Impact of heparin-binding domain of recombinant human osteocalcin-fibronectinIII9-14 on the osteoblastic cell response

Fibronectin (FN) containing a heparin-binding domain (HBD) and an Arg-Gly-Asp (RGD) domain can promote cell adhesion and proliferation compared to FN that contained only RGD. Here, we have engineered recombinant human osteocalcin (rhOC) with FN type III9-14 (rhOC-FNIII9-14) containing RGD and HBD to promote the cellular activity of MC3T3-E1 cells, including adhesion, proliferation, and differentiation. RhOC-FNIII9-14 significantly increased cell adhesion and proliferation of MC3T3-E1 cells compared to rhOC-FNIII9-10 (P < 0.05). Moreover, rhOC-FNIII9-14 showed osteogenic differentiation of MC3T3-E1 cells in mineralization activity and osteogenic gene expression.     

Fibronectin fragment promotes osteoblast-associated gene expression and biological activity of human osteoblast-like cell

A fibronectin fragment, which spans from the ninth to tenth type III domain, showed > 95% biological activity in promoting osteoblast adhesion, proliferation and differentiation level compared with those of native fibronectin (FN) molecule. This suggests that the FN type III ninth-tenth domain fragment serves a good substitute for native FN in biomimetic application for osteogenesis.     

Adhesion of lymphoid cells to the carboxyl-terminal heparin-binding domains of fibronectin

Previously, we have shown that some lymphoid cell lines adhere to fibronectin (FN)-coated substratum, whereas others do not. In this study, the adhesion of five adherent lymphoid cell lines to different FN domains was examined. These cell lines ranged in their adherence to substratum coated with FN, the cell-binding domain (CBD) fragment, or the heparin-binding domain (HBD) fragments. None of the cell lines adhered to substratum coated with the gelatin-binding domain fragment. Three of the lymphoid cell lines adhered preferentially to HBD over CBD, whereas two other lymphoid cell lines and BHK fibroblasts adhered preferentially to CBD. These results suggest that two distinct adhesive interactions occur between cells and FN and that the pattern of interaction varies among cell types. Using MOPC 315 (which adheres preferentially to HBD) as a cell model to study the cell-HBD interaction, the HBD-promoted adhesion was found to be independent of the RGD sequence and could be inhibited by anti-FN antibodies. Moreover, the MOPC 315-HBD interaction had the following characteristics: (1) adhesion was temperature dependent, (2) presence of divalent cations was necessary, (3) integrity of cellular microfilaments but not microtubules was required, (4) inhibition of protein synthesis abolished adhesion, (5) pretreatment of cells with trypsin inhibited adhesion, and (6) the adhesion was mediated by the carboxyl-terminal HBD.     

SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis

Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B β-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid “multimerization sequence” (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and β-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated β-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.     

The role of the ninth and tenth type III domains of human fibronectin in cell adhesion

Fibronectins (FN) contain sites, in addition to the cell recognition site RGD in the tenth type III domain (FIII10), that are required for adhesive activity. The role of FIII10 and the adjacent FIII9 was analysed in functional cell adhesion assays recombinant FIII domains in which the domain boundaries were strictly conserved. FIII9 had no adhesive activity. FIII10, and FIII9 plus FIII10 had less activity than FN, whereas the activity of FIII9-10 was similar to FN. We conclude that FIII9 acts synergistically with FIII10 in cell adhesion, and that this synergy is dependent upon the structural integrity of the FIII9-10 pair of domains.     

Modulation of Cell-adhesive Activity of Fibronectin by the Alternatively Spliced EDA Segment

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA⁺ FN was significantly more potent than EDA⁻ FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA⁺ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin α5 and β1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin α5β1. In support of this conclusion, purified integrin α5β1 bound more avidly to EDA⁺ FN than to EDA⁻ FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA⁺ FN and EDA⁻ FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin α5β1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180° at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III₁₀ module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.     

