REGULATION OF INTEGRIN FUNCTION - EVIDENCE THAT BIVALENT-CATION-INDUCED CONFORMATIONAL-CHANGES LEAD TO THE UNMASKING OF LIGAND-BINDING SITES WITHIN INTEGRIN ALPHA-5-BETA-1

The molecular mechanisms that regulate integrin-ligand binding are unk nown; however, bivalent cations are essential for integrin activity. A ccording to recent models of integrin tertiary structure, sites involv ed in ligand recognition are located on the upper face of the seven-bl aded beta-propeller formed by the N-terminal repeats of the alpha subu nit and on the von Willebrand factor A-domain-like region of the beta subunit. The epitopes of function-altering monoclonal antibodies (mAbs ) cluster in these regions of the alpha and beta 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-alpha 5 mAbs JES5 and 16, the inhibitory anti-beta 1 mAb 13, and the stimulatory anti-b eta 1 mAb 12G10 to alpha 5 beta 1. Analysis of the binding of these mA bs to alpha 5 beta 1 over a range of Mn2+, Mg2+ or Ca2+ concentrations demonstrated that there was a concordance between the ability of cati ons to elicit conformational changes and the ligand-binding potential of alpha 5 beta 1. Competitive ELISA experiments provided evidence tha t the domains of the alpha 5 and beta 1 subunits recognized by mAbs JB S5/16 and 13/12G10 are spatially close, and that the distance between these two domains is increased when alpha 5 beta 1 is occupied by biva lent cations. Taken together, our findings suggest that bivalent catio ns induce a conformational relaxation in the integrin that results in exposure of ligand-binding sites, and that these sites lie near an int erface between the alpha subunit beta-propeller and the beta subunit p utative A-domain.