U.S. patent application number 10/151431 was filed with the patent office on 2003-03-06 for mammalian adrenocorticotropic hormone receptors and uses.
This patent application is currently assigned to State of Oregon. Invention is credited to Cone, Roger D., Mountjoy, Kathleen G..
Application Number | 20030044973 10/151431 |
Document ID | / |
Family ID | 25347876 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030044973 |
Kind Code |
A1 |
Cone, Roger D. ; et
al. |
March 6, 2003 |
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 ACTH.sup.R agonists and antagonists in vitro using
preparations of receptor from such cultures of eukaryotic cells
transformed with a recombinant eukaryotic expression construct
comprising the ACTH.sub.R receptor gene. The invention specifically
provides human and bovine ACTH.sup.R genes.
Inventors: |
Cone, Roger D.; (Oregon
City, OR) ; Mountjoy, Kathleen G.; (Portland,
OR) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
State of Oregon
|
Family ID: |
25347876 |
Appl. No.: |
10/151431 |
Filed: |
May 20, 2002 |
Related U.S. Patent Documents
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10151431 |
May 20, 2002 |
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09542122 |
Apr 4, 2000 |
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6392027 |
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09542122 |
Apr 4, 2000 |
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09105298 |
Jun 26, 1998 |
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6046011 |
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09105298 |
Jun 26, 1998 |
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08478992 |
Jun 7, 1995 |
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5773229 |
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08478992 |
Jun 7, 1995 |
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08077673 |
Jun 15, 1993 |
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5554729 |
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08077673 |
Jun 15, 1993 |
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07866560 |
Apr 10, 1992 |
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5280112 |
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Current U.S.
Class: |
435/325 ;
536/23.5 |
Current CPC
Class: |
C12Q 1/6876 20130101;
G01N 2333/72 20130101; G01N 33/566 20130101; G01N 2333/695
20130101; G01N 33/74 20130101; G01N 2500/00 20130101; C12Q 2600/158
20130101; G01N 2333/726 20130101; C07K 14/723 20130101 |
Class at
Publication: |
435/325 ;
536/23.5 |
International
Class: |
C12N 005/06; C07H
021/04 |
Goverment Interests
[0001] This invention was made with government support under
1R01DK41921-03, 1R01DK43859-01, and 1P01DK44239-10A1 by the
National Institutes of Health. The government has certain rights in
the invention.
Claims
We claim:
1. An isolated nucleic acid encoding a mammalian
adrenocorticotropic hormone receptor having an amino acid sequence
that is 39% identical and co-linear, excluding the third
intracellular loop and carboxy-terminal tail, to human melanocyte
stimulating hormone receptor.
2. A cell membrane preparation comprising a mammalian
adrenocorticotropic hormone receptor having an amino acid sequence
identified by SEQ ID NO.4 or SEQ ID NO.6 and produced by a cell
that expresses a recombinant expression vector encoding the
mammalian adrenocorticotropic hormone receptor.
3. The cell membrane preparation of claim 2, wherein the mammalian
adrenocorticotropic hormone receptor has an amino acid sequence
identified by SEQ ID NO.4.
4. The cell membrane preparation of claim 2, wherein the mammalian
adrenocorticotropic hormone receptor has an amino acid sequence
identified by SEQ ID NO.6.
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 (.alpha.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 (ACTH.sup.R) gene. The invention comprises the
nucleotide sequence of these genes encoding the mammalian
ACTH.sup.Rs 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
ACTH.sup.R, the gene corresponding to this receptor, a nucleic acid
hybridization probe comprising DNA sequences of the human
ACTH.sup.R, a recombinant eukaryotic expression construct capable
of expressing the human ACTH.sup.R in cultures of transformed
eukaryotic cells and such cultures of transformed eukaryotic cells
that synthesize the human ACTH.sup.R, a homogeneous composition of
the human ACTH.sup.R, and antibodies against and epitopes of the
human ACTH.sup.R.
[0013] The present invention is also directed toward the isolation,
characterization and pharmacological use of the bovine ACTH.sup.R,
the gene corresponding to this receptor, a nucleic acid
hybridization probe comprising DNA sequences of the bovine
ACTH.sup.R, a recombinant eukaryotic expression construct capable
of expressing the bovine ACTH.sup.R in cultures of transformed
eukaryotic cells and such cultures of transformed eukaryotic cells
that synthesize the bovine ACTH.sup.R, a homogeneous composition of
the bovine ACTH.sup.R, and antibodies against and epitopes of the
bovine ACTH.sup.R.
