Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function

M Obara, MS Kang, KM Yamada - Cell, 1988 - cell.com
M Obara, MS Kang, KM Yamada
Cell, 1988cell.com
Polypeptide sequences required for function of the cell-binding domain of human fibronectin
were analyzed by site-directed mutagenesis. Site-specific deletion of the putative
recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive
activity of fibronectin fusion proteins expressed in E. coli by 297%. A second functional site
over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a
296% loss of activity, indicating cooperativity between sites. The two classes of mutant …
Summary
Polypeptide sequences required for function of the cell-binding domain of human fibronectin were analyzed by site-directed mutagenesis. Site-specific deletion of the putative recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive activity of fibronectin fusion proteins expressed in E. coli by 297%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a 296% loss of activity, indicating cooperativity between sites. The two classes of mutant protein displayed synergism of activity in a trans complementation assay. Effective actin microfilament bundle organization was also dependent on the combined function of both sites. Thus, fibroblast adhesion and intracellular response to the fibronectin cell-binding domain involve two synergistic sites, each of major quantitative importance.
cell.com