Characterization of the interactions of an amino-terminal fibronectin fragment with the native molecule: Implications for polymerization of fibronectin

Plasma fibronectin is a 450-kD glycoprotein composed of two similar subunits connected by interchain disulfide bridges that may fold over in solution, allowing the amino terminus of each chain to bind the carboxyl terminus of the same subunit or a different subunit, thereby allowing polymerization. In order to study the characteristics of the fold-over interaction, the interaction between the amino terminal 29-kD fragment of fibronectin with native fibronectin has been studied in detail. One 29-kD molecule bound per fibronectin subunit, the apparent equilibrium dissociation constant was 40 nM, and the half-times for association and dissociation at 22°C were, respectively, 16 h and 23 days. Complexation could be inhibited by high concentrations of salt, but not by 8M urea. Amino terminal 20-kD and carboxyl terminal 8-kD subfragments of the 29-kD fragment also bound fibronectin and the activity was dependent on the integrity of the type 1 loop structures. The kinetics of the interaction of 29-kD fragment with fibronectin were unaffected by the presence of ligands, but were affected by detergents such as sodium dodecyl sulfate or deoxycholate, which enhanced the rate of interaction over 100-fold or 6-fold, respectively. Therefore, the interaction of fibronectin with ionic cell membrane components such as deoxycholate in vivo may trigger polymerization.     

Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.     

Interaction of fibronectin with its receptor on platelets

We report that the 12,000 dalton domain of fibronectin that interacts with fibroblast cell surfaces also binds specifically to thrombin-inducible, saturable receptors on platelets. Furthermore, we have used chemical cross-linking and monoclonal antibodies to show that the 12,000 dalton domain of fibronectin interacts directly with glycoprotein IIIa at the platelet cell surface. Both binding and cross-linking of this domain to platelets are competed by a hexapeptide previously shown to block fibroblast adhesion to fibronectin. Finally, we show that a complex of the platelet glycoproteins IIIa and IIb binds to affinity columns of a cell-attachment fragment of fibronectin. These results localize a major fibronectin-platelet interaction to a specific domain of fibronectin and to a specific platelet glycoprotein.     

Interaction of gelatin with a fluorescein-labeled 42-kDa chymotryptic fragment of fibronectin

A 42-kDa gelatin binding fragment of human plasma fibronectin was labeled with fluorescein and its fluid-phase interaction with gelatin was investigated. At 25 degrees C in 0.1 M Tris, 0.15 M NaCl, pH 7.3, a dissociation constant, Kd = 0.6 microM, was obtained from the dependence of fluorescence polarization on gelatin concentration. An identical value was obtained for the unlabeled fragment by competition. Binding was unaffected by higher concentrations of NaCl up to 1.0 M, but increased as much as 20-fold at low ionic strength. The dependence of Kd on temperature revealed that dissociation of the complex is accompanied by an increase in entropy. Thus, the interaction is not dominated by either hydrophobic or electrostatic forces; an important role for hydrogen binding is proposed.     

An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor.

Retrovirus entry into cells is mediated by specific binding of the envelope glycoprotein to a cell membrane receptor. Constitutive envelope gene expression prevents infection by interfering with the binding of viruses which recognize the same receptor. We have used this property to investigate the receptor binding capacities of deleted or truncated murine leukemia virus ecotropic envelope glycoproteins. Friend murine leukemia virus envelope glycoproteins bearing internal amino-terminal deletions, or a soluble 245-amino-acid gp70 amino-terminal fragment, were expressed in NIH 3T3 cells. The susceptibility of these cells to ecotropic and amphotropic virus infection was determined. We observed that both membrane-bound and soluble forms of the gp70 245-amino-acid amino-terminal domain induced resistance to ecotropic virus, indicating that this fragment binds the ecotropic receptor. Binding occurs both at the cell surface and in the endoplasmic reticulum, as shown by the use of soluble envelope fragments either secreted in the culture supernatants or retained in the endoplasmic reticulum lumen by a KDEL sequence. These results suggest that the gp70 amino-terminal domain folds into a structure which recognizes the ecotropic receptor regardless of the carboxy-terminal part of the molecule.     

