Role for p40phox 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, p67phox, 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 p67phox is mediated by p47phox, which can be isolated from the cytoplasm prior to oxidase activation as a complex along with a third protein, p40phox. Although p40phox also translocates to the membrane of activated cells, its role in regulation of the NADPH oxidase has been unclear. p40phox 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 p40phox 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 p40phox 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, p47phox, and p67phox from stable transgenes. As previously shown by a number of groups for parental COS7 cells, heterologous expression of FcγIIA enabled “COSphox” 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 COSphoxFcγR cells upon transient or stable transfection of an expression plasmid for p40phox. NBT-positive phagosomes were detected within five minutes in a synchronized phagocytosis assay. Upon transient expression of derivatives of p40phox 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 COSphoxFcγR cells ingesting IgG-SRBC. Transient expression of p40phox derivatives with point mutations in the SH3 domain, which disrupts a potential binding site for p47phox, or in the PC motif, which should disrupt binding to p67phox, resulted in a ~2-fold reduction in the numbers of NBT-positive phagosomes. Introduction of both the SH3 and PC mutations into p40phox 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 p40phox in coupling NADPH oxidase activation to phagocytosis of IgG-coated particles. Results of experiments using mutant derivatives of p40phox suggest that its ability to interact with both PI(3)P and p47phox/p67phox are critical determinants in this process.