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J. Biol. Chem., Vol. 281, Issue 23, 16006-16015, June 9, 2006

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A Conformational Switch in Vinculin Drives Formation and Dynamics of a Talin-Vinculin Complex at Focal Adhesions^*

Daniel M. Cohen, Brett Kutscher, Hui Chen, Douglas B. Murphy^¶, and Susan W. Craig¹

From the Departments of Biological Chemistry and ^¶Cell Biology, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205 and the Department of Electrical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin–cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or -actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to K_d 10^–7 is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.

Received for publication, January 25, 2006 , and in revised form, March 9, 2006.

^* This work was supported by a Howard Hughes predoctoral fellowship (to D. M. C.), an American Heart Association postdoctoral fellowship (to H. C.), and National Institute of Health Grant GM41605 (to S. W. C.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Dept. of Biological Chemistry, The Johns Hopkins School of Medicine, 503 WBSB, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3666; Fax: 410-955-5759; E-mail: scraig{at}jhmi.edu.

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