.

The present invention also includes protein preparations of mammalian, preferably bovine or human ACTH ^R, and preparations of membranes containing mammalian ACTH^R, derived from culture of transformed eukaryotic cells. In a preferred embodiment, cell membranes containing bovine ACTH^Rprotein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of bovine ACTH^R. In another preferred embodiment, cell membranes containing human ACTH^Rprotein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of human ACTH^R.

It also an object of this invention to provide mammalian, preferably bovine or human ACTH ^Rfor use in the in vitro screening of novel adenosine agonist and antagonist compounds. In a preferred embodiment, membrane preparations containing the bovine ACTH^R, derived from culture of transformed eukaryotic cells, are used to determine the drug dissociation properties of various novel adenosine agonist and antagonist compounds in vitro. In another preferred embodiment, membrane preparations containing the human ACTH^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 vitro. These properties are then used to characterize such novel compounds by comparison to the binding properties of known bovine or human ACTH^Ragonists and antagonists.

The present invention is also useful for the in vitro detection of analogues of agonists or antagonists of ACTH ^R, 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 ACTH ^R, 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.

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

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the nucleotide sequence of the human (SEQ ID NO:3) adrenocorticotropic hormone receptor. [0036]
FIG. 2 illustrates the nucleotide sequence of the bovine (SEQ ID NO:5) adrenocorticotropic hormone receptor. [0037]
FIG. 3 presents an amino acid sequence comparison between the human adrenocorticotropic hormone receptor protein and the mouse and human melanocyte stimulating hormone receptor proteins. [0038]
FIG. 4 illustrates the tissue distribution of human adrenocorticotropic hormone receptor gene expression by Northern blot hybridization. [0039]
FIG. 5 illustrates localization of the putative ACTH receptor mRNA to the adrenal cortex by in situ hybridization (brightfield illumination). [0040]
FIG. 6 illustrates localization of the putative ACTH receptor mRNA to the adrenal cortex by in situ hybridization (darkfield illumination).[0041]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term “adrenocorticotropic 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 FIGS. [0042] 1 (SEQ ID No:3) and 2 (SEQ ID No.:5). This definition is intended to encompass natural allelic variations in the adrenocorticotropic hormone receptor sequence. Cloned genes of the present invention may code for ACTH^Rs of any species of origin, including, for example, bovine, rat, rabbit, cat, and human, but preferably code for receptors of mammalian, most preferably bovine and human, origin.
Nucleic acid hybridization probes provided by the invention comprise DNA sequences that are substantially homologous to the DNA sequences in FIG. 1 (SEQ ID NO:3) and FIG. 2 (SEQ ID NO:5). Nucleic acid probes are useful for detecting ACTH[0043] ^Rgene 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) associated with certain genetic disorders.
The production of proteins such as the ACTH[0044] ^Rfrom cloned genes by genetic engineering is well known. See, e.g., U.S. Pat. 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 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 ACTH[0045] ^Rmay 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 probes generated from the ACTH^Rgene 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, ACTH^Rgene sequences may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the ACTH^RR gene sequence provided herein. See U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis.
The ACTH[0046] ^Rmay be synthesized in host cells transformed with a recombinant expression construct comprising a DNA sequence encoding the ACTH^R. 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 ACTH^Rand/or to express DNA which encodes the ACTH^R. For the purposes of this invention, a recombinant expression construct is a replicable DNA construct in which a DNA sequence encoding the ACTH^Ris operably linked to suitable control sequences capable of effecting the expression of the ACTH^Rin 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 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 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 transformants.
Vectors useful for practicing the present invention include plasmids, viruses (including phage), retroviruses, and intergratable DNA fragments (i.e., fragments intergratable into the host genome by homologous recombination). The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host. A preferred vector is 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 ACTH[0047] ^R. Transformed host cells may ordinarily express the mammalian ACTH^R, but host cells transformed for purposes of cloning or amplifying nucleic acid hybridization probe DNA need not express the receptor. When expressed, the mammalian ACTH^Rwill 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 means contiguous and, in the case of leaders sequences, contiguous and in the same translational reading frame. [0048]
