An allosteric Ca2+ binding site on the beta 3 integrins that regulates the dissociation rate for RGD ligands

Here we use a model RGD-containing ligand to study how Ca2+ and Mg2+ regulate ligand binding to beta 3-integrins, Fab-9, an antibody that contains an optimized RGD loop in its antigen binding site (Barbas, C. F., Languino, L., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10003-10007), was used as the model ligand. Across a physiologic range of Mg2+, Fab-9 bound to both alpha v beta 3 and alpha IIb beta 3 with a monophasic binding isotherm. Across the same range of Ca2+, the binding of Fab-9 to the beta 3-integrins was biphasic. Low concentrations of Ca2+ (mu M) promoted the binding of Fab-9. Higher concentrations of Ca2+ (mM) blocked Fab-9 binding. These data suggest that Ca2+ binds to two distinct classes of sites on the beta 3-integrins, with the low affinity Ca2+ binding site(s) being an inhibitory site. We designate this inhibitory site(s) as the I site. Further biochemical characterization showed that the I site has the following characteristics: 1) it is specific for Ca2+; 2) it is allosteric to the ligand binding site; 3) its occupation increases the dissociation rate between integrin and RGD ligand; and 4) occupation of the I site can induce cellular deadhesion.