Affiliation:
1. Departments of Molecular Physiology and Biological Physics, Pathology and Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011; and
2. INSERM U452, Faculte de Medicine, Nice 06107, Cedex 2, France
Abstract
Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10−6 M, 48 h; Aullo et al., 1993 ; Boquet et al. 1995 ) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)γS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPγS-induced translocation of cytosolic RhoA ( Gonget al., 1997a ) to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPγS, which prevents immunoprecipitation of non-ADP–ribosylated RhoA. Dissociation of cytosolic RhoA–rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop ( Weiet al., 1997 ) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.
Publisher
American Society for Cell Biology (ASCB)
Subject
Cell Biology,Molecular Biology
Cited by
140 articles.
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