Amino acid residues required for binding of vascular cell adhesion molecule-1 to integrin alpha 4 beta 7.
The homologous Ig-like domains 1 and 4 of vascular cell adhesion molecule (VCAM)-1 present binding sites to the leukocyte integrins alpha 4 beta 1 and alpha 4 beta 7 . In the present study, amino acid substitution mutants were used to identify sequence motifs mediating binding of integrin alpha 4 beta 7 to the first domain of VCAM-1. We demonstrate that binding of integrin alpha 4 beta 7 to VCAM-1 containing the D40A mutation located in the loop between beta 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 alpha 4 beta 7. Analysis of the E66A mutation demonstrated that the G(64)NEH sequence, which is exposed on the loop structure between beta strands E1 and F1, represents an additional recognition site for alpha 4 beta 7 integrin. However, the inhibitory effect of the E66A mutation on cell binding was not specific for alpha 4 beta 7 but was also observed for integrin alpha 4 beta 1. In contrast to the I(39)DSP and G(64)NEH sequences, the K(79)LEK motif present in beta strand G1 was involved in binding to alpha 4 beta 1 but not alpha 4 beta 7. The function of G(64)NEH and K(79)LEK motifs in alpha 4++-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 alpha 4 beta 1 and alpha 4 beta 7 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 alpha 4 beta 1 and alpha 4 beta 7 integrins, whereas the K(79)LEK sequence appears to confer specificity for alpha 4 beta 1 binding.
Kilger
G
G
Institute for Medical Microbiology, Immunology & Hygiene, Technical University, Munich, Germany.
Clements
J
J
Holzmann
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002448:Cell Adhesion / Q000235:genetics / Q000276:immunology; D039041:Integrin alpha4beta1; D016023:Integrins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D016297:Mutagenesis, Site-Directed / Q000276:immunology; D010957:Plasmids; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000596:Amino Acids; D039041:Integrin alpha4beta1; D016023:Integrins; D016201:Receptors, Lymphocyte Homing; D019010:Vascular Cell Adhesion Molecule-1; C071777:integrin alpha4beta7