The assembly of cytosolic subunits p47(
phox), p67(
phox), and
p40(phox) with
flavocytochrome b(558) at the membrane is required for activating the neutrophil
NADPH oxidase that generates
superoxide for microbial killing. The p47(
phox) subunit plays a critical role in
oxidase assembly. Recent studies showed that the p47(
phox)
Phox homology (PX) domain mediates
phosphoinositide binding in vitro and regulates
phorbol ester-induced
NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47(
phox) PX domain in neutrophils is unclear, we investigated its role using p47(
phox) knock-out (KO) mouse neutrophils to express human p47(
phox) and derivatives harboring R90A mutations in the PX domain that result in loss of
phosphoinositide binding. Human p47(
phox)
proteins were expressed at levels similar to endogenous murine p47(
phox), with the exception of a
chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47(
phox) rescued p47(
phox) KO mouse neutrophil
NADPH oxidase activity. Plasma membrane NAPDH
oxidase activity was reduced in neutrophils expressing p47(
phox) with Arg(90) substitutions, with substantial effects on responses to either
phorbol ester or formyl-
Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47(
phox) Arg(90) mutants supported normal levels of intracellular
NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47(
phox) PX domain for neutrophil
NADPH oxidase activation.