CA2388198A1

CA2388198A1 - Adrenocorticotropic hormone receptor and uses

Info

Publication number: CA2388198A1
Application number: CA002388198A
Authority: CA
Inventors: Roger D. Cone; Kathleen G. Mountjoy
Original assignee: Individual
Current assignee: Oregon Health and Science University
Priority date: 1992-04-10
Filing date: 1993-04-07
Publication date: 1993-10-28

Abstract

The present invention rotates to a mammalian adrenocorticotropic hormone receptor. The invention is directed toward the isolation, characterization and phar-macological use of mammalian adrenocorticotropic hor-mone 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 syn-thesize mammalian adrenocorticotropic hormone recep-tor. The invention also provides methods for screening ACTH R agonists and antagonists in vitro using prepar-ations of receptor from such cultures of eukaryotic cells transformed with a recombinant eukaryotic expression construct comprising the ACTH R receptor gene. The in-vention specifically provides human and bovine ACTH R genes.

Description

ADItENOCOItTTCOTROPIC HORIVhONE RECEPTOR
AND USES
BACKGRO~JND OF THE IZW1;NTION
This invention was made with government support under 1RO1DK41921-03, IRO1DK43g59-0I, and 1P41DK44239-10A1 by the National Institutes of Health. The United States government has certain~rights in the invention.
I0 1. ~'11~ o,~the Inoention This invention relates to adrenocorticotmpic hormone receptors from mammalian specias~ and the genes corjesponding t0 SUCit receptors.
Specifically, the invention relates to the isolation, cloning and sequencing of ~ a human adrenocorticotropic hormone receptor gene. The invention also relates to the IS isolation, cloning and sequencing of a bovine adrenocorticotropic hormone receptor gene. The inveadon relates to the construction of eukaryotic recombinant expression constructs capable of expressing those adrenocorticotrapic hormone nxeptors in cultturs of transformed eukaryotic cells, and the production of the adrmocorticotropic hormone receptor in such cultures. The invention 20 relates to the use of such cultures of transformed eukaryotic cells to produce homogeneous compositions of such adrenocorticotropic hormone receptors. The invention also provides culturr of such cells producing adrenocorticotropic hormone receptor for the characterization of novel and u~fui drugs.
25 2. ~~~~-~ncl of t11~ Iave tiou The proopiomelartocortin (POMC) gene product is processed to produce a large number of biologically active peptide. Two of these peptides, alpha-meIanocyte stimulating hormone («MSI-i~, and adrenocorticotropic hormone (ACTH) have well understood roles in control of melanocytc and adrenocortical 30 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 outer tissues, including the brain, and immune system, and bind to specific ttcrptors there with a distinct pharmacology [.rte, Hanneman er al., in Peptide Hormone as Prohormones, G. Martinez, ed. (Ellis Horwood Ltd.: Chichester, UK) pp. 53-82; DeWied & Jolles, 1982, Physiol.
Rev. ø~: 976-1059 for reviews].
A complete understanding of these peptides and their diverse biological 5 activities requires the isolation and characterization of their corresponding retxptors. Some biochemical studies have been reported on the prior art.
Oelofsen & Rarnachandrast, 1983, Arch. Biochem. Biophys. ~: 414-421 dixlose recepwr binding studies on ACTH rxeptors on nt adipo~ytes.
Matz do Catt, 1991, Pros. Natl. Acad. Sci. USA $$: 8525-8529 disclose 10 functional expression of ACTH rx~ton in Xerropus lat»s oocytes following injection of total cellular RNA from adtrstal tissue.
Moore et al., 1991, Endocrinology ~: 107-114 relates to Allgrove syndrome, an autosomal reoe~ssive syndrome characterizod by ACTH
insensitivity.
The pi~xnt invention comprises a hutnact adtenocorticotcopic hormone 15 roc~t~or gale, the nucleotide sequatce of this gene and the deduced amino acid sequatce of its cognate protein, a homogeneous composition of the adratocorriootrnpic hormone raeptor, nucleic acid hybridization probes and a method for ~ietamining the tissue distribution of e~zpr~ion of the gene, a recombinsnt acptession construct capable of expressing the gate in cultures of 20 transformed eukaryotie cells, and such cultures of transforntcd eukaryotic cells useful in the characocrirstion of rove! and uxful drugs. The present invention also oontprises the bovine adratocorticotropic honnorte raxptor gene.

DESCRIPTION OF THE DRAWINGS
", Figure 1 A-C illustrates the nucleotide sequence of the human (SEQ ID N0:

