Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes.
NADPH oxidase is the enzyme complex responsible for the production of oxygen radicals in phagocytes. On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox and p47phox, as well as the Ras-related G-protein rac 2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b to form a functional complex. Besides rac 2, rac 1 G-protein is also involved in the activation of the NADPH oxidase, but, to date, it has not been documented whether it is also translocated in activated neutrophils. In this paper we show that: (a) in neutrophils stimulated with formylmethionyl-leucylphenylalanine, concanavalin A or phorbol 12-myristate 13-acetate, both rac 1 and rac 2 are translocated from cytosol to the membranes; (b) in neutrophils from a patient with a form of chronic granulomatous disease in which p67phox is absent, rac 2 and p47phox were translocated as in normal neutrophils on stimulation with the above agonists, but rac 1 failed to be translocated from the cytosol to the membranes. This is the first demonstration that, in activated neutrophils, rac 1 is translocated from the cytosol to the membranes and this translocation requires p67phox. These results, coupled with those showing that rac 2 is not translocated in activated neutrophils lacking p47phox [El Benna, Ruedi and Babior (1994) J. Biol. Chem. 269, 6729-6734], may suggest that the assembly of the cytosolic components of NADPH oxidase on the plasma membrane takes place through selective coupling of activated rac 1 and rac 2 with p67phox and p47phox respectively.
Dusi
S
S
Institute of General Pathology, University of Verona, Italy.
Donini
M
M
Rossi
F
F
D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006105:Granulomatous Disease, Chronic / Q000378:metabolism; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010750:Phosphoproteins / Q000172:deficiency / Q000378:metabolism / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020744:rac GTP-Binding Proteins
D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D003208:Concanavalin A; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate