Rap1 Activation Is Required for Fcγ Receptor-Dependent Phagocytosis

Abstract

Phagocytosis of IgG-opsonized microbes via the Fcγ receptor (FcγR) requires the precise coordination of a number of signaling molecules, including the low-molecular mass GTPases. Little is known about the Ras-family GTPase Rap1 in this process. We therefore investigated its importance in mediating FcγR-dependent phagocytosis in NR8383 rat alveolar macrophages. Pulldown of active Rap1 and fluorescence microscopic analysis of GFP-RalGDS (Ral guanine dissociation stimulator)-transfected macrophages revealed that Rap1 is indeed activated by FcγR crosslinking. Inhibition of Rap1 activity, both by Rap1GAP (GTPase-activating protein) expression and liposome-delivered blocking Ab, severely impaired the ability of cells to ingest IgG-opsonized targets. FcγR-induced Rap1 activation was found to be independent of both cAMP and Ca2+, suggesting a role for the second messenger-independent guanosine exchange factor, C3G. This was supported by the facts that 1) liposome-delivered blocking Ab against C3G inhibited both FcγR-dependent phagocytosis and Rap1 activation, and 2) both active Rap1GTP and C3G were found to translocate to the phagosome. Taken together, our data demonstrate a novel role for Rap1 and its exchange factor C3G in mediating FcγR-dependent phagocytosis.