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

• Published in: Biochemical journal Vol. 355; no. Pt 3; pp. 851 - 858
• Main Authors: Geiszt, M, Dagher, M C, Molnár, G, Havasi, A, Faure, J, Paclet, M H, Morel, F, Ligeti, E
• Format: Journal Article
• Language: English
• Published: England Portland Press 01.05.2001
• Subjects: Animals; Cattle; Cell Membrane; Cell Membrane - enzymology; Cytosol; Cytosol - enzymology; GTPase-Activating Proteins; GTPase-Activating Proteins - metabolism; Guanine Nucleotide Exchange Factors; Humans; Immunology; In Vitro Techniques; Innate immunity; Leukocytes, Mononuclear; Leukocytes, Mononuclear - metabolism; Life Sciences; NADPH Oxidase; NADPH Oxidases - metabolism; Neutrophils; Neutrophils - cytology; Neutrophils - metabolism; Nuclear Proteins; Nuclear Proteins - metabolism; Oxygen; Oxygen - metabolism; Phosphoproteins; Phosphoproteins - metabolism; Precipitin Tests; Protein Prenylation; ras GTPase-Activating Proteins; ras GTPase-Activating Proteins - metabolism; ras-GRF1; Repressor Proteins
• DOI: 10.1042/bj3550851
• ISSN: 0264-6021

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.