3) adrenocorticotrophic hormone receptor.
Figure 2 A-B, illustrates the nucleotide sequence of the bovine (SEQ ID N0: 5) adrenocorticotrophic hormone receptor.
Figure 3 presents an amino acid sequence comparison between the human adrenocorticotrophic hormone receptor protein and the mouse and human melanocyte stimulating hormone receptor proteins.
Figure 4 illustrates the tissue distribution of mammalian ACTH receptor gene expression by Northern blot hybridization of a panel of tissues from the rhesus macaque performed 0 under high stringency conditions demonstrating the existence of a cross-reacting 4Ø kb species specific to the rhesus adrenal gland.
Figure 5 illustrates localization of the human ACTH receptor mRNA to the rhesus macaque adrenal cortex by in situ hybridization. These results show a Iightfield micrograph of hematoxylin and eosin stained section of rhesus adrenal showing capsule ~s (C), zone glomerulosa (G), zone fasciculata (F), zone reticulate (R), and medulla (M).
Figure 6 depicts darkfield micrograph of the hematoxylin and eosin stained section of rhesus adrenal showing capsule (C), zone glomerulosa {G), zone fasciculata (F), zone reticulate (R.), and~medulla (M).
I
Figure 7apresents thenucleotide sequence of thehumanACZ Hreceptor (SEQ IDNo:3) alongside its corresponding amino acidsequence {SEQ ID No:4) andFigure 7b presents only its corresponding amino acid sequence (SEQ ID No:4).
Figure 8a presents the nucleotide sequence ofthe bovineACTH receptor (SBQ TD
No: 5) alongside its corresponding amino acid sequence (SEQ ID No:6)and Figure 8b presents only its corresponding amino acid sequence (SEQ ID No:6).

SUMMARY OF THE INVENTION
The present invention relates to the cloning, expression and functional characterization of mammalian adrenocvrticotropic hormone receptor (ASR) genes. The invention comprises the nucleotide sequence of these genes encoding the mammalian ACTH's and the deduced amino acid sequences of the cognate proteins, as well as tissue distribution patterns of expression of these genes.
In particular, the present invention is directed toward the isolation, characterization and pharmacological use of the human ACTHR, the gene cornsponding to this rxeptot, a nucleic acid hybridization probe comprising DNA sequences of the human ACTH', a recombinant eukaryotic expression construct capable of expressing the human ACTH' in cultures of transformed eukaryotic cells and such cultures of transformed eulcaryotic cells that synthesize the human ACTHR, a homogeneous composition of the human ACTH', and antibodies against and epitopes of the human ACTH'.
The present invention is also directed toward the isolation, characterization and pharmacological use of the bovine ACTH', the gene corresponding to this rx~tor, a nucleic acid hybridization probe comprising DNA sequences of the bovine ACTH', a rxombinant eukaryotic exprrxsion construct capable of expressing the bovine ACTH' in cultures of transformed eukaryotic cells and such cultures of transformed eukaryoac cells that synthesize the bovine ACTH', a homogeneous composition of the bovine ACTHR, and antibodies against and epit~pes of the bovine AG'TH~'.
It is an object of the invention to provide a nucleic acid comprising a nucleotide sequence encoding a mammalian ACTH'. In a preferred embodiment of the invaition, the nucleotide sequence encodes the human ACTH'. In another prefernd embodiment, the nucleotide sequence encodes the bovine ACTH'.
The present invention includes' a nucleic acid comprising a nucleotide sequence encoding a human ACTH' receptor derived from a DNA molecule isolated from a human genomic library (SEQ ID N0:3). In this embodiment of the invention, the nucleotide sequence includes 2028 nucleotides of the human ACTH' gene 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 nucleic acid comprising a nucleotide sequence encoding a bovine ACTHRR (SEQ ID NO:S) derived from a cDNA
molecule isolated from a cDNA library constructed with bovine RNA. In this 5 embodiment of the invention, the nucleotide sequence includes 1 I06 nucleotides of the bovine AC'THR gene comprising 893 nucleotides of coding sequence, 133 nucleotides of 5' untranslated sequence and 82 nucleotides of 3' untranslated saluenct.
The invention includts nucleic acids comprising nucleotide sequences of 10 mammalian ACTH's, most preferably bovine and human ACTH"s (SEQ ID
NOs:S&3), and includes allelic variations of these nucleotide sequences and the corresponding At;'TFiR 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 sequtna of the corresponding AC'TH'~
15 disclosed herdn, wherein the resulting AG'THR molecule has substantially the same biological properties a the At'TH" molecule corresponding to the nuciootide xqumce described herein. The term "substantially homologous w' as used in this invention encompasses such allelic variability as dexribed in this paragraph.
The invention also includes a protein comprised of a predicted amino acid 20 sequence for the bovine ~SEQ ID N0:6) and human (SEQ IS N0:4)~ AG'TH'~
deduced from the nucleotide saltrax,~ comprising the complete coding seqtterux of the bovine (SEQ ID NO:S) grid human (5EQ ID N0:3) 11C'TH"R gale as In another aspect, the invention comprises a homogeneous composition of 25 a 34 bbdalnon bovine AG'fH'~ or derivative thereof, wherein the amino acid seqtxnct of the ACTH" or derivative ther~aof comprixs a sequence shown in Figure 2 (SEQ DJ N0:6).
In another aspect, the invention comprises a honiog~ous composition of a 34 Icilodalton human AG'fHa or derivative thereof, wherein the amino acid 30 sequence of the ACTH" yr derivative thcrnof comprises a sequence shown in Figure 2 (SEQ ID N0:4).

