α5β1 integrin can occupy several distinct conformational states which support different strengths of binding to fibronectin [García, A. J., et al. (1998) J. Biol. Chem. 273, 34710-34715]. Using a model system in which specific activating monoclonal antibodies were used to achieve uniform activated states, the binding of α5β1 to full-length wild-type fibronectin and mutants of fibronectin in the defined RGD and PHSRN synergy sites was analyzed using a novel method that measures the strength of the coupling between integrin and its ligand. Neither TS2/16- nor AG89-activated α5β1 showed significant mechanical coupling to RGD-deleted fibronectin. However, peptide competition assays demonstrated a 6-fold difference in the binding affinities of these two states for RGD. The mutant synergy site reduced the AG89 (low)-activated state to background levels, but the TS2/16-activated state still retained approximately 30% of the wild-type activity. Thus, these two active binding states of α5β1 interact differently with both the RGD and synergy domains. The failure of the AG89-activated state to show mechanical coupling to either the RGD or synergy domain mutants was unexpected and implies that the RGD domain itself does not contribute significant mechanical strength to the α5β1-fibronectin interaction. The lack of RGD alone to support α5β1 coupling was further confirmed using a synthetic polymer presenting multiple copies of the RGD loop. These results suggest a model in which the RGD domain serves to activate and align the α5β1-fibronectin interface, and the synergy site provides the mechanical strength to the bond.
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