A REQUIREMENT FOR ARF6 IN FC-GAMMA RECEPTOR-MEDIATED PHAGOCYTOSIS IN MACROPHAGES

Phagocytosis requires extension of F-actin-rich pseudopods and is acco mpanied by membrane fusion events. Members of the ARF family of GTPase s are essential for many aspects of membrane trafficking, To test a ro le for this family of proteins in Fc gamma receptor-mediated phagocyto sis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 mu M BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Gels apparatus as indicated by altered immunofluorescent distribution of P-COP and reduced efflux of BODIPY C -5-ceramide, a phospholipid that normally accumulates in the Gels appa ratus. In contrast, BFA had no effect on phagocytosis of IgG-coated er ythrocytes. These results suggested that activation of BFA-sensitive A RFs is not required for phagocytosis, ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) o r binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particl es. These results indicate a requirement for ARF6 in Fc gamma receptor -mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fc gamma receptor activation.