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 5 limited to oligonucleotide, nick-translated, random primed, or in vitro amplified probes made using cDNA or genomie clone embodying the invention, and oligonucleotide and other synthetic probes svnchesized chemically using the nucleotide sequence information of cDNA or genomic clone embodiments of the invention.
10 It is a further object of this invention to provide sequences of mammalian AG'TH', preferably the bovine or human ACTFi'~, for use as nucleic acid hybridization probes to determine the panern, amount and extent of expression of this receptor in various tissues of mammals, including humans. It is also an objxt of the praatt invention to provide nucleic acid hybridization probes IS derived from the sequatxs of the bovine or human AG'TIi'~ to be used for the deceaion and diag<tosis of ga>etic diseases. It is an object of this invention to provide nucleic acid hybridization probes derived from the DNA soquencGS of the bovine or human AC'fH'~ to be used for the detection of novel related receptor genes.
20 The present invention also includes synthetic peptides made using the nucleotide soqttenoe infonnatiat comprising cDNA or genomic clone embodiments of the invattion. The invention includes either naturally occurring or synthetic peptides which may be used as antigens for the production of AGTHR-specific antibodies, or used for competitors of the ACT'Ii'~ molecule for drug binding, or 25 to be used for the production of inhibitors of the binding of agonists or antagonists or analogues therrof to ACTH" molecule.
The present invention also provides antibodies against and epitopes of mammalian ACTH"s, preferably bovine or human ACTH" proteins. It is an object of the praatt invention to provide antibodies that is immunologically 30 reactive to a mammalian ACTH" protein. It is a particular object of the invention to provide a monoclonal antibodies to mammalian ACTH's protein, most preferably bovine or human AC Tfl' protein.
It is also an object of the gresent invention to provide a hybridoma cell line that produces such an antibody. It is a particular object of the invention to provide a hybri~ma 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 AG"TH'~
antigen.
The present invention also provides a hybridoma ceU 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 prestnt invention also provides a pharmaceudral composition comprising a therapeutically effective amount of a monoclonal antibody that is immunologically ractive to a mamtnaiian AG'I'fi", preferably a bovine or human ACTH'R, and in a pharmaceutically acceptable canitr.
It is a further object of the pr~asent invention to provide an epitope of a mammalian ACTH" ptntein wherein the epitope is immunologically reactivt to an antibody specific for the mammalian ACTH'. In preferred embodiments, the epitope is derived from bovine of human ACTH' protatt.
It is another object of the invention to provide a chimeric antibody that is immunologially ractive to a mammalian ACTH" protein. In a preferred embodiment, the chimerie antibody is a monoclonal antibody. In a preferred embodiment, the ACTFI' is a bonrine of human AC'Tfi'.
The prdmt invention provides a recombinant exp~ion construct comprising the nucleotide sequence of a mammalian ACTH', preferably the banrine cc human ACTH' and sequaxes sufficient to direct the synthesis of bovine or human ACTH' in cultures of transformed eukaryotic cells. In a preferred embodiment, the recombinant expression construct is comprised of ptasmid seqtra»t derived from the plasmid pcDNAUneo and cDNA or genomic DNA of bovine or human ACTH' gene. This invention includes a recombinant expression constntct comprising essentially tlu nucleotide xqua~es of genomic or cDNA
clones of bovine or human ACTH' in an embodiment that provides for their expression in cultures of transformed eulcaryotic cells.
_7_ CA 02388198 2002-06-26 _ _ It is also an object of this invention to provide cultures of transformed eukaryotic cells that have ban transformed with such a recombinant expression construct and that synthesize mamrttalian, preferably bovine or human, ACTHR
protein. In a prtferrai embodiment, the invention provides human 293 cells that synthesize bovine ACTHi". In an additional preferred embodiment, the invention provides human 293 ceps that synthesize human ACT'FiR protein.
'Ihe ptesatt invention also includes protein preparations of mammalian, preferably bovine or human ACTH", and preparations of membranes containing mammalian ACTH", derived from cultures of transformed eukaryotic cells. In a preferred embodiment, cell membranes containing bovine ACTHR protein art isolated from 293 call cultures transformed with a recombinant expression construct that directs the synthesis of boYine AG'TH". In another preferred embodiment, cell membruta cattaining human ACTH" protein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of human ACT'H'~.
It also an object of this invention to provide mammalian, preferably bovine or human ACTH" for ux in the rte vittn screening of novel adatosine agonist and antagonist compounds. In a preferred embodiment, membrane preparations costtaiuing the bovine AGTH", derived from cultures of transformed eukaryotic cells, arc used -to deoecmine the drug dissociation properties of various navel adenosine agoctist and antagonist compo<u>ds in vitro. In anochcr preferred embodimatt, membrane pre~a~xtions containing the human ACTH", derived from cultures of transformed wkaryotie cells, are used to determine the drug dissoaation properties of variau novel adenosine agonist and antagonist oolrrpounds in »tro. Thex properties are that used to characterize such novel compounds by compuison to the binding properties of known bovine or human ACTH' agvnists and antagonists.
The present invention is also uxful for the ire viv~o detection of analogues of agonises or antagonists of ACTH", known or unknown, either naturally ocxurring or as the embodiments of a drug.
It is an object of the present invention to provide a method for the .g.

