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J. Biol. Chem., Vol. 283, Issue 5, 2858-2870, February 1, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/5/2858    most recent M708306200v1 Alert me when this article is cited 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 Google Scholar Articles by Shinde, A. V. Articles by Van De Water, L. PubMed PubMed Citation Articles by Shinde, A. V. Articles by Van De Water, L.

Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin 9β1-dependent Cellular Activities^*

Arti V. Shinde, Christopher Bystroff, Chunyu Wang^¶1, Mariette G. Vogelezang^||, Peter A. Vincent^**, Richard O. Hynes^||, and Livingston Van De Water²

From the Center for Cell Biology and Cancer Research and ^**Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, the Department of Biology and ^¶Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, and the ^||Howard Hughes Medical Institute, Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Alternative splicing of the fibronectin (FN) gene transcript provides an efficient mechanism for generating functionally appropriate forms of this adhesive glycoprotein in situ. Cellular FNs that include the EIIIA and/or EIIIB FN-III segments are prominently expressed during embryogenesis, wound healing, tumor progression, and inflammation. However, the roles of this domain in altering overall FN protein structure and regulating cellular function remain unclear. We previously reported that two integrins, 9β1 and 4β1, ligate the EIIIA segment ( Liao, Y. F., Gotwals, P. J., Koteliansky, V. E., Sheppard, D., and Van De Water, L. (2002) J. Biol. Chem. 277, 14467-14474[Abstract/Free Full Text] ) and that the epitopes for function-blocking monoclonal antibodies lie within the C-C' loop of EIIIA ( Liao, Y. F., Wieder, K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876-17884[Abstract/Free Full Text] ). We have now performed site-directed mutagenesis within the EIIIA segment and carried out cell adhesion assays on these mutant EIIIAs. We find that the Asp⁴¹ and Gly⁴² residues within the C-C' loop of EIIIA are necessary for integrin 9β1 binding. Synthetic peptides based on the predicted important amino acid sequence from the C-C' loop encode sufficient information to completely inhibit 9β1-mediated cell adhesion. We also report that EIIIA promotes filopodial formation in 9β1-expressing cells accompanied by Cdc42 activation. Our data provide a cellular activity for the EIIIA segment, evidence for conformational lability, and peptide sequences for probing EIIIA functions in vitro and in vivo.

Received for publication, October 5, 2007

This paper is dedicated to the memory of Dr. Charles Lowry, a gifted teacher and generous colleague.

^* This work was supported in part by National Institutes of Health Grants GM-56442 (to L. V. D. W.) and P01 HL066105 (to R. O. H.) and American Heart Predoctoral Fellowship Award 0415545T (to A. S.). 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. 1 and 2.

¹ Supported by the Alzheimer's Association and the James D. Watson Young Investigator Program of NYSTAR.

² To whom correspondence should be addressed: Center for Cell Biology and Cancer Research, MC-165, Albany Medical College, Albany, NY 12208. Tel.: 518-262-9945; Fax: 518-262-9189; E-mail: vandewl{at}mail.amc.edu.