Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-02-20
2001-08-28
Ulm, John (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S007200, C435S069100, C435S252300, C435S320100
Reexamination Certificate
active
06281346
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to rat ob receptor proteins, to DNA and RNA sequences encoding them, and to assays using rat receptor proteins.
BACKGROUND OF THE INVENTION
Recently the identification of mutations in several genes involved in the onset of obesity in rodents have been identified. Of particular interest are mutations discovered in the peptide hormone, leptin, which is a component of a novel signal transduction pathway that regulates body weight (Zhang et al. 1994,
Nature
372:425-432; Chen et al. 1996,
Cell
84:491-495). Leptin was initially discovered by the positional cloning of the obesity gene, ob, in mice. Two different ob alleles have been identified: one mutation causes the premature termination of the leptin peptide resulting in a truncated protein, and the other mutation changes the transcriptional activity of the obesity (ob) gene, resulting in a reduced amount of circulating leptin.
There is a correlation between a decrease in the levels of biologically active leptin and the overt obese phenotype observed in ob/ob mice. Recombinant leptin has been shown to induce weight loss in the ob/ob mouse but not in the diabetic phenotype db/db mouse (Campfield et al. 1995,
Science
269: 546-549; Halaas et al. 1995,
Science
269: 543-546; Pellymounter et al. 1995,
Science
269:540-543; Rentsch et al. 1995,
Biochem. Biophys. Res. Comm.
214:131-136; and Weigle et al. 1995,
J. Clin. Invest.
96:2065-2070).
Although the synthesis of leptin occurs in the adipocyte, its ability to decrease food intake and increase metabolic rate appears to be mediated centrally by the hypothalamus. Injection of recombinant leptin into the third ventricle of the brain elicits a similar response as peripheral administration of leptin. Furthermore, the recent cloning of the human receptor for the leptin, the ob-receptor (OB-R), reveals that it is transcribed in the hypothalamus (Tartaglia et al. 1995, Cell 83:1263-1271; Stephens et al. 1995,
Nature
377: 530-532). In addition, a mutation that results in premature termination of the long-form of the mouse OB-R, which is preferentially expressed in the hypothalamus, appears to be responsible for the obese phenotype of the db/db mouse (Lee et al. 1996,
Nature
379:632-635; Chua et al. 1996,
Science
271:994-996; and Chen et al. 1996,
Cell
84:491-495).
The fa mutation is a recessive allele that arose spontaneously in the 13M rat strain and was first reported in 1961 (Zucker et al. 1961,
J. Heredity
52: 275-278. The onset of obesity in the fa/fa Zucker rat is at 5-7 weeks of age and progresses with age. The mature fatty rat is approximately twice the weight of lean litter mates and over 40% of its body weight is adipose tissue (Zucker et al. 1962,
Proc. Soc. Exp. Biol. Med.
110:165-171; Zucker et al. 1963,
J. Nutrition
80:6-19). The fa/fa Zucker rat exhibits hypercholesterolemia, hyperlipemia, and hyperglycemia and has been used extensively as an animal model for human cardiovascular disease and diabetes. Most of the fatty Zucker rat colonies have been maintained by outbreeding in order to retain heterozygousity at as many loci as possible. However, certain stocks have been inbred to produce animals such as the Zucker diabetic fatty (ZDF) rat which exhibits a more profound diabetic phenotype than the outbred fa/fa Zucker rat (Clark, et al. 1983,
Proc. Soc. Exp. Biol. Med.
173: 68-75).
The fa mutation maps to rat chromosome 5 in a region that is syntenic with the db allele on mouse chromosome 4 (Truett, et al. 1991,
Proc. Natl. Acad. Sci.
88: 7806-7809). This observation, in conjunction with the similar phenotypes of the fa/fa rat and the db/db mouse, led to the proposal that the fa gene was the rat homologue of the db gene. Higher resolution genetic mapping supports the contention that the fa mutation is located in the gene encoding the rat OB-R (Chua et al.
Science
271: 994).
