Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function |
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Authors: | M Obara M S Kang K M Yamada |
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Institution: | Membrane Biochemistry Section, National Cancer Institute, Bethesda, Maryland 20892. |
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Abstract: | 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 greater than or equal to 97%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a greater than or equal to 96% 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. |
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