Isolation of an amino-terminal fibronectin-binding protein on human U937 cells and rat peritoneal macrophages.

Several cell-mediated activities for the amino terminus of fibronectin have been documented. In the present study we describe a macrophage surface protein with binding activity directed to the amino terminus of the fibronectin molecule. The binding of a 29-kDa amino-terminal fibronectin fragment to macrophages reached steady state by 30 min and was half-maximal at approximately 2 x 10(-8) M. This binding was specifically inhibited by excess unlabeled 29-kDa fragment or intact fibronectin but not by a 180-kDa fibronectin fragment which lacks the amino terminus. Competitive binding studies of the 70-kDa amino-terminal fibronectin fragment to macrophages revealed a single binding site with KD = 7.14 x 10(-8) M and approximately 8 x 10(4) binding sites/cell. Radiolabeled surface proteins extracted from rat peritoneal macrophages and from the human U937 cell line were applied to an affinity column comprised of the 70-kDa amino-terminal fragment of fibronectin coupled to a solid support. A single trypsin-sensitive radiolabeled protein of 67 kDa, from either cell type, was eluted from this column with urea. This protein showed no immunologic identity with fibronectin, fibrin(ogen), or albumin. The 67-kDa protein exhibited identical apparent molecular weight under reducing and nonreducing conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. We have localized the fibronectin binding activity of this protein to within the 29-kDa amino-terminal domain of fibronectin. The 67-kDa protein eluted from the 70-kDa column failed to bind to a column comprised of the 45-kDa gelatin-binding fragment of fibronectin. Additionally, the 67-kDa protein was specifically eluted from the 70-kDa column by the 29-kDa amino-terminal fragment but not by the 45-kDa gelatin-binding fragment. These data suggest that this 67-kDa protein is a macrophage cell surface binding protein for the amino terminus of fibronectin.     

Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells

The active migration of tumor cells through extracellular matrices has been proposed to play a role in certain aspects of metastasis. Metastatic tumor cells migrate in vitro in response to substratum-bound adhesive glycoproteins such as fibronectin. The present studies use affinity-purified proteolytic fragments of fibronectin to determine the nature of adhesion- and/or motility-promoting domains within the protein. Two distinct fragments were identified with cell adhesion-promoting activities. By a number of criteria, the adhesive activity promoted by these two fragments was distinct. One fragment, a 75-kD tryptic fragment purified by monoclonal antibody chromatography, promoted the adhesion, spreading, and haptotactic motility of melanoma cells. Experiments using a synthetic cell attachment peptide in solution indicated that at least part of the attachment activity exhibited by the 75-kD fragment is mediated by the sequence arg-gly-asp-ser. It was not possible to demonstrate migration-stimulating activity using a small (11.5 kD) peptic fragment containing this sequence (Pierschbacher, M.D., E. G. Hayman, and E. Ruoslahti, 1981, Cell, 26:259-267) suggesting that another cell-binding activity within the 75 kD fragment distinct from arg-gly-asp-ser might be required for motility. The second fragment that stimulated melanoma adhesion was a 33-kD tryptic/catheptic carboxyl-terminal heparin-binding fragment, which is localized to the A chain of fibronectin. This fragment promotes adhesion and spreading but not the motility of these cells. Melanoma adhesion to this heparin-binding fragment was sensitive to the effects of cycloheximide, which contrasted adhesion to the haptotaxis-promoting fragment. Importantly, these studies illustrate that haptotaxis in response to fibronectin is not due to simple adhesion gradients of this protein. The results are discussed in light of a model for multiple distinct cell surface constituents mediating cell adhesion and motility on fibronectin.     

Inhibition of binding of fibronectin to matrix assembly sites by anti- integrin (alpha 5 beta 1) antibodies

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.     

Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH₂-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH₂-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124–128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30–35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.     

Transglutaminase-sensitive glutamine residues of human plasma fibronectin revealed by studying its proteolytic fragments

The sites of transglutamination of fibronectin and fibronectin fragments, by coagulation factor XIIIa and tissue transglutaminase, were studied. It was shown that the intact fibronectin molecule has two sites sensitive to coagulation factor XIIIa and four sites sensitive to tissue transglutaminase: 180--190-kDa gelatin/heparin-binding fragments, 2 and 5--6 sites; 29-kDa heparin-I/fibrin-I-binding N-terminal fragments, 1 and 2 sites; 70-kDa gelatin-binding fragments, 0 and 1 site; 60-kDa cell-binding central fragments, 1 and 3--4 sites; 60-kDa, 45-kDa, 30-kDa heparin-II-binding C-terminal fragments, 1 and 2 sites. Thus, we have found a new coagulation-factor-XIIIa-sensitive site localized in the cell-binding central fragment, inaccessible to enzyme in the intact fibronectin molecule. Tissue transglutaminase appeared to interact with all of the three coagulation-factor-XIIIa-sensitive sites and, in addition, some others which are either available on the intact molecule or can be revealed only in proteolytic fragments of the fibronectin. We suggest that interdomain and intersubunit interactions in the intact fibronectin molecule account for the masking of glutamine residues potentially accessible to transglutaminases.     

Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin

Mechanisms of cell interaction with fibronectin have been studied with proteolytic fibronectin fragments that have well-defined ligand binding properties. Results of a previous study (Rogers, S. L., J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, 1985, J. Neurosci., 5:369-378) demonstrated that (a) central (CNS) and peripheral (PNS) nervous system neurons adhere to, and extend neurites on a 33-kD carboxyl terminal fibronectin fragment that also binds heparin, and (b) neurons from the PNS, but not the CNS, have stable interactions with a 75-kD cell-binding fragment and with intact fibronectin. In the present study domain-specific reagents were used in inhibition assays to further differentiate cell surface interactions with the two fibronectin domains, and to define the significance of these domains to cell interactions with the intact fibronectin molecule. These reagents are (a) a soluble synthetic tetrapeptide Arg-Gly-Asp-Ser (RGDS; Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) representing a cell-binding determinant in the 75-kD fragment, and (b) an antibody raised against the 33-kD fragment that binds specifically to that fragment. Initial cell attachment to, and neurite extension upon, fibronectin and the two different fragments was evaluated in the presence and absence of the two reagents. Attachment of both PNS and CNS cells to intact fibronectin was reduced in the presence of RGDS, the former more so than the latter. In contrast, the antibody to the 33-kD fragment did not affect attachment of PNS cells to fibronectin, but significantly decreased attachment of CNS cells to the molecule. RGDS inhibited attachment of CNS cells to the molecule. RGDS inhibited attachment of both cell types to the 75-kD fragment to a greater degree than it did attachment to the intact molecule. Cell interaction with the 33-kD fragment was not affected by RGDS. Reduction of neurite lengths (determined after 24 h of culture) by the domain-specific reagents paralleled the reduction in initial adhesion to each substratum. Therefore, it appears that (a) both PNS and CNS cells have receptors for each cell-binding domain of fibronectin, (b) the receptor(s) for the two domains are distinct, with attachment to the 33-kD fragment being independent of RGDS, and (c) the relative importance of each domain to cell interaction with intact fibronectin is different for CNS and PNS cells.     

Isolation and biological characterization of active fragments of the adhesive glycoprotein fibronectin

Cellular fibronectin is a major cell surface glycoprotein that can mediate the adhesion of cells to collagen in vitro. To analyze its mechanism of action, we have undertaken experiments to isolate fragments of fibroblast fibronectin that retain different active sites. In this paper, we describe the purification of three chymotryptic fragments with apparent molecular weights of 40,000, 160,000 and 205,000 from chicken cellular fibronectin. These fragments were electrophoretically pure and retained different biologically active sites, as determined by a series of bioassays and competitive inhibition experiments. The 40K fragment was identified as the collagen-binding fragment. The 160K fragment was found to contain the cell surface-binding site(s) of cellular fibronectin. The 205K fragment contained both collagen-binding and cell surface-binding sites, and apparently represents the sum of the 40K and 160K fragments. When native fibronectin is cleaved to the 205K fragment, a polypeptide region containing all interchain disulfide bonds is lost. This alteration was accompanied by decreased hemagglutinating activity and loss of the capacity to restore a normal morphology to transformed cells, whereas cell attachment to collagen and cell spreading activities remained. Our results directly support the idea that the fibronectin molecule consists of separate structural domains containing different biological characteristics.     

Location of a fibronectin domain involved in newt epidermal cell migration

The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33- and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxy-terminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using IB10, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which IB10 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.     

A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide

The active form of fibronectin is its extracellular matrix form, which allows for the attachment of cells and influences both the growth and migration of cells. The matrix form is assembled by cells; however, many cells are defective in this regard. Several regions within fibronectin have been shown to play a role in matrix assembly by cells. One such region has been localized into the first type III repeat of fibronectin (Chernousov, M. A., F. J. Fogerty, V. E. Koteliansky, and D. F. Mosher. J. Biol. Chem. 266:10851-10858). We have identified this site as a fibronectin-fibronectin binding site and reproduced it as a synthetic peptide. This site is contained in a 14-kD fragment that corresponds to portions of the first two type III repeats. The 14-kD fragment was found to bind to cell monolayers and to inhibit fibronectin matrix assembly. The 14-kD fragment only slightly reduced the binding of fibronectin to cell surfaces but it significantly inhibited the subsequent incorporation of fibronectin into the extracellular matrix. The 14-kD fragment also bound to purified fibronectin and inhibited fibronectin-fibronectin binding. A synthetic 31-amino acid peptide (P1) representing a segment of the 14-kD fragment retained the ability to inhibit fibronectin-fibronectin binding. Peptide P1 specifically bound fibronectin from plasma in affinity chromatography, whereas a column containing another peptide from the 14-kD fragment did not. These results define a fibronectin-fibronectin binding site that appears to promote matrix assembly by allowing the assembly of fibronectin molecules into nascent fibrils. The 14-kD fragment and the P1 peptide that contain this site inhibit matrix assembly by competing for the fibronectin-fibronectin binding.     

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.     

Functional mapping of cytotactin: proteolytic fragments active in cell- substrate adhesion

Cytotactin is an extracellular matrix glycoprotein with a restricted distribution during development. In electron microscopic images, it appears as a hexabrachion with six arms extending from a central core. Cytotactin binds to other extracellular matrix proteins including a chondroitin sulfate proteoglycan (CTB proteoglycan) and fibronectin. Although cytotactin binds to a variety of cells including fibroblasts and neurons, in some cases it causes cells in culture to round up and it inhibits their migration. To relate these various effects of cytotactin on cell behavior to its binding regions, we have examined its ability to support cell-substrate adhesion and have mapped its cell- binding function onto its structure. In a cell-substrate adhesion assay, fibroblasts bound to cytotactin but remained round. In contrast, they both attached and spread on fibronectin. Neither neurons nor glia bound to cytotactin in this assay. In an assay in which cell-substrate contact was initiated by centrifugation, however, neurons and glia bound well to cytotactin; this binding was blocked by specific anti- cytotactin antibodies. The results suggest that neurons and glia can bind to cytotactin-coated substrates and that these cells, like fibroblasts, possess cell surface ligands for cytotactin. After applying methods of limited proteolysis and fractionation, these assays were used to map the binding functions of cytotactin onto its structure. Fragments produced by limited proteolysis were fractionated into two major pools: one (fraction I) contained disulfide-linked oligomers of a 100-kD fragment and two minor related fragments, and the second (fraction II) contained monomeric 90- and 65-kD fragments. The 90- and 65-kD fragments in fraction II were closely related to each other and were structurally and immunologically distinct from the fragments in fraction I. Only components in fraction I were recognized by mAb M1, which binds to an epitope located in the proximal portion of the arms of the hexabrachion and by a polyclonal antibody prepared against a 75-kD CNBr fragment of intact cytotactin. A mAb (1D8) and a polyclonal antibody prepared against a 35-kD CNBr fragment of cytotactin only recognized components present in fraction II. In cell- binding experiments, fibroblasts, neurons, and glia each adhered to substrates coated with fraction II, but did not adhere to substrates coated with fraction I. Fab fragments of the antibody to the 35-kD CNBr fragment strongly inhibited the binding of cells to cytotactin, supporting the conclusion that fraction II contains a cell-binding region. In addition, Fab fragments of this antibody inhibited the binding of cytotactin to CTB pr     

Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.

Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.     

Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid

Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet- poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino- terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.     

Extracellular matrix assembly of cell-derived and plasma-derived fibronectins by substrate-attached fibroblasts

Using a previously described model system for the incorporation of plasma fibronectin into the extracellular matrix (McKeown-Longo, P.J. and Mosher, D.F., 1985. J. Cell Biol., 100:364-374), we compared the binding of cell-derived and plasma-derived fibronectins to human fibroblast cell layers. Binding was measured in time course experiments using metabolically labeled cell-derived, iodinated cell-derived, and iodinated plasma-derived fibronectins. The kinetics of matrix assembly of cell- and plasma-derived fibronectins were the same. Competitive binding curves using intact fibronectin or the 70-kD amino-terminal fragment of fibronectin suggested that cell surface binding sites have equal affinity for cell- and plasma-derived fibronectins. Iodinated fibronectins did not bind to isolated matrices containing collagen type I, fibronectin, and thrombospondin. These results suggest that fibroblasts do not distinguish between cell-derived and plasma-derived fibronectins when assembling exogenous fibronectin into extracellular matrix.     

