IDENTIFICATION OF LIGAND-BINDING SITES ON INTEGRIN ALPHA-4-BETA-1 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 r ecently described leucine-aspartic acid-valine (LDV)-based small molec ule inhibitor of alpha 4 beta 1 (BIO-1494), that contained a single re active amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ab ility to block the interaction with a competitive inhibitor lacking th e reactive group, (ii) the absolute requirement of divalent cations fo r cross-linking, and (iii) the lack of cross-linking to the functional ly 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 f lexible cross-linker DSS both the alpha 4 and beta 1 chains were modif ied. 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 loca lized by CNBr peptide mapping within residues 130-146, a region that c ontains 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 liga nd and published data for the RGD family supports the notion of a comm on ligand binding pocket formed by both integrin chains. The cross-lin king strategy developed here should serve as a useful tool for studyin g alpha 4 beta 1 function.