Fibronectin enhances viability and alters cytoskeletal functions (with effects on the phosphatidylinositol 3-kinase pathway) in small cell lung cancer

Small cell lung cancer (SCLC) is a rapidly progressive disease with ultimate poor outcome. SCLC has been shown to interact closely with the stromal and extracellular matrix (ECM) components of the diseased host. ECM consists of type I/IV collagen, laminin, vitronectin, and fibronectin (FN) among others. Herein, we investigated the behavior of a SCLC cell line (NCI-H446) on FN-coated surface. Over a course of 72 h, FN (10 micro g/ml) caused both increased survival and proliferation of NCI-H446 cells. Survival under serum-starved conditions increased 1.44-fold and proliferation in the presence of fetal calf serum increased by 1.30-fold. The phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 reduced both survival and proliferation of NCI-H446 cells (0.48- and 0.27-fold, respectively), even on FN-coated surface. We next determined the effects of FN on cytoskeletal function such as cell motility/morphology and adhesion. Over a course of 24 h, FN reduced aggregation of NCI-H446 cells and induced flattened cellular morphology with neurite-like projections after 1 h, however, in the presence of LY294002, the cells rounded up. Adhesion of NCI-H446 cells also increased with FN (4.47-fold) which was abrogated with LY294002 treatment. This correlated with phosphorylation of the cytoskeletal protein p125FAK, on Tyr397, Tyr861 and Ser843 residues with FN. Even in the presence of LY294002, these serine/tyrosine residues were still phosphorylated on FN-coated surface. In contrast, the focal adhesion protein paxillin was not phosphorylated at Tyr31 with FN. In summary, FN stimulation of SCLC cells leads to enhancement of viability and changes in cytoskeletal function that are partially mediated through the PI3-K pathway.     

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.     

Fibronectin matrix composition and organization can regulate cell migration during amphibian development

Fibronectin (FN) is an adhesive extracellular matrix component that is essential for vertebrate development. It forms a fibrillar matrix at the cell surface which controls cell morphology, migration, proliferation, and other important cellular processes. To address specific functions of FN matrix structure during early vertebrate development, we introduced normal and mutant recombinant FNs (recFNs) into the blastocoel cavity of embryos of the amphibian Pleurodeles waltl. Here we show that a native recFN FN(A-B-) as well as recFNs with specific mutations in the cell-binding domain, FN(RGD-) and FN(syn-), or in a FN-binding region, FNDeltaIII(1), are assembled into fibrillar matrix. A recFN (FNDeltaIII(1-7)) that forms a structurally distinct matrix in cultured cells was assembled into aggregates at the cell periphery and was able to inhibit assembly of endogenous amphibian FN matrix in a dose-dependent manner. Cell adhesion, spreading, and migration were perturbed in vitro and in vivo on chimeric matrices containing FN(RGD-), FN(syn-), or FNDeltaIII(1-7) co-assembled with amphibian FN. Developmentally, this perturbation resulted in defects in mesoderm patterning and inhibition of gastrulation. These results indicate that FN matrix fibrillar structure and composition are important determinants of cell adhesion and migration during development.     

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro⁵¹⁰-Tyr⁵¹¹-Ser⁵¹² (PYS) and Gln⁵¹⁶-Val⁵¹⁷-Gln⁵¹⁸ (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, and replacing the α-helix or QVQ shifts polysialic acid to FN1 O-glycans in full-length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, a homologous but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro⁵¹⁰-Tyr⁵¹¹ eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM- olfactory cell adhesion molecule chimera, and inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 α-helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.     

A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.     

Fibronectin and proteolytic fragments of fibronectin interfere with the adhesion of Staphylococcus epidermidis to plastic

W.M. DUNNE AND E.M. BURD. 1993. The adhesion of five strains of slime-positive Staphylococcus epidermidis to plastic microwells was significantly diminished ( P < 0.005) in a concentration-dependent fashion when wells were previously coated with increasing concentrations (1.6–13.1 μg cm^-2) of human fibronectin (FN). The adhesion of four of five strains was significantly reduced when wells were coated with 3.2 μg cm^-2 of FN and at concentrations ≥6.5 μg cm^-2 the adhesion of all slime-positive strains was significantly reduced. The coating of microwells with chymotryptic fragments of FN containing the heparin-binding, gelatin-binding, or cell-binding domains also reduced bacterial adhesion but none of the fragments exceeded the anti-adhesive activity of intact FN. A comparison of FN-coated or albumin-coated microwells showed that both proteins caused a significant reduction in the adhesion of test strains to plastic but that the anti-adhesive activity of FN was greater than albumin at all concentrations tested. The adhesion of the slime-negative phase variant of one of the test strains to plastic was neither enhanced nor reduced by FN coating indicating that the production of an exopolysaccharide by Staph. epidermidis influences interactions with protein-coated surfaces. These results support the contention that FN does not mediate the adhesion of all strains of Staph. epidermidis to plastic surfaces.     