[0014] It is an object of the invention to provide a nucleotide
sequence encoding a mammalian ACTH.sup.R. In a preferred embodiment
of the invention, the nucleotide sequence encodes the human
ACTH.sup.R. In another preferred embodiment, the nucleotide
sequence encodes the bovine ACTH.sup.R.
[0015] The present invention includes a nucleotide sequence
encoding a human ACTH.sup.R 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 ACTH.sup.R 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 ACTH.sup.RR derived from a cDNA molecule isolated
from a cDNA library construct with bovine RNA (SEQ ID NO:3). In
this embodiment of the invention, the nucleotide sequence includes
1106 nucleotides of the bovine ACTH.sup.R 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
ACTH.sup.Rs, most preferably bovine and human ACTH.sup.Rs (SEQ ID
NOS:3&5), and includes allelic variations of these nucleotide
sequences and the corresponding ACTH.sup.R 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
ACTH.sup.R disclosed herein, wherein the resulting ACTH.sup.R
molecule has substantially the same biological properties as the
ACTH.sup.R 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) ACTH.sup.R
deduced from the nucleotide sequence comprising the complete coding
sequence of the bovine (SEQ ID NO:3) and human (SEQ ID NO:5)
ACTH.sup.RR gene as described herein.
[0019] In another aspect, the invention comprises a homogeneous
composition of a 34 kilodalton bovine ACTH.sup.R or derivative
thereof, wherein the amino acid sequence of the ACTH.sup.R 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 ACTH.sup.R or derivative
thereof, wherein the amino acid sequence of the ACTH.sup.R 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 ACTH.sup.R, preferably the bovine or human
ACTH.sup.R, for use as nucleic acid hybridization probes to
determine the pattern, amount and extent of expression of this
receptor in various tissues of mammals, including humans. It is
also an object of the present invention to provide nucleic acid
hybridization probes derived from the sequences of the bovine or
human ACTH.sup.R 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 ACTH.sup.R 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 ACTH.sup.R-specific antibodies,
or used for competitors of the ACTH.sup.R molecule for drug
binding, or to be used for the production of inhibitors of the
binding of agonists or antagonist or analogues thereof to
ACTH.sup.R molecule.
[0024] The present invention also provides antibodies against and
epitopes of mammalian ACTH.sup.Rs, preferably bovine or human
ACTH.sup.R proteins. It is an object of the present invention to
provide antibodies that is immunologically reactive to a mammalian
ACTH.sup.R protein. It is a particular object of the invention to
provide a monoclonal antibodies to mammalian ACTH.sup.R protein,
most preferably bovine or human ACTH.sup.R 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 ACTH.sup.R
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
ACTH.sup.R, preferably a bovine or human ACTH.sup.RR, and in a
pharmaceutically acceptable carrier.
[0027] It is a further object of the present invention to provide
an epitope of a mammalian ACTH.sup.R protein wherein the epitope is
immunologically reactive to an antibody specific for the mammalian
ACTH.sup.R. In preferred embodiments, the epitope is derived from
bovine of human ACTH.sup.R protein.
[0028] It is another object of the invention to provide a chimeric
antibody that is immunologically reactive to a mammalian ACTH.sup.R
protein. In a preferred embodiment, the chimeric antibody is a
monoclonal antibody. In a preferred embodiment, the ACTH.sup.R is a
bovine or human ACTH.sup.R.
[0029] The present invention provides a recombinant expression
construct comprising the nucleotide sequence of a mammalian
ACTH.sup.R, preferably the bovine or human ACTH.sup.R and sequences
sufficient to direct the synthesis of bovine or human ACTH.sup.R 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 ACTH.sup.R gene. This invention
includes a recombinant expression construct comprising essentially
the nucleotide sequences of genomic or cDNA clones of bovine or
human ACTH.sup.R 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, ACTH.sup.R protein. In a
preferred embodiment, the invention provides human 293 cells that
synthesize bovine ACTH.sup.R. In an additional preferred
embodiment, the invention provides human 293 cells that synthesize
human ACTH.sup.R protein.
