Phosphoinositide-dependent activation of Rho A involves partial opening ofthe RhoA/Rho-GDI complex

Rho GTPases have two interconvertible forms and two cellular localizations. In their GTP-bound conformation, they bind to the cell membrane and are ac tivated. In the inactive GDP-bound conformation, they associate with a cyto solic protein called GDP dissociation inhibitor (GDI). We previously report ed that the RhoA component of the RhoA/Rho-GDI complex was not accessible t o the Clostridium botulinum C3 ADP-ribosyl transferase, unless the complex had been incubated with phosphoinositides. We show here that PtdIns, PtdIns (4)P. PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdInsP(3) enhance not only the C3-dependent ADP-ribosylation, but also the GDP/GTP exchange in the RhoA co mponent of the prenylated RhoA/Rho-GDI complex. In contrast, in the nonpren ylated RhoA/Rho-GDI complex, the levels of ADP-ribosylation and GDP/GTP exc hange are of the same order as those measured on free RhoA acid are not mod ified by phosphoinositides. In both cases, phosphoinositides partially open ed, but did not fully dissociate the complex. Upon treatment of the prenyla ted RhoA/Rho-GDI complex with phosphoinositides, a GTP-dependent transfer t o neutrophil membranes was evidenced. Using an overlay assay with the preny lated RhoA/Rho-GDI complex pretreated with PtdIns(4)P and labeled with [alp ha(32)P]GTP. three membrane proteins with molecular masses between 26 and 3 2 kDa were radiolabeled. We conclude that in the presence of phosphoinositi des, the prenylated RhoA/Rho-GDI complex partially opens, which allows RhoA to exchange GDP for GTP. The opened GTP-RhoA/Rho-GDI complex acquirer the capacity to target specific membrane proteins.