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

Abstract

We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [γ-32P]GTP-bound Rac revealed dominant GAP activity related to a 50kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. 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 and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase.

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

“…Antibodies against ARHGAP1 and ARHGAP25 were raised in rabbits and characterized as described in ref. [40,44]. Isotype control polyclonal rabbit-Ab [γ-32 P]GTP was from Izotóp Intézet, Hungary.…”

“…Immunoblots revealed the presence of all identified RacGAPs both in the cytosolic and in the membrane fractions [40,44]. To mimic the potential effect of cytosolic RacGAPs, we first added these proteins to the semirecombinant cell-free O2 •--generation system.…”

“…To mimic the potential effect of cytosolic RacGAPs, we first added these proteins to the semirecombinant cell-free O2 •--generation system. In previous studies it has been shown that addition of Bcr, ARHGAP1 or ARHGAP35 decreased O2 •--production in crude cell-free systems [39,40]. However, these studies were carried out under different experimental conditions so that a direct comparison of individual RacGAP effects was not possible.…”

“…Due to their complex domain structure [32,34] and extensive regulation [30,35], RacGAPs could fine tune Nox activity. In fact, a role for RacGAPs in the regulation of assembly and function of the Nox2 complex has been indicated in previous in vitro studies [36][37][38][39][40] and in genetically modified animals [41][42][43]. These were all isolated studies and comparative aspects have not been raised.…”

“…In human neutrophils the RacGAPs Bcr, ARHGAP35 and ARHGAP1 have been identified by immunoblotting [40] and all three proteins decreased O2 •--production when they were added to a cell-free system [39,40]. However, the contribution of endogenous, membrane-bound RacGAPs has not been investigated.…”