STRUCTURAL REQUIREMENTS FOR BIOLOGICAL-ACTIVITY OF THE 9TH AND 10TH FIII DOMAINS OF HUMAN FIBRONECTIN

The ninth and tenth type III domains of fibronectin each contain speci fic cell binding sequences, RGD in FIII10 and PHSRN in FIII9, that act synergistically in mediating cell adhesion, We investigated the relat ionship between domain-domain orientation and synergistic adhesive act ivity of the FIII9 and FIII10 pair of domains. The interdomain interac tion of the FIII9-10 pair was perturbed by introduction of short flexi ble linkers between the FIII9 and FIII10 domains. Incremental extensio ns of the interdomain link between FIII9 and FIII10 reduced the initia l cell attachment, but had a much more pronounced effect on the downst ream cell adhesion events of spreading and phosphorylation of focal ad hesion kinase. The extent of disruption of cell adhesion depended upon the length of the interdomain linker, Nuclear magnetic resonance spec troscopy of the wild type and mutant FIII9-10 proteins demonstrated th at the structure of the RGD-containing loop is unaffected by domain-do main interactions, We conclude that integrin-mediated cell adhesion to the central cell binding domain of fibronectin depends not only upon specific interaction sites, but also on the relative orientation of th ese sites. These data have implications for the molecular mechanisms b y which integrin-ligand interactions are achieved.