Evidence that ligand and metal ion binding to integrin alpha(4)beta(1) axeregulated through a coupled equilibrium

We have used the highly selective alpha (4)beta (1) inhibitor 2S-[(1-benzen esulfonyl-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 o f 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 ana lysis of the metal ion dependences of ligand binding, several unexpected fi ndings 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 BIO766 2. From solution binding studies on purified alpha (4)beta (1), two types o f Ca2+-binding sites were identified, one dependent upon and the other inde pendent of BIO7662 binding. Second, we observed that the metal ion dependen ce 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 BIO77 62 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 o f mum Ca2+ closely resembled the effects of Mn2+ on alpha (4)beta (1) funct ion. Third, we observed that the rate of BIO7662 binding was dependent on t he metal ion concentration and that the ED50 for the metal ion dependence o f BIO7662 binding was affected by the concentration of the BIO7662. These s tudies point to an even more complex interplay between metal ion and ligand binding than previously appreciated and provide evidence for a three-compo nent coupled equilibrium model for metal ion-dependent binding of ligands t o alpha (4)beta (1).