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J. Biol. Chem., Vol. 282, Issue 23, 17280-17288, June 8, 2007

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The Integrin Binding Site 2 (IBS2) in the Talin Rod Domain Is Essential for Linking Integrin Subunits to the Cytoskeleton^*

Michèle Moes¹, Sophie Rodius, Stacey J. Coleman, Susan J. Monkley, Erik Goormaghtigh^¶, Laurent Tremuth, Corinne Kox, Patrick P. G. van der Holst, David R. Critchley, and Nelly Kieffer²

From the Laboratoire de Biologie et Physiologie Intégrée (CNRS/GDRE-ITI), University of Luxembourg, L-1511 Luxembourg, Grand Duchy of Luxembourg, Biochemistry Department, University of Leicester, Leicester LE1 9HN, United Kingdom, and ^¶Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, B-1050 Bruxelles, Belgium

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this helix, which disrupted the -helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to IIb3 integrin in focal adhesions and to inhibit in vitro this association as shown by an IIb3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1^-/- cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.

Received for publication, December 27, 2006 , and in revised form, March 16, 2007.

^* This work was accomplished within a CNRS-sponsored European network "Integrins and Transfer of Information" (GDRE-ITI) and was supported by European Community Project HPRN-CT-2002-00253, the University of Luxembourg, and grants from the "Fonds National de la Recherche," Luxembourg, and the "Fondation Luxembourgeoise contre le Cancer," Luxembourg. 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 Recipient of a doctoral fellowship from the Ministère de la Culture, de l'Enseignement Supérieur et de la Recherche, Luxembourg. Data presented here were obtained as part of a doctoral thesis to be submitted to the "Université Libre de Bruxelles", Belgium.

² To whom correspondence should be addressed: Laboratoire de Biologie et Physiologie Intégrée, (CNRS/GDRE-ITI), Université du Luxembourg, 162A, Ave. de la Faïencerie, L-1511 Luxembourg. Tel.: 352-4666446440; Fax: 352-4666446442; E-mail: nelly.kieffer{at}uni.lu.

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This article has been cited by other articles:

				S. Rodius, O. Chaloin, M. Moes, E. Schaffner-Reckinger, I. Landrieu, G. Lippens, M. Lin, J. Zhang, and N. Kieffer The Talin Rod IBS2 {alpha}-Helix Interacts with the {beta}3 Integrin Cytoplasmic Tail Membrane-proximal Helix by Establishing Charge Complementary Salt Bridges J. Biol. Chem., August 29, 2008; 283(35): 24212 - 24223. [Abstract] [Full Text] [PDF]