Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins

The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and n onprenylated small GTPases were expressed in Sf9 insect cells and Escherich ia coli. respectively. Nucleotide binding to and hydrolysis by prenylated a nd nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs acceler ate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50 RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than wit h the prenylated protein, whereas no difference was observed in the reactio n of isolated GAP domains of either p50RhoGAP or Bcr with the different typ es of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bet. The effect of fluoride is not influenced by addition or chelation o f Al3+. This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GA Ps. It is suggested that both intramolecular interactions and membrane targ eting of GAP proteins represent potential mechanisms regulating Rac signali ng.