ACAPs are Arf6 GTPase-activating proteins that function in the cell periphery

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosoma l membrane trafficking and the actin cytoskeleton in the cell periphery. GT Pase-activating proteins (GAPs) are critical regulators of Arf function, co ntrolling the return of Arf to the inactive GDP-bound state. Here, we repor t the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2 . Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motif s including coiled coil, pleckstrin homology, Arf GAP, and three complete a nkyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cul tured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recrui ted to and, when overexpressed. inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblast s. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs . In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP 1 and ACAP2 were recruited to peripheral, tubular membranes, where activati on of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by A rf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced r uffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.