Characterization of membrane-localized and cytosolic Rac-GTPase-activatingproteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase

We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunobl ot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosal. An overlay assay performed with [gamma-P-32]GTP-bound Rac revealed dominant GAP activity related to a 50 kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunop recipitation with specific antibody directed against the GAP domain of p50R hoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized anti soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent sui table targets for both the up-regulation and down-regulation of the NADPH o xidase.