C-terminal heparin-binding domain of fibronectin regulates integrin-mediated cell spreading but not the activation of mitogen-activated protein kinase

Fibronectin (FN) stimulates multiple signalling events including mitogen-ac tivated protein kinase (MAPK) activation. During cell spreading, both the c ell-binding domain and the C-terminal heparin-binding domain (HepII) of FN co-operatively regulate cytoskeleton organization. However, in comparison w ith the large number of studies on the functions of cell-binding domain, th ere is little information about the role of HepII. We therefore investigate d the effect of HepII on integrin-mediated cell spreading and adhesion on F N and MAPK activation. In contrast with cells on FN substrates, rat embryo fibroblasts on FN120, which lacks HepII, were less spread, had weaker adhes ion to FN and failed to form focal adhesions and actin stress fibres. Phosp hotyrosine was present in the focal contacts of rat embryo fibroblasts on F N within 30 min but was absent from cells on FN120. Overall, tyrosine phosp horylation was much less in cell lysates from cells on FN120, with decrease d phosphorylation of focal adhesion kinase ('pp125FAK') on tyrosine-397, im plying additional regulation of tyrosine phosphorylation by HepII. Neverthe less, adhesion-mediated MAPK activity was similar in cells on FN and on FN1 20. Furthermore, cells spread on FN and on FN120 substrates showed similar MAPK activation in response to treatment with epidermal growth factor and w ith platelet-derived growth factor. Consistently, overexpression of syndeca n-4, which binds to HepII, enhanced cell spreading and adhesion on FN but d id not affect integrin-mediated MAPK activation. We therefore conclude that both HepII and syndecan-4 regulate integrin-mediated cell spreading but no t MAPK activation.