Multiple activation states of integrin α4β1 detected through their different affinities for a small molecule ligand

Abstract

We have used the highly specific α4β1 inhibitor 4-((N'- 2- methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study α4β1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of α4β1. Observed K(D) values for BIO1211 binding ranged from a value of 20-40 nM in the nonactivated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to 100 pM in the activated state seen in 2 mM Mn2+ to 18 pM when binding was measured after coactivation by 2 mM Mn2+ plus 10 μg/ml of the integrin-activating monoclonal antibody TS2/16. The large range in K(D) values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 x 10-4 s-1 to >140 x 10-4 s-1. Association rate constants varied only slightly under the same conditions, all failing in the narrow range from 0.9 to 2.7 x 106 M-1 s-1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on α4β1 that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule- 1 and CS1 to α4β1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.