Quantitative detection of agonise yr antagonists, or analogues thereof, of ACZ'FiR, lrnown 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 theraof in blood, saliva, semen, cerebrospinal 5 fluid, plasma, lymph, or any other bodily fluid.
Specific preferred embodiments of the present invention will become evident fmm the following more detailed description of certain prefcrxed embodiments and the claims.
1O DETAILED DESGRII'TION OF THE PREE:~~ EMBODIMENTS
The term "a~drcnocorticotmpic hormone receptor" as used herein refers w protons substantially homologous to, and having substantially the same biological activity as, the proteins coded For by the nucleotide sequences depicted in Figure 1 & 2 (SEQ ID N0:3&5). This definition is intended to encompass natural allelic IS variations in the adrenocorti~tropic hormona receptor setluence. Cloned genes of the pn~att invention may code for ACTIi"s of any species of origin, including, for example, bovine, rat, rabbit, cat, and human, but preferably code .
for racxpton of mammalian, most preferably bovine and human, origin.
Nucleic acid hybridization probes provided by the invention comprise 24 DNA , sequences that are substantially homologous to the DNA sequences in Figure 1 (SEQ ID~ N0:3) and 2 (SEQ ID NO:S). Nucleic acid probes arc uxful for ducting AC"TT'H'~ gene expression in cells and tissues using techniques well-latoWm in the art, including but not limited to Northern blot hybridization, in situ hybridization and Southern hybridisation to reverse transcriptase -ZS polymarase chain reaction product DNAs. The probai provided by the present invtation, including oligonucleosidts probes derived therefrom, are useful arc also useful for Southern hybridizuion of mammalian, preferably human, genomic DNA for scnenirtg for restriction fragment length polymorphism (RFLP}
associated with cabin genetic disorders.
34 The peoduction of proteins such as the AG'TH" from cloned genes by genetic engineering is well known. See, e.g., U.S. Patrnt No. 4,761,371 to &eil _g_ et al. at Col. 6 line 3 to Col. 9 line 65. 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.
S DNA which encodes the ACTFiR may be obtained, in view of the instant dixlosure, by chemical synthesis, by screening reverse transcripts of mRNA
from appropriate cells or cell line cultures, by scrxning genomic libraries from appropriate cells, or by combinations of these procures, as illustrated below.
Scrxning of mRNA or genomic DNA may be carried out with oliganucleotide 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 chcmiluminexatt group in accordance with know procedures and used in conventional hybridization assays, as described in greater detail in the F~amplts below. In the aiternative, ACTI~i'~ gene sequences may be obtained by use of the polymerise chain reaction (PCR) procedure, with the PCR
oligonucleotide primers being produced from the ACTHRR gent sequence provided herein. See U.S. Patent Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis.
The ACTH's may be synth~sizai in host ells transformed with a recombinant expression construct comprising a DNA sequence encoding the ACTH". Such a recombinant expression construct can also be comprixd of a vector that is a rcplicable DNA co<tstruct. Vector arc used herein either to amplify DNA encoding the ACTH" andlor to express DNA which encodes the AG'I~i'~. For the purposes of this invention, a recombinant expression construct is a repliable DNA construct in which a DNA sequence encoding the ACTHR is operably linked to suitable control scquertces capable of effecting the ezprcssion ' of the AG'rH'~ in a suitable host. The raced for such control sequences will vary depending upon the host selected and the transformation method chosen.
Generally, control soqua~s include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA
ribosomal binding sifts, and scquatccs which control the termination of transcription and translation. Amplification voctors do not require expression control domains. All that is noedad is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of aansformants.
5 Vectors useful for pracxicing the present invention include plasmids, viruses (including phage), retroviruses, and integrata,ble DNA fragments (i.e., fiagmcnts incegramble into the host genome by homologous recombination). The vector replicates and functions indepaydattly of the host genome, or may, in some insranas, integrate into the gaame itself. Suitable vectors will contain replicon 10 and control sequences which arc dtrivod from spocies compatible with the intendod expression host. A preferred vector is the plasmid pcDNAI/neo.
Transformed host cells are cells which have been transformed or transfected with recombinant expression constructs made using rxombinant DNA tochniques and comprising a mammalian ACTH'. Transformed host calls may ordinarily expras 15 the mammalian ACTH", but host cells transformed for purposes of cloning or amplifying nucleic acid hybridization probe DNA need race express the receptor.
What acpressed, the mammalian ACTH' will typically be located in the host call membrane.
DNA regions are opaably linked when they are functionally related to 20 each other. For example: a promoter is operably lit>loed to a coding sequencx if it controls the transcription of the sequencx; a ribosome binding site is operably linbed to a coding sequence if it is positioned so a to permit translation.
Generally, operably linked mans contiguous and, in the case of leaders sequances, contiguous and in the same translational reading frame.
25 Cultures of cells derived from multicellular organisms are a desirable host for recombinant AG'IHt' synthesis. In prit>cipal, any higher eukaryotic cell culture is workable, whether from vertebrate or inverteeraZe culture. However, mamtttalian cells are preferred, as illustrated in the Examples. Propagation of such ctlls in cell culture has bxonx a routine procedure. See Tjssue Cy,lture, 30 Academic Press, Kruse do Paitersort, editors (1973). Examples of useful host cell lira are human 293 cells, VERO and HeLa calls. Chinese hamster ovary (CHO) cell lines, and WI138, BHK, CaS-7, CV, and MDCK cell lines. Human 293 cells are preferred. Expression vectors for such cells ordinarily include (if nexssary) an origin of replication, a promoter lob upstream from the gene to be expressed, along with a ribosome binding site, RNA splice sites (if intron-containing gcnomic 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 acogenau origin, such as may be derivod from SV40 or other viral source (e.g., polyoma, adenwirus, 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.
The invention provides homogeneous compositions of mammalian ACTH"
pmtein produced by transformed eukaryotic cells as provided herein. Such homogeneous compositions are intended to be comprised of mammalian ACT'R'~
protein that comprises 9096 of the protein in such homogenous composition.
Mammalian ACTH" prooan made from cloned gems in accordance with the prexnt invention may be uxd for screening agonist compounds for ACTH"
activity, or for determining the amount of a AL'TH'~ agonist or antagonist drug in a solution (e.g., blood plasma or serum). For example, host calls may be transformed with a recombinant expression construct of the prtsent invention, AC"rH" expresxd in that host, the cells lysed, arid the membranes from those cells used to scat oompouutds far AC'TH'~ binding activity. Competitive binding assays in which such procedures may be carried out are welt known in the art.
By selection of host cells which do not ordinarily express AGTH'~s, pure preparations of membranes containing ACTH"s an be obtained. Further, ACTH"
agonises and antagonists ran be identified by transforming host cells with v~ors of the pc~tnt invention. Membranes obtained from such cells cut be used in binding studies wherein the drug dissociation activity is monitored.
The r~ocombinant expression constructs of the ptesart invention are useful in molecular biology to transform cells which do not ordinarily express the ACTH" to therr~fta~ express this rx~Or. Such cells are useful as intermediates for making cell membrane preparations useful for receptor binding assays, which are in turn uxful for drug screening. Further, genes and vectors comprising the recombinant expression construct of the present invention are useful in gene therapy. For such purposes, reuoviral vector as described in U.S. Patent No.
4,650,7b4 to Temin & Watanabe or U.S. Patent No. 4,861,719 to Miller may be employed. Cloned gates of the present invention, or fragments thereof, may also be used in gene therapy carried out homologous recombinati~ or site-directed mutagenGSis. Set gtntrolly Thomas do Capexhi, 1987, Cell ~,1,: 503-S 12;
Bertling, 1987, Bioscience Reports 7: 10?-112; Smithies a al., 1985, Nature ~:
230-234.
Oligonucleotides of the prGSa~t invention are uxful as diagnostic tools for probing ACTH-receptor gene expr~ion in tissues. For example, tissues can be probed in situ with oligon~leotide probes carrying detestable groups by conventional autoradiography techniques, as explained in greater detail in the Examples below. to investigate native expression of this recxptor or pathologial conditions rr~lating thereto. Further, chromosomes can be pTObed to investigate the prasatx or absence of the ACTH" gate, 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 AGZ'li". The antibodies provided by the invention can be raised in animals by inoculation with cells that mcpress a mammalian ACTHRR or tpitopa of a mammalian ACTH's using methods well latown in the art. Animals that can be uxd 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 mica. rats, hamsters) and rabbits.
The most prrefared animal is the mouse.
Cells that an be used for such iitoctrlations, or for any of the other means used in the invention, include arty cell line which naturally expresses a mammalian AG'TH", or any cell or cell line that expresses a mammalian AC1 H'~
or any epicope therein a a result of molecular or genetic engineering, or that has been tr~tod to increase the expr~ion of a mammalian ACTH" 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 AC1"HR and that express the mammalian ACT'H'~ gate product.
5 The present invention provides monoclonal antibodies that are immunologically reactive with an epitape that is a mammalian ACTHR present on the surface of mammalian etlls, preferably human or bovine cells. These antibodies are made using methods utd techniques well known to those of skill in the art.
10 Monoclonal antibodies provided 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 calls expressing a mammalian ACTH', including human cells, 15 as dextibod above. The myeloma cell lines a sed in the invention include lines derived from myelomas of mice, rats, ttanisters, primates and humans.
Preferred myeloma cell lines are from bavirK, and the most preferred bovine myeloma cell tine is P3XE,3-Ag8.653. The animals from whom spleens are obtained after immunization are raze, mice and hamsters, preferably mice, most preferably 20 Halb/c mice. Spleat cells and myeloma cells are fused using a number of mat>ads well known in the art, including but not limited to incubation with inactivated SGtdai virus and incubation in the prtesatce of polyethylene glycol (PEG). The most preferred tttetltod for call fusion is incubation in the presence of a solution of 4596 (wlv) PEG-1450. Monoclonal antibodies produced by 25 hybridotrta all lines can be harvested from cell culture supernatant fluids from in virro cell growth; alternatively, hybridoma cells can be injected subcutaneousiy andlor into the peritoneal cavity of an animal, most preferably a bovine, and the monoclonal antibodies obtained from blood andlor ascites fluid.
Monoclonal antibodies provi~d by the persatt invention can also be 30 produced by recombinant genetic methods well brown to those of skill in the art.
and the present invention atcompasses antibodies made by such methods that are immunologically reactive with an epitope of a mammalian AC'THR.
The present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a mammalian AC'I'HR. Such fragments can be produced by any number of methods, including but not limited 5 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 immunologirally reactive with an epitope of a mammalian ACTH" made by methods known to those of skill in the art.
The present invention also encompasses an epitope of a mammalian 10 ACTH" that is comprised of sequences and/or a conformation of sequences present in the mammalian ACTH" molecule. This epitope may be naturally occurring, or may be the result of proteolytic cleavage of the mammalian A(.'I'~i' motecule and isolation of an epatope-containing peptide or may be obtained by synthesis of an epitop~-containing peptide using methods well known to those 15 sldllod in the art. The present invention also encompasses epitope peptides produced as a result of genetic engineering technology and synthesized by genetically enBir»ed prokaryotic or eukaryotic calls.
The invention also inciudes chimeric antibodies, comprised of immtmologically reactive light chain and heavy chain peptides to an epitope that 20 is a mammalian ACTH'. The chirneric antibodies embodied in the present invention include those that are derived from naturally occurring antibodies as well as chimeric antibodies made by mans of genetic engineering technology well b>own to those of skill in the art.
The F~campies which follow are illustrative of specific embodiments of the 25 invattion, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.

