GIT proteins, a novel family of phosphatidylinositol 3,4,5-trisphosphate-stimulated GTPase-activating proteins for ARF6

ADP-ribosylation factor (ARF) proteins are key players in numerous vesicula r trafficking events ranging from the formation and fusion of vesicles in t he Golgi apparatus to exocytosis and endocytosis. To complete their GTPase cycle, ARFs require a guanine nucleotide-exchange protein to catalyze repla cement of GDP by GTP and a GTPase-activating protein (GAP) to accelerate hy drolysis of bound GTP. Recently numerous guanine nucleotide-exchange protei ns and GAP proteins have been identified and partially characterized. Every ARF GAP protein identified to date contains a characteristic zinc finger m otif. GIT1 and GIT2, two members of a new family of G protein-coupled recep tor kinase-interacting proteins, also contain a putative zinc finger motif and display ARF GAP activity. Truncation of the amino-terminal region conta ining the zinc finger motif prevented GAP activity of GIT1, One zinc molecu le was found associated per molecule of purified recombinant ARF-GAP1, GIT1 , and GIT2 proteins, suggesting the zinc finger motifs of ARF GAPs are func tional and should play an important role in their GAP activity. Unlike ARF- GAP1, GIT1 and GIT2 stimulate hydrolysis of GTP bound to ARF6. Accordingly we found that the phospholipid dependence of the GAP activity of ARF-GAP1 a nd GIT proteins was quite different, as the GIT proteins are stimulated by phosphatidylinositol 3,4,5-trisphosphate whereas ARF-GAP1 is stimulated by phosphatidylinositol 4,5-bisphosphate and diacylglycerol, These results sug gest that although the mechanism of GTP hydrolysis is probably very similar in these two families of ARF GAPs, GIT proteins might specifically regulat e the activity of ARF6 in cells in coordination with phosphatidylinositol 3 -kinase signaling pathways.