The Journal of Experimental Medicine
IN Cell Analyzer 2000
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Published online 16 April 2001.
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© The Rockefeller University Press, 0022-1007/2001/4/955/ $5.00
The Journal of Experimental Medicine, Volume 193, Number 8, April 16, 2001 955-966

Original Article

Pag3/Pap{alpha}/Kiaa0400, a Gtpase-Activating Protein for Adp-Ribosylation Factor (Arf), Regulates Arf6 in Fc{gamma} Receptor–Mediated Phagocytosis of Macrophages

Hiroshi Uchidaa,b, Akiko Kondoa, Yasunori Yoshimurab, Yuichi Mazakia, and Hisataka Sabea,c

a Department of Molecular Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan
b Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo 160-8582, Japan
c Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
Department of Molecular Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.81-6-6871-668681-6-6872-4814

sabe{at}obi.or.jp

The Fc{gamma} receptor (Fc{gamma}R)-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 GTPases are involved. We have isolated a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF), paxillin-associated protein with ARFGAP activity (PAG)3/Pap{alpha}/KIAA0400, from mature monocytes and macrophage-like cells. Mammalian ARFs fall into three classes, and the class III isoform (ARF6) has been shown to be involved in Fc{gamma}R-mediated phagocytosis. Here we report that PAG3 is enriched together with ARF6 and F-actin at phagocytic cups formed beneath immunoglobulin G–opsonized beads in P388D1 macrophages, in which overexpression of ARF6, but not ARF1 (class I) or ARF5 (class II), inhibits the phagocytosis. Overexpression of PAG3, but not its GAP-inactive mutant, attenuated the focal accumulation of F-actin and blocked phagocytosis, although surface levels of the Fc{gamma}Rs were not affected. Other ubiquitously expressed ARFGAPs, G protein–coupled receptor kinase interactors GIT2 and GIT2-short/KIAA0148, which we have shown to exhibit GAP activity 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 propose that PAG3 acts as a GAP for ARF6 and is hence involved in Fc{gamma}R-mediated phagocytosis in mouse macrophages.

Key Words: actin remodeling • ARF family GTPases • ARFGAP • membrane remodeling • Rho family GTPases

Abbreviations used in this paper: ARF, ADP-ribosylation factor; BFA, brefeldin A; DIC, differential interference contrast; EGFP, enhanced green fluorescent protein; GAP, GTPase-activating protein; HA, hemagglutinin; PAG, paxillin-associated protein with ARFGAP activity.

© 2001 The Rockefeller University Press


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