Cultures of cells derived from multicellular organisms are a desirable host for recombinant ACTH[0049] ^Rsynthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. However, mammalian cells are preferred, as a illustrated in the Examples. Propagation of such cells in cell culture has become a routine procedure See Tissue Culture, 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 be expressed, along with a ribosome binding site, RNA splice sites (if intron-containing 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 (e.g., polyoma, adenovirus, VSV, or MPV), or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. [0050]
The invention provides homogeneous compositions of mammalian ACTH[0051] ^Rprotein produced by transformed eukaryotic cells as provided herein. Such homogeneous compositions are intended to be comprised of mammalian ACTH^Rprotein that comprises 90% of the protein in such homogeneous composition.
Mammalian ACTH[0052] ^Rprotein made from cloned genes in accordance with the present invention may be used for screening agonist compounds for ACTH^Ractivity, or for determining the amount of a ACTH^Ragonist or antagonist drug in a solution (e.g., blood plasma or serum). For example, host cells may be transformed with a recombinant expression construct of the present invention, ACTH^Rexpressed in that host, the cells lysed, and the membranes from those cells used to screen compounds for ACTH^Rbinding 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 ACTH^Rs, pure preparations of membranes containing ACTH^Rs can be obtained. Further, ACTH^Ragonists 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 in molecular biology to transform cells which do not ordinarily express the ACTH[0053] ^Rto thereafter express this receptor. Such cells are useful as intermediates for 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 therapy. For such purpose, retroviral vectors as described in U.S. Pat. No. 4,650,764 to Temin & Watanabe or U.S. Pat. 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; Bertling, 1987, Bioscience Reports 7: 107-112; Smithies et al., 1985, Nature 317: 230-234.
Oligonucleotides of the present invention are useful as diagnostic tools for probing ACTH-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 ACTH[0054] ^Rgene, 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 ACTH[0055] ^R. The antibodies provided by the invention can be raised in animals by inoculation with cells that express a mammalian ACTH^RR or epitopes of a mammalian ACTH^Rusing methods will 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 mammalian ACTH[0056] ^R, or any cell or cell line that expresses a mammalian ACTH^Ror any epitope therein as a result of molecular or genetic engineering, or that has been treated to increase the expression of a mammalian ACTH^Rby physical, biochemical or genetic means. Preferred cells are human cells, most preferably human 293 cells that have been transformed with a recombinant expression construct comprising DNA sequences encoding a mammalian ACTH^Rand that express the mammalian ACTH^Rgene product.
The present invention provides monoclonal antibodies that are immunologically reactive with an epitope that is a mammalian ACTH[0057] ^Rpresent on the surface of mammalian cells, preferably human or bovine cells. These antibodies are made using methods and techniques well known to those of skill in the art.
Monoclonal antibodies by the present invention are produced by hybridoma 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 ACTH[0058] ^R, 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 bovine, and the most preferred bovine myeloma cell line is P3X63-Ag8.653. The animals from whom spleens are obtained after immunization are rats, mice and hamster, 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 presence of polyethylene glycol (PEG). The most preferred method for cell fusion is incubation in the presence of a solution of 45% (w/v) 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 bovine, and the monoclonal antibodies obtained from blood and/or ascites fluid.
Monoclonal antibodies provided by the present invention can also be produced by recombinant genetic methods well known to those of skill in the art, and the present invention encompasses antibodies made by such methods that are immunologically reactive with an epitope of a mammalian ACTH[0059] ^R.
The present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a mammalian ACTH[0060] ^R. Such fragments can be produced by any number of methods, including but not limited to proteolytic cleavage, chemical synthesis or preparation of such fragments by means of genetic engineering technology. The present invention also encompasses single-chain antibodies that are immunologically reactive with an epitope of a mammalian ACTH^Rmade by methods known to those of skill in the art.
The present invention also encompasses an epitope of a mammalian ACTH[0061] ^Rthat is comprised of sequences and/or a conformation of sequences present in the mammalian ACTH^Rmolecule. This epitope may be naturally occurring, or may be the result of proteolytic cleavage of the mammalian ACTH^Rmolecule 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 ACTH[0062] ^R. 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. [0063]

EXAMPLE 1

Isolation of an ACTH Receptor Probe by Random PCR Amplification of Human Melanoma cDNA Using Degenerate Oligonucleotide Primers

In order to clone novel G-protein coupled receptors, human melanoma 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 347: 76-80). The PCR products obtained in this experiment were characterized by nucleotide sequencing. Two novel sequences representing novel G-protein-coupled were identified. [0064]

