Two-stage activation for α5β1 integrin binding to surface-adsorbed fibronectin

Abstract

By analyzing the functional binding of α5β1 integrin to adsorbed fibronectin in intact cells, we demonstrate that integrin activation results in linear increases in adhesion strength as a function of ligand density, suggesting that modulation of the receptor-ligand interaction is the dominant mechanism for adhesion during the initial stages of adhesion and that cooperative binding contributes little to initial adhesion strength. Using this experimental framework, we show the existence of three distinct activation states for α5β1 integrin binding to adsorbed fibronectin for both passive, antibody-induced and active, cell-controlled activation. During the initial phase of adhesion, α5β1 integrin is activated in an energy- dependent process from the nonbinding ground state to an intermediate state in which the receptor binds fibronectin and provides significant mechanical coupling. In later stages of adhesion maturation, α5β1 integrin is activated to a higher binding state, which provides significant increases in adhesion strength compared with the intermediate state. These multiple binding states most likely result from different integrin conformations and reflect distinct interactions between α5β1 and sites on adsorbed fibronectin. Multiple activation states for α5β1 suggest the existence of distinct stages in adhesion signaling and strengthening and can provide a versatile mechanism for the regulation of adhesive interactions.