Identification of the peptide sequences within the EIIIA (EDA) segment of fibronectin that mediate integrin α9β1-dependent cellular activities

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, α9β1 and α4β1, 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) 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). We have now performed site-directed mutagenesis within the EIIIA segment and carried out cell adhesion assays on the semutant EIIIAs. We find that the Asp⁴¹ and Gly⁴² residues within the C-C′ loop of EIIIA are necessary for integrin α9β1 binding. Synthetic peptides based on the predicted important amino acid sequence from the C-C′ loop encode sufficient information to completely inhibit α9β1-mediated cell adhesion. We also report that EIIIA promotes filopodial formation in α9β1-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.