Fellowship for Yunjie Huang: Arf6 Regulation of Rac1 and Cdc42 in Platelets: A Role for the GIT1/Beta-PIX Complex

Cytoskeletal rearrangement is one of the central events for platelet hemostatic function. ADPribosylation factor 6 (Arf6) is the most divergent member of Arf family of Ras superfamily GTPases and plays an important role in cytoskeletal reorganization and membrane trafficking. Previously, we showed that Arf6 is important for platelet function. Arf6 is found in the GTP]bound state in resting platelets and is converted to GDP]bound form upon activation. In platelets, Arf6 regulates F]actin formation and the loss of Arf6]GTP is required for activation of the Rho family members, Rac1 and Cdc42. Like other Ras superfamily members, Arf6 functions through its GTP]GDP cycle. However, its regulators/effectors in platelets are unknown. One candidate, G protein]coupled receptor kinase interacting protein 1 (GIT1) has GTPase activating protein (GAP) activity toward Arf6 and forms a constitutive complex with p21]activated kinase interacting exchange factor (PIX). The GIT1/PIX complex is associated with integrin upon platelet activation. PIX is a GTP exchange factor (GEF) for Rac1 and Cdc42 in nucleated cells. Hydrogen peroxide]inducible clone 5 (Hic]5) is a member of paxillin family which are signaling and adaptor proteins in focal adhesion complexes. The central hypothesis of this proposal is that Arf6, through the GIT1/ƒÀ]PIX complex, regulates activation of Rac1 and Cdc42 and thus activation of platelets. To address this hypothesis, three specific aims are proposed.