HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 11, 7458-7467, March 17, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/11/7458    most recent M508058200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Patel, B. Articles by Critchley, D. R. Search for Related Content PubMed PubMed Citation Articles by Patel, B. Articles by Critchley, D. R. Social Bookmarking                      What's this?

The Activity of the Vinculin Binding Sites in Talin Is Influenced by the Stability of the Helical Bundles That Make Up The Talin Rod^*

Bipin Patel, Alexandre R. Gingras, Audrey A. Bobkov, L. Miya Fujimoto, Man Zhang, Robert C. Liddington, Daniela Mazzeo, Jonas Emsley^¶, Gordon C. K. Roberts, Igor L. Barsukov, and David R. Critchley¹

From the Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom, The Burnham Institute, La Jolla, California 92037, and ^¶School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom

The talin rod contains 11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, talin failed to compete for binding of the vinculin Vd1 domain to an immobilized talin polypeptide containing a constitutively active VBS. However, talin did bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a K_d of 9 µM. Interestingly, Vd1 binding exposed a trypsin cleavage site in the talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, talin 482-889 binds Vd1 with high affinity (K_d 0.14 µM), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact talin. In support of this, hemagglutinin-tagged talin 482-889 localized efficiently to focal adhesions, whereas 482-655 did not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold bound Vd1 much less well than wild type. We conclude that the stability of the helical bundles that make up the talin rod is an important factor determining the activity of the individual VBSs.

Received for publication, July 22, 2005 , and in revised form, December 20, 2005.

^* The work at the University of Leicester was supported by grants from Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom and the Wellcome Trust, and that at the Burnham Institute was supported by National Institutes of Health Grant 5 U54 GM64346 (to the Cell Migration Consortium). 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 Fig. 1.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, University of Leicester, University Rd., Leicester, LE1 7RH, UK. Tel.: 116-252-3477; Fax: 116-252-5097; E-mail: drc{at}le.ac.uk.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Himmel, A. Ritter, S. Rothemund, B. V. Pauling, K. Rottner, A. R. Gingras, and W. H. Ziegler Control of High Affinity Interactions in the Talin C Terminus: HOW TALIN DOMAINS COORDINATE PROTEIN DYNAMICS IN CELL ADHESIONS J. Biol. Chem., May 15, 2009; 284(20): 13832 - 13842. [Abstract] [Full Text] [PDF]

				A. R. Gingras, W. H. Ziegler, A. A. Bobkov, M. G. Joyce, D. Fasci, M. Himmel, S. Rothemund, A. Ritter, J. G. Grossmann, B. Patel, et al. Structural Determinants of Integrin Binding to the Talin Rod J. Biol. Chem., March 27, 2009; 284(13): 8866 - 8876. [Abstract] [Full Text] [PDF]

				A. del Rio, R. Perez-Jimenez, R. Liu, P. Roca-Cusachs, J. M. Fernandez, and M. P. Sheetz Stretching Single Talin Rod Molecules Activates Vinculin Binding Science, January 30, 2009; 323(5914): 638 - 641. [Abstract] [Full Text] [PDF]

				J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem Vinculin controls focal adhesion formation by direct interactions with talin and actin J. Cell Biol., December 3, 2007; 179(5): 1043 - 1057. [Abstract] [Full Text] [PDF]

				M. Moes, S. Rodius, S. J. Coleman, S. J. Monkley, E. Goormaghtigh, L. Tremuth, C. Kox, P. P. G. van der Holst, D. R. Critchley, and N. Kieffer The Integrin Binding Site 2 (IBS2) in the Talin Rod Domain Is Essential for Linking Integrin beta Subunits to the Cytoskeleton J. Biol. Chem., June 8, 2007; 282(23): 17280 - 17288. [Abstract] [Full Text] [PDF]

				H. Chen, D. M. Choudhury, and S. W. Craig Coincidence of Actin Filaments and Talin Is Required to Activate Vinculin J. Biol. Chem., December 29, 2006; 281(52): 40389 - 40398. [Abstract] [Full Text] [PDF]

				D. M. Cohen, B. Kutscher, H. Chen, D. B. Murphy, and S. W. Craig A Conformational Switch in Vinculin Drives Formation and Dynamics of a Talin-Vinculin Complex at Focal Adhesions J. Biol. Chem., June 9, 2006; 281(23): 16006 - 16015. [Abstract] [Full Text] [PDF]