It would be desirable to be able to further experiment with the rodent model system for obesity, and to be able to clone and produce purified rat ob receptor to use in assays for the identification of ligands which may be useful in understanding obesity and for its prevention and treatment.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to a rat ob receptor which is substantially free from associated rat membrane proteins. It also relates to substantially purified rat ob receptor (“rat OB-R” or “rat OB-receptor”) protein. One of the rat OB-Rs of this invention is obtained from a rat which has a wild-type OB-R. Another rat OB-R of this invention is obtained from a rat which has the fa mutation.
Another aspect of this invention is to nucleic acids which encode a rat OB receptor. The nucleic acid may be any nucleic acid which can encode a protein, such as genomic DNA, cDNA, or any of the various forms of RNA. Preferably, the nucleic acid is cDNA.
This invention also includes vectors containing a rat OB-R gene, host cells containing the vectors, and methods of making susbstantially pure rat OB-R protein comprising the steps of introducing a vector comprising a rat OB-R gene into a host cell, and cultivating the host cell under appropriate conditions such that rat OB-R is produced. The rat OB-R so produced may be harvested from the host cells in conventional ways.
Yet another aspect of this invention are assays which employ a rat OB-R. In these assays, various molecules, suspected of being rat OB-R ligands are contacted with a rat OB-R, and their binding is detected. In this way agonists, antagonists, and ligand mimetics may be identified. A further aspect of this invention are the ligands so indentified.
REFERENCES:
patent: 5643748 (1997-07-01), Snodgrass et al.
patent: WO 96/08510 (1996-03-01), None
patent: WO 96/35787 (1996-11-01), None
patent: 9726335 (1997-07-01), None
Iida et alBBRC 2221996, p. 19-26.*
Bennett et al,Current Biology6(9) 1996, p. 1170-80.*
Phillips, Michael S., et al. “Leptin receptor missense mutation in the fatty Zucker rat”, 1996, Nature Genetics, vol. 13, No. 1, pp. 18-19.
Murakami, Takashi, et al. “Cloning of Rat Obese cDNA and its Expression in Obese Rats+”, 1995 Biochem and Biophy., No. 3, pp. 944-952.
Guan et al., “Differential Expression of mRNA for Leptin Receptor Isoforms in the Rat Brain”, Molec. and Cell Endocrinology, vol. 133, pp. 1-7, 1997.
Chen et al. “Evidence That the Diabetes Gene Encodes the Leptin Receptor: Identification of a Mutation in the Leptin Receptor Gene in db/db Mice,” 1996 Cell 84:491-495.
Chua et al. “Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor,” 1996 Science 271:994-996.
Chua et al. “Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr),” 1996 Diabetes 45:1141-1143.
Lee et al. “Abnormal splicing of the leptin receptor in diabetic mice”, 1996 Nature 379:632-635.
Phillips et al. “Leptin receptor missense mutation in the fatty Zucket rat”, 1996 Nature Genetics 13:19-20.
Rosenblum et al. “Functional STAT 1 and 3 signaling by the leptin receptor (OB-R): reduced expression of the rat fatty leptin receptor in transfected cells”, 1996 Endocrinology 137:5178-5181.
Spiegelman et al. “Adipogenesis and Obesity: Rounding out the big picture”, 1996 Cell 87:377-389.
Stephens et al. “The role of neuropeptide Y in the antiobesity action of the obese gene product”, 1995 Nature 377:530-532.
Takaya et al. “Molecular cloning of rat leptin receptor isoform complementary DNAs—identification of a missense mutation in Zucker fatty (fa/fa) rats”, 1996 Biochem Biophys Res Comm 225:75-83.
Tartaglia et al. “Identification and Expression Cloning of a Leptin Receptor, OB-R”, 1995 Cell 83:1263-1271.
Wang et al. “A novel leptin receptor isoform in rat”, 1996 FEBS Lett. 392:87-90.
Caskey C. Thomas
Hess John W.
Liu Qingyun
Phillips Michael Sean
Cocuzzo Anna L.
Merck & Co. , Inc.
Tribble Jack L.
Ulm John
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