Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

Fibronectin(Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins alpha IIb beta 3 and alpha 5 beta 1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin alpha 3 beta 1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu -> Pro) stabilizing mutant (FnIII9'10), (ii) an Arg to Ala synergy site mutation (FnIII9(R)->(A)10), (iii) a two-Gly (FnIII9(2G)10) insertion, and (iv) a four-Gly (FNIII9(4G)10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin alpha 3 beta 1 to the Fn fragments. Integrin alpha 3 beta 1 had the highest affinity for FnIII9'10 and FnIII9(2G)10. Mutation within the synergy site decreased integrin alpha 3 beta 1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9'10. Cell attachment studies demonstrate that alpha 3 beta 1-mediated epithelial cell binding is greater on FnIII9'10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin alpha 3 beta 1 binding to Fn.