Amino acid residues required for binding of vascular cell adhesion molecule-1 to integrin α4β7

The homologous Ig-like domains 1 and 4 of vascular cell adhesion molecule (VCAM)-1 present binding sites to the leukocyte integrins α4β1 and α4β7. In the present study, amino acid substitution mutants were used to identify sequence motifs mediating binding of integrin α4β7 to the first domain of VCAM-1. We demonstrate that binding of integrin α4β7 to VCAM-1 containing the D40A mutation located in the loop between β strands C1 and D1 was completely abrogated and was not restored by activating integrin binding functions with Mn²⁺. Thus, the I(39)DSP motif functions as a central recognition site for integrin α4β7. Analysis of the E66A mutation demonstrated that the G(64)NEH sequence, which is exposed on the loop structure between β strands E1 and F1, represents an additional recognition site for α4β7 integrin. However, the inhibitory effect of the E66A mutation on cell binding was not specific for α4β7 but was also observed for integrin α4β1. In contrast to the I(39)DSP and G(64)NEH sequences, the K(79)LEK motif present in β strand G1 was involved in binding to α4β1 but not α4β7. The function of G(64)NEH and K(79)LEK motifs in α4-integrin interactions was confirmed by divalent cation titration assays and peptide inhibition studies. Integrin binding to E66A or E81A;K82A mutants was restored by activation with saturating concentrations of Mn²⁺. Binding of both α4β1 and α4β7 integrins was not affected by E29A, R36A, E50A or E87A mutations. Together, these results identify the I(39)DSP and G(64)NEH motifs as common recognition sites for both α4β1 and α4β7 integrins, whereas the K(79)LEK sequence appears to confer specificity for α4β1 binding.