PCR amplification was performed as follows. Total RNA was isolated from a human melanoma tumor sample by the guanidinium thiocyanate method (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, N.Y.), 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):
GAGTCGACCTTG(C/T)G(C/T)(C/G)AT(C/T)(A/G)CIIT(G/T)GAC(C/A)G(C/G)TAC	(SEQ ID NO:1)

and

Primer VI (antisense):
CAGAATTCAG(T/A)AGGGCAICCAGCAGAI(G/C)(G/A)(T/C)GA	(SEQ ID NO:2)

in 100 μl of a solution containing 50 mM Tris-HCl (pH 8.3), 2.5 mM MgCl[0066] ₂, 0.01% gelatin, 200 μM each dNTP, and 2.5 units of Toq polymerase (Saiki et ea., 1988, Science 239: 487-491). These primers were commercially synthesized by Research Genetics Inc. (Huntsville, Ala.). Each PCR amplification cycle consisted of incubations at 94° C. for 1 min (denaturation), 45° 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 SalI, the PCR products were separated on a 1.2% agarose gel. A slice of this gel, corresponding to PCR products of 300 basepairs (bp) in size, was cut out and purified using glass beads and sodium iodide, and then the insert was cloned into a pBKS cloning vector (Stratagene, LaJolla, Calif.). [0067]
A total of 172 of such pBKS clones containing inserts were sequenced using Sequenase (U.S. Biochemical Corp., Cleveland, Ohio) 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 G-protein coupled receptors were identified. [0068]

EXAMPLE 2

Isolation and Characterization of Human ACTH^RGenomic Clones

In order to isolate the human gene corresponding to one of the two G-protein coupled receptor probes of Example 1, a human genomic library was at high stringency (50% formamide, 1M NaCl, 50 nM Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 100 μg/ml salmon sperm DNA, 10×Denhardt's solution, 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 1. The nucleotide sequence of this clone is shown in FIG. 1 (SEQ ID NO:3). Nucleotide sequence analysis and homology comparisons were done on the OHSU computer system with software provided by Intelligenetics Inc. (Mountain View, Calif.) [0069]
One of these genomic clones was determined to encode an human MSH receptor (see copending U.S. patent application Ser. No. 07/866,979). The human MSH receptor has a predicted amino acid sequence that is 75% identical and colinear with a mouse αMSH receptor cDNA sequence. [0070]
The second human genomic clone encodes a highly related G-coupled receptor protein (SEQ ID NO:3). The predicted amino acid sequence (SEQ ID NO:4) of this clone (FIG. 3, represented as human ACTH-R) is 39% identical and also colinear, excluding the third intracellular loop and carboxy-terminal tail, with the human MSH receptor gene product (FIG. 3; represented as human MSH-R). The predicted molecular weight of this putative ACTH[0071] ^Ris 33.9 kilodaltons (kD). Based on its high degree of homology to the murine (mouse MSH-R; FIG. 3) and human MSH receptors, and the pattern of expression in different tissue types, as described in Example 3 below, this gene is a believed to encode a human ACTH receptor.
As bovine genomic DNA clone was isolated from a bovine genomic library, essentially as described above, and its nucleotide sequence determined (FIG. 2; SEQ ID NO.5). [0072]
The predicted amino acid sequences of the mouse αMSH[0073] ^R, human MSH^R, and the putative human ACTH^Rare aligned in FIG. 3. These sequences define the melanocortin receptors as a novel subfamily of the G protein-coupled 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. 11: 1-20), including the proline residues 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., 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 appear highly related to the other G protein-coupled receptors which recognize peptide ligands, such as the receptors for bombesin (see Spindel et al., 1990, Mol. Endocrinol 4: 1956-1963) or substance K (see Masu et al., 1987, Nature 329: 836-838), but rather, are more closely related to the receptor for Δ⁹-tetradhydrocannabinol (see Matsuda et al., 1990, Nature 346: 561-564). For example, the human ACTH^Rand rat cannabinoid receptors are about 30% identical in predicted transmembrane and intracellular loop amino acid sequences. 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 FIG. 3 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 3

