Role for p40^phox in Fcγ-Receptor-Induced NADPH Oxidase Activation.

The phagocyte NADPH oxidase catalyzes the production of superoxide, the precursor to microbicidal oxidants that are essential for normal host defense. The active enzyme complex includes a membrane-bound flavocytochrome b along with p47^−phox, p67^phox, and Rac-GTP, which translocate from the cytosol to the membrane to activate electron transport through the flavocytochrome upon cellular stimulation with soluble or particulate agonists. Translocation of p67^phox is mediated by p47^phox, which can be isolated from the cytoplasm prior to oxidase activation as a complex along with a third protein, p40^phox. Although p40^phox also translocates to the membrane of activated cells, its role in regulation of the NADPH oxidase has been unclear. p40^phox is not required for reconstitution of high-level NADPH oxidase activity in cell-free systems or in heterologous whole cell models based in COS7 or K562 cell lines. It has recently been recognized that p40^phox binds phosphatidylinositol-3-phosphate (PI(3)P), a product of type III phosphatidylinositol 3-kinase, via an N-terminal PX domain, and that PI(3)P is generated in high levels in the phagosomal membrane, where it recruits proteins important for phagosomal maturation. To examine whether p40^phox might play a role in NADPH oxidase activation during phagocytosis, we introduced a cDNA for the human FcγIIA receptor via retroviral-mediated gene transfer into COS7 cells expressing the phagocyte flavocytochrome b, p47^phox, and p67^phox from stable transgenes. As previously shown by a number of groups for parental COS7 cells, heterologous expression of FcγIIA enabled “COS^phox” cells to ingest IgG-coated sheep red blood cells (IgG-SRBC). However, NADPH oxidase activity was not detected, using nitroblue tetrazolium as a probe to detect superoxide production, which is otherwise evident by the accumulation of intraphagosomal formazan deposits in macrophages and neutrophils undergoing phagocytosis. However, numerous NBT-positive (formazan-stained) phagosomes were seen in COS^phoxFcγR cells upon transient or stable transfection of an expression plasmid for p40^phox. NBT-positive phagosomes were detected within five minutes in a synchronized phagocytosis assay. Upon transient expression of derivatives of p40^phox which had point missense mutations in the PX domain predicted to prevent binding of PI(3)P, only rare NBT-positive phagosomes were detected in COS^phoxFcγR cells ingesting IgG-SRBC. Transient expression of p40^phox derivatives with point mutations in the SH3 domain, which disrupts a potential binding site for p47^phox, or in the PC motif, which should disrupt binding to p67^phox, resulted in a ~2-fold reduction in the numbers of NBT-positive phagosomes. Introduction of both the SH3 and PC mutations into p40^phox resulted in only rare NBT-positive phagosomes following transient transfection of the double mutant and subsequent phagocytosis of IgG-SRBC. Thus, this study identifies a role for p40^phox in coupling NADPH oxidase activation to phagocytosis of IgG-coated particles. Results of experiments using mutant derivatives of p40^phox suggest that its ability to interact with both PI(3)P and p47^phox/p67^phox are critical determinants in this process.