Evidence that ligand and metal ion binding to integrin α4β1 axe regulated through a coupled equilibrium

We have used the highly selective alpha (4)beta (1) 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 alpha (4)beta (1), integrin-ligand interactions on Jurkat cells. Binding of [S-35]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. K-D values for the binding of BIO7662 to Mn2+-activated alpha (4)beta (1) and to the nonactivated state of the integrin that exists in I mm Mg2+, I mm Ca2+ were < 10 pm, indicating that it has a high affinity for both activated and nonactivated integrin. No binding was observed on alpha (4)beta (1) negative cells. Through an analysis of the metal ion dependences of ligand binding, several unexpected findings about alpha (4)beta (1) function were made. First, we observed that Ca2+ binding to alpha (4)beta (1), was stimulated by the addition of BIO7662. From solution binding studies on purified alpha (4)beta (1), two types of Ca2+-binding sites were identified, one dependent upon and the other independent of BIO7662 binding. Second, we observed that the metal ion dependence of ligand binding was affected by the affinity of the ligand for beta (4)beta (1). ED50 values for the metal ion dependence of the binding of BIO7762 and the binding of a lower affinity ligand, BIO1211, differed by 2-fold for Mn2+, 30-fold for Mg2+, and > 1000-fold for Ca2+. Low Ca2+ (ED50 = 5-10 muM) stimulated the binding of BIO7662 to alpha (4)beta (1). The effects of mum Ca2+ closely resembled the effects of Mn2+ on alpha (4)beta (1) 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 alpha (4)beta (1).