Abstract
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 [(35)S]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 Mn(2+)-activated alpha(4)beta(1) and to the nonactivated state of the integrin that exists in 1 mm Mg(2+), 1 mm Ca(2+) 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 Ca(2+) 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 Ca(2+)-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 alpha(4)beta(1). ED(50) 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 Mn(2+), 30-fold for Mg(2+), and >1000-fold for Ca(2+). Low Ca(2+) (ED(50) = 5-10 microm) stimulated the binding of BIO7662 to alpha(4)beta(1). The effects of microm Ca(2+) closely resembled the effects of Mn(2+) 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 ED(50) 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).
Highlights
Integrins are a large family of heterodimeric cell surface receptors that mediate cell-cell and cell-matrix interactions in diverse settings
BIO7662 is a member of a family of highly selective ␣41 inhibitors that was derived by rational design from the LDV sequence of CS-1 fibronectin and that directly competes with vascular cell adhesion molecule-1 (VCAM-1), CS-1, and BIO1211 for binding to the integrin
Because cation-binding sites map to the same regions on the ␣and -chains as the ligand-binding sites, various models have been proposed in which the cations bind ligands or compete with ligand for binding [51,52,53,54] or in which cation binding regulates the opening of the integrin dimer to expose the ligand-binding site [29]
Summary
Synthesis of [35S]BIO7662—[35S]BIO7662 (1, 241 Ci/mmol) was synthesized at PerkinElmer Life Sciences by reacting [35S]phenylsulfonyl chloride with the proline hydrochloride methyl ester precursor under basic conditions. The cells were pelleted by centrifugation, washed two times with TBS (50 mM Tris-HCl, 150 mM NaCl, 0.1% bovine serum albumin, 2 mM glucose, 10 mM HEPES, pH 7.4), suspended at ϳ2 ϫ 106 cells/ml in TBS, and counted using a Neubauer hemocytometer. The cells were further diluted with TBS to the concentration indicated and treated with [35S]BIO7662 at room temperature. Pervanadate was made fresh at the time of use by adding 0.09% hydrogen peroxide (final concentration) to 20 mM sodium orthovanadate and incubating the solution at room temperature for 15 min. The cells were collected by centrifugation and lysed in 1% Nonidet P-40, 25 mM Tris-HCl, pH 7.4, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 2% bovine serum albumin, 1 mM phenylmethanesulfonyl fluoride at 1 ϫ 108 cells/ml lysis buffer. All solutions were prepared with metal ion-free water (Fluka)
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