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Objective-Vascular endothelial growth factor (VEGF) exerts proangiogenic action and induces activation of a variety of proangiogenic signaling pathways, including the Rho family small G proteins. However, regulators of the Rho family small G proteins in vascular endothelial cells (ECs) are poorly understood. Here we attempted to clarify the expression, subcellular localization, downstream effectors, and proangiogenic role of FGD5, a member of the FGD family of guanine nucleotide exchange factors. Methods and Results-FGD5 was shown to be selectively expressed in cultured human vascular ECs. Immunofluorescence microscopy showed that the signal for FGD5 was observed at peripheral membrane ruffles and perinuclear regions in human umbilical vein ECs. Overexpression of FGD5 increased Cdc42 activity, whereas knockdown of FGD5 by small interfering RNAs inhibited the VEGF-induced activation of Cdc42 and extracellular signal-regulated kinase. VEGF-promoted capillary-like network formation, permeability, directional movement, and proliferation of human umbilical vein ECs and the reorientation of the Golgi complex during directional cell movement were attenuated by knockdown of FGD5. Conclusion-This

Cyclical activation and inactivation of Rho family small G proteins, such as Rho, Rac, and Cdc42, are needed for moving cells to form leading edge structures in response to chemoattractants. However, the mechanisms underlying the dynamic regulation of their activities are not fully understood. We recently showed that another small G protein, Rap1, plays a crucial role in the platelet-derived growth factor (PDGF)-induced formation of leading edge structures and activation of Rac1 in NIH3T3 cells. We showed here that knockdown of afadin, an actin-binding protein, in NIH3T3 cells resulted in a failure to develop leading edge structures in association with an impairment of the activation of Rap1 and Rac1 and inactivation of RhoA in response to PDGF. Overexpression of a constitutively active mutant of Rap1 (Rap1-CA) and knockdown of SPA-1, a Rap1 GTPase-activating protein that was negatively regulated by afadin by virtue of binding to it, in afadin-knockdown NIH3T3 cells restored the formation of leading edge structures and the reduction of the PDGF-induced activation of Rac1 and inactivation of RhoA, suggesting that the inactivation of Rap1 by SPA-1 is responsible for inhibition of the formation of leading edge structures. The effect of Rap1-CA on the restoration of the formation of leading edge structures and RhoA inactivation was diminished by additional knockdown of ARAP1, a Rap-activated Rho GAP, which localized at the leading edges of moving NIH3T3 cells. These results indicate that afadin regulates the cyclical activation and inactivation of Rap1, Rac1, and RhoA through SPA-1 and ARAP1.