The alpha(5)beta(1) integrin is a cell surface receptor for fibronectin implicated in several cellular activities including cell proliferation, differentiation, and migration. The primary site at which the alpha(5)beta(1) integrin interacts with fibronectin is the RGD (Arg-Gly-Asp) amino acid sequence. In general, the sites on the integrin alpha subunits involved in ligand binding are not well characterized. Eased on previous cross-linking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (positions 223-567) on the alpha(5) subunit as a putative binding region and divided it into four subdomains named domains I, II, III, and IV. Chimeric receptors were prepared in which sequences on the alpha(5) subunit were exchanged with the corresponding sequences on the alpha(6) subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human alpha(5) integrin gene was transfected into CHO B2 cells, which are deficient in alpha(5) expression. Only chimeras of domain III or IV express on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal adhesion assembly, and migration on fibronectin, The adhesion of the chimeric receptors to fibronectin remained sensitive to the RGD peptide, and antibodies that inhibit interaction with the fibronectin synergy site and RGD loop remain inhibitory for the chimeras, indicating that our chimeras do not inhibit binding to either the RGD or synergy sites. Finally, the affinity of soluble fibronectin to cells via the alpha(5)beta(1) receptor decreased only about S-fold, This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly, The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type alpha(5) receptor adhere to fibronectin mere inhibited in cells expressing the chimeric receptors, Therefore, our results suggest that the chimeras of these domains likely interrupt alpha(5)-mediated conformational signaling.
