Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function.
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.
Obara
M
M
Membrane Biochemistry Section, National Cancer Institute, Bethesda, Maryland 20892.
Kang
M S
MS
Yamada
K M
KM
D000199:Actins / Q000502:physiology; D000595:Amino Acid Sequence; D002448:Cell Adhesion; D002478:Cells, Cultured; D002872:Chromosome Deletion; D005347:Fibroblasts; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000235:genetics / Q000502:physiology*; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology
D000199:Actins; D005353:Fibronectins; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C046617:arginyl-glycyl-aspartyl-serine