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J. Biol. Chem., Vol. 280, Issue 39, 33610-33619, September 30, 2005

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A New Functional Role of the Fibrinogen RGD Motif as the Molecular Switch That Selectively Triggers Integrin IIb3-dependent RhoA Activation during Cell Spreading^*

From the Laboratoire de Biologie et Physiologie Intégrée (CNRS/GDRE-ITI), Université du Luxembourg, 162A Avenue de la Faïencerie, L-1511 Luxembourg, Grand-Duchy of Luxembourg

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin IIb3 occurs exclusively through the synergistic ^400-411 sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen ^400-411 sequence and the RGD motif in the molecular events leading to ligand-induced IIb3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the ^400-411 sequence (fragment D), and CHO cells expressing resting wild type (IIb3wt), constitutively active (IIb3T562N), or non-functional (IIb3D119Y) receptors. Our data provide evidence that the ^400-411 site by itself is able to initiate IIb3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the 3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the 3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, IIb3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.

Received for publication, January 5, 2005 , and in revised form, May 11, 2005.

^* This work was supported in part by the University of Luxembourg, as well as grants from the Fonds National de la Recherche, Luxembourg, the Fondation "Aide aux Enfants atteints d'un Cancer," Luxembourg and the EC Project HPRN-CT-2002-00253, and accomplished within a CNRS-sponsored European network Groupement de Recherche Européen-Intégrines et Transfert d'Information (GDRE-ITI). Data presented here were obtained by A. S. as part of his doctoral thesis, to be submitted to the University Henry Poincaré, Nancy, France. 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 Both authors contributed equally to this work.

² Recipient of a doctoral fellowship from the Ministère de la Culture, de l'Enseignement Supérieur et de la Recherche, Luxembourg.

³ To whom correspondence should be addressed. Tel.: 352-466-644-440; Fax: 352-466-644-442; E-mail: nelly.kieffer{at}uni.lu.

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