SUBSTRATE-SPECIFIC BINDING OF THE AMINO-TERMINUS OF FIBRONECTIN TO ANINTEGRIN COMPLEX IN FOCAL ADHESIONS

The assembly of fibronectin fibrils involves the amino terminal and ce ll adhesion domains of fibronectin as well as alpha(5) beta(1) integri ns. Efficient binding of biotinylated or radioiodinated 70-kDa amino-t erminal fragments occurred only if fibroblasts were plated on fibronec tin or on 180- or 85-kDa cell adhesion fragments of fibronectin. On an 11.5-kDa fragment of fibronectin that included the Arg-Gly-Asp (RGD) sequence, but not the synergy site, binding was reduced 50-fold. Confo rmation of the 180-kDa fragment was important for direct binding inter actions with the amino terminus of fibronectin. No binding was seen if cells were plated on type I collagen, vitronectin, RGD peptides or an tibodies to alpha(5) beta(1) inte grins. High affinity interactions be tween invasin and alpha(5) beta(1) integrin promoted low levels of bin ding. Monoclonal antibodies that blocked the function of either the RG D or the synergy site inhibited binding of I-125-labeled 70-kDa fragme nts to cells by similar to 60%. By fluorescence and interference refle ction microscopy, biotinylated 70 kDa fragments were shown to co-local ize with alpha(5) beta(1) integrins in focal adhesions. We propose tha t cell-mediated binding of the amino terminus of fibronectin involves interactions with both fibronectin and its alpha(5) beta(1) integrin r eceptor in an activated complex.