Tissue Distribution of ACTH Receptor Gene Expression

To further gain insight into this receptor, we have examined the tissue distribution of its corresponding mRNA 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 FIG. 4. [0074]
A panel of tissue samples was examined by Northern hybridization analysis performed under high stringency conditions. The nitrocellulose filter was hybridized with a putative human ACTH receptor probe to determine the distribution of receptor mRNA. In two primary human melanocyte cultures examined, the ACTH[0075] ^Ris encoded by two mRNA species of approximately equal stoichiometry, one at 3.0 kb, and one which co-migrates with murine αMSH^RmRNA at 3.9 kb.
The putative human ACTH receptor is encoded predominantly by a single mRNA species of approximately 4.0 kb in the human adrenal gland, although several minor species are present as well. Northern analysis of a panel of tissues from the rhesus macaque performed under high stringency conditions demonstrated the existence of a cross-reacting 4.0 kb species to the rhesus adrenal gland (FIG. 4). In situ hybridization of a fragment of the putative human ACTH receptor to sections of rhesus adrenal tissue localized the expression of this receptor solely to the cortex, with no apparent hybridization to the medulla or capsule, as would be predicted for this receptor (FIGS. 5 & 6). Adrenal tissue from a juvenile rhesus macaque was fixed for 24 hours in 10% formalin in phosphate buffered saline, then incubated for 24 hours in 20% sucrose in PBS. Sections were prepared and hybridized with a 600 nucleotide [0076] ³⁵S-labelled RNA antisense probe complementary to coding sequence spanning transmembrane domains 1-6 of the putative human ACTH receptor. Hybridizations were performed at 65° C. in 2×SSC and washed at 65° C. with 0.1×SSC.
The results of these experiments are shown in FIGS. 5 & 6. FIG. 5 illustrates lightfield micrograph of an hematoxylin and eosin stained section of rhesus adrenal showing capsule (C), zona glomerulosa (G), zona fasciculata (F), zona reticulata (R), and medulla (M). FIG. 6 depicts darkfield micrograph of the same field. Within the cortex, receptor expression was found across the entire zona fasciculata, the site of glucocorticoid production, and in the cortical half of the zona glomerulosa, the site of aldosterone synthesis. The zona resticulata was largely negative, except for a small band of hybridization adjacent to the medulla, which might result from a cross-reaction between the putative ACTH[0077] ^Rprobe and a receptor for γ₃MSH, which is known to bind to this region of the adrenal cortex.
Additionally, we have been unable to detect expression in the brain of ACTH receptor described here, despite extensive documentation of ACTH 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 issue. [0078]
