The eighth FIII domain of human fibronectin promotes integrin alpha(5)beta(1) binding via stabilization of the ninth FIII domain

Binding of the extracellular matrix molecule fibronectin to the integrin re ceptor alpha (5)beta (1) elicits downstream signaling pathways that modulat e cell function. Fibronectin-alpha (5)beta (1) interaction occurs via the c onserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A s ynergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FI II domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin bind ing, cell adhesion, and protein denaturation. Mutation of the FIII9 synergi stic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for int egrin alpha (5)beta (1) to levels observed for FIII10 alone, but the corres ponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhes ion assays also demonstrated enhanced functional activity of constructs con taining FIII8. Equilibrium chemical denaturation studies indicated that FII I8 confers conformational stability upon FIII9, but only if the exposed loo ps, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These re sults demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism f or integrin alpha (5)beta (1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activati on of downstream signaling occurs.