ADP Ribosylation Factor 6 ( Arf6) Acts through FilGAP Protein to Down- regulate Rac Protein and Regulates Plasma Membrane Blebbing*

Background: FilGAP is a Rac GTPase-activating protein, but how it is regulated remains unclear. Results: GTPase Arf6 binds to FilGAP and stimulates its RacGAP activity to induce plasma membrane blebbing. Conclusion: GTPase Arf6 is a novel physiological regulator of FilGAP. Significance: This study establishes a novel molecular link between Arf6 and FilGAP that may have a role in Arf6-dependent inactivation of Rac. The small GTP-binding protein Arf6 reorganizes the actin cytoskeleton through the regulation of Rac activity. We identified FilGAP, a Rac-specific Rho GTPase-activating protein that is recruited to plasma membranes by binding to activated Arf6. FilGAP binds to Arf6 through its pleckstrin homology domain. Activated Arf6 stimulated RacGAP activity of FilGAP, and knockdown of endogenous Arf6 by siRNA suppresses FilGAP-mediated bleb formation. Mutant FilGAP lacking phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding (FilGAP R39C) binds to activated Arf6 and induces bleb formation. Moreover, bleb formation induced by wild-type FilGAP occurs in the presence of phosphatidylinositol 3-kinase inhibitors, suggesting a PIP3-independent interaction between FilGAP and Arf6. We propose that FilGAP may function as a mediator of the regulation of Rac by Arf6.