Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins 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. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [gamma-(32)P]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 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.
Geiszt
M
M
Department of Physiology, Semmelweis University, P.O. Box 259, H-1444 Budapest, Hungary.
Dagher
M C
MC
Molnár
G
G
Havasi
A
A
Faure
J
J
Paclet
M H
MH
Morel
F
F
Ligeti
E
E
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D020690:GTPase-Activating Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D017368:Protein Prenylation; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*; D020666:ras-GRF1
C074560:ARHGAP35 protein, human; C513043:ARHGAP5 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; C513039:RASGRF1 protein, human; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; C085379:rho GTPase-activating protein; D019255:NADPH Oxidases; D010100:Oxygen