Purification of ARAP3 and characterization of GAP activities

ARAP3 is a dual Arf and Rho GTPase activating protein (GAP) that was identified from pig leukocyte cytosol using a phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns[3,4,5]P3) affinity matrix in a targeted proteomics study. ARAP3's domain structure includes five PH domains, an Arf GAP domain, three ankyrin repeats, a Rho GAP domain, and a Ras association domain. ARAP3 is a PtdIns(3,4,5)P3-dependent GAP for Arf6 both in vitro and in vivo. It acts as a Rap-GTP-activated RhoA GAP in vitro, and this activation depends on a direct interaction between ARAP3 and Rap-GTP; in vivo PtdIns(3,4,5)P3 seems to be required to allow ARAP3's activation as a RhoA GAP by Rap-GTP. Overexpression of ARAP3 in pig aortic endothelial (PAE) cells causes the PI3K-dependent loss of adhesion to the substratum and interferes with lamellipodium formation. This overexpression phenotype depends on ARAP3's intact abilities to bind PtdIns(3,4,5)P3, to interact with Rap-GTP, and to be a catalytically active RhoA and Arf6 GAP.