Mammalian adrenocorticotropic hormone receptor nucleic acids

Details Mammalian adrenocorticotropic hormone receptor nucleic acids Mammalian adrenocorticotropic hormone receptor nucleic acids

2000-04-04

2002-05-21

Gambel, Phillip (Department: 1644)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100

Reexamination Certificate

active

06392027

ABSTRACT:

This invention was made with government support under 1R
0
1DK41921-03, 1R
0
1DK43859-01, and 1P
0
1DK44239-10A1 by the National Institutes of Health. The government has certain rights in the invention.
1. Field of the Invention
This invention relates to adrenocorticotropic hormone receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of a human adrenocorticotropic hormone receptor gene. The invention also relates to the isolate, cloning and sequencing of a bovine adrenocorticotropic hormone receptor gene. The invention relates to the construction of eukaryotic recombinant expression constructs capable of expressing these adrenocorticotropic hormone receptors in cultures 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 adrencorticotripic hormone receptor for the characterization of novel and useful drugs.
2. Background of the Invention
The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, alpha-melanoctye stimulating hormone (&agr;MSH), and adrencorticotripic hormone (ACTH) have well understood roles in control of melanocyte and adrenocortical function, respectively. Both of the hormones, however, are found in a variety of forms with unknown functions. The melanocortin peptides also have a diverse array of biological activities in other tissues, including the brain, and immune system and bind to specific receptors there with a distinct pharmacology [see, Hanneman et al., in
Peptide Hormone as Prohormones
, G. Martinez, ed. Ellis Horwood Ltd.: Chichester, UK pp. 53-82; Dewied & Jolles, 1982, Physiol. Rev. 62: 976-1059 for reviews].
A complete understanding of these peptides and their diverse biological activities requires the isolation and characterization of their corresponding receptors. Some biochemical studies have been reported in the prior art.
Oelofsen & Ramachandran, 1983, Arch. Biochem. Biophys. 225: 414-421 disclose receptor binding studies on ACTH receptors on rat adipocytes.
Mertz & Catt, 1991, Proc. Natl. Acad. Sci. USA 88: 8525-8529 disclose functional expression of ACTH receptors in
Xenopus laevis
oocytes following injection of total cellular RNA from adrenal tissue.
Moore et al., 1991, Endocrinology 34: 107-114 relates to Allgrove syndrome, an autosomal recessive syndrome characterized by ACTH insensitivity.
The present invention comprises a human adrencorticotropic 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 expression the gene in cultures of transformed eukaryotic cells, and such cultures of transformed eukaryotic cells useful in the characterization of novel and useful drugs. The present invention also comprises the bovine adrenocorticotropic hormone receptor gene.
SUMMARY OF THE INVENTION
The present invention relates to the cloning, expression and functional characterization of mammalian adrenocorticotropic hormone receptor ACTH-R genes. The invention comprises the nucleotide sequence of these genes encoding the mammalian ACTH-RS and the deduced amino acid sequences of the cognate proteins, as well as tissue distribution patterns of expression of these genes.
In particular, the present invention is directed toward the isolation, characterization and pharmacological use of the human ACTH
R
, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the human ACTH-R, a recombinant eukaryotic expression construct capable of expressing the human ACTH-R in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the human ACTH-R, a homogeneous composition of the human ACTH
R
, and antibodies against and epitopes of the human ACTH-R.
The present invention is also directed toward the isolation, characterization and pharmacological use of the bovine ACTH-R, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the bovine ACTH-R, a recombinant eukaryotic expression construct capable of expressing the bovine ACTH-R in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the bovine ACTH-R, a homogeneous composition of the bovine ACTH
R
, and antibodies against and epitopes of the bovine ACTH-R.
It is an object of the invention to provide a nucleotide sequence encoding a mammalian ACTH-R. In a preferred embodiment of the invention, the nucleotide sequence encodes the human ACTH-R. In another preferred embodiment, the nucleotide sequence encodes the bovine ACTH-R.
The present invention includes a nucleotide sequence encoding a human ACTH-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-R gene comprising 893 nucleotides of coding sequence, 696 nucleotides 5′ untranslated sequence and 439 nucleotides of 3′ untranslated sequence.
The present invention also includes a nucleotide sequence encoding a bovine ACTH-R derived from a cDNA molecule isolated for a cDNA library constructed with bovine RNA (SEQ ID NO:3). In this embodiment of the invention, the nucleotide sequence includes 1106 nucleotides of the bovine ACTHR gene comprising 893 nucleotides of coding sequence, 133 nucleotides of 5′ untranslated sequence and 82 nucleotides of 3′ untranslated sequence.
The invention includes nucleotide sequences of mammalian ACTH-R, most preferably bovine and human ACTH-R (SEQ ID NOs:
3&5
), and includes allelic variations of these nucleotide sequences and the corresponding ACTH-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-R disclosed herein, wherein the resulting ACTH-R molecule has substantially the same biological properties as the ACTH-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.
The invention also includes a predicted amino acid sequence for the bovine (SEQ ID NO:4) and human (SEQ ID NO:6) ACTH-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-R gene as described herein.
In another aspect, the invention comprises a homogeneous composition of 34 kilodalton bovine ACTH-R or derivative thereof, wherein the amino acid sequence of the ACTH
R
or derivative thereof comprises a sequence shown in
FIG. 3
(SEQ ID NO:4).
In another aspect, the invention comprises a homogeneous composition of a 34 kilodalton human ACTH-R or derivative thereof, wherein the amino acid sequence of the ACTH
R
or derivative thereof comprises a sequence shown in
FIG. 3
(SEQ ID NO:6).
This invention provides both nucleotide and amino acid probes derived from these sequences. The invention includes probes isolated from either cDNA or genomic DNA clones, as well as probes made synthetically with the sequence information derived therefrom. The invention specifically includes but is not limited to olignonucleotide, nick-translated, random primed, or in vitro amplified probes made using cDNA or genomic clone embodying the invention, and oligonucleotide and o

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