Abstract

An α₄β₁/α₄β₇ 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 α₄ integrins. In the presence of 1 mM each Ca²⁺/Mg²⁺, ³⁵S-compound 1 bound to several cell lines expressing both α₄β₁ and α₄β₇, 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 α₄β₁ antagonist, completely inhibited ³⁵S-compound 1 binding, suggesting that α₄β₁ was responsible for the observed binding. ³⁵S-Compound 1 bound RPMI-8866 cells expressing predominantly α₄β₇ with a K_D of 1.9 nM in the presence of 1 mM Mn²⁺, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated α₄β₇. With Ca²⁺/Mg²⁺, ³⁵S-compound 1 bound Jurkat cells expressing primarily α₄β₁ with a K_D of 18 nM. In contrast, the binding of ³⁵S-compound 1 to Mn²⁺-activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four α₄β₁/α₄β₇ antagonists to block binding of activated α₄β₁ or α₄β₇ to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to ³⁵S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of ³⁵S-compound 1 binding to α₄β₁ in Ca²⁺/Mg²⁺ was used to identify nonselective antagonists among these four. These studies demonstrate that α₄β₁ and α₄β₇ have distinct binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.