The molecular mechanisms that regulate integrin-ligand binding are unknown; however, bivalent cations are essential for integrin activity. According to recent models of integrin tertiary structure, sites involved in ligand recognition are located on the upper face of the seven-bladed β-propeller formed by the N-terminal repeats of the α subunit and on the von Willebrand factor A-domain-like region of the β subunit. The epitopes of function-altering monoclonal antibodies (mAbs) cluster in these regions of the α and β subunits; hence these mAbs can be used as probes to detect changes in the exposure or shape of the ligand-binding sites. Bivalent cations were found to alter the apparent affinity of binding of the inhibitory anti-α5 mAbs JBS5 and 16, the inhibitory anti-β1 mAb 13, and the stimulatory anti-β1 mAb 12G10 to α5β1. Analysis of the binding of these mAbs to α5β1 over a range of Mn2+, Mg2+ or Ca2+ concentrations demonstrated that there was a concordance between the ability of cations to elicit conformational changes and the ligand-binding potential of α5β1. Competitive ELISA experiments provided evidence that the domains of the α5 and β1 subunits recognized by mAbs JBS5/16 and 13/12G10 are spatially close, and that the distance between these two domains is increased when α5β1 is occupied by bivalent cations. Taken together, our findings suggest that bivalent cations induce a conformational relaxation in the integrin that results in exposure of ligand-binding sites, and that these sites lie near an interface between the α subunit β-propeller and the β subunit putative A-domain.
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