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

1994; Elsevier BV; Volume: 269; Issue: 29 Linguagem: Inglês

10.1016/s0021-9258(17)32292-5

1083-351X

Denise C. Hocking, Jane Sottile, Paula J. McKeown‐Longo,

Protease and Inhibitor Mechanisms

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 '261-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 '%labeled 70-kDa fragment by ligand blot assay and affinity chromatography.AU of the fibronectin fragments that bound the 70-kDa fragment contained the 111-1 module.In solid phase binding assays, the '261-labeled 70-kDa fragment bound preferentially to reduced fibronectin as compared with unreduced fibronectin fragments.Binding of the '"I-labeled 70-kDa fragment to reduced fibronectin was inhibited by a monoclonal antibody directed against the 111-1 domain.Isolated III-1, however, did not bind the 1z61labeled 70-kDa fragment when adsorbed to plastic tissue culture wells.Heat denaturation of 111-1 prior to adsorption conferred 70-kDa fragment binding properties on the isolated module.The laaI-labeled 70-kDa fragment did not bind to heat-denatured 111-2, suggesting that 70-kDa fragment binding was a property of the 111-1 module and not a general characteristic of all type I11 modules.The binding of 'asI-labeled 70-kDa fragment to 111-1 was of high affinity (K, = 1.8 x 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 I11 module of fibronectin.Unfolding of the 111-1 module on the cell surface may control matrix assembly site expression and represent an important step in the initiation of cell-dependent fibronectin polymerization.Fibronectins are a family of high molecular weight multidomain glycoproteins composed of two structurally similar subunits that are joined at the carboxyl terminus by disulfide bonds (1).The primary structure of each subunit is organized into three types of repeating homologous units, termed types I, 11, and I11 (1, 2).Twelve type I modules are found grouped at