A Rho exchange factor mediates thrombin and Gα12-induced cytoskeletal responses

Thrombin induces astrocytoma cell rounding through a Rho-dependent pathway (Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P,, and Brown, J. H. (1998) J. Biol. Chem. 273, 10099-10106), The involvement of the G(12) family of G proteins and the role of specific Rho exchange factors in transducing signals from the thrombin receptor to Rho-dependent cytoskeletal responses was examined. Microinjection of cDNAs for activated G alpha(12) or G alpha(13) induced cell rounding, and antibodies to G alpha(12) or G alpha(13) blocked the response to thrombin, in contrast, activation or inhibition of G alpha(q) function had relatively little effect. The cytoskeletal response to G alpha(12) was inhibited by microinjection of C3 exoenzyme, indicating Rho dependence. Two Rho-specific guanine nucleotide exchange factors (GEFs), oncogenic lbc and p115, increased the percentage of rounded cells 4-5-fold, and this was inhibited by C3. Mutant GEFs lacking the Dbl homology (DH) domain required for exchange factor activity failed to induce cell rounding. However, the DH mutants of Ibc and p115 were efficacious inhibitors off rounding induced by thrombin or G alpha(12). The effects of lbc were dependent on an intact pleckstrin homology domain, which may be required for appropriate targeting of the Rho-GEF, These findings identify the G alpha(12) protein family as transducers of thrombin signaling to the cytoskeleton and provide the first evidence that a Rho-GEF transduces signals between G protein-coupled receptors and Rho-mediated cytoskeletal responses.