Isolation of an ACTH Receptor Probe by Random PCR Amplification of Human Melanoma cDNA Using In order to clone novel G-protein coupled r~oceptors, human melanoma cDNA was used as template for a polymerise chain reaction (PCR)-based random cloning experiment. PCR was performed using a pair of degenerate 10 oligonucleotide primers cort~ponding to the putative third and sixth transmembrane regions of G-prooran coupled rocxptors (Libeit er al., 1989, Science ~: 569-72; Zhou a at., 1990, Nature 4~7: 76-80). The PCR products obtained in this experiment were characterized by nucleotide sequencing. Two novel sequa>oes sting novel G-protein-coupled rerxptors were identified.
15 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 1$: 5294-5299). Double-saanded cDNA
was synd>esized from total RNA with murine reverse transcriptase (BRL, Gaithersburg, MD) by oGgo-dT priming [Maniacs a al., ~j~,~~
20 ~, (Cold Spring Harbor Laboratory Press, Cold Spring Ha:bor, NY), 1990j. The melanoma cDNA mixture was then subjected to 45 cycles of PCR amplification using 500 picotnoles of degaxrate oligonucleotide primers having the following seqtacnce:
25 Primer III (xnx):
GAGTCGACCTGTG(CII~G(Cll7(C!G)AT(C!'~(AIG)CI1T(G!T)GAC(ClA)G(CIG)TAC
(SEQ ID NO: l ) and Primer VI (antisenx):
35 CAGAATTCAGCT/A)AGGGCA1CCAGCAGAI(GICxGIAxTICX'rAA
(SEQ ID N0:2) in 100 ~d of a solution containing 50 mM Tris-HCl (pH 8.3), 2.5 mM MgCh, 0.0196 gelatin, 200 ~cM each dNTP, and 2.5 units of Taq polymerase (Saiki et al., 1988, Science ~.: 487-491). These primers were commercially synthesized by Research Genetics Ine. (Huntsville, AL). Each PCR amplification cycle consisted of incubations at 94°C for 1 min (dataturation), 45°C for 2 min (annealing), and 72°C for 2 min (extension).
Amplified products of the PCR reaction were extracted with phenoUchloroform and precipitated with ethanol. After digestion with F.coRI
and ,Sail, the PCR products wen separated on a 1.296 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 (Stratagate, 1-aJolla, CA).
A total of In of such pHKS clones containing inserts were sequenced using Se~ucnase * (U. S. Biochemical Corp., Cleveland, OH) by the didcoxynucltotide chain termination method (Sanger et al., 1977, Proc. Nad.
Acad. Sci. USA ~: 5463-54bT). Two typo of sequences homologous to other G-protein coupled recxptors were identified.
ERAN~LE 2 In order to isolate the human gene corresponding to one of the two G-protazt coupled raxptox probes of Example 1, a human genomic library was scrar>~ at high stringency (5096 formamide, 1M NaCI, 50nM Tris-HCI, pH 7.5, ~ O. I % sodium pyrophosphate, 0.2 % sodium dodecyl sulfate, 104~cg1m1 salmon sperm DNA, 10X .Dathardt's solution, 42°C), using the human PCR
fragments isoLatcd as described in Eacample 1. Two different types of scqucitces were isolated, corresponding to the two PCR fragments, and were found to encode highly related G protein couplet rarepwts. 'These genomic clones were ~ xqua»d as described in Example 1. The nucleotide sequence of this clone is shown in Figure 1 (SEQ ID N0:3). Nucleotide sequence analysis and homology comparisons were done on the OHSU computer system with software providcc3 by Inteliigcnctics Inc. f Mountain Vicw, CA).

