Activation of the integrin αvβ3 involves a discrete cation-binding site that regulates conformation

Abstract

"Activation" of integrins is involved in the dramatic transition of leukocytes and platelets from suspension to adhesion. The integrin αvβ3 is not known to take part in this sort of transition, even though it shares its β subunit with αIIbβ3, the activable integrin on platelets. In the context of a constitutively adhered cell, changes in activation state may be more subtle in their effects, but nonetheless important in regulating cell behavior. We hypothesized that αvβ3 can undergo conformational changes analogous to those associated with αIIbβ3 activation. Accordingly, we examined αvβ3 on the surface of M21 cells (a human melanoma cell line) and found that, like αIIbβ3, it can undergo conformational changes upon binding of a ligand analog and can be activated for ligand binding and migration by a monoclonal antibody directed against β3. Modulation of the binding of this activating antibody, AP5, ligand binding, and antibody-mediated activation all are associated with a discrete cation-binding site shared in both αIIbβ3 and αvβ3. Based on a measured Ki, this site has an apparent Kd for calcium of approximately 20 μM. At physiological levels of calcium, about 40% of the total αvβ3 on a cell's surface is in a conformation detected by AP5. The data suggest a model for both αvβ3 and αIIbβ3 function in which the molecule can exist in either of (at least) two conformational states, one stabilized either by AP5 or ligand binding, refractory to calcium binding, and enhanced for ligand recognition, the other stabilized by calcium binding and refractory to AP5 and ligand binding. Functional analysis suggests that AP5 activates αvβ3 by preventing occupation of this calcium site, and that the activated form of αvβ3 differs functionally from the basal form. The active form is more conducive to migration and the basal to tight adhesion.