[0031] The present invention also includes protein preparations of
mammalian, preferably bovine or human ACTH.sup.R, and preparations
of membranes containing mammalian ACTH.sup.R, derived from culture
of transformed eukaryotic cells. In a preferred embodiment, cell
membranes containing bovine ACTH.sup.R protein are isolated from
293 cell cultures transformed with a recombinant expression
construct that directs the synthesis of bovine ACTH.sup.R. In
another preferred embodiment, cell membranes containing human
ACTH.sup.R protein are isolated from 293 cell cultures transformed
with a recombinant expression construct that directs the synthesis
of human ACTH.sup.R.
[0032] It also an object of this invention to provide mammalian,
preferably bovine or human ACTH.sup.R for use in the in vitro
screening of novel adenosine agonist and antagonist compounds. In a
preferred embodiment, membrane preparations containing the bovine
ACTH.sup.R, derived from culture of transformed eukaryotic cells,
are used to determine the drug dissociation properties of various
novel adenosine agonist and antagonist compounds in vitro. In
another preferred embodiment, membrane preparations containing the
human ACTH.sup.R, derived from cultures of transformed eukaryotic
cells, are used to determine the drug dissociation properties of
various novel adenosine agonist and antagonist compounds in vitro.
These properties are then used to characterize such novel compounds
by comparison to the binding properties of known bovine or human
ACTH.sup.R agonists and antagonists.
[0033] The present invention is also useful for the in vitro
detection of analogues of agonists or antagonists of ACTH.sup.R,
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 ACTH.sup.R, known or unknown, either
naturally occurring or as the embodiments of a drug. It is an
additional object of the invention to provide a method to detect
such agonists, antagonists, or analogues thereof in blood, saliva,
semen, cerebrospinal fluid, plasma, lymph, or any other bodily
fluid.
[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 ACTH.sup.Rs of any species of origin, including, for example,
bovine, rat, rabbit, cat, and human, but preferably code for
receptors of mammalian, most preferably bovine and human,
origin.
[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 ACTH.sup.R 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 ACTH.sup.R 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 ACTH.sup.R 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 ACTH.sup.R 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, ACTH.sup.R gene sequences may
be obtained by use of the polymerase chain reaction (PCR)
procedure, with the PCR oligonucleotide primers being produced from
the ACTH.sup.R R gene sequence provided herein. See U.S. Pat. Nos.
4,683,195 and 4,683,202 to Mullis.
[0046] The ACTH.sup.R may be synthesized in host cells transformed
with a recombinant expression construct comprising a DNA sequence
encoding the ACTH.sup.R. Such a recombinant expression construct
can also be comprised of a vector that is a replicable DNA
construct. Vectors are used herein either to amplify DNA encoding
the ACTH.sup.R and/or to express DNA which encodes the ACTH.sup.R.
For the purposes of this invention, a recombinant expression
construct is a replicable DNA construct in which a DNA sequence
encoding the ACTH.sup.R is operably linked to suitable control
sequences capable of effecting the expression of the ACTH.sup.R 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 ACTH.sup.R. Transformed host cells may
ordinarily express the mammalian ACTH.sup.R, but host cells
transformed for purposes of cloning or amplifying nucleic acid
hybridization probe DNA need not express the receptor. When
expressed, the mammalian ACTH.sup.R 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 ACTH.sup.R 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
ACTH.sup.R protein produced by transformed eukaryotic cells as
provided herein. Such homogeneous compositions are intended to be
comprised of mammalian ACTH.sup.R protein that comprises 90% of the
protein in such homogeneous composition.
[0052] Mammalian ACTH.sup.R protein made from cloned genes in
accordance with the present invention may be used for screening
agonist compounds for ACTH.sup.R activity, or for determining the
amount of a ACTH.sup.R 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, ACTH.sup.R expressed in that host, the cells lysed, and
the membranes from those cells used to screen compounds for
ACTH.sup.R 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
ACTH.sup.Rs, pure preparations of membranes containing ACTH.sup.Rs
can be obtained. Further, ACTH.sup.R agonists and antagonists can
be identified by transforming host cells with vectors of the
present invention. Membranes obtained from such cells can be used
in binding studies wherein the drug dissociation activity is
monitored.
