Two-stage activation for alpha(5)beta(1) integrin binding to surface-adsorbed fibronectin

By analyzing the functional binding of alpha(5)beta(1) integrin to adsorbed fibronectin in intact cells, we demonstrate that integrin activation resul ts in linear increases in adhesion strength as a function of ligand density , suggesting that modulation of the receptor-ligand interaction is the domi nant 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 activa tion states for alpha(5)beta(1) integrin binding to adsorbed fibronectin fo r both passive, antibody-induced and active, cell-controlled activation. Du ring the initial phase of adhesion, alpha(5)beta(1) integrin is activated i n an energy-dependent process from the nonbinding ground state to an interm ediate state in which the receptor binds fibronectin and provides significa nt mechanical coupling. In later stages of adhesion maturation, alpha(5)bet a(1) integrin is activated to a higher binding state, which provides signif icant increases in adhesion strength compared with the intermediate state. These multiple binding states most likely result from different integrin co nformations and reflect distinct interactions between alpha(5)beta(1) and s ites on adsorbed fibronectin, Multiple activation states for alpha(5)beta(1 ) suggest the existence of distinct stages in adhesion signaling and streng thening and can provide a versatile mechanism for the regulation of adhesiv e interactions.