LOCALIZATION OF RHOA GTPASE TO ENDOTHELIAL CAVEOLAE-ENRICHED MEMBRANEDOMAINS

Caveolae are small microdomains of the plasma membrane that are though t to play important roles in signal transduction processes. In this wo rk, we have investigated the association of Rho proteins with caveolae -enriched membrane domains isolated from cultured endothelial cells. F ractionation of ECV304 cells by sucrose gradient density centrifugatio n in the absence of detergent resulted in the co-sedimentation of a si gnificant proportion of RhoA and Cdc42 with known caveolae marker prot eins, including caveolin, but not with other non-caveolae membrane pro teins such as the angiotensin-converting enzyme. Immunoprecipitation e xperiments carried on crude endothelial cell lysates as well as with s olubilized caveolae-enriched membrane domains showed the coimmunopreci pitation of caveolin with RhoA but not with Cdc42. Incubation of endot helial cell lysates with a glutathione-S-transferase (GST)-RhoA fusion protein resulted in the specific precipitation of caveolin, while add ition of GST-caveolin-1 to the lysates promoted the precipitation of R hoA. Moreover, incubation of bacterially expressed RhoA with GST-caveo lin-1 resulted in the precipitation of RhoA, indicating that RhoA dire ctly interacts with caveolin-1. This interaction was found to be nucle otide-independent and was not affected by prior modification of RhoA w ith the C3 exoenzyme from C. botulinium or with the cytotoxic necrotin izing factor from E. coli. Taken together, these results suggest the a ssociation of RhoA with endothelial caveolae-enriched membrane domains , likely through physical interaction with caveolin-1. These findings may provide new insights into the functions played by Rho proteins and caveolae in signal transduction events. (C) 1998 Academic Press.