Fibronectin's amino-terminal matrix assembly site is located within the 29-kDa amino-terminal domain containing five type I repeats.
Fibronectin is organized into disulfide cross-linked, insoluble pericellular matrix fibrils by fibroblasts in vitro. Two sites, the Arg-Gly-Asp-Ser-containing cell attachment domain and a site located in the first 70 kDa of fibronectin, are required for matrix assembly. The first 70 kDa of fibronectin contain two structural motifs termed type I and type II homologies, which are repeated nine and two times, respectively. Previous work has implicated the amino-terminal region and the carboxyl terminus containing three type I repeats in matrix assembly, suggesting that type I repeats possess binding activity essential for fibronectin matrix assembly. To test this hypothesis, we developed a sensitive capture immunoassay to quantify insoluble matrix fibronectin and tested a panel of fibronectin fragments, containing all of the type I repeats found in the intact protein, for their ability to inhibit matrix assembly. Only fragments containing the first five type I repeats inhibited fibronectin matrix assembly, although sequences carboxyl-terminal to this domain enhanced this activity. Additional evidence for the specific recognition of the amino-terminal type I repeats by matrix assembling cells was found when the reversible, detergent-sensitive binding of a 125I-labeled fragment containing the first five type I repeats (29 kDa) to cell monolayers was studied. Only monolayers of cell lines that incorporate fibronectin into a fibrillar matrix specifically bound 125I-labeled 29 kDa. Binding of the radiolabeled amino-terminal fragment to matrix-forming cells was inhibited by unlabeled fragments containing the first five type I repeats but not by unlabeled fragments containing the remaining seven type I repeats. Matrix assembly is therefore not a generalized property of type I repeats. Rather, a critical site is located within the first 29 kDa of fibronectin.
Quade
B J
BJ
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
McDonald
J A
JA
D000595:Amino Acid Sequence; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D004220:Disulfides / Q000032:analysis; D005169:Factor VIII / Q000302:isolation & purification; D005353:Fibronectins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000378:metabolism
D004220:Disulfides; D005353:Fibronectins; D010446:Peptide Fragments; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; D005169:Factor VIII; D003094:Collagen