The rapid activation of N-Ras by α-thrombin in fibroblasts is mediated by the specific G-protein Gαi2–Gβ1–Gγ5 and occurs in lipid rafts

α-thrombin is a potent mitogen for fibroblasts and initiates a rapid signal transduction pathway leading to the activation of Ras and the stimulation of cell cycle progression. While the signaling events downstream of Ras have been studied in significant detail and appear well conserved across many species and cell types, the precise molecular events beginning with thrombin receptor activation and leading to the activation of Ras are not as well understood. In this study, we examined the immediate events in the rapid response to α-thrombin, in a single cell type, and found that an unexpected degree of specificity exists in the pathway linking α-thrombin to Ras activation. Specifically, although IIC9 cells express all three Ras isoforms, only N-Ras is rapidly activated by α-thrombin. Further, although several Gα subunits associate with PAR1 and are released following stimulation, only Gαi2 couples to the rapid activation of Ras. Similarly, although IIC9 cells express many Gβ and Gγ subunits, only a subset associates with Gαi2, and of those, only a single Gβγ dimer, Gβ1γ5, participates in the rapid activation of N-Ras. We then hypothesized that co-localization into membrane microdomains called lipid rafts, or caveolae, is at least partially responsible for this degree of specificity. Accordingly, we found that all components localize to lipid rafts and that disruption of caveolae abolishes the rapid activation of N-Ras by α-thrombin. We thus report the molecular elucidation of an extremely specific and rapid signal transduction pathway linking α-thrombin stimulation to the activation of Ras.