Indicates trademark One of these genomic clones was determined to encode an human MSH receptor.
The human MSH receptor has a predicted amino acid sequence that is 75 %
identical and collinear with a mouse aMSH receptor cDNA.
The second human genomic clone isolated encodes a highly related G-coupled receptor protein (SEQ ID N0:3). The predicted amino acid sequence (SEQ ID
N0:4) of this clone (Figure lA-C) represented as human ACTH-R) is 39% identical and also collinear, excluding the third intracellular loop and carboxyterminal tail, with the human MSH receptor gene product (Figure 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; Figure 3) and human MSH
receptors, and the pattern of expression in different tissue types, as described in Example 3 below, this gene is believed to encode a human ACTH receptor.
A bovine genomic DNA clone was isolated from a bovine genomic library, essentially as described above, and its nucleotide sequence determined (Figure 2; SEQ ID
N0:5).
The predicted amino acid sequences of the mouse aMSHR, human MSHR, and the putative human ACTHR are aligned in Figure 3. These sequences define the melanocortin receptors as a novel subfamily of the G protein-coupled receptors with a number of unusual features. The melanocortin receptors are the smallest G protein-coupled 2 0 receptors identified to date (297-317aa) resulting from a short _ amino terminal extracelluar domain, a short carboxy-terminal intracellular domain, and a very small third intracellular loop. The melanocortin receptors 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 r in the alpha helical structure of the. transmembrane domains and thought to be involved in i the formation of the binding pocket (see Applebury & Hargrage, 1986, Vision Res.
i 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 1. ~: 3269-3275 and Karnik et al., 1988, 1'roc. Natl. Acad. Set.
USA $~,: 8459-8463). Remarkably, the metanocortin reccptars do not appear highly related to the other G protein-coupled receptors which recognize peptide ligands, such as the rxeptors for bombesin (see Spindel et al., 1990, Mol.
Endocrinol. 4: 1956-1953) or substance K (see Masu et u1., 1987, Nature ~Q:
836-838), but rathei, are more closely related to the receptor for d'-tetradhydrocannabinol (set Matsuda et al., 1990, Nature ~ø: 561-564). For j example, the human AG'THs and rat- cartnahinoid raxptors are about 30 %
identical in predicted transmembrane and intracellular loop amino acid sequences.
The cannabinoid receptor also lacks the conserved pmline in transmembrane 5 and the cysteine in the firm extzacellular loop nxasary for disulfide bond formation.
Least parsimony analysis with the recxptor sesluences shown in Figuie 3 suggests I .
the cannabinoid and melanocortir< receptors may be evolutionarily reIatai and form a subfamily distinct from the peptide receptors and the amine reccpwrs.
Regardless of whether the similarities are the result of evolutionary conservation or convergence, the soqua~e and putative structural similarities between the meiartocarrin and cannabinond rxepton may be informative in the search for the endogenous cannabinoid-liloe ligand.
EaIJPrMPZE 3 ' To furiha gain insight into this receptor, we have examined the tissue distribution of its corr~poading mRNA from various tissues by performing Northan hybridization experiments on RNA isolated from various tissues (see Maniaris a al., ibid.). The results of there experiments are shown in Figure 4.
A panel of tissue samples was examined by Northern hybridization analysis performed undez 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 meIanocytc cultures examined, the ACTFiR is encoded by two mRNA species of approximately equate stoichiometry, one at 3.0kb, and one which co-migrates with murine aMSHR
mRNA at 3.9kb.
The putative human ACTH receptor is encoded predominantly by a single S mRNA species of approximately 4.Okb 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.Okb species specific to the rhesus adrenal gland (Fig a 4 ) In situ hybridization of a fragment of the putative human ~p AGTH receptor to sections of rhesus adrenal tissue loarlizai the expression of this rocxptor solely to the cortex, with no apparent hybridization to the medulla or capsule, as would be predicted for this receptor (Figures 5 and 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%
15 sucrose in PBS. Sections were prepared and hybridized with a 600 nucleotide 3sS-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 2xSSC and washed at 65°C with O.IxSSC.
. 20 The results of these experiments are shown in Figures 5 and 6.
l Figure S 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). Figure 6 depicts darkfield micrograph of the same field. Within the cortex, 25 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 zone reticulate 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 l 3p for y3MSH, which is known to bind to this region of the adrenal cortex.
l Additionally, we have boon unable to detect expression in the brain of AG'TH roceptordescribed here, despite extensive documentation of ACTH binding l sites there as well as in other tissues. These findings suggest the existence of alternate forms of these or related receptors that may be specifically expressed in brain tissue.
It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

