A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase

In activated neutrophils NADPH oxidase is regulated through various signali ng intermediates, including heterotrimeric G proteins, kinases, GTPases, an d phospholipases, ADP-ribosylation factor (ARF) describes a family of GTPas es associated with phospholipase D (PLD) activation. PLD is implicated in N ADPH oxidase activation, although it is unclear whether activation of PLD b y ARF is linked to receptor-mediated oxidase activation. We explored whethe r ARF participates in NADPH oxidase activation by formyl-methionine-leucine -phenylalanine (fMLP) and whether this involves PLD. Using multicolor forwa rd angle light scattering analyses to measure superoxide production in diff erentiated neutrophil-like PLB-985 cells, we tested enhanced green fluoresc ent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts . The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased supero xide production, whereas the ARF6(T27N) mutant defective in GTP binding cau sed diminished responses to fMLP. The ARF1 mutants had no effect on fMLP re sponses, and none of the ARF proteins affected phorbol 12-myristate 13-acet ate-elicited oxidase activity. PLD inhibitors 1-butanol and 2,3-diphosphogl ycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation , blocked fMLP-elicited oxidase activity in transfected cells. The data sug gest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activ ation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase acti vation also appears to be PLD-dependent, but ARF-independent, ARF6 and prot ein kinase C may act through distinct pathways, both involving PLD.