OB receptor isoforms and nucleic acids encoding them

Details OB receptor isoforms and nucleic acids encoding them OB receptor isoforms and nucleic acids encoding them

1997-05-06

2001-07-10

Fitzgerald, David L. (Department: 1646)

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

Organic compounds

Carbohydrates or derivatives

C530S350000, C435S320100, C435S325000, C435S252300, C435S252330, C435S254110, C435S007100, C435S007210, C435S069100, C514S002600

Reexamination Certificate

active

06258944

ABSTRACT:

STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not Applicable
REFERENCE TO MICROFICHE APPENDIX
Not Applicable
FIELD OF THE INVENTION
This invention relates to ob receptor protein isoforms, to DNA and RNA sequences encoding them, and to assays using the receptor isoform 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 oblob 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 94:491-495).
The OB-R from wild type (lean) rats and from rats having the fatty mutation (both heterozygous and homozygous fa ) have been isolated and sequenced. (patent application Ser. Nos. 60/146,928 and 60/013,969, now 08/803,346, pending filed Feb. 22, 1996 and Mar. 22, 1996, which are hereby incorporated by reference.) The gene encoding the OB-R of the fatty rat bears a missense mutation such that its expression product differs from the wild-type rat OB-R amino acid sequence shown in SEQ ID NO: 15 by the substitution Gln
269
→Pro, as shown in SEQ ID NO: 20.
Various isoforms of the OB-Rs have also been identified. These isoforms are due to alternative splicing. For example, in the mouse the a form has 5 amino acids following the Lysine at 889; the b form has 273 amino acids after Lysine 889; the c form has 3 amino acids after Lysine 889; and the d form contains 11 amino acids after Lysine 889.
It would be desirable to be able to further experiment with various isoforms in order to better understand obesity, and to be able to clone and produce novel oh receptor isoforms 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
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to novel ob receptor isoforms designated c′, f and g which are substantially free from associated membrane proteins. It also relates to substantially purified ob receptor isoform c′, f and g proteins. These isoforms are present in various species, including rat, mouse and human.
Another aspect of this invention is to nucleic acids which encode OB receptor isoforms c′, f or g. 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 OB-R isoform c′, f or g gene, host cells containing the vectors, and methods of making susbstantially pure OB-R isoform c′, f or g protein comprising the steps of introducing a vector comprising a OB-R isoform c′, f or g gene into a host cell, and cultivating the host cell under appropriate conditions such that OB-R isoform c′, f or g is produced. The OB-R isoform c′, f or g so produced may be harvested from the host cells in conventional ways.
Yet another aspect of-this invention are assays which employ OB-R isoform c′, f or g. In these assays, various molecules, suspected of being OB-R isoform c′, f or g ligands are contacted with a OB-R isoform c′, f or g, 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.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 is the amino acid sequence of residues Lys
889
through Asn
895
of the rat OB-R isoform f, corresponding to the alternate exon of that isoform.
SEQ ID NO: 2 is the N-terminal amino acid sequence of the rat OB-R isoform g, corresponding to the alternate exon of that isoform.
SEQ ID NOS: 3, 4, and 7-10 are the sequences of PCR primers, and SEQ ID NOS: 5, 6, and 11-14, respectively, are the amino acid sequence fragments to which they correspond.
SEQ ID NO: 15 shows the amino acid sequence of the wild-type rat OB-R, and SEQ ID NO: 16 provides the corresponding cDNA sequence.
SEQ ID NO: 17 is the sequence of a cDNA encoding the rat OB-R isoform f.
SEQ ID NO: 18 is the sequence of a cDNA encoding the rat OB-R isoform c′.
SEQ ID NO: 19 shows the amino acid sequence of the isoform f protein corresponding to the wild-type rat OB-R.
SEQ ID NO: 20 provides the amino acid sequence of the fatty rat OB-R.
SEQ ID NO: 21 shows the amino acid sequence of the isoform f protein corresponding to the fatty rat OB-R.
As used througout the specification and claims, the following definitions apply:
“Substantially free from associated membrane proteins” means that the receptor protein is not in physical contact with any membrane proteins.
“Substantially purified OB-receptor isoform c′, f or g” means that the protein isoform is at least 90% and preferably at least 95% pure.
“Wild type” means that the gene or protein is substantially the same as that found in an animal which is not considered to have a mutation for that gene or protein.
“fa” means that the gene or protein is substantially the same as that found in a rat homologous for the fatty mutation.
“Substantially the same” when referring to a nucleic acid or amino acid sequence means either it is the same as the reference sequence, or if not exactly the same, contains changes which do not affect its biological activity or function.
It has been suprisingly found, in accordance with this invention that the OB-R exists in a large variety of isoforms, including three novel ones, form c′, f and g. These isoforms apply to all species, but for convenience, throughout the specification and claims, numberings of amino acids and nucleotides will use the rat wild type sequences (SEQ ID NOS: 15 and 16) as a reference. However, it is to be understood that this invention is not limited to rat wild type proteins and nucleic acids and specifically includes rat (wild type and fatty), mouse, and human OB-R isoform c′, f and g proteins and nucleic acids.
OB-R isoform f differs from wild type protein in that after the Lysine at position 889 (referring to the rat sequence in SEQ ID NO: 15 there are six amino acids, ending at an Asparagine residue at position 895. In the cDNA, the codons are then followed b

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