N-TERMINAL TYPE-I MODULES REQUIRED FOR FIBRONECTIN-BINDING TO FIBROBLASTS AND TO FIBRONECTINS III1 MODULE

Assembly of fibronectin fibrils occurs at the surface of substrate-att ached cells and is mediated by the first to the fifth type I modules i n the N-terminal 70 kDa portion of the molecule. The first type III mo dule (III1) of fibronectin, not present in the 70 kDa portion, contain s a conformation-dependent binding site for the 70 kDa N-terminal regi on of fibronectin, suggesting that the III1 module on cell-surface fib ronectin may serve as a binding site for fibronectin's N-terminus on s ubstrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblast s and to III1. Proteins containing the fourth and fifth type I modules (70K Delta 1(1-3)) bound specifically to III1 in solid-phase binding assays; proteins lacking I-4 and I-5 did not bind. N-terminal molecule s containing the fourth and fifth type I modules also bound to fibrobl asts, suggesting that III1-like binding sites are present on the cell surface. However, the high-affinity binding sites on fibroblasts for f ibronectin or the 70 kDa protein displayed more complex determinants, inasmuch as 70 kDa deletion mutants lacking I-4 and I-5 also bound to the cell surface, and deletion mutants lacking I1-3 and I4-5 both comp eted only partially for binding of I-125-labelled fibronectin or 70 kD a protein. These data indicate that the N-terminal part of fibronectin binds to III1 via I-4 and I-5 and that interactions in addition to th at of I-4 and I-5 with III1 are important for cell-surface-mediated fi bronectin polymerization.