Claims

1. A nucleic acid comprising a nucleotide sequence encoding a mammalian adrenocorticotropic hormone receptor.

2. A nucleic acid according to Claim 1 wherein the mammalian adrenocorticotropic hormone receptor is the bovine adrenocorticotropic hormone

3. A nucleic acid according to Claim 1 wherein the nucleotide sequence is substantially homologous to the sequence in Figure 1A (SEQ ID
NO:3).

4. A nucleic acid according to Claim 1 wherein the mammalian adrenocorticotropic hormone receptor is the human adrenocorticotropic hormone receptor.

5. A nucleic acid according to Claim 1 wherein the nucleotide sequence is substantially homologous to the sequence in Figure 1B (SEQ ID
NO:5).

6. A nucleic acid according to Claim 1 wherein the mammalian adrenocorticotropic hormone receptor end therein has the biological properties of a native adrenocorticotropic hormone receptor.

7. A homogeneous composition of a 33.9 kilodalton adrenocorticotropic hormone receptor or derivative thereof, wherein the amino acid sequence of the adrenocorticotropic hormone receptor or derivative thereof comprises the sequence shown in Figure 2 (SEQ ID NO:4).

8. A homogeneous composition of a 33.9 kilodalton adrenocorticotropic hormone receptor or derivative thereof, wherein the amino acid sequence of the adrenocorticotropic hormone raptor or derivative thereof comprises the sequence shown as SEQ ID NO:6.

9. A nucleic acid hybridization probe for the detection of mammalian adrenocorticotropic hormone receptor expression comprising the nucleotide sequence of Claim 3.

