High Affinity Ligand Binding by Integrins Does Not Involve Head Separation

Bing-Hao Luo,Timothy A Springer,Junichi Takagi

doi:10.1074/jbc.m301516200

Abstract

Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha- and beta-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha-subunit beta-propeller domain and the beta-subunit I-like domain. Basal and activation-dependent ligand binding by alpha(IIb)beta(3) and alpha(V)beta(3) was unaffected. The covalent linkage prevented dissociation of alpha(IIb)beta(3) into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type alpha(IIb)beta(3) on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between alpha(IIb) and beta(3) headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated alphabeta headpiece.

Highlights

Integrins are major metazoan adhesion receptors that play a fundamental role in cellular organization
An alternative model for integrin activation has been proposed that is supported by high resolution EM, physicochemical studies, ligand binding assays, introduction of disulfide bonds that lock in the bent conformation, and localization of epitopes that become exposed after integrin activation [5, 26]
Introduction of Disulfide Bonds between the ␤-Propeller and I-like Domains—To make mutant integrins that were unable to undergo head separation, we mutationally introduced cysteine residues at the interface between the ␣IIb ␤-propeller and ␤3 I-like domains (Fig. 1)

Summary

High Affinity Ligand Binding by Integrins Does Not Involve Head Separation*

Many experiments support the idea that the inter-subunit association at the cytoplasmic region maintains integrins in low affinity state [7,8,9,10] This notion led to a “hinge hypothesis,” where association/dissociation of the cytoplasmic tails caused hinging between the two subunits, and changed the conformation of the ligand-binding extracellular segments [11]. An alternative model for integrin activation has been proposed that is supported by high resolution EM, physicochemical studies, ligand binding assays, introduction of disulfide bonds that lock in the bent conformation, and localization of epitopes that become exposed after integrin activation [5, 26]. We have used mutagenesis to introduce disulfide bonds between the ␣-subunit ␤-propeller domain and the ␤-subunit I-like domain, and we have tested the effect of preventing head separation on activation of ␣IIb␤3 and ␣V␤3 integrins on the cell surface. We test the effect of ligand-mimetic compounds on the association between the ␣and ␤-subunits in native integrins on the cell surface and in soluble integrin fragments that contain only the headpiece

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION