Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts.

Abstract

Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P. J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol-wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix.In vivo, fibronectin is a large, dimeric glycoprotein of plasma and most body fuids, and an insoluble constituent of loose connective tissue and basement membranes. It is synthesized by a wide variety of cell types in culture and is found in culture medium and in the insoluble extracellular matrix around cultured cells (22, 34,47). The fibronectin in the matrix of cultured fibroblasts is found in fibrillar structures that are 5-10 nm in diameter (7,12,19,23,51). Similar structures have been identified in granulation tissue of skin wounds (52). Although there are structural differences between fibronectin purified from plasma and fibronectin synthesized by cultured cells (2, 16), fibronectin of the extracellular matrix is derived from both plasma (or serum) and local cells (l 8, 32, 38). Furthermore, both plasma (32) and cellular (5,8,21, 25, 31,57) flbronectin are present in the matrix as high molecular weight, disulfide-bonded multimers. Multimerization of plasma flbronectin probably occurs by disulfide 364 exchange in the 70,000-mol-wt amino-terminal region of the molecule (33).Fibronectin is believed to mediate cell-matrix adhesion. When coated onto plastic or glass substrata, fibronectin promotes the attachment and spreading of cells. The cell adhesive activity of fibronectin has been localized, first to increasingly smaller regions within the fibronectin subunit (15, 43, 44), and then to a specific tetrapeptide sequence (42). It has been postulated that cells in suspension interact with this region of the fibronectin molecule by means of ...

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

“…The 70K N-terminal fragment of FN, however, binds to cells with the same affinity and distribution as FN and is a potent inhibitor of FN assembly (Bultmann et al, 1998;Magnusson and Mosher, 1998;McKeown-Longo and Mosher, 1985). 70K binding to fibroblasts thus mimics the characteristics of initial FN binding.…”

“…The linear arrays formed by 70K remained stable for up to 24h and bound 70K did not become deoxycholate-insoluble or form SDS-non-dissociable multimers. In contrast, bound FITC-FN entered the deoxycholate-non-extractable and SDSinsoluble fraction over time, just as FN does when bound to FN-secreting fibroblasts (Choi and Hynes, 1979;McKeown-Longo and Mosher, 1985), and formed an extensive network of fibrils after 24h of incubation with cells. Thus, 70K mimics the high affinity binding of FN that initiates assembly but lacks the interactions that lead to detergent-insoluble fibril formation, i.e., fibril progression.…”

“…70K was radiolabeled with 125 I as described previously (McKeown-Longo and Mosher, 1985). Vitronectin was isolated from human plasma (Bittorf et al, 1993).…”

“…125 I-70K binding to cells was carried out in the absence of serum utilizing previously described methods (McKeown-Longo and Mosher, 1985;Tomasini-Johansson et al, 2001). Tissue culture plastic wells in 24-well plates were coated with laminin at 10-20μg/ml in PBS overnight at 4°C.…”

“…The five N-terminal type I modules are essential for fibrillogenesis (McDonald et al, 1987;McKeown-Longo and Mosher, 1985;Schwarzbauer, 1991;Sottile et al, 1991). The Nterminal 70-kDa catheptic fragment (70K), containing these modules and the adjacent gelatinbinding region, binds to cell monolayers with the same affinity as intact fibronectin and blocks assembly of intact fibronectin, but does not become incorporated into detergent-insoluble extracellular matrix (McKeown-Longo and Mosher, 1985). Bound 70K co-localizes with preexisting FN fibrils (Chernousov et al, 1985), albeit incompletely (Zhang et al, 1994).…”