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

We have used the highly specific alpha(4)beta(1) inhibitor 4-((N'-2-methylphenyl)ureido)- phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha(4)beta(1). 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+, I 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 mu g/ml of the integrin-activating monoclonal antibody TS2/16. The large range in RD 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 falling in the narrow range from 0.9 to 2.7 x 10(6) 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 alpha(4)beta(1) 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 alpha(4)beta(1) were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.