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J. Biol. Chem., Vol. 280, Issue 6, 4674-4683, February 11, 2005

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Uncoupling of Inhibitory and Shuttling Functions of Rho GDP Dissociation Inhibitors^*

Estelle Dransart, Annie Morin, Jacqueline Cherfils, and Birgitta Olofsson

From the Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR 9063, F-91198 Gif-sur-Yvette, France

Rho GDP dissociation inhibitors (rhoGDIs) are postulated to regulate the activity of small G proteins of the Rho family by a shuttling process involving the extraction of Rho from donor membranes, the formation of the inhibitory cytosolic Rho/rhoGDI complexes, and delivery of Rho to target membranes. However, the role of rhoGDIs in site-specific membrane targeting or extraction of Rho is still poorly understood. Here we investigated the molecular functions of two rhoGDIs, the specific rhoGDI-3 and the less specific but well studied rhoGDI-1, in HeLa cells using structure-based mutagenesis of the rhoGDI protein. We identified two sites in rhoGDI, which form conserved interactions with their Rho target, whose mutation results in the uncoupling of inhibitory and shuttling functions of rhoGDIs: D66^GDI-3 (equivalent to D45^GDI-1), a conserved residue in the helix-loop-helix^GDI/switch 1^Rho interface, and D206^GDI-3 (equivalent to D185^GDI-1) in the -sandwich^GDI/switch 2^Rho interface. Mutations of both sites result in the loss of rhoGDI-3 or rhoGDI-1 inhibitory activity but not of their ability to form cytosolic complexes with RhoG or Cdc42 in vivo. Remarkably, the mutants were detected at Rho-induced membrane ruffles or protrusions where they co-localized with RhoG or Cdc42, likely identifying for the first time the site of extraction of a Rho protein by a rhoGDI in vivo. We propose that these mutations act by modifying the steady-state kinetics of the shuttling process regulated by rhoGDIs, such that transient steps at the cell membranes now become detectable. They should provide valuable tools for future investigations of the dynamics of membrane extraction or delivery of Rho proteins and their regulation by cellular partners.

Received for publication, August 24, 2004 , and in revised form, October 22, 2004.

^* This work was supported by the CNRS and by grants from the "Association pour la Recherche sur le Cancer." 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.

A fellow of the French Ministry of Education and member of the Doctoral School "Therapeutic Innovation," University of Paris-XI.

To whom correspondence should be addressed: Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), CNRS UPR 9063, Batiment 34, Ave. de la Terrasse, F-91198 Gif-sur-Yvette, France. Tel.: 33-1-69-82-35-07; E-mail: olofsson{at}lebs.cnrs-gif.fr.

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