The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways

We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-I, In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-I transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-I cooperated with activated Raf-l and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA, Finally, PAR-I transforming activity was blocked by pertussis toxin and by coexpression of the RGS domain of Lsc, implicating G alpha (i) and G alpha (12)/G alpha (13) subunits, respectively, as mediators of PAR-I transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.