The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin

Abstract

Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5min following ligand addition at concentrations as low as 5nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with α 6 and β 1 integrin subunits at the base of the extensions. α 5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectinfibronectin interactions during progression of assembly.

Fibronectin fibrillogenesis regulates three-dimensional neovessel formation

“…Here, we demonstrate that endothelial cell function within the specific confines of the 3D ECM is unexpectedly regulated by Fn fibrillogenesis. Fn, a disulfide-linked dimer of subunits composed of three types of repeating modules (i.e., type I, II, and III repeats), binds to the endothelial cell surface by displaying a dominant cell-adhesive domain (module III 9,10 ), a C-terminal heparin-binding domain , and a 70-kDa Nterminal domain that are recognized by integrins, syndecans, and Fn matrix assembly sites, respectively (Mao and Schwarzbauer 2005;Tomasini-Johansson et al 2006). Once the soluble Fn dimer engages adhesion molecules on the endothelial cell surface, signaling cascades are triggered that initiate the unfolding of the globular glycoprotein and the consequent exposure of cryptic domains, which then serve to support Fn polymerization and matrix assembly (Geiger et al 2001;Mao and Schwarzbauer 2005;Tomasini-Johansson et al 2006).…”

“…Fn, a disulfide-linked dimer of subunits composed of three types of repeating modules (i.e., type I, II, and III repeats), binds to the endothelial cell surface by displaying a dominant cell-adhesive domain (module III 9,10 ), a C-terminal heparin-binding domain , and a 70-kDa Nterminal domain that are recognized by integrins, syndecans, and Fn matrix assembly sites, respectively (Mao and Schwarzbauer 2005;Tomasini-Johansson et al 2006). Once the soluble Fn dimer engages adhesion molecules on the endothelial cell surface, signaling cascades are triggered that initiate the unfolding of the globular glycoprotein and the consequent exposure of cryptic domains, which then serve to support Fn polymerization and matrix assembly (Geiger et al 2001;Mao and Schwarzbauer 2005;Tomasini-Johansson et al 2006). As observed during both vasculogenesis and angiogenesis in vivo (Clark et al 1982;Risau and Lemmon 1988;Kim et al 2000;Francis et al 2002;Hynes 2002;Neri and Bicknell 2005), endothelial cells embedded in 3D fibrin or collagen-rich gels rapidly assemble a pericellular Fn matrix following stimulation with proangiogenic growth factors.…”

Calcium indirectly regulates immunochemical reactivity and functional activities of the N-domain of thrombospondin-1

“…Some integrin ligands may also be subject to conformational regulation of activity similar to that described for their receptors. For example, conformational regulation of fibronectin controls its ability to assemble into fibrils (Mao and Schwarzbauer, 2005;Tomasini-Johansson et al, 2006).…”

“…Recent crystal structures of C-terminal constructs based on TSP1 and TSP2 identified a total of 30-31 calcium binding sites, one in the second EGF repeat, 26 in the so called wire modules, and three or four in the lectin-like G module (Kvansakul et al, 2004;Carlson et al, 2005). Removal of calcium alters the hydrodynamic properties and circular dichroism spectrum of TSP1 (Lawler and Simons, 1983;Vuillard et al, 1991), alters its structure as visualized by rotary shadowing electron microscopy (Galvin et al, 1985;Lawler et al, 1985), and produces local conformational changes in the wire modules as assessed by electron spin resonance (Slane et al, 1988), intrinsic fluorescence , and exposure of calcium-dependent epitopes recognized by two TSP1 antibodies recognizing epitopes in the wire module, A6.1 and D4.6 (Dixit et al, 1986;Annis et al, 2006) (Annis et al, 2007). Removing calcium markedly enhances binding of TSP1 to type V collagen (Galvin et al, 1987).…”

Fibrillin Assembly Requires Fibronectin

“…The new FNsuper70K construct spans the N-terminus through module FNIII 3 plus 17 residues (GNFFKKTLPMLSYQDCS) from the following intron and is the human homolog to a splice variant found in zebrafish (Liu et al, 2003). It was produced recombinantly by modification of the pCOCO baculovirus expression vector encoding FN70K Tomasini-Johansson et al, 2006). DNA for encoding the 17-residue sequence was produced by annealing overlapping oligonucleotides, 5Ј-ACGCTCCGGAAACTTCTTCAAGAAGA-CACTTCCTATGTTATC-3Ј and 5Ј-CCATCTGCAGAACAATCCTGATAA-GATAACATAGGAAGTGTCTTC-3Ј, and extending with Taq polymerase.…”