ALPHA-4 INTEGRIN BINDING INTERFACES ON VCAM-1 AND MADCAM-1 - INTEGRINBINDING FOOTPRINTS IDENTIFY ACCESSORY BINDING-SITES THAT PLAY A ROLE IN INTEGRIN SPECIFICITY

Integrins are a family of heterodimeric adhesion receptors that mediat e cellular interactions with a range of matrix components and cell sur face proteins. Vascular cell adhesion molecule-l (VCAM-1) is an endoth elial cell ligand for two leukocyte integrins (alpha 4 beta 1 and alph a 4 beta 7). A related CAM, mucosal addressin cell adhesion molecule-l (MAdCAM-1) is recognized by alpha 4 beta 7 but is a poor ligand for a lpha 4 beta 1. Previous studies have revealed that all alpha 4 integri n-ligand interactions are dependent on a key acidic ligand motif cente red on the CAM domain 1 C-D loop region. By generating VCAM-1/MAdCAM-1 chime ras and testing recombinant proteins in cell adhesion assays we have found that alpha 4 beta 1 binds to the MAdCAM-1 adhesion motif w hen present in VCAM-1, but not when the VCAM-1 motif was present in MA dCAM-1, suggesting that this region does not contain all of the inform ation necessary to determine integrin binding specificity. To characte rize integrin-CAM specificity further we measured alpha 4 beta 1 and a lpha 4 beta 7 binding to a comprehensive set of mutant VCAM-1 construc ts containing amino acid substitutions within the predicted integrin a dhesion face. These data revealed the presence of key ''regulatory res idues'' adjacent to integrin contact sites and an important difference in the ''footprint'' of alpha 4 beta 1 and alpha 4 beta 7 that was as sociated with an accessory binding site located in VCAM-1 Ig domain 2. The analogous region in MAdCAM-1 is markedly different in size and se quence and when mutated abolishes integrin binding activity.