Identification of a novel thrombin receptor sequence required for activation-dependent responses.
Thrombin receptor (TR) activation by alpha-thrombin requires proteolytic cleavage, although synthetic peptides modeled after the new N-terminus directly effect receptor activation without cleavage, presumably by interacting with an unidentified region of the receptor. To further define critical residues responsible for receptor activation, we performed epitope mapping of anti-TR1-160, a previously described polyclonal antibody that inhibits peptide ligand-induced receptor activation in various cell types expressing a functional TR. An enzyme-linked immunosorbent assay (ELISA) using overlapping decapeptides derived from the TR extracellular domains identified four immunodominant peaks within the long N-terminal extension centered between amino acids 34-44, 48-67, 65-79, and 87-94. Soluble peptides derived from regions 83-94, but not those from other regions of the receptor, neutralized the ability of anti-TR1-160 to inhibit peptide ligand-induced platelet aggregation, suggesting that antibodies directed against this region of the TR are important in ligand-mediated activation. Thrombin receptor mutants lacking discrete regions of the TR were subsequently evaluated using microinjected Xenopus oocytes. Whereas a TR mutant lacking amino acid residues Thr67-Lys82 (TR delta 67-82) showed normal to exaggerated responses to either alpha-thrombin or synthetic peptide ligands, only TR mutants with limited deletions spanning the residues Gln83-Ser93 exhibited dysfunctional responses to either agonist (200 nmol/L alpha-thrombin or 200 mumol/L TR42-47). These data provide a model for receptor activation that implicates a discrete and previously uncharacterized sequence within the TR N-terminal extension that is necessary for initiation of signal transduction events independent of the initiating agonist.
Bahou
W F
WF
Department of Medicine, State University of New York at Stony Brook.
Kutok
J L
JL
Wong
A
A
Potter
C L
CL
Coller
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D018179:Receptors, Thrombin / Q000235:genetics* / Q000276:immunology; D016415:Sequence Alignment; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D014981:Xenopus
D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D018179:Receptors, Thrombin; D013917:Thrombin