Received for publication December 5, 2002.
Revised January 15, 2003.
Accepted for publication May 22, 2003.

A Small Molecule 41/47 Antagonist Differentiates Between the Low Affinity States of 41 and 47: Characterization of Divalent Cation-Dependence

Abstract

An 41/47 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 4 integrins. In the presence of 1 µM each Ca⁺⁺/Mg⁺⁺, ³⁵S-compound 1 bound to several cell lines expressing both 4 1 and 47, but BIO7662, a specific 41 antagonist, completely inhibited ³⁵S-compound 1 binding, suggesting that 41 was responsible for the observed binding. ³⁵S-compound 1 bound RPMI-8866 cells expressing predominantly 47 with a K_D of 1.9 nM in the presence of 1 mM Mn⁺⁺, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated 47. With Ca⁺⁺/Mg⁺⁺, ³⁵S-compound 1 bound Jurkat cells expressing primarily 4 1 with a K_D of 18 nM. In contrast, the binding of ³⁵S-compound 1 to Mn⁺⁺- activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four 41/ 47 antagonists to block binding of activated 41 or 47 to vascular cell adhesion molecule-1 (VCAM-1) or mucosal addressin cell adhesion molecule-1 (MAdCAM-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 41 in Ca⁺⁺/Mg⁺⁺ was used to identify nonselective antagonists among these four. These studies demonstrate that 41 and 47 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.

Key words: MAdCAM-1, VCAM-1, alpha4beta1, alpha4beta7, cation, integrin