Mammalian adrenocorticotropic hormone receptors and uses

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 ACTHR agonists and antagonists in vitro using preparations of receptor from such cultures of eukaryotic cells transformed with a recombinant eukaryotic expression construct comprising the ACTHR receptor gene. The invention specifically provides human and bovine ACTHR genes.

Description

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] 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.

[0004] 2. Background of the Invention

[0005] The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, alpha-adrenocorticotropic hormone (&agr;MSH), 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].

[0006] 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.

[0007] Oelofsen & Ramachandran, 1983, Arch. Biochem. Biophys. 225: 414-421 disclose receptor binding studies on ACTH receptors on rat adipocytes.

[0008] 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.

[0009] Moore et al., 1991, Endocrinology 34: 107-114 relates to Allgrove syndrome, an autosomal recessive syndrome characterized by ACTH insensitivity.

[0010] 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

[0011] The present invention relates to the cloning, expression and functional characterization of mammalian adrenocorticotropic hormone receptor (ACTHR) gene. The invention comprises the nucleotide sequence of these genes encoding the mammalian ACTHRs and the deduced amino acid sequences of the cognate proteins, as well as tissue distribution patterns of expression of these genes.

[0012] In particular, the present invention is directed toward the isolation, characterization and pharmacological use of the human ACTHR, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the human ACTHR, a recombinant eukaryotic expression construct capable of expressing the human ACTHR in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the human ACTHR, a homogeneous composition of the human ACTHR, and antibodies against and epitopes of the human ACTHR.

[0013] The present invention is also directed toward the isolation, characterization and pharmacological use of the bovine ACTHR, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the bovine ACTHR, a recombinant eukaryotic expression construct capable of expressing the bovine ACTHR in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the bovine ACTHR, a homogeneous composition of the bovine ACTHR, and antibodies against and epitopes of the bovine ACTHR.

[0014] It is an object of the invention to provide a nucleotide sequence encoding a mammalian ACTHR. In a preferred embodiment of the invention, the nucleotide sequence encodes the human ACTHR. In another preferred embodiment, the nucleotide sequence encodes the bovine ACTHR.

[0015] The present invention includes a nucleotide sequence encoding a human ACTHR 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 2028 nucleotides of the human ACTHR gene comprising 893 nucleotides of coding sequence, 696 nucleotides of 5′ untranslated sequence and 439 nucleotides of 3′ untranslated sequence.

[0016] The present invention also includes a nucleotide sequence encoding a bovine ACTHRR 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 ACTHR gene comprising 893 nucleotides of coding sequence, 133 nucleotides of 5′ untranslated sequence and 82 nucleotides of 3′ untranslated sequence.

[0017] The invention includes nucleotide sequences of mammalian ACTHRs, most preferably bovine and human ACTHRs (SEQ ID NOS:3&5), and includes allelic variations of these nucleotide sequences and the corresponding ACTHR 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 ACTHR disclosed herein, wherein the resulting ACTHR molecule has substantially the same biological properties as the ACTHR 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.

[0018] The invention also includes a predicted amino acid sequence for the bovine (SEQ ID NO:4) and human (SEQ ID NO:6) ACTHR deduced from the nucleotide sequence comprising the complete coding sequence of the bovine (SEQ ID NO:3) and human (SEQ ID NO:5) ACTHRR gene as described herein.

[0019] In another aspect, the invention comprises a homogeneous composition of a 34 kilodalton bovine ACTHR or derivative thereof, wherein the amino acid sequence of the ACTHR or derivative thereof comprise a sequence shown in FIG. 3 (SEQ ID NO:4).

[0020] In another aspect, the invention comprises a homogeneous composition of a 34 kilodalton human ACTHR or derivative thereof, wherein the amino acid sequence of the ACTHR or derivative thereof comprises a sequence shown in FIG. 3 (SEQ ID NO:6).

[0021] 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.

[0022] It is a further object of this invention to provide sequences of mammalian ACTHR, preferably the bovine or human ACTHR, 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 ACTHR 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 bovine or human ACTHR to be used for the detection of novel related receptor genes.

[0023] 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 ACTHR-specific antibodies, or used for competitors of the ACTHR molecule for drug binding, or to be used for the production of inhibitors of the binding of agonists or antagonist or analogues thereof to ACTHR molecule.

[0024] The present invention also provides antibodies against and epitopes of mammalian ACTHRs, preferably bovine or human ACTHR proteins. It is an object of the present invention to provide antibodies that is immunologically reactive to a mammalian ACTHR protein. It is a particular object of the invention to provide a monoclonal antibodies to mammalian ACTHR protein, most preferably bovine or human ACTHR protein.

[0025] 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 ACTHR antigen. 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.

[0026] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody that is immunologically reactive to a mammalian ACTHR, preferably a bovine or human ACTHRR, and in a pharmaceutically acceptable carrier.

[0027] It is a further object of the present invention to provide an epitope of a mammalian ACTHR protein wherein the epitope is immunologically reactive to an antibody specific for the mammalian ACTHR. In preferred embodiments, the epitope is derived from bovine of human ACTHR protein.

[0028] It is another object of the invention to provide a chimeric antibody that is immunologically reactive to a mammalian ACTHR protein. In a preferred embodiment, the chimeric antibody is a monoclonal antibody. In a preferred embodiment, the ACTHR is a bovine or human ACTHR.

[0029] The present invention provides a recombinant expression construct comprising the nucleotide sequence of a mammalian ACTHR, preferably the bovine or human ACTHR and sequences sufficient to direct the synthesis of bovine or human ACTHR 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 bovine or human ACTHR gene. This invention includes a recombinant expression construct comprising essentially the nucleotide sequences of genomic or cDNA clones of bovine or human ACTHR in an embodiment that provides for their expression in cultures of transformed eukaryotic cells.

[0030] 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, ACTHR protein. In a preferred embodiment, the invention provides human 293 cells that synthesize bovine ACTHR. In an additional preferred embodiment, the invention provides human 293 cells that synthesize human ACTHR protein.

[0031] The present invention also includes protein preparations of mammalian, preferably bovine or human ACTHR, and preparations of membranes containing mammalian ACTHR, derived from culture of transformed eukaryotic cells. In a preferred embodiment, cell membranes containing bovine ACTHR protein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of bovine ACTHR. In another preferred embodiment, cell membranes containing human ACTHR protein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of human ACTHR.

[0032] It also an object of this invention to provide mammalian, preferably bovine or human ACTHR for use in the in vitro screening of novel adenosine agonist and antagonist compounds. In a preferred embodiment, membrane preparations containing the bovine ACTHR, 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 ACTHR, 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 ACTHR agonists and antagonists.

[0033] The present invention is also useful for the in vitro detection of analogues of agonists or antagonists of ACTHR, known or unknown, either naturally occurring or as the embodiments of a drug.

[0034] It is an object of the present invention to provide a method for the quantitative detection of agonists or antagonists, or analogues thereof, of ACTHR, 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.

[0035] 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

[0036] FIG. 1 illustrates the nucleotide sequence of the human (SEQ ID NO:3) adrenocorticotropic hormone receptor.

[0037] FIG. 2 illustrates the nucleotide sequence of the bovine (SEQ ID NO:5) adrenocorticotropic hormone receptor.

[0038] 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.

[0039] FIG. 4 illustrates the tissue distribution of human adrenocorticotropic hormone receptor gene expression by Northern blot hybridization.

[0040] FIG. 5 illustrates localization of the putative ACTH receptor mRNA to the adrenal cortex by in situ hybridization (brightfield illumination).

[0041] FIG. 6 illustrates localization of the putative ACTH receptor mRNA to the adrenal cortex by in situ hybridization (darkfield illumination).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] 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. 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 ACTHRs 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.

[0043] 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 ACTHR 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) associated with certain genetic disorders.

[0044] The production of proteins such as the ACTHR from 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

[0045] DNA which encodes the ACTHR 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 probes generated from the ACTHR 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, ACTHR gene sequences may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the ACTHR R gene sequence provided herein. See U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis.

[0046] The ACTHR may be synthesized in host cells transformed with a recombinant expression construct comprising a DNA sequence encoding the ACTHR. 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 ACTHR and/or to express DNA which encodes the ACTHR. For the purposes of this invention, a recombinant expression construct is a replicable DNA construct in which a DNA sequence encoding the ACTHR is operably linked to suitable control sequences capable of effecting the expression of the ACTHR 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 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.

[0047] 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 ACTHR. Transformed host cells may ordinarily express the mammalian ACTHR, but host cells transformed for purposes of cloning or amplifying nucleic acid hybridization probe DNA need not express the receptor. When expressed, the mammalian ACTHR will typically be located in the host cell membrane.

[0048] 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.

[0049] Cultures of cells derived from multicellular organisms are a desirable host for recombinant ACTHR synthesis. 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.

[0050] 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.

[0051] The invention provides homogeneous compositions of mammalian ACTHR protein produced by transformed eukaryotic cells as provided herein. Such homogeneous compositions are intended to be comprised of mammalian ACTHR protein that comprises 90% of the protein in such homogeneous composition.

[0052] Mammalian ACTHR protein made from cloned genes in accordance with the present invention may be used for screening agonist compounds for ACTHR activity, or for determining the amount of a ACTHR agonist 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, ACTHR expressed in that host, the cells lysed, and the membranes from those cells used to screen compounds for ACTHR 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 ACTHRs, pure preparations of membranes containing ACTHRs can be obtained. Further, ACTHR 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.

[0053] The recombinant expression constructs of the present invention are useful in molecular biology to transform cells which do not ordinarily express the ACTHR to 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.

[0054] 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 ACTHR gene, and potential pathological conditions related thereto, as also illustrated by the Examples below.

[0055] The invention also provides antibodies that are immunologically reactive to a mammalian ACTHR. The antibodies provided by the invention can be raised in animals by inoculation with cells that express a mammalian ACTHR R or epitopes of a mammalian ACTHR using 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.

[0056] 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 ACTHR, or any cell or cell line that expresses a mammalian ACTHR or any epitope therein as a result of molecular or genetic engineering, or that has been treated to increase the expression of a mammalian ACTHR by 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 ACTHR and that express the mammalian ACTHR gene product.

[0057] The present invention provides monoclonal antibodies that are immunologically reactive with an epitope that is a mammalian ACTHR present 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.

[0058] 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 ACTHR, 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.

[0059] 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 ACTHR.

[0060] The present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a mammalian ACTHR. 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 ACTHR made by methods known to those of skill in the art.

[0061] The present invention also encompasses an epitope of a mammalian ACTHR that is comprised of sequences and/or a conformation of sequences present in the mammalian ACTHR molecule. This epitope may be naturally occurring, or may be the result of proteolytic cleavage of the mammalian ACTHR 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.

[0062] The invention also includes chimeric antibodies, comprised of immunologically reactive light chain and heavy chain peptides to an epitope that is a mammalian ACTHR. 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.

[0063] 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 ACTH Receptor Probe by Random PCR Amplification of Human Melanoma cDNA Using Degenerate Oligonucleotide Primers

[0064] 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.

[0065] 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: 1 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)

[0066] in 100 &mgr;l of a solution containing 50 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 0.01% gelatin, 200 &mgr;M 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).

[0067] 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.).

[0068] 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.

EXAMPLE 2

Isolation and Characterization of Human ACTHR Genomic Clones

[0069] 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 &mgr;g/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.)

[0070] 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 &agr;MSH receptor cDNA sequence.

[0071] 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 ACTHR is 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.

[0072] 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).

[0073] The predicted amino acid sequences of the mouse &agr;MSHR, human MSHR, and the putative human ACTHR are 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 &Dgr;9-tetradhydrocannabinol (see Matsuda et al., 1990, Nature 346: 561-564). For example, the human ACTHR and 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

[0074] 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.

[0075] 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 ACTHR is encoded by two mRNA species of approximately equal stoichiometry, one at 3.0 kb, and one which co-migrates with murine &agr;MSHR mRNA at 3.9 kb.

[0076] 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 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.

[0077] 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 ACTHR probe and a receptor for &ggr;3MSH, which is known to bind to this region of the adrenal cortex.

[0078] 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.

[0079] 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.

Claims

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.