Interaction of a fragment of the cannabinoid CB1 receptor C-terminus with arrestin-2

Desensitization of the cannabinoid CB1 receptor is mediated by the interaction with arrestin. In this study, we report the structural changes of a synthetic diphosphorylated peptide corresponding to residues 419-439 of the CB1 C-terminus upon binding to arrestin-2. This segment is pivotal to the desensitization of CB1. Using high-resolution proton NMR, we observe two helical segments in the bound peptide that are separated by the presence a glycine residue. The binding we observe is with a diphoshorylated peptide, whereas a previous study reported binding of a highly phosphorylated rhodopsin fragment to visual arrestin. The arrestin bound conformations of the peptides are compared.     

Interaction of heparin with fibronectin and isolated fibronectin domains.

Fluorescence polarization, gel exclusion chromatography and affinity chromatography were used to characterize the interaction of heparins of different size with human plasma fibronectin (Fn) and several of its isolated domains. The fluid-phase interaction of Fn with heparin was dominated by the 30 kDa and 40 kDa Hep-2 domains located near the C-terminal ends of the A and B chains respectively. The 30 kDa Hep-2A domain from the heavy chain was indistinguishable from the 40 kDa Hep-2B domain in this respect; the presence of an additional type III homology unit in the latter had no effect on the binding. Evidence was provided that each Hep-2 domain has two binding sites for heparin. The N-terminal Hep-1 domain reacted weakly in fluid phase even though it binds strongly to immobilized heparin. Fn and Hep-2 fragments were rather undiscriminating in their reaction with fluoresceinamine-labelled heparins of different sizes. However, oligosaccharides smaller than the tetradecasaccharide (14-mer) bound Fn with a 5-10-fold lower affinity. These results suggest that the Hep-2 domains of Fn are able to recognize a broad spectrum of oligosaccharides that presumably vary significantly with respect to the amount and spatial distribution of charge.     

Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.     

Augmentation of phagocytosis by a specific fibronectin fragment that links particulate activators to the fibronectin adherence receptor of human monocytes

Intact human plasma fibronectin of 44,000 m.w. and a fibronectin fragment of 180,000 m.w. promote dose-dependent adherence of gelatin-coated particles to human monocytes without phagocytosis. Both of these proteins, however, augment monocyte ingestion of gelatin-coated targets that are particulate activators of the alternative complement pathway or of nonactivators bearing IgG. Unlike intact fibronectin, the 180,000 m.w. fragment also binds directly to particulate activators that lack gelatin to augment their phagocytosis by human monocytes. Prior attachment to monocytes of gelatin-coated sheep erythrocytes bearing increasing concentrations of intact fibronectin decreases in a dose-dependent fashion the capacity of these monocytes to engage in augmented phagocytosis of particulate activators opsonized with the 180,000 m.w. fibronectin. Occupation of the monocyte fibronectin receptors with particle-bound, intact fibronectin does not decrease monocyte phagocytosis of plain particulate activators or of IgG-coated particles. Thus, the 180,000 m.w. fibronectin fragment both directly opsonizes particulate activators and interacts with monocyte fibronectin receptors to promote particle adherence, thereby enhancing phagocytosis through a concerted action with the distinct receptors for particulate activators.     

Interaction of swine lipoproteins with the low density lipoprotein receptor in human fibroblasts.

HDLc, a cholesterol-rich lipoprotein that accumulates in the plasma of cholesterol-fed swine, was shown to resemble functionally human and swine low density lipoprotein in its ability to bind to the low density lipoprotein receptor in monolayers of cultured human fibroblasts. This binding occurred even though HDLc lacked detectable apoprotein B, which is the major protein of low density lipoprotein. After it was bound to the low density lipoprotein receptor, HDLc, like human and swine low density lipoprotein, delivered its cholesterol to the cells, and this, in turn, caused a suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, an activation of the cholesterol-esterifying system, and a net accumulation of free and esterified cholesterol within the cells. Swine HDLc, like human high density lipoprotein, did not bind to the low density lipoprotein receptor nor did it elicit any of the subsequent metabolic events. HDLc, like human low density lipoprotein, was incapable of producing a metabolic effect in fibroblasts derived from a subject with the homozygous form of familial hypercholesterolemia, which lack low density lipoprotein receptors. These results indicate that two lipoproteins that have been associated with athersclerosis--low density lipoprotein in humans and HDLc in cholesterol-fed swine--both can cause the accumulation of cholesterol and cholesteryl esters within cells through an interaction with the low density lipoprotein receptor.     

