title: Cell surface molecules that bind fibronectin's matrix assembly domain.
abstract: The assembly of fibronectin into disulfide cross-linked extracellular matrices requires the interaction of mesenchymal cells with two distinct sites on fibronectin, the Arg-Gly-Asp cell adhesive site and an amino-terminal site contained within the first five type I homologous repeats (Quade, B. J., and McDonald, J. A. (1988) J. Biol. Chem. 263, 19602-19609). Proteolytically derived 29-kDa fragments of fibronectin (29kDa) containing these repeats bind to monolayers of cultured fibroblasts and inhibit fibronectin matrix assembly. The cell surface molecules interacting with fibronectin's 29-kDa matrix assembly domain have resisted purification using conventional methods such as affinity chromatography. Accordingly, in order to identify molecules which bind this fragment, 125I-labeled 29kDa was allowed to bind to fibroblast monolayers and chemically cross-linked to the cell surface with bis(sulfosuccinimidyl) suberate. Extraction of the cross-linked cell layer yielded radiolabeled complexes of 56, 150, and 280 kDa. Formation of these cross-linked complexes was specifically inhibited by the addition of excess unlabeled 29kDa but was unaffected by the presence of fibronectin fragments containing other type I repeats outside of the 29kDa matrix assembly domain. The cross-linked complexes were insoluble in nondenaturing detergents but soluble when denatured and reduced, suggesting that 29kDa may be cross-linked to components of the pericellular matrix. Immunoprecipitation of cross-linked cell extracts with a polyclonal antibody to fibronectin that does not recognize the amino terminus demonstrate that the 280-kDa band contains 29kDa cross-linked to fibronectin present on the cell surface. Formation of the 150-kDa complex was inhibited by EDTA, suggesting that divalent cations are required for its formation. Although the molecular mass and divalent cation requirement suggest that the 150-kDa complex may be related to an integrin, this complex was not immunoprecipitated by polyclonal antibodies generated to the alpha 5 beta 1 integrin fibronectin receptor.
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: A H
authors.initials: AH
authors.lastname: Limper
authors.forename: B J
authors.initials: BJ
authors.lastname: Quade
authors.forename: R M
authors.initials: RM
authors.lastname: LaChance
authors.forename: T M
authors.initials: TM
authors.lastname: Birkenmeier
authors.forename: T S
authors.initials: TS
authors.lastname: Rangwala
authors.forename: J A
authors.initials: JA
authors.lastname: McDonald
mesh_terms: D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D002642:Chick Embryo; D003432:Cross-Linking Reagents; D003571:Cytochalasin B / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D011233:Precipitin Tests
chemical_list: D003432:Cross-Linking Reagents; D005353:Fibronectins; D016023:Integrins; D003571:Cytochalasin B; D004492:Edetic Acid