It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternative equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims. [0079]
1 6 33 base pairs nucleic acid single linear DNA (genomic) misc_feature 1..33 /function= “Degenerate oligonucleotide primer (sense)” 1 GAGTCGACCT GTGYGYSATY RCTKGACMGS TAC 33 31 base pairs nucleic acid single linear DNA (genomic) YES misc_feature 1..31 /function= “Degenerate oligonucleotide primer (antisense)” 2 CAGAATTCAG WAGGGCACCA GCAGASRYGA A 31 2012 base pairs nucleic acid single linear DNA (genomic) CDS 694..1587 /product= “Human adrenocorticotropic hormone receptor” 5′UTR 1..693 3′UTR 1588..2012 3 ACAACACTTT ATATATATTT TTATAAATGT AAGGGGTACA AAGGTGCCAT TTTGTTACAT 60 GGATATACCG TGTAGTGGTG AAGCCTGGGC TTTTAGTGTA TCTGTCATCA GAATAACATA 120 CGTGTTACCC ATAGGAATTT CTCATCACCC GCCCCCTCCA CCCTTCGAGT CTCCAATGTC 180 CATTCCACAC TCTATATCCA CGTGTATGCA TATAGCTCCA CATATAAGTG AGAACATGTA 240 GTATTTGACT TCCTCTTTCT GAGTTATTTC ACTTTGATAA TGGCCTCCAC TTCCATCCAT 300 GTTGCTGCAA AAGACATGAC CTTATTCTTT TTGATAGCTG GGGAGTACTC CATTGTGTAT 360 ATGTACCACA TTTCTTTATC CATTCACCCA TTGAGAACAC TTAGTTGATT CCATATCTTT 420 GCTATTGTCA CTAGTGCTGC AATAAACATA CATGTGCAGG CTCCTTCTAA TATACTGATT 480 TATATTTTAT GGAGAGAGAT AGAGTTCTTA GCGAGTGTGC TGTTTATTTC TAGTGTACTT 540 GCAACTAATA TTCTGTATAC TCCCTTTAGG TGATTGGAGA TTTAACTTAG ATCTCCAGCA 600 AGTGCTACAA GAAGAAAAGA TCCTGAAGAA TCAATCAAGT TTCCGTGAAG TCAAGTCCAA 660 GTAACATCCC CGCCTTAACC ACAAGCAGGA GAA ATG AAG CAC ATT ATC AAC TCG 714 Met Lys His Ile Ile Asn Ser 1 5 TAT GAA AAC ATC AAC AAC ACA GCA AGA AAT AAT TCC GAC TGT CCT CGT 762 Tyr Glu Asn Ile Asn Asn Thr Ala Arg Asn Asn Ser Asp Cys Pro Arg 10 15 20 GTG GTT TTG CCG GAG GAG ATA TTT TTC ACA ATT TCC ATT GTT GGA GTT 810 Val Val Leu Pro Glu Glu Ile Phe Phe Thr Ile Ser Ile Val Gly Val 25 30 35 TTG GAG AAT CTG ATC GTC CTG CTG GCT GTG TTC AAG AAT AAG AAT CTC 858 Leu Glu Asn Leu Ile Val Leu Leu Ala Val Phe Lys Asn Lys Asn Leu 40 45 50 55 CAG GCA CCC ATG TAC TTT TTC ATC TGT AGC TTG GCC ATA TCT GAT ATG 906 Gln Ala Pro Met Tyr Phe Phe Ile Cys Ser Leu Ala Ile Ser Asp Met 60 65 70 CTG GGC AGC CTA TAT AAG ATC TTG GAA AAT ATC CTG ATC ATA TTG AGA 954 Leu Gly Ser Leu Tyr Lys Ile Leu Glu Asn Ile Leu Ile Ile Leu Arg 75 80 85 AAC ATG GGC TAT CTC AAG CCA CGT GGC AGT TTT GAA ACC ACA GCC GAT 1002 Asn Met Gly Tyr Leu Lys Pro Arg Gly Ser Phe Glu Thr Thr Ala Asp 90 95 100 GAC ATC ATC GAC TCC CTG TTT GTC CTC TCC CTG CTT GGC TCC ATC TTC 1050 Asp Ile Ile Asp Ser Leu Phe Val Leu Ser Leu Leu Gly Ser Ile Phe 105 110 115 AGC CTG TCT GTG ATT GCT GCG GAC CGC TAC ATC ACC ATC TTC CAC GCA 1098 Ser Leu Ser Val Ile Ala Ala Asp Arg Tyr Ile Thr Ile Phe His Ala 120 125 130 135 CTG CGG TAC CAC AGC ATC GTG ACC ATG CGC CGC ACT GTG GTG GTG CTT 1146 Leu Arg Tyr His Ser Ile Val Thr Met Arg Arg Thr Val Val Val Leu 140 145 150 ACG GTC ATC TGG ACG TTC TGC ACG GGG ACT GGC ATC ACC ATG GTG ATC 1194 Thr Val Ile