Identification of ligand binding sites on integrin α4β1 through chemical cross-linking

We have used chemical cross-linking to identify sequences in integrin alpha 4 beta 1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha 4 beta 1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha 4 beta 7. With ANB-NOS as the cross-linker, only the beta 1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha 4 and beta 1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha 4 beta 1 but not on K562 cells expressing alpha 2 beta 1 The site of cross-linking on the beta 1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alpha IIb beta 3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha 4 beta 1 function.