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J. Biol. Chem. 278 (7): 4854-4861

© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac
"THE EMPEROR'S NEW CLOTHES"*

Natalia SigalDagger §, Yara GorzalczanyDagger §, Rive SarfsteinDagger §, Carolyn Weinbaum, Yi Zheng||, and Edgar PickDagger **

From the Dagger  Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel, the || Division of Experimental Hematology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, and the  Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b559 and four cytosolic components as follows: p47phox, p67phox, p40phox, and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b559 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 p67phox but in the absence of amphiphile and p47phox. 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 [p67phox-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 Mg2+ 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.

* This work was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grant 128/01 (to E. P.), and National Institutes of Health Grants GM60523 (to Y. Z.) and GM46372 (to C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

** To whom correspondence and should be addressed: Dept. of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-3-640-7872; Fax: 972-3-642-9119; E-mail: epick@post.tau.ac.il.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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