10. The nucleic acid hybridization probe according to Claim 9 whereby the probe is adapted for use in the detection and diagnosis of genetic disease in a human.

11. The nucleic acid hybridization probe according to Claim 9 whereby the probe is adapted for use in the detection, isolation and characterization of novel mammalian receptor genes.

12. A nucleic acid hybridization probe for the detection of mammalian adrenocorticotropic hormone receptor expression comprising the nucleotide sequence of Claim 5.

13. The nucleic acid hybridization probe according to Claim 12 whereby the probe is adapted for use in the detection and diagnosis of generic disease in a human.

14. The nucleic acid hybridization probe warding to Claim 12 whereby the probe is adapted for use in the decoction, isolation and characterization of novel mammalian receptor genes.

15. A recombinant expression construct comprising a nucleotide sequence encoding a mammalian adrenocorticotropic hormone receptor.

16. A recombinant expression construct comprising the DNA sequence of Claim 3, wherein the construct is capable of expressing the bovine adrenocorticotropic hormone receptor in a transformed eukaryotic cell culture.

17. A recombinant expression construct comprising the DNA sequence of Claim 5, what the construct is capable of expressing the human adrenocorticotropic hormone receptor in a transformed eukaryotic cell culture.

18. The recombinant expression construct of Claim 15 comprising pcDNAI/neo sequences.

19. A eukaryotic cell culture transformed with the expression construct of Claim 16, wherein the transformed eukaryotic cell culture is capable of expressing bovine adrenocorticotropic hormone receptor.

20. A eukaryotic cell culture transformed with the expression construct of Claim 17, wherein the transformed eukaryotic cell culture is capable of expressing the human adrenocorticotropic hormone receptor.

21. A method of screening a compound as an inhibitor of agonist binding to a mammalian adrenocorticotropic hormone receptor, the method comprising the following steps:
(a) transforming a eukaryotic cell culture with an expression construct as in Claim 15 capable of expressing the adrenocorticotropic hormone receptor in a eukaryotic cell; and (b) assaying for ability of the compound to inhibit the binding of a detectable adrenocorticotropic hormone receptor agonist.

22. The method of Claim 21 wherein the mammalian adrenocorticotropic hormone receptor is the bovine adrenocorticotropic hormone receptor.

23. The method of Claim 21 wherein the mammalian adrenocorticotropic hormone receptor is the human adrenocorticotropic hormone receptor.

24. A method of quantitatively detecting a compound as an inhibitor of agonist binding to a mammalian adrenocorticotropic hormone receptor, the method comprising the following steps:
(a) transforming a eukaryotic cell culture with an expression construct as in Claim 15 capable of expressing the mammalian adrenocorticotropic hormone rotor in a eukaryotic call; and (b) assaying for amount of a compound by measuring the extent of inhibition of binding of a detectable receptor agonist.

25. The method of Claim 24 wherein the mammalian adrenocorticotropic hormone receptor is the bovine adrenocorticotropic hormone receptor.

26. The method of Claim 24 wherein the mammalian adrenocorticotropic hormone receptor is the human adrenocorticotropic hormone receptor.

27. The method of Claim 24 wherein the compound to be tested is present in a human.

28. The method of Claim 24 wherein the compound is present in human blood.

29. The method of Claim 24 wherein the compound is present in human cerebrospinal fluid.

30. The method of Claim 24 wherein the compound is unknown.

31. An antibody or fragment thereof that is immunologically reactive to a mammalian adrenocorticotropic hormone rector.

32. The antibody according to Claim 31, wherein the antibody is a monoclonal antibody.

33. The antibody according to Claim 31, wherein the mammalian adrenocorticotropic hormone rear is the bovine adrenocorticotropic hormone receptor.

34. The antibody according to Claim 31, wherein the mammalian adrenocorticotropic hormone receptor is the human adrenocorticotropic hormone receptor.

35. A cell line which produces an antibody or fragment thereof that is immunologically reactive be a mammalian adrenocorticotropic hormone receptor.

36. The cell line according to Claim 35, wherein the antibody is a monoclonal antibody.

37. The cell line according to Claim 35, wherein the mammalian adrenocorticotropic hormone receptor is the bovine adrenocorticotropic hormone receptor.

38. The cell line according to Claim 35, wherein the mammalian adrenocorticotropic hormone receptor is the human adrenocorticotropic hormone receptor.

39. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or fragment thereof according to claim 31 in a pharmaceutically acceptable carrier.

40. An epitope of a mammalian adrenocorticotropic hormone receptor wherein the epitope is immunologically reactive to the antibody or fragment thereof according to claim 31.

41. The epitope according to claim 40 wherein the mammalian adrenocorticotropic hormone receptor is the bovine adrenocorticotropic hormone receptor.

42. The epitope according to claim 44 wherein the mammalian adrenocorticotropic hormone receptor is the human adrenocorticotropic hormone receptor.

43. The antibody of Claim 31 that is a chimeric antibody.

44. The chimeric antibody according to claim 43 wherein the mammalian adrenocorticotropic hormone receptor is the bovine adrenocorticotropic hormone receptor.

45. The chimeric antibody wording to claim 43 wherein the mammalian adrenocorticotropic hormone receptor is the human adrenocorticotropic hormone receptor.