Substrate-specific binding of the amino terminus of fibronectin to an integrin complex in focal adhesions.

The assembly of fibronectin fibrils involves the amino-terminal and cell adhesion domains of fibronectin as well as alpha 5 beta 1 integrins. Efficient binding of biotinylated or radioiodinated 70-kDa amino-terminal fragments occurred only if fibroblasts were plated on fibronectin 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. Conformation of the 180-kDa fragment was important for direct binding interactions with the amino terminus of fibronectin. No binding was seen if cells were plated on type I collagen, vitronectin, RGD peptides or antibodies to alpha 5 beta 1 integrins. High affinity interactions between invasin and alpha 5 beta 1 integrin promoted low levels of binding. Monoclonal antibodies that blocked the function of either the RGD or the synergy site inhibited binding of 125I-labeled 70-kDa fragments to cells by approximately 60%. By fluorescence and interference reflection microscopy, biotinylated 70-kDa fragments were shown to co-localize with alpha 5 beta 1 integrins in focal adhesions. We propose that cell-mediated binding of the amino terminus of fibronectin involves interactions with both fibronectin and its alpha 5 beta 1 integrin receptor in an activated complex.