FIBRONECTINS III-1 MODULE CONTAINS A CONFORMATION-DEPENDENT BINDING-SITE FOR THE AMINO-TERMINAL REGION OF FIBRONECTIN

Cultured fibroblasts express binding sites for the amino-terminal regi on of fibronectin on their cell surface that mediate the assembly of s oluble fibronectin into disulfide-stabilized fibrils. These binding si tes have been termed matrix assembly sites and have been studied in bi nding assays using a I-125-labeled 70-kDa fragment derived from the am ino terminus of fibronectin. In an attempt to isolate the protein(s) r esponsible for binding the 70-kDa fragment, cell surface proteins were cleaved from fibroblast monolayers by mild trypsinization. Trypsiniza tion of monolayers generated a series of fibronectin fragments that bo und the I-125-labeled 70-kDa fragment by ligand blot assay and affinit y chromatography. All of the fibronectin fragments that bound the 70-k Da fragment contained the III-1 module. In solid phase binding assays, the I-125-labeled 70-kDa fragment bound preferentially to reduced fib ronectin as compared with unreduced fibronectin fragments. Binding of the I-125-Iabeled 70-kDa fragment to reduced fibronectin was inhibited by a monoclonal antibody directed against the III-1 domain. Isolated III-1, however, did not bind the I-125-labeled 70-kDa fragment when ad sorbed to plastic tissue culture webs. Heat denaturation of III-1 prio r to adsorption conferred 70 kDa fragment binding properties on the is olated module. The I-125-labeled 70-kDa fragment did not bind to heat- denatured III-2, suggesting that 70-kDa fragment binding was a propert y of the III-1 module and not a general characteristic of all type III modules. The binding of I-125-labeled 70-kDa fragment to III-1 was of high affinity (K-D = 1.8 x 10(-8) M). These results indicate that a b inding site for the 70-kDa amino terminus of fibronectin is contained within a cryptic site found in the first type III module of fibronecti n. Unfolding of the III-1 module on the cell surface may control matri x assembly site expression and represent an important step in the init iation of cell-dependent fibronectin polymerization.