The guanine nucleotide exchange factor trio activates the phagocyte NADPH oxidase in the absence of GDP to GTP exchange on Rac. "The emperor's nw clothes".
The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b(559) and four cytosolic components as follows: p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b(559) and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67(phox) but in the absence of amphiphile and p47(phox). We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67(phox)-Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg(2+) from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.
Sigal
Natalia
N
Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Gorzalczany
Yara
Y
Sarfstein
Rive
R
Weinbaum
Carolyn
C
Zheng
Yi
Y
Pick
Edgar
E
D000818:Animals; D052158:Aptamers, Peptide; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism* / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
D052158:Aptamers, Peptide; D020662:Guanine Nucleotide Exchange Factors; D029701:Saccharomyces cerevisiae Proteins; C465200:TRIPalpha protein, S cerevisiae; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein