Recombinant production of glucagon receptors
First Claim
1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a glucagon receptor polypeptide having seven transmembrane domains, an N-terminal extracellular domain, three extracellular loop domains, and three intracellular loop domains,wherein the receptor polypeptide is capable of binding glucagon with a KD no greater than 100 nM and is capable of transducing signal in a suitable host cell in response to such binding,and wherein the nucleic acid molecule hybridizes under high stringency hybridization conditions to a probe having the nucleotide sequence of the complement of the sequence shown in SEQ ID NO:
- 14, from nucleotide 145 to 1599, or the sequence shown in SEQ ID NO;
24, from nucleotide 53 to 1483.
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Abstract
The present invention provides isolated DNA molecules comprising a DNA segment encoding a glucagon receptor. Also provided are DNA constructs comprising a first DNA segment encoding a glucagon receptor operably linked to additional DNA segments required for the expression of the first DNA segment, as well as host cells containing such DNA constructs. The present invention also provides a method for detecting the presence of glucagon antagonists, comprising the steps of (a) exposing a compound in the presence of a glucagon against to a recombinant glucagon receptor coupled to a response pathway under conditions and for time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting a reduction in the stimulation of the response pathway resulting from the binding of the compound to the glucagon receptor, relative to the stimulation of the response pathway by the glucagon agonist alone and therefrom determining the presence of a glucagon antagonist.
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Citations
38 Claims
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1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a glucagon receptor polypeptide having seven transmembrane domains, an N-terminal extracellular domain, three extracellular loop domains, and three intracellular loop domains,
wherein the receptor polypeptide is capable of binding glucagon with a KD no greater than 100 nM and is capable of transducing signal in a suitable host cell in response to such binding, and wherein the nucleic acid molecule hybridizes under high stringency hybridization conditions to a probe having the nucleotide sequence of the complement of the sequence shown in SEQ ID NO: - 14, from nucleotide 145 to 1599, or the sequence shown in SEQ ID NO;
24, from nucleotide 53 to 1483. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
- 14, from nucleotide 145 to 1599, or the sequence shown in SEQ ID NO;
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24. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
- 15 from glutamine, amino acid 28, to tyrosine, amino acid 142.
- View Dependent Claims (25, 26)
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27. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a fragment of a glucagon receptor polypeptide having the sequence shown as residues 1 to 485 of SEQ ID NO:
- 15 or residues 1 to 477 of SEQ ID NO;
25, the fragment comprising at least(a) the complete sequence of an extracellular or intracellular domain of the receptor, or (b) ten contiguous amino acids from the sequence of the corresponding mature receptor. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
- 15 or residues 1 to 477 of SEQ ID NO;
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38. A polynucleotide probe which is capable of hybridizing under conditions of high stringency with the coding strand of a cDNA encoding a glucagon receptor or the complement thereof,
wherein the sequence of the coding strand is: -
(a) SEQ ID NO;
14;(b) SEQ ID NO;
24 ;
or(c) an allelic variant of the coding sequence of (a) or (b); and wherein the probe comprises at least 18 contiguous nucleotides from the sequence of; the coding or noncoding strand of the cDNA, or an RNA molecule complementary to either of said strands; provided that the at least 18 contiguous nucleotides are not contained entirely within a nucleotide sequence encoding a transmembrane domain of said receptor or a noncoding sequence complementary thereto.
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Specification