Mutant OB receptors and nucleotides encoding them

Details Mutant OB receptors and nucleotides encoding them Mutant OB receptors and nucleotides encoding them

1997-12-02

2003-10-14

Eyler, Yvonne (Department: 1646)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving luciferase

C430S350000, C536S023500, C435S007200, C435S007210, C435S320100, C435S325000, C435S252300

Reexamination Certificate

active

06632625

ABSTRACT:

STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not Applicable.
REFERENCE TO MICROFICHE APPENDIX
Not Applicable.
FIELD OF THE INVENTION
This invention relates to mutant ob receptor proteins, to nucleotides encoding them, and to assays using the mutant 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).
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.
SUMMARY OF THE INVENTION
Not Applicable.
BRIEF DESCRIPTION OF THE DRAWINGS
Not Applicable.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to mutant ob receptors (“OB-Rs”), also referred to as “leptin receptors” 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 “lack a functional” 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.


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Lee et al., “Abnormal splicing of the leptin receptor in diabetic mice,” 1996 Nature 379:632-635.
Wang et al., “A novel leptin receptor isoform in rat,” 1996 FEBS Letters 392:87-90.
White et al., “Leptin Receptor (OB-R) Signaling,” 1997 J. Biol. Chem. 272(7):4065-4071.
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Tartaglia et al., “Identification and Expression Cloning of a Leptin Receptor, OB-R,” 1995 Cell 83:1263-1271.
Shields et al., “The Evolution of Haematopoietic Cytokine/Receptor Complexes,” 1995 Cytokine 7(7):679-688.
Guan et al., “Differential Expression of mRNA for Leptin Receptor Isoforms in the Rat Brain,” 1997 Molec. and Cell Endocrin. 133:1-7.
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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.
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Rosenblum et al. “Functional STAT 1 and 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.
Iida et al., “Phenotype-Linked Amino Acid Alteration in Leptin Receptor cDNA from Zucker Fatty (fa/fa) Rat,” 1996 Biochem. and Biophys. Res. Comm. 222:19-26.

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