Activation of alpha(v)beta 3-vitronectin binding is a multistage process in which increases in bond strength are dependent on Y747 and Y759 in the cytoplasmic domain of beta 3

Integrin receptors serve as mechanical links between the cell and its struc tural environment. Using alpha (v)beta3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between alp ha (v)beta3 and vitronectin develops was analyzed by measuring the resistan ce of these bonds to mechanical separation. Three distinct stages of activa tion, as defined by increases in the alpha (v)beta3-vitronectin binding str ength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the beta3 subunit. Stage 2 is characterized by a 4-fold increase in binding st rength and is dependent on stage 1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction w ith cellular proteins. The Y747F mutant blocked the transition from stage I to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of alpha (v)beta3 i ntegrin.