Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils.

Precise spatiotemporal regulation of O2(-)-generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1-3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes. In structural studies we revealed that simultaneous interaction of Rac with its effector protein p67(phox) and regulatory protein RacGAP was sterically possible. The effect of RacGAPs was experimentally investigated in a cell-free O2(-)-generating system consisting of isolated membranes and recombinant p47(phox) and p67(phox) proteins. Addition of soluble RacGAPs decreased O2(-) production and there was no difference in the effect of four RacGAPs previously identified in neutrophils. Depletion of membrane-associated RacGAPs had a selective effect: a decrease in ARHGAP1 or ARHGAP25 level increased O2(-) production but a depletion of ARHGAP35 had no effect. Only membrane-localized RacGAPs seem to be able to interact with Rac when it is assembled in the Nox2 complex. Thus, in neutrophils multiple RacGAPs are involved in the control of O2(-) production by Nox2, allowing selective regulation via different signaling pathways.