Evidence That Ligand and Metal Ion Binding to Integrin α4β1 Are Regulated through a Coupled Equilibrium

We have used the highly selective α4β1 inhibitor 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid (BIO7662) as a model ligand to study α4β1 integrin-ligand interactions on Jurkat cells. Binding of [35S]BIO7662 to Jurkat cells was dependent on the presence of divalent cations and could be blocked by treatment with an excess of unlabeled inhibitor or with EDTA.KD values for the binding of BIO7662 to Mn2+-activated α4β1 and to the nonactivated state of the integrin that exists in 1 mmMg2+, 1 mm Ca2+ were 1000-fold for Ca2+. Low Ca2+ (ED50 = 5–10 μm) stimulated the binding of BIO7662 to α4β1. The effects of μm Ca2+ closely resembled the effects of Mn2+ on α4β1 function. Third, we observed that the rate of BIO7662 binding was dependent on the metal ion concentration and that the ED50 for the metal ion dependence of BIO7662 binding was affected by the concentration of the BIO7662. These studies point to an even more complex interplay between metal ion and ligand binding than previously appreciated and provide evidence for a three-component coupled equilibrium model for metal ion-dependent binding of ligands to α4β1.