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J. Biol. Chem., Vol. 281, Issue 28, 19204-19219, July 14, 2006

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Liposomes Comprising Anionic but Not Neutral Phospholipids Cause Dissociation of Rac(1 or 2)·RhoGDI Complexes and Support Amphiphile-independent NADPH Oxidase Activation by Such Complexes^*

Yelena Ugolev, Shahar Molshanski-Mor, Carolyn Weinbaum, and Edgar Pick¹

From the 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 and the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b₅₅₉ with the cytosolic components p47^phox, p67^phox, p40^phox, and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2)·RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP)·RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP)·RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2)·RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP)·RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP)·RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67^phox, without the need for an anionic amphiphile, as activator. Both Rac1(GDP)·RhoGDI and Rac1(GMPPNP)·RhoGDI complexes elicited amphiphile-independent, p67^phox-dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.

Received for publication, January 3, 2006 , and in revised form, May 12, 2006.

^* 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 19/05, the Roberts-Guthman Chair in Immunopharmacology (to E. P.), and National Institutes of Health Grant GM46372 (to C. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence 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{at}post.tau.ac.il.

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