Real-time monitoring of hematopoietic cell interaction with fibronectin fragment

Real-time cell analysis (RTCA) system based on measurement of electrical microimpedance has been introduced to monitor adherent cell cultures. We describe its use for real-time analysis of hematopoietic cell adhesion to bone marrow stroma proteins. Cells growing in suspension do not generate any significant change in the microimpedance signal until the surface with embedded microelectrodes is coated with a cell-binding protein. We show that in this case, the microimpedance signal specifically reflects cell binding to the coated surface. The optimized method was used to monitor the effect of two histone deacetylase inhibitors, suberoylanilide hydroxamic acid (SAHA) and tubastatin A, on JURL-MK1 cell adhesion to cell-binding fragment of fibronectin (FNF). Both compounds were used in non-toxic concentrations and induced an increase in the cell adhesivity. The kinetics of this increase was markedly slower for SAHA although tubulin hyperacetylation occurred rapidly for any of the two drugs. The strengthening of cell binding to FNF was paralleled with a decrease of Lyn kinase activity monitored using an anti-phospho-Src family antibody. The inhibition of Src kinase activity with PP2 accordingly enhanced JURL-MK1 cell interaction with FNF. Actin filaments were present at the proximity of the plasma membrane and in numerous membrane protrusions. In some cells, F-actin formed clusters at membrane regions interacting with the coated surface and these clusters colocalized with active Lyn kinase. Our results indicate that the role of Src kinases in the regulation of hematopoetic cell adhesion signaling is similar to that of c-Src in adherent cells.     

Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts

Transforming growth factors of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung fibroblasts (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to 20-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.     

Interaction of enzymatically modified low-density lipoproteins with fibronectin

Interaction of human low-density lipoproteins (LDL) with homologous fibronectin fixed on collagen-Sepharose was studied. LDL were digested with pepsin, the degree of hydrolysis amounting to 10%. Upon passing modified LDL through a fibronectin-collagen-Sepharose column the desorption of fibronectin occurred. Addition of the increasing amount of fibronectin to the pepsin-treated LDL solution in the presence of Ca2+ ions led to the formation of LDL-fibronectin insoluble complexes. Interaction of native LDL with fibronectin was not observed. The data suggest that enzymatic modification of LDL increasing interaction of modified LDL with fibronectin, a component of extracellular matrix, could promote the accumulation of such LDL in arterial walls.     

Surface activation of the cell adhesion fragment of fibronectin

One of the earliest events in the adhesion of fibroblasts to a substratum is the recognition by the cells of macromolecular adhesive factors, such as fibronectin. This early event is followed by a complex series of cell alterations leading to adhesion and spreading. To identify cell surface components involved in the initial cell-fibronectin recognition step, we have employed an assay involving latex particles coated with radiolabelled plasma Fibronectin (Fn). In previous studies from this laboratory (Harper & Juliano , J cell biol 87 (1980) 755) [28], we demonstrated that Fn-mediated adhesion of CHO cells is temperature-dependent, cation-dependent and sensitive to cytoskeletal disrupting agents; by contrast, binding of 3H-Fn beads was unaffected by these factors, indicating that this process reflects binding and recognition events at the cell surface which are independent of cytoskeletal and metabolic activity. Biological specificity of 3H-Fn bead-to-cell binding was confirmed by the ability of anti-Fn antisera to completely block the process. To examine surface components which may mediate binding we treated Fn beads with purified glycosaminoglycans (GAGs) or glycolipids prior to incubation with cells. Among the GAGs tested, heparin, heparan sulfate and dermatan sulfate blocked bead binding in a dose-related fashion with heparin being most potent. The gangliosides GT1, and GM1, also inhibited bead binding. However, treatment of cells with neuraminidase had no effect on bead binding while subsequent analysis of treated cells by thin layer chromatography revealed a drastic reduction in the amount of GM3, the predominant CHO cell ganglioside. CHO cells were also incubated with a panel of proteolytic enzymes to study the possible role of cell surface proteins or glycoproteins in Fn bead binding. We found 3H-Fn bead binding to be quite sensitive to pretreatment with thermolysin, pronase, and papain but only moderately sensitive to treatment with trypsin. From our findings we suggest: (1) binding of Fn beads to CHO cells reflects an early step in the adhesion process; (2) glycolipids may block bead binding but are probably not the endogenous binding site for Fn; (3) protease sensitive components (glycoproteins or proteoglycans) may be more likely candidates as cell surface-binding sites for Fn.     

