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J Biol Chem. 2010 Oct 1;285(40):30698-707. doi: 10.1074/jbc.M110.107458. Epub 2010 Jul 2.

The guanine nucleotide exchange protein for ADP-ribosylation factor 6, ARF-GEP100/BRAG2, regulates phagocytosis of monocytic phagocytes in an ARF6-dependent process.

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Department of Host Defense and Biochemical Research, Juntendo University, School of Medicine, Tokyo 113-8421, Japan.


Phagocytosis is a complex multistep process requiring diverse signaling and regulatory molecules. ADP-ribosylation factor 6 (ARF6), a small GTPase, is known to regulate membrane trafficking and the actin cytoskeketon at the plasma membrane and functions as a regulatory molecule of phagocytosis. ARF activity is regulated by cycling between GDP-bound and GTP-bound forms. ARF activation is catalyzed by guanine nucleotide exchange factors (GEFs) that facilitate GTP binding. We had earlier reported a 100-kDa ARF-GEF, termed ARF-guanine nucleotide exchange protein 100, GEP100, that preferentially activates ARF6 and was also described by Dunphy et al. (Dunphy, J. L., Moravec, R., Ly, K., Lasell, T. K., Melancon, P., and Casanova, J. E. (2006) Curr. Biol. 16, 315-320) as brefeldin A-resistant ARF-GEF2 (BRAG2). We have now examined a role for GEP100 in phagocytosis. Stable depletion of GEP100 decreased phagocytosis of serum-treated zymosan and IgG-coated latex beads by human monocyte-macrophage-like U937 cells differentiated with phorbol 12-myristate 13-acetate. Decrease of phagocytic activity by RNAi was not rescued by GEP100ΔSec7, a deletion mutant lacking the ARF-activating domain. GEP100-depleted cells also exhibited reduced F-actin fibers around internalized particles. Attachment of these particles to cells and amounts of C3bi and Fcγ receptors, however, were not affected by GEP100 depletion. On immunofluorescence microscopy, GEP100 and ARF6 were concentrated and partially colocalized around internalized particles. Phagocytosis by GEP100-depleted cells was not further affected by depletion of ARF6. Phagocytic activity of GEP100-depleted cells was, however, rescued by expression of the constitutively active ARF6Q67N mutant but not by the dominant-negative ARF6T27N mutant. These data are consistent with the conclusion that GEP100 functions in phagocytosis via its role in ARF6-dependent actin remodeling.

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