Role of the third intracellular loop and of the cytoplasmic tail in the mitogenic signaling of the protease-activated receptor 1.
The activation of the protease-activated receptor 1 (PAR-1) by thrombin has been shown to induce an activation of the MAP kinase cascade and to stimulate cell proliferation. To examine the mechanisms of signal transduction by PAR-1, we constructed several PAR-1 mutants which were stably expressed in CHO cells. When compared to wild-type PAR-1, mutation of Ser306-->Ala (S306A) in the third intracellular loop of PAR-1 inhibited MAP kinase activation and cell proliferation stimulated by thrombin. The thrombin activation of MAP kinase was inhibited by pertussis toxin, suggesting a role for a Gi-like protein. As shown by calcium signaling and inosotol trisphosphate generation, the Ser306-mutated PAR-1 induced a strong activation of phospholipase C after thrombin addition. Deletion of the cytoplasmic tail of PAR-1 also inhibited thrombin-induced DNA synthesis but the MAP kinase pathway was activated as with wild-type PAR-1. In contrast, the deletion of the C-tail of PAR-1 prevented almost completely the activation of the phospholipase C pathway. Taken together these results suggest that the C-tail of PAR-1 is a critical site for PAR-1 coupling to phospholipase C activation, while the third intracellular loop of PAR-1 is implicated in PAR-1 coupling to Gi and MAP kinase activation. In addition, these results also show that MAP kinase activation is necessary but not sufficient for thrombin to induce cell proliferation.
Chen
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INSERM U 489, and Association Claude Bernard, Hôpital Tenon, 4 rue de la Chine, 75020 Paris, France.
Berrou
J
J
Vigneau
C
C
Rondeau
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E
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007295:Inositol Phosphates / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D037342:Pertussis Toxin; D010957:Plasmids / Q000235:genetics; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007295:Inositol Phosphates; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin; D002118:Calcium