Arsenite-induced changes in methylation of the 70,000 dalton heat shock proteins in chicken embryo fibroblasts

Methylated lysyl and arginyl residues are present in the two major heat shock proteins, hsp70A and hsp70B, of chicken embryo fibroblasts. Here, we demonstrate that this methylation can be modulated by sodium arsenite, a chemical that increases the synthesis of hsp70. In particular, in hsp70A the amount of epsilon-N-trimethyl-lysine significantly decreases and the amount of epsilon-N-dimethyl-lysine and epsilon-N-monomethyl-lysine increases, while in hsp70B, the quantity of NG-monomethyl-arginine is reduced fivefold after arsenite treatment. To determine the specificity of these changes in methylation the pool size of S-adenosyl-L-methionine (AdoMet) and the total cellular level of methylated protein was measured. After arsenite treatment, no significant change in AdoMet pool size and the level of protein methylation was observed with the exception of an apparent increase in NG-monomethyl-arginine in total cellular protein. Thus, the arsenite-induced changes in methylation of hsp70 polypeptides are not a generalized phenomenon and may reflect a modulation in the structure or function of these two polypeptides after their induced synthesis by this chemical.     

Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers. Cross-linking of cellular and plasma fibronectin and of amino-terminal fibronectin fragments

Factor XIIIa cross-links plasma fibronectin as it is being assembled into the extracellular matrix of cultured human skin fibroblasts (Barry, E. L. R., and Mosher, D. F. (1988) J. Biol. Chem. 262, 10464-10469). We have further characterized this process. Fibroblasts were metabolically labeled with proline in the presence or absence of ascorbate and Factor XIIIa. Endogenous fibronectin in the extracellular matrix was cross-linked by Factor XIIIa. There was no evidence for cross-linking of collagenous proteins. Fibro-blast cell layers were incubated with iodinated 27-kDa heparin-binding or 70-kDa collagen- and heparin-binding amino-terminal fibronectin fragments. Factor XIIa cross-linked the fragments into high molecular weight aggregates. The amounts of cross-linked fragments reaches a steady state after 1 to 2 h, whereas intact fibronectin continues to be cross-linked for 24 h. When fibroblast cell layers were pulsed with iodinated fibronectin or amino-terminal fragments and Factor XIIIa was included in the chase media, the high molecular weight aggregates were formed in a step-wise manner. The smallest cross-linking steps were to high molecular weight extracellular matrix molecules forming approximately 270-, 300-, and 440-kDa complexes for the 27-kDa fragment, 70-kDa fragment, and intact fibronectin, respectively. When iodinated fibronectin was bound to fibroblast cell layers and chased into the matrix pool in the absence of Factor XIIIa, it could also be cross-linked into high molecular weight complexes when Factor XIIIa was added to the media. These results, therefore, indicate that both cellular and plasma fibronectin and amino-terminal fragments are cross-linked specifically by Factor XIIIa, that the cross-linking is probably to other fibronectin molecules rather than to collagenous proteins, and that both assembling and assembled fibronectin are substrates for Factor XIIIa.