Recombinant thrombin receptors and assays using them
AssigneesCOR Therapeutics, Inc., The Regents of the University of California
InventorShaun R. Coughlin
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Abstract
The DNA encoding the cell surface receptor for thrombin has been cloned and sequenced. The availability of this DNA permits the recombinant production of thrombin receptor which can be produced at cell surfaces and is useful in assay systems both for the detection of thrombin and for the evaluation of candidate thrombin agonists and antagonists. Further, the elucidation of the structure of the thrombin receptor permits the design of agonist and antagonist compounds which are useful diagnostically and therapeutically. The availability of the thrombin receptor also permits production of antibodies specifically immunoreactive with the receptor per se or with specific regions thereof which are also useful diagnostically or therapeutically.
Claims73
An isolated nucleic acid molecule comprising a sequence encoding a mammalian thrombin receptor, wherein the thrombin receptor has seven transmembrane domains and is activated by thrombin-mediated proteolytic cleavage of an N-terminal activation peptide;
- wherein the nucleic acid molecule has the sequence of:
- (a) a cDNA present in a mammalian library, or
- (b) a nucleic acid molecule degenerate with a cDNA according to (a),
- wherein microinjection into Xenopus oocytes of cRNA corresponding to said cDNA affords cells which express active thrombin receptors on their surfaces;
- and wherein a polyclonal antiserum raised against a peptide immunogen consisting essentially of the sequence shown in SEQ ID NO:
- 101 is immunoreactive with the thrombin receptor.
An isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:220.
An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:219.
An isolated nucleic acid molecule comprising about residues 350 to about 1501 of SEQ ID NO:
- 219 or the full length complement thereof.
An isolated nucleic acid molecule encoding a polypeptide comprising an antigenic sequence fragment of SEQ ID NO:
- 220, wherein the polypeptide is useful for making an antibody specific for a human thrombin receptor.
A nucleic acid molecule comprising a recombinant expression system capable, when transformed into a recombinant host cell, of producing a thrombin receptor at the cell surface of the host, which expression system comprises a nucleotide sequence encoding said thrombin receptor operatively linked to a control sequence operable in said host cell, wherein said nucleotide sequence encoding the thrombin receptor comprises the nucleotide sequence of:
- (a) a cDNA present in a mammalian library, or
- (b) a nucleic acid molecule degenerate with a cDNA according to (a),
- wherein microinjection into Xenopus oocytes of cRNA corresponding to said cDNA affords cells which express active thrombin receptors on their surfaces;
- wherein the thrombin receptor has seven transmembrane domains and is activated by thrombin-mediated proteolytic cleavage of an N-terminal activation peptide;
- and wherein a polyclonal antiserum raised against a peptide immunogen consisting essentially of the sequence shown in SEQ ID NO:
- 101 is immunoreactive with the thrombin receptor.
A method to assess thrombin receptor agonist or antagonist activity of a candidate substance, which method comprises:
- (a) incubating recombinant host cells expressing mammalian thrombin receptors in the presence of thrombin, a thrombin receptor agonist, or a thrombin receptor antagonist and in the presence of the candidate substance,
- wherein said recombinant host cells have been transformed with an expression system that produces thrombin receptor at the cell surface of the host cells, which expression system comprises the nucleotide sequence of:
- (i) a cDNA present in a mammalian library, or
- (ii) a nucleic acid molecule degenerate with a cDNA according to (a),
- wherein microinjection into Xenopus oocytes of cRNA corresponding to said cDNA affords cells which express active thrombin receptors on their surfaces;
- wherein the thrombin receptor has seven transmembrane domains and is activated by thrombin-mediated proteolytic cleavage of an N-terminal activation peptide;
- and wherein a polyclonal antiserum raised against a peptide immunogen consisting essentially of the sequence shown in SEO ID NO:
- 101 is immunoreactive with the thrombin receptor;
- and
- (b) measuring the binding of said thrombin, thrombin receptor agonist, or thrombin receptor antagonist to the surface of said cells in the presence of said candidate substance as compared to binding in the absence of said candidate substances whereby the agonist or antagonist activity of the candidate substance is determined.
Dependencies are inferred from the claim text.