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The Journal of biological chemistry

Fibronectin's III-1 module contains a conformation-dependent binding site for the amino-terminal region of fibronectin.


PMID 8034677

Abstract

Cultured fibroblasts express binding sites for the amino-terminal region of fibronectin on their cell surface that mediate the assembly of soluble fibronectin into disulfide-stabilized fibrils. These binding sites have been termed matrix assembly sites and have been studied in binding assays using a 125I-labeled 70-kDa fragment derived from the amino terminus of fibronectin. In an attempt to isolate the protein(s) responsible for binding the 70-kDa fragment, cell surface proteins were cleaved from fibroblast monolayers by mild trypsinization. Trypsinization of monolayers generated a series of fibronectin fragments that bound the 125I-labeled 70-kDa fragment by ligand blot assay and affinity chromatography. All of the fibronectin fragments that bound the 70-kDa fragment contained the III-1 module. In solid phase binding assays, the 125I-labeled 70-kDa fragment bound preferentially to reduced fibronectin as compared with unreduced fibronectin fragments. Binding of the 125I-labeled 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 125I-labeled 70-kDa fragment when adsorbed to plastic tissue culture wells. Heat denaturation of III-1 prior to adsorption conferred 70-kDa fragment binding properties on the isolated module. The 125I-labeled 70-kDa fragment did not bind to heat-denatured III-2, suggesting that 70-kDa fragment binding was a property of the III-1 module and not a general characteristic of all type III modules. The binding of 125I-labeled 70-kDa fragment to III-1 was of high affinity (KD = 1.8 x 10(-8) M). These results indicate that a binding site for the 70-kDa amino terminus of fibronectin is contained within a cryptic site found in the first type III module of fibronectin. Unfolding of the III-1 module on the cell surface may control matrix assembly site expression and represent an important step in the initiation of cell-dependent fibronectin polymerization.