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

Abstract

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 supraphysiological 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.