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 (approximately 2-4 microM) 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) (GTPgammaS). GDI also inhibited Ca2+ sensitization by GTP. G14V RhoA, by alpha-adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTPgammaS 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.
Gong
M C
MC
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, USA.
Gorenne
I
I
Read
P
P
Jia
T
T
Nakamoto
R K
RK
Somlyo
A V
AV
Somlyo
A P
AP
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004928:Escin / Q000494:pharmacology; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D007082:Ileum / Q000166:cytology / Q000378:metabolism; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D011169:Portal Vein / Q000166:cytology / Q000378:metabolism; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D001743:Urinary Bladder / Q000033:anatomy & histology / Q000737:chemistry; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D015003:Yeasts / Q000235:genetics / Q000502:physiology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D000577:Amides; D004791:Enzyme Inhibitors; D020730:Guanine Nucleotide Dissociation Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; D011994:Recombinant Proteins; D014662:Vasoconstrictor Agents; C108830:Y 27632; D010656:Phenylephrine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004928:Escin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D002118:Calcium