Melanocyte stimulating hormone receptor and uses

Details Melanocyte stimulating hormone receptor and uses Melanocyte stimulating hormone receptor and uses

1998-12-01

2001-07-31

Eyler, Yvonne (Department: 1646)

Chemistry: analytical and immunological testing

Biospecific ligand binding assay

C436S503000, C436S504000, C435S007100, C435S007200, C435S069100, C435S007210

Reexamination Certificate

active

06268221

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to melanocyte stimulating 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 melanocyte stimulating hormone receptor gene. The invention also relates to the isolation, cloning and sequencing of a mouse melanocyte stimulating hormone receptor gene. The invention relates to the construction of eukaryotic recombinant expression constructs capable of expressing these melanocyte stimulating hormone receptors in cultures of transformed eukaryotic cells, and the production of the melanocyte stimulating hormone receptor in such cultures. The invention relates to the use of such cultures of transformed eukaryotic cells to produce homogeneous compositions of such melanocyte stimulating hormone receptors. The invention also provides cultures of such cells producing melanocyte stimulating hormone receptor for the characterization of novel and useful drugs. Antibodies against and epitopes of these melanocyte stimulating hormone receptor proteins are also provided by the invention.
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, &agr;-melanocyte stimulating hormone (&agr;MSH), and adrenocorticotropic hormone (ACTH) have well understood roles in control of melanocyte and adrenocortical function, respectively. Both of these hormones, however, are found in a variety of forms with unknown functions. The melanocortin peptides also have a diverse array of biological activities in other tissues, including the brain, and immune system, and bind to specific receptors there with a distinct pharmacology (see, Hanneman et al., in
Peptide Hormone as Prohormones
, G. Martinez, ed. (Ellis Horwood Ltd.: Chichester, UK) pp. 53-82; DeWied & Jolles, 1982
, Physiol. Rev
. 62: 976-1059 for reviews).
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 on the prior art.
Shimuze, 1985
, Yale J. Biol. Med
. 58: 561-570 discusses the physiology of melanocyte stimulating hormone.
Tatro & Reichlin, 1987
, Endocrinology
121: 1900-1907 disclose that MSH receptors are widely distributed in rodent tissues.
Solca et al., 1989
, J. Biol. Chem
. 264: 14277-14280 disclose the molecular weight characterization of mouse and human MSH receptors linked to radioactivity and photoafinity labeled MSH analogues.
Siegrist et al., 1991
, J. Receptor Res
. 11: 323-331 disclose the quantification of receptors in mouse melanoma tissue by receptor autoradiography.
The present invention comprises a human melanocyte stimulating hormone receptor gene, the nucleotide sequence of this gene and the deduced amino acid sequence of its cognate protein, a homogeneous composition of the melanocyte stimulating 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 eukaryotic cells, and such cultures of transformed eukaryotic cells useful in the characterization of novel and useful drugs. The present invention also comprises the homologue of the human melanocyte stimulating hormone receptor gene from the mouse.
SUMMARY OF THE INVENTION
The present invention relates to the cloning, expression and functional characterization of mammalian melanocyte stimulating hormone receptor (MSH-R) genes. The invention comprises the nucleotide sequence of these genes encoding the mammalian MSH-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 MSH-R, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the human MSH-R, a recombinant eukaryotic expression construct capable of expressing the human MSH-R in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the human MSH-R, a homogeneous composition of the human MSH-R, and antibodies against and epitopes of the human MSH-R.
The present invention is also directed toward the isolation, characterization and pharmacological use of the mouse MSH-R, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the mouse MSH-R, a recombinant eukaryotic expression construct capable of expressing the mouse MSH-R in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the mouse MSH-R, a homogeneous composition of the mouse MSH-R, and antibodies against and epitopes of the mouse MSH-R.
It is an object of the invention to provide a nucleotide sequence encoding a mammalian MSH-R. In a preferred embodiment of the invention, the nucleotide sequence encodes the human MSH-R. In another preferred embodiment, the nucleotide sequence encodes the mouse MSH-R.
The present invention includes a nucleotide sequence encoding a human MSH-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 1635 nucleotides of the human MSH-R gene comprising 953 nucleotides of coding sequence, 462 nucleotides of 5′ untranslated sequence and 220 nucleotides of 3′ untranslated sequence.
The present invention also includes a nucleotide sequence encoding a mouse MSH-R derived from a cDNA molecule isolated from a cDNA library constructed with RNA from mouse Cloudman melanoma cells (SEQ ID NO:3). In this embodiment of the invention, the nucleotide sequence includes 1260 nucleotides of the mouse MSH-R gene comprising 947 nucleotides of coding sequence, 15 nucleotides of 5′ untranslated sequence and 298 nucleotides of 3′ untranslated sequence.
The invention includes nucleotide sequences of mammalian MSH-Rs, most preferably mouse and human MSH-Rs (SEQ ID NOs:3&5), and includes allelic variations of these nucleotide sequences and the corresponding MSH-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 MSH-R disclosed herein, wherein the resulting MSH-R molecule has substantially the same biological properties as the MSH-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 mouse (SEQ ID NO:4) and human (SEQ ID NO:6) MSH-R deduced from the nucleotide sequence comprising the complete coding sequence of the mouse (SEQ ID NO:3) and human (SEQ ID NO:5) MSH-R gene as described herein.
In another aspect, the invention comprises a homogeneous composition of a 35.3 kilodalton mouse MSH-R or derivative thereof, wherein the amino acid sequence of the MSH-R or derivative thereof comprises the mouse MSH-R sequence shown in
FIG. 2
(SEQ ID NO:4).
In another aspect, the invention comprises a homogeneous composition of a 34.7 kilodalton human MSH-R or derivative thereof, wherein the amino acid sequence of the MSH-R or derivative thereof comprises the human MSH-R sequence shown in
FIG. 2
(SEQ ID NO:6).
This invention provides both nucleotide and amino acid probes derived from these sequences. The invention includes probes isolated from either cDNA or genomic DNA clones, as well as probes made synthetically with the sequence information derived therefrom. The invention specifically includes but is not limited to oligonucleotide, nick-translated

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