INHIBITION OF RHOA TRANSLOCATION AND CALCIUM SENSITIZATION BY IN-VIVOADP-RIBOSYLATION WITH THE CHIMERIC TOXIN DC3B

Pretreatment of intact rabbit portal vein smooth muscle with the chime ric 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 co ntraction and the Ca2+-sensitization of force by phenylephrine, endoth elin and guanosine triphosphate (GTP)gamma S, but did not inhibit Ca2-sensitization by phorbol dibutyrate. DC3B also inhibited GTP gamma S- induced translocation of cytosolic RhoA (Gong et al., 1997a) to the me mbrane fraction. In DC3B-treated muscles the small fraction of membran e-associated RhoA could be immunoprecipitated, even after exposure to GTP gamma S, which prevents immunoprecipitation of non-ADP-ribosylated RhoA. Dissociation of cytosolic RhoA-rhoGDI complexes with SDS restor ed the immunoprecipitability and ADP ribosylatability: of RhoA, indica ting that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that Rho A plays a significant role in G-protein-, but not protein kinase C-med iated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo t he Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.