Not Applicable.
Not Applicable.
This invention relates to mutant ob receptor proteins, to nucleotides encoding them, and to assays using the mutant receptor proteins.
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).
It would be desirable to clone and produce mutant ob receptor proteins to use in assays for the identification of ligands which may be useful in understanding obesity and for its prevention and treatment.
Not Applicable.
Not Applicable.
This invention relates to mutant ob receptors (xe2x80x9cOB-Rsxe2x80x9d), also referred to as xe2x80x9cleptin receptorsxe2x80x9d which differ from wild type receptors by having a mutation selected from the group consisting of:
a) lacking a functional first CK-F3 module;
b) lacking a functional second CK-F3 module; and
c) lacking a functional intracellular domain.
The expression xe2x80x9clack a functionalxe2x80x9d module or domain means that the receptor no longer has biological function associated with structural portion of the receptor molecule. This may be accomplished by deletion of the module or domain, or by substitutions the amino acids which make up one domain by other amino acids such that function is lost.
This invention also relates to mutant ob receptors, which are selected from the group consisting of:
a) receptors which contain a deletion of amino acid residues which comprise all or substantially all of the first CK-F3 module;
b) receptors which contain a substitution of all or substantially all of a F3 domain for a CK domain in a second CK-F3 module, resulting in a F3-F3 module; and
c) receptors which have a deletion of all or substantially all of the intracellular domain.
Another aspect of this invention is to nucleic acids which encode a mutant OB-R of this invention. Preferably, the nucleic acid is DNA.
This invention also includes vectors containing a mutant OB-R gene, host cells containing the vectors, and methods of making mutant OB-R protein comprising the steps of introducing a vector comprising a mutant OB-R gene into a host cell, and cultivating the host cell under appropriate conditions such that mutant OB-R is produced. The mutant 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 mutant OB-R. In these assays, various molecules, suspected of being wild-type OB-R ligands are contacted with a mutant OB-R, and their binding is detected. A particularly preferred type of assay is a transactivation assay, wherein the binding of the ligand to a mutant OB-R initiates a cascade of intracellular events, resulting in transcription and/or translation of a reporter gene which can be detected. Using the assays of this invention, agonists, antagonists, and ligand mimetics may be identified. A further aspect of this invention are the ligands so indentified.
Still another aspect of this invention is the use of the mutant receptors of this invention in gene therapy. Genes encoding the mutant receptors may be transferred into an animal in need of treatment, and the regulation of weight gain may be influenced.