CONTRIBUTIONS OF THE I AND EF HAND DOMAINS TO THE DIVALENT CATION-DEPENDENT COLLAGEN-BINDING ACTIVITY OF THE ALPHA(2)BETA(1) INTEGRIN

The alpha(2) beta(1) integrin binds collagen in a Mg2+-dependent manne r that is inhibited by Ca2+, Like the intact integrin, purified recomb inant proteins containing the or, integrin I domain, either alone or w ith variable numbers of cu, integrin EF hand metal binding sites, boun d collagen in a Mg2+-dependent manner, and Ca2+ did not support bindin g, However, unlike the intact integrin, Ca2+ did not inhibit the Mg2+- dependent binding of any of the fusion proteins to collagen. Binding t o collagen was saturable and blocked by the alpha(2) beta(1) function blocking antibody 6F1. Deletional analysis demonstrated that residues present within the amino-terminal 35 amino acids contribute to the 6F1 epitope and are required for Mg2+-dependent collagen binding, The res ults indicate that the I domain contains a Mg2+ binding site that is e ssential for collagen binding and that the I domain alone is sufficien t for collagen binding. Binding is markedly enhanced in a divalent cat ion dependent manner by the addition of the first EF hand motif, Mutat ion of the EF hand to an inactive form completely abrogated the effect , The sites necessary for Ca2+ inhibition are not present within the I domain or the adjacent region containing the three EF hand sites.