Trp Thr Phe Cys Thr Gly Thr Gly Ile Thr Met Val Ile 155 160 165 TTC TCC CAT CAT GTG CCC ACA GTG ATC ACC TTC ACG TCG CTG TTC CCG 1242 Phe Ser His His Val Pro Thr Val Ile Thr Phe Thr Ser Leu Phe Pro 170 175 180 CTG ATG CTG GTC TTC ATC CTG TGC CTC TAT GTG CAC ATG TTC CTG CTG 1290 Leu Met Leu Val Phe Ile Leu Cys Leu Tyr Val His Met Phe Leu Leu 185 190 195 GCT CGA TCC CAC ACC AGG AAG ATC TCC ACC CTC CCC AGA GCC AAC ATG 1338 Ala Arg Ser His Thr Arg Lys Ile Ser Thr Leu Pro Arg Ala Asn Met 200 205 210 215 AAA GGG GCC ATC ACA CTG ACC ATC CTG CTC GGG GTC TTC ATC TTC TGC 1386 Lys Gly Ala Ile Thr Leu Thr Ile Leu Leu Gly Val Phe Ile Phe Cys 220 225 230 TGG GCC CCC TTT GTG CTT CAT GTC CTC TTG ATG ACA TTC TGC CCA AGT 1434 Trp Ala Pro Phe Val Leu His Val Leu Leu Met Thr Phe Cys Pro Ser 235 240 245 AAC CCC TAC TGC GCC TGC TAC ATG TCT CTC TTC CAG GTG AAC GGC ATG 1482 Asn Pro Tyr Cys Ala Cys Tyr Met Ser Leu Phe Gln Val Asn Gly Met 250 255 260 TTG ATC ATG TGC AAT GCC GTC ATT GAC CCC TTC ATA TAT GCC TTC CGG 1530 Leu Ile Met Cys Asn Ala Val Ile Asp Pro Phe Ile Tyr Ala Phe Arg 265 270 275 AGC CCA GAG CTC AGG GAC GCA TTC AAA AAG ATG ATC TTC TGC AGC AGG 1578 Ser Pro Glu Leu Arg Asp Ala Phe Lys Lys Met Ile Phe Cys Ser Arg 280 285 290 295 TAC TGG TAGAATGGCT GATCCCTGGT TTTAGAATCC ATGGGAATAA CGTTGCCAAG 1634 Tyr Trp TGCCAGAATA GTGTAACATT CCAACAAATG CCAGTGCTCC TCACTGGCCT TCCTTCCCTA 1694 ATGGATGCAA GGATGACCCA CCAGCTAGTG TTTCTGAATA CTATGGCCAG GAACAGTCTA 1754 TTGTAGGGGC AACTCTATTT GTGACTGGAC AGATAAAACG TGTAGTAAAA GAAGGATAGA 1814 ATACAAAGTA TTAGGTACAA AAGTAATTAG GTTTGCATTA CTTATGACAA ATGCATTACT 1874 TTTGCACCAA TCTAGTAAAA CAGCAATAAA AATTCAAGGG CTTTGGGCTA AGGCAAAGAC 1934 TTGCTTTCCT GTGGACATTA ACAAGCCAGT TCTGAGGCGG CCTTTCCAGG TGGAGGCCAT 1994 TGCAGCCAAT TTCAGAGT 2012 297 amino acids amino acid linear protein 4 Met Lys His Ile Ile Asn Ser Tyr Glu Asn Ile Asn Asn Thr Ala Arg 1 5 10 15 Asn Asn Ser Asp Cys Pro Arg Val Val Leu Pro Glu Glu Ile Phe Phe 20 25 30 Thr Ile Ser Ile Val Gly Val Leu Glu Asn Leu Ile Val Leu Leu Ala 35 40 45 Val Phe Lys Asn Lys Asn Leu Gln Ala Pro Met Tyr Phe Phe Ile Cys 50 55 60 Ser Leu Ala Ile Ser Asp Met Leu Gly Ser Leu Tyr Lys Ile Leu Glu 65 70 75 80 Asn Ile Leu Ile Ile Leu Arg Asn Met Gly Tyr Leu Lys Pro Arg Gly 85 90 95 Ser Phe Glu Thr Thr Ala Asp Asp Ile Ile Asp Ser Leu Phe Val Leu 100 105 110 Ser Leu Leu Gly Ser Ile Phe Ser Leu Ser Val Ile Ala Ala Asp Arg 115 120 125 Tyr Ile Thr Ile Phe His Ala Leu Arg Tyr His Ser Ile Val Thr Met 130 135 140 Arg Arg Thr Val Val Val Leu Thr Val Ile Trp Thr Phe Cys Thr Gly 145 150 155 160 Thr Gly Ile Thr Met Val Ile Phe Ser His His Val Pro Thr Val Ile 165 170 175 Thr Phe Thr Ser Leu Phe Pro Leu Met Leu Val Phe Ile Leu Cys Leu 180 185 190 Tyr Val His Met Phe Leu Leu Ala Arg Ser His Thr Arg Lys Ile Ser 195 200 205 Thr Leu Pro Arg Ala Asn Met Lys Gly Ala Ile Thr Leu Thr Ile Leu 210 215 220 Leu