Fibronectin regulates latent transforming growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding protein-1

Latent transforming growth factor-beta-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGF beta in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-beta (TGF beta) into the ECM of osteoblasts and fibroblasts. Immunolocalization studies suggested that fibronectin provides an initial scaffold that precedes and patterns LTBP1 deposition but that LTBP1 and fibronectin are later localized in separate fibrillar networks, suggesting that the initial template is lost. Treatment of fetal rat calvarial osteoblasts with a 70-kDa N-terminal fibronectin fragment that inhibits fibronectin assembly impaired incorporation of LTBP1 and TGFbeta into the ECM. Consistent with this, LTBP1 failed to assemble in embryonic fibroblasts that lack the gene for fibronectin. LTBP1 assembly was rescued by full-length fibronectin and superfibronectin, which are capable of assembly into fibronectin fibrils, but not by other fibronectin fragments, including a 160-kDa RGD-containing fragment that activates alpha5beta1 integrins. This suggests that the critical event for LTBP1 assembly is the formation of a fibronectin fibrillar network and that integrin ligation by fibronectin molecules alone is not sufficient. Not only was fibronectin essential for the initial incorporation of LTBP1 into the ECM, but the continued presence of fibronectin was required for the continued assembly of LTBP1. These studies highlight a nonredundant role for fibronectin in LTBP1 assembly into the ECM and suggest a novel role for fibronectin in regulation of TGF beta via LTBP1 interactions.     

Matrix assembly sites for exogenous fibronectin are decreased on human fibroblasts after treatment with agents which increase intracellular cAMP

Studies with cultured fibroblasts have shown that plasma as well as cellular fibronectin can be organized into fibrillar structures and that this organization is mediated by sites at the cell surface. Treatment of human skin fibroblasts with cholera toxin resulted in a prompt decrease in the number of binding sites for 125I-labeled plasma fibronectin and a 125I-labeled 70-kDa amino-terminal fragment of fibronectin. This decrease was accompanied by less incorporation of labeled fibronectin into deoxycholate-insoluble extracellular matrix. Binding of 125I-fibronectin was also decreased in cultures treated with epinephrine, isoproterenol, or forskolin. These results, therefore, indicate that G proteins and the adenylate cyclase system are involved in regulation of fibronectin matrix assembly sites may be one mechanism whereby hormones or growth factors can modify extracellular matrix characteristics.     

Identification of novel and distinct binding sites within tenascin-C for soluble and fibrillar fibronectin

Interactions between fibronectin and tenascin-C within the extracellular matrix provide specific environmental cues that dictate tissue structure and cell function. The major binding site for fibronectin lies within the fibronectin type III-like repeats (TNfn) of tenascin-C. Here, we systematically screened TNfn domains for their ability to bind to both soluble and fibrillar fibronectin. All TNfn domains containing the TNfn3 module interact with soluble fibronectin. However, TNfn domains bind differentially to fibrillar fibronectin. This distinct binding pattern is dictated by the fibrillar conformation of FN. TNfn1-3, but not TNfn3-5, binds to immature fibronectin fibrils, and additional TNfn domains are required for binding to mature fibrils. Multiple binding sites for distinct regions of fibronectin exist within tenascin-C. TNfn domains comprise a binding site for the N-terminal 70-kDa domain of fibronectin that is freely available and a binding site for the central binding domain of fibronectin that is cryptic in full-length tenascin-C. The 70-kDa and central binding domain regions are key for fibronectin matrix assembly; accordingly, binding of several TNfn domains to these regions inhibits fibronectin fibrillogenesis. These data highlight the complexity of protein-protein binding, the importance of protein conformation on these interactions, and the implications for the physiological assembly of complex three-dimensional matrices.     

Biochemical characterization of the fibronectin binding sites for IgG

Plasma fibronectin (Fn) is a constituent of cryoglobulins and has been shown to interact with immune complexes. In a previous report we demonstrated that Fn specifically bound to IgG immobilized on a solid matrix. To localize and biochemically characterize the sites on the Fn molecule involved in this interaction, Fn was enzymatically cleaved with subtilisin and subjected to IgG affinity chromatography. Three major polypeptide fragments of 16 kDa, 22 kDa, and a triplet of 26- to 29-kDa bound IgG. They were localized to three separate regions of the molecule by Western blot analysis using antisera to specific regions of the Fn molecule, by amino acid sequencing, and by their previously described heparin binding affinities. The 22-kDa fragment interacted with IgG under physiologic conditions and it is localized at the N-terminal of the Fn molecule. The 16-kDa and 26- to 29-kDa fragments bound to IgG under conditions of lower ionic strength; the former commences at residue 588, carboxyl-terminal to the collagen binding region and the latter begins at residue 1597, carboxyl-terminal to the cell binding domain. The interaction of Fn with Ig has significant implications in host defense and also in immune complex disease where basement membrane Fn may sequester immune complexes from the circulation.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.