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 I (PAR-1) by thrombin has been shown to induce an activation of the MAP kinase cascade and to stimul ate 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 Ser(306)--> Ala ( S306A) in the third intracellular loop of PAR-1 inhibited MAP kinase activa tion 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 gen eration, the Ser(306)-mutated PAR-1 induced a strong activation of phosphol ipase C after thrombin addition. Deletion of the cytoplasmic tail of PAR-1 also inhibited thrombin-induced DNA synthesis but the MAP kinase pathway wa s activated as with wild-type PAR-1. In contrast, the deletion of the C-tai l 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 th e third intracellular loop of PAR-1 is implicated in PAR-1 coupling to Gi a nd MAP kinase activation. In addition, these results also show that MAP kin ase activation is necessary but not sufficient for thrombin to induce cell proliferation.