Gly Val Phe Ile Phe Cys Trp Ala Pro Phe Val Leu His Val Leu 225 230 235 240 Leu Met Thr Phe Cys Pro Ser Asn Pro Tyr Cys Ala Cys Tyr Met Ser 245 250 255 Leu Phe Gln Val Asn Gly Met Leu Ile Met Cys Asn Ala Val Ile Asp 260 265 270 Pro Phe Ile Tyr Ala Phe Arg Ser Pro Glu Leu Arg Asp Ala Phe Lys 275 280 285 Lys Met Ile Phe Cys Ser Arg Tyr Trp 290 295 1108 base pairs nucleic acid single linear DNA (genomic) CDS 133..1026 5′UTR 1..132 3′UTR 1027..1106 5 GGGGCCAGAA AGTTCCTGCT TCAGAGCAGA AGATCTTCAG CAAGAACTAC AAAGAAGAAA 60 AGATTCTGGA GAATCAATCA AGTTTCCTGT CAAGTTCCAG TAACGTTTCT GTCTTAACTG 120 CACACAGGAA AG ATG AAA CAC ATT CTC AAT CTG TAT GAA AAC ATC AAC 168 Met Lys His Ile Leu Asn Leu Tyr Glu Asn Ile Asn 1 5 10 AGT ACA GCA AGA AAT AAC TCA GAC TGT CCT GCT GTG ATT TTG CCA GAA 216 Ser Thr Ala Arg Asn Asn Ser Asp Cys Pro Ala Val Ile Leu Pro Glu 15 20 25 GAG ATA TTT TTC ACA GTA TCC ATT GTT GGG GTT TTG GAG AAC CTG ATG 264 Glu Ile Phe Phe Thr Val Ser Ile Val Gly Val Leu Glu Asn Leu Met 30 35 40 GTC CTT CTG GCT GTG GCC AAG AAT AAG AGT CTT CAG TCG CCC ATG TAC 312 Val Leu Leu Ala Val Ala Lys Asn Lys Ser Leu Gln Ser Pro Met Tyr 45 50 55 60 TTT TTC ATC TGC AGC TTG GCT ATT TCC GAT ATG CTG GGG AGC CTG TAC 360 Phe Phe Ile Cys Ser Leu Ala Ile Ser Asp Met Leu Gly Ser Leu Tyr 65 70 75 AAG ATT TTG GAA AAC GTT CTG ATC ATG TTC AAA AAC ATG GGT TAC CTC 408 Lys Ile Leu Glu Asn Val Leu Ile Met Phe Lys Asn Met Gly Tyr Leu 80 85 90 GAG CCT CGA GGC AGT TTT GAA AGC ACA GCA GAT GAT GTG GTG GAC TCC 456 Glu Pro Arg Gly Ser Phe Glu Ser Thr Ala Asp Asp Val Val Asp Ser 95 100 105 CTG TTC ATC CTC TCC CTT CTC GGC TCC ATC TGC AGC CTG TCT GTG ATT 504 Leu Phe Ile Leu Ser Leu Leu Gly Ser Ile Cys Ser Leu Ser Val Ile 110 115 120 GCC GCT GAC CGC TAC ATC ACA ATC TTC CAC GCT CTG CAG TAC CAC CGC 552 Ala Ala Asp Arg Tyr Ile Thr Ile Phe His Ala Leu Gln Tyr His Arg 125 130 135 140 ATC ATG ACC CCC GCA CCG TGC CCT CGT CAT CTG ACG GTC CTC TGG GCA 600 Ile Met Thr Pro Ala Pro Cys Pro Arg His Leu Thr Val Leu Trp Ala 145 150 155 GGC TGC ACA GGC AGT GGC ATT ACC ATC GTG ACC TTC TCC CAT CAC GTC 648 Gly Cys Thr Gly Ser Gly Ile Thr Ile Val Thr Phe Ser His His Val 160 165 170 CCC ACA GTG ATC GCC TTC ACA GCG CTG TTC CCG CTG ATG CTG GCC TTC 696 Pro Thr Val Ile Ala Phe Thr Ala Leu Phe Pro Leu Met Leu Ala Phe 175 180 185 ATC CTG TGC CTC TAC GTG CAC ATG TTC CTG CTG GCC CGC TCC CAC ACC 744 Ile Leu Cys Leu Tyr Val His Met Phe Leu Leu Ala Arg Ser His Thr 190 195 200 AGG AGG ACC CCC TCC CTT CCC AAA GCC AAC ATG AGA GGG GCC GTC ACA 792 Arg Arg Thr Pro Ser Leu Pro Lys Ala Asn Met Arg Gly Ala Val Thr 205 210 215 220 CTG ACT GTC CTG CTC GGG GTC TTC ATT TTC TGT TGG GCA CCC TTT GTC 840 Leu Thr Val Leu Leu Gly Val Phe Ile Phe Cys Trp Ala Pro Phe Val 225 230 235 CTT CAT GTC CTC TTG ATG ACA TTC TGC CCA GCT GAC CCC TAC TGT GCC 888 Leu His Val Leu Leu Met Thr Phe Cys Pro Ala Asp Pro Tyr Cys Ala 240 245 250 