PAG3/Pap alpha/KIAA0400, a GTPase-activating protein for ADP-ribosylation factor (ARF), regulates ARF6 in Fc gamma receptor-mediated phagocytosis of macrophages

The Fc gamma receptor (Fc gammaR)-mediated phagocytosis of macrophages is a complex process where remodeling of both the actin-based cytoskeleton and plasma membrane occur coordinately. Several different families of small GTP ases are involved. We have isolated a GTPase-activating protein (GAP) for A DP-ribosylation factor (ARF), paxillin-associated protein with ARFGAP activ ity (PAG)3/Pap alpha /KIAA0400, from mature monocytes and macrophage-like c ells. Mam malian ARFs fall into three classes, and the class III isoform (A RF6) has been shown to be involved in Fc gammaR-mediated phagocytosis. Here we report that PAG3 is enriched together with ARF6 and F-actin at phagocyt ic cups formed beneath immunoglobulin G-opsonized beads in P388D1 macrophag es, in which overexpression of ARF6, but not ARF1 (class I) or ARF5 (class II), inhibits the phagocytosis. Overexpression of PAG3, but riot its GAP-in active mutant, attenuated the focal accumulation of F-actin and blocked pha gocytosis, although surface levels of the Fc gamma Rs were not affected. Ot her ubiquitously expressed ARFGAPs, G protein-coupled receptor kinase inter actors GIT2 and GIT2-short/KIAA0148, which we have shown to exhibit GAP act ivity for ARF1 in COS-7 cells, did not accumulate at the phagocytic cups or inhibit phagocytosis. Moreover, cooverexpression of ARF6, but not ARF1 or ARF5, restored the phagocytic activity of PAG3-overexpressing cells. We pro pose that PAG3 acts as a GAP for ARF6 and is hence involved in Fc gammaR-me diated phagocytosis in mouse macrophages.