A NOVEL ROLE FOR THE INTEGRIN-BINDING III-10 MODULE IN FIBRONECTIN MATRIX ASSEMBLY

Fibronectin matrix assembly is a cell-dependent process which is upreg ulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentia tion. Previous studies have demonstrated that the alpha(5) beta(1) int egrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin 's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McK eown-Longo. 1994. J. Biol. Chem. 269: 19183-19191), The present study was undertaken to define the relationship between the alpha(5) beta(1) integrin and fibronectin polymerization. Solid phase binding assays u sing recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent bin ding site for the III-1 module of fibronectin. Unfolded III-10 could s upport the formation of a ternary complex containing both III-1 and th e amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded II I-10 and unfolded III-1 could support fibronectin binding, but only II I-10 could promote the formation of disulfide-bonded multimers of fibr onectin in the absence of cells. III-10-dependent multimer formation w as inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amin o-terminal fragments of fibronectin. A fragment of III-10, termed III- 10/A, was able to block matrix assembly in fibroblast monolayers. Simi lar results were obtained using the III-1OA/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-10/A was blocki ng matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta(1 )-containing sites of focal adhesions on cells plated on fibronectin o r the III-9,10 modules of fibronectin. Monoclonal antibodies against t he III-1 or the III-9,10 modules of fibronectin blocked binding of III -1,2 to cells without disrupting focal adhesions. These data suggest t hat a role of the alpha(5) beta(1) integrin in matrix assembly is to r egulate a series of sequential self-interactions which result in the p olymerization of fibronectin.