TGC TAC ATG TCC CTC TTC CAG GTG AAT GGT GTG TTG ATC ATG TGT AAT 936 Cys Tyr Met Ser Leu Phe Gln Val Asn Gly Val Leu Ile Met Cys Asn 255 260 265 GCC ATC ATC GAC CCC TTC ATA TAT GCC TTT CGG AGC CCA GAG CTC AGG 984 Ala Ile Ile Asp Pro Phe Ile Tyr Ala Phe Arg Ser Pro Glu Leu Arg 270 275 280 GTC GCA TTC AAA AAG ATG GTT ATC TGC AAC TGT TAC CAG TAGAATGATT 1033 Val Ala Phe Lys Lys Met Val Ile Cys Asn Cys Tyr Gln 285 290 295 GGTCCCTGAT TTTAGGAGCC ACAGGGATAT ACTGTCAGGG ACAGAGTAGC GTGACAGACC 1093 AACAACACTA GGACT 1108 297 amino acids amino acid linear protein 6 Met Lys His Ile Leu Asn Leu Tyr Glu Asn Ile Asn Ser Thr Ala Arg 1 5 10 15 Asn Asn Ser Asp Cys Pro Ala Val Ile Leu Pro Glu Glu Ile Phe Phe 20 25 30 Thr Val Ser Ile Val Gly Val Leu Glu Asn Leu Met Val Leu Leu Ala 35 40 45 Val Ala Lys Asn Lys Ser Leu Gln Ser Pro Met Tyr Phe Phe Ile Cys 50 55 60 Ser Leu Ala Ile Ser Asp Met Leu Gly Ser Leu Tyr Lys Ile Leu Glu 65 70 75 80 Asn Val Leu Ile Met Phe Lys Asn Met Gly Tyr Leu Glu Pro Arg Gly 85 90 95 Ser Phe Glu Ser Thr Ala Asp Asp Val Val Asp Ser Leu Phe Ile Leu 100 105 110 Ser Leu Leu Gly Ser Ile Cys Ser Leu Ser Val Ile Ala Ala Asp Arg 115 120 125 Tyr Ile Thr Ile Phe His Ala Leu Gln Tyr His Arg Ile Met Thr Pro 130 135 140 Ala Pro Cys Pro Arg His Leu Thr Val Leu Trp Ala Gly Cys Thr Gly 145 150 155 160 Ser Gly Ile Thr Ile Val Thr Phe Ser His His Val Pro Thr Val Ile 165 170 175 Ala Phe Thr Ala Leu Phe Pro Leu Met Leu Ala Phe Ile Leu Cys Leu 180 185 190 Tyr Val His Met Phe Leu Leu Ala Arg Ser His Thr Arg Arg Thr Pro 195 200 205 Ser Leu Pro Lys Ala Asn Met Arg Gly Ala Val Thr Leu Thr Val Leu 210 215 220 Leu Gly Val Phe Ile Phe Cys Trp Ala Pro Phe Val Leu His Val Leu 225 230 235 240 Leu Met Thr Phe Cys Pro Ala Asp Pro Tyr Cys Ala Cys Tyr Met Ser 245 250 255 Leu Phe Gln Val Asn Gly Val Leu Ile Met Cys Asn Ala Ile Ile Asp 260 265 270 Pro Phe Ile Tyr Ala Phe Arg Ser Pro Glu Leu Arg Val Ala Phe Lys 275 280 285 Lys Met Val Ile Cys Asn Cys Tyr Gln 290 295

Claims

We claim:

1. An isolated nucleic acid encoding a mammalian adrenocorticotropic hormone receptor having an amino acid sequence that is 39% identical and co-linear, excluding the third intracellular loop and carboxy-terminal tail, to human melanocyte stimulating hormone receptor.

2. A cell membrane preparation comprising a mammalian adrenocorticotropic hormone receptor having an amino acid sequence identified by SEQ ID NO.4 or SEQ ID NO.6 and produced by a cell that expresses a recombinant expression vector encoding the mammalian adrenocorticotropic hormone receptor.

3. The cell membrane preparation of claim 2, wherein the mammalian adrenocorticotropic hormone receptor has an amino acid sequence identified by SEQ ID NO.4.

4. The cell membrane preparation of claim 2, wherein the mammalian adrenocorticotropic hormone receptor has an amino acid sequence identified by SEQ ID NO.6.