IGD Motifs, Which Are Required for Migration Stimulatory Activity of Fibronectin Type I Modules, Do Not Mediate Binding in Matrix Assembly

Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K) of fibronectin (FN) stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD) motifs in four of the nine FN type 1 (FNI) modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (FNI)-F-3 and (FNI)-F-5; (FNI)-F-7 and (FNI)-F-9; or (FNI)-F-3, (FNI)-F-5, (FNI)-F-7, and (FNI)-F-9. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(FNIII)-F-3, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (FNI)-F-9, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the beta-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.