Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II

We characterized the role of guanine nucleotide dissociation inhibitor (GDI ) in RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth muscle. Endogeno us contents (similar to2-4 muM) of RhoA and RhoGDI were near stoichiometric , whereas a supraphysiological GDI concentration was required to relax Ca2 sensitization of force by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTP gammaS). GDI also inhibited Ca2+ sensitization by GTP.G14V RhoA, by alpha -adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTP gS translocated Rho-kinase to a Triton X-114-extractable membrane fraction. GTP.G14V RhoA complexed with GDI also induced Ca2+ sensitization, probably through in vivo dissociation of GTP. RhoA from the complex, because it was reversed by addition of excess GDI. GDI did not inhibit Ca2+ sensitization by phorbol ester. Constitutively active Cdc42 and Rac1 inhibited Ca2+ sens itization by GTP.G14V RhoA. We conclude that 1) the most likely in vivo fun ction of GDI is to prevent perpetual "recycling" of GDP.RhoA to GTP.RhoA; 2 ) nucleotide exchange (GTP for GDP) on complexed GDP. RhoA/GDI can precede translocation of RhoA to the membrane; 3) activation of Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and Rac1 can inhibit Ca2+ sensitization by activated GTP.RhoA.