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

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908 We characterized the role of guanine nucleotide dissociation inhibitor (GDI) in RhoA/Rho-kinase-mediated Ca 2+ 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 Ca 2+ sensitization of force by GTP and guanosine 5'- O -(3-thiotriphosphate) (GTP S). GDI also inhibited Ca 2+ sensitization by GTP · G14V RhoA, by -adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTP S translocated Rho-kinase to a Triton X-114-extractable membrane fraction. GTP · G14V RhoA complexed with GDI also induced Ca 2+ 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 Ca 2+ sensitization by phorbol ester. Constitutively active Cdc42 and Rac1 inhibited Ca 2+ 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 Ca 2+ sensitization by activated GTP · RhoA. calcium sensitization; Cdc42; Rac; RhoGDI; Y-27632; smooth muscle * M. C. Gong, I. Gorenne, and P. Read contributed equally to this work.