[0053] The recombinant expression constructs of the present
invention are useful in molecular biology to transform cells which
do not ordinarily express the ACTH.sup.R 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
ACTH.sup.R 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 ACTH.sup.R. The antibodies
provided by the invention can be raised in animals by inoculation
with cells that express a mammalian ACTH.sup.R R or epitopes of a
mammalian ACTH.sup.R 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 ACTH.sup.R, or any cell or cell
line that expresses a mammalian ACTH.sup.R or any epitope therein
as a result of molecular or genetic engineering, or that has been
treated to increase the expression of a mammalian ACTH.sup.R 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 ACTH.sup.R and that express the mammalian
ACTH.sup.R gene product.
[0057] The present invention provides monoclonal antibodies that
are immunologically reactive with an epitope that is a mammalian
ACTH.sup.R 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 ACTH.sup.R, including human cells, as
described above. The myeloma cell lines used in the invention
include lines derived from myelomas of mice, rats, hamsters,
primates and humans. Preferred myeloma cell lines are from bovine,
and the most preferred bovine myeloma cell line is P3X63-Ag8.653.
The animals from whom spleens are obtained after immunization are
rats, mice and hamster, preferably mice, most preferably Balb/c
mice. Spleen cells and myeloma cells are fused using a number of
methods well known in the art, including but not limited to
incubation with inactivated Sendai virus and incubation in presence
of polyethylene glycol (PEG). The most preferred method for cell
fusion is incubation in the presence of a solution of 45% (w/v)
PEG-1450. Monoclonal antibodies produced by hybridoma cell lines
can be harvested from cell culture supernatant fluids from in vitro
cell growth; alternatively, hybridoma cells can be injected
subcutaneously and/or into the peritoneal cavity of an animal, most
preferably a bovine, and the monoclonal antibodies obtained from
blood and/or ascites fluid.
[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 ACTH.sup.R.
[0060] The present invention encompasses fragments of the antibody
that are immunologically reactive with an epitope of a mammalian
ACTH.sup.R. Such fragments can be produced by any number of
methods, including but not limited to proteolytic cleavage,
chemical synthesis or preparation of such fragments by means of
genetic engineering technology. The present invention also
encompasses single-chain antibodies that are immunologically
reactive with an epitope of a mammalian ACTH.sup.R made by methods
known to those of skill in the art.
[0061] The present invention also encompasses an epitope of a
mammalian ACTH.sup.R that is comprised of sequences and/or a
conformation of sequences present in the mammalian ACTH.sup.R
molecule. This epitope may be naturally occurring, or may be the
result of proteolytic cleavage of the mammalian ACTH.sup.R molecule
and isolation of an epitope-containing peptide or may be obtained
by synthesis of an epitope-containing peptide using methods well
known to those skilled in the art. The present invention also
encompasses epitope peptides produced as a result of genetic
engineering technology and synthesized by genetically engineered
prokaryotic or eukaryotic cells.
[0062] The invention also includes chimeric antibodies, comprised
of immunologically reactive light chain and heavy chain peptides to
an epitope that is a mammalian ACTH.sup.R. The chimeric antibodies
embodied in the present invention include those that are derived
from naturally occurring antibodies as well as chimeric antibodies
made by means of genetic engineering technology well known to those
of skill in the art.
[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 .mu.l of a solution containing 50 mM Tris-HCl (pH
8.3), 2.5 mM MgCl.sub.2, 0.01% gelatin, 200 .mu.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.degree. C. for 1 min (denaturation),
45.degree. C. for 2 min (annealing), and 72.degree. 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 ACTH.sup.R 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 .mu.g/ml salmon sperm DNA, 10.times.Denhardt's
solution, 42.degree. 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 .alpha.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 ACTH.sup.R 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
.alpha.MSH.sup.R, human MSH.sup.R, and the putative human
ACTH.sup.R 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
.DELTA..sup.9-tetradhydrocannabinol (see Matsuda et al., 1990,
Nature 346: 561-564). For example, the human ACTH.sup.R 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 ACTH.sup.R is
encoded by two mRNA species of approximately equal stoichiometry,
one at 3.0 kb, and one which co-migrates with murine
.alpha.MSH.sup.R 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
.sup.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.degree. C. in
2.times.SSC and washed at 65.degree. C. with 0.1.times.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 ACTH.sup.R probe
and a receptor for .gamma..sub.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.
Sequence CWU 1
1
* * * * *