A Small Molecule alpha_4 beta_1/alpha_4beta_7 Antagonist Differentiates between the Low-Affinity States of alpha_4 beta_1 and alpha_4 beta_7: Characterization of Divalent Cation Dependence

摘要

An alpha_4 beta_1/alpha_4beta_7 dual antagonist, ~35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of alpha_4 integrins. In the presence of 1 mM each Ca~2+/Mg~2+, ~35S-compound 1 bound to several cell lines expressing both alpha_4 beta_1/alpha_4beta_7, but 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), a specific alpha_4 beta_1 antagonist, completely inhibited ~35S-compound 1 binding, suggesting that alpha_4beta_1 was responsible for the observed binding. ~35S-Compund 1 bound RPMI-8866 cells expressing predominantly alpha_4 beta_7 with a K_D of .19 nM in the presence of 1 mM Mn~2+, and binding was inhibited only 20% by BIO7662, suggesting tiat the probe is a potent antagonist of activated alpha_4 beta_7. With Ca~2+/Mg~2+, ~35S-compound 1 bound Jurkat cells expressing primarily alpha_4 beta_1 with a K_D of 18 nM. In contrast, the binding of ~35S-compound 1 to Mn~2+ -activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four alpha_4 beta_1/alpha_4beta_7 antagonists to block binding of activated alpha_4 beta_1 or alpha_4beta_7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to ~35S-compound 1 was measured, an a similar rank order of potency was observed for native ligand and rpobe. Inhibition of ~35S-compound 1 binding to alpha_4 beta_1 in Ca~2+/Mg~2+ was used to identify nonselective antagonists among these four. These studies demonstrate that alpha_4 beta_1 and alpha_4 beta_1 have distince binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.