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. Endogenous contents (∼2-4 μM) of RhoA and RhoGDI were near stoichiometric, whereas a supra-physiological GDI concentration was required to relax Ca²⁺ sensitization of force by GTP and guanosine 5′-O-(3-thiotriphosphate) (GTPγS). GDI also inhibited Ca²⁺ sensitization by GTP·G14V RhoA, by α-adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTPγS 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⁺ sensitization by GTP·G14V RhoA. We conclude that 1) the most likely in vivo function 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.