Characterization of regions of fibronectin besides the arginine-glycine-aspartic acid sequence required for adhesive function of the cell-binding domain using site-directed mutagenesis.
Previous studies of adhesion mediated by the central cell-binding domain of fibronectin suggest that additional polypeptide information besides the Arg-Gly-Asp sequence is required for full activity. We analyzed this putative second, synergistic region of fibronectin more extensively by deletion analysis and oligonucleotide-based site-directed mutagenesis. Resulting mutated fusion proteins expressed using lambda gt11 were assayed for baby hamster kidney fibroblast cell spreading activity. Deletion mutants truncating from the amino terminus showed a decrease of activity in two apparently discrete steps. Complementary studies using a series of overlapped internal deletions designed to retain the repetitive fibronectin structure also indicated that two distinct peptide regions besides the RGD sequence were necessary for full activity. Removal of the carboxyl-terminal region resulted in the greatest loss of activity (greater than or equal to 20- versus 3-5-fold). Very similar results were obtained with HT-1080 cells dependent on the alpha 5 beta 1 integrin receptor for adhesion to fibronectin. An anti-fibronectin monoclonal antibody that inhibits cell adhesion was found to bind to the carboxyl-terminal functional region, and a point mutation caused specific loss of its epitope. These studies reveal unexpected complexity in the organization of these functional regions, which contrasts with adhesion models based only on simple, short peptide recognition sequences.
Aota
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Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Nagai
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Yamada
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KM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001120:Arginine / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics*; D001665:Binding Sites; D002448:Cell Adhesion; D006224:Cricetinae; D005353:Fibronectins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005998:Glycine / Q000235:genetics*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D001224:Aspartic Acid; D001120:Arginine; D005998:Glycine