Fibronectin promotes cervical cancer tumorigenesis through activating FAK signaling pathway

Cervical cancer is a cancer arising from the cervix, and it is the fourth most common cause of death in women. Overexpression of fibronectin 1 (FN1) was observed in many tumors and associated with the survival and metastasis of cancer cells. However, the mechanism by which FN1 promotes cervical cancer cell viability, migration, adhesion, and invasion, and inhibits cell apoptosis through focal adhesion kinase (FAK) signaling pathway remains to be investigated. Our results demonstrated that FN1 was upregulated in patients with cervical cancer and higher FN1 expression correlated with a poor prognosis for patients with cervical cancer. FN1 knockdown by small interfering RNA (siRNA) inhibited SiHa cell viability, migration, invasion, and adhesion, and promoted cell apoptosis. FN1 overexpression in CaSki cell promoted cell viability, migration, invasion, and adhesion, and inhibited cell apoptosis. Further, phosphorylation of FAK, a main downstream signaling molecule of FN1, and the protein expression of Bcl-2/Bax, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), and N-cadherin was upregulated in CaSki cells with FN1 overexpression, but caspase-3 protein expression was downregulated. The FAK phosphorylation inhibitor PF573228 inhibited FN1 overexpression-induced expression of those proteins in CaSki cells with FN1 overexpression. In vivo experiment demonstrated that FN1 knockdown significantly inhibited FN1 expression, phosphorylation of FAK, and tumor growth in xenograft from the nude mice. These results suggest that FN1 regulates the viability, apoptosis, migration, invasion, and adhesion of cervical cancer cells through the FAK signaling pathway and is a potential therapeutic target in the treatment of cervical cancer.     

A Peptide Motif from the Second Fibronectin Module of the Neural Cell Adhesion Molecule,NCAM, NLIKQDDGGSPIRHY,is a Binding Site for the FGF Receptor

The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analyses been demonstrated to be involved in NCAM–FGFR interactions. An FGFR activation motif (FRM) in the first NCAM FN3 module also has been suggested to take part in NCAM interactions with FGFR. Here, we show for the first time that a peptide motif in the second NCAM FN3 module, different from the previously described FGL motif (NLIKQDDGGSPIRHY; termed BCL) binds and activates FGFR and induces FGFR-dependent neurite outgrowth in cultures of cerebellar granule neurons. Our results provide evidence that the BCL motif is one of the multiple FGFR binding sites in NCAM. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA⁺). Here, we investigated if the FN EDA⁺ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA^-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.     

材料表面化学刺激对纤连蛋白构象及细胞的影响

生物材料的表面化学性质通过改变吸附蛋白的构象,影响细胞粘附铺展,产生不同的细胞行为.采用金-硫自组装单分子层技术(alkanethiol self-assembled monolayers,SAMs)构建了不同化学基团(-NH2、-COOH、-OH和-CH3)修饰的基底材料表面.运用X射线光能质谱(XPS)和接触角仪表...     

Fibronectin type III domain based monobody with high avidity

Significant efforts have been made to improve the target-binding strength of numerous affinity molecules that are widely used in biomedical research and clinical applications. While many antibody-like fragments have been successfully optimized through affinity maturation, the process is time-consuming and restricted by the stability, solubility, and expression level of the protein sequences. To generate a fibronectin type III domain (FN3) based monobody that binds to the tumor-related biomarkers with exceptional high strength and stability, we developed a multivalent strategy by fusing an alphavbeta3-binding FN3 monobody with a short COMP pentamerization domain through a linker that facilitates appropriate display of multivalent FN3 domains. The fusion protein was highly expressed in the soluble fraction of Escherichia coli and efficiently self-assembled into a pentameric molecule that could be readily purified. Compared to the monomeric form, the pentameric monobody bound to alphavbeta3 integrin much more tightly with significantly slower off-rate, while still maintaining its excellent specificity toward alphavbeta3 when tested by using purified integrins and integrin-expressing cell lines. The multivalent strategy we describe here could be applied to engineer other FN3 monobodies to acquire significantly improved targeting-binding strengths.     

Fibronectin augments anti-CD3-mediated IL-2 receptor (CD25) expression on human peripheral blood lymphocytes.

The extracellular matrix (ECM) is composed of a number of macromolecules that promote cell adhesion, cell migration, and differentiation. Receptors for these molecules have been identified and belong to a superfamily of cell surface proteins, collectively known as the integrins. In this study, we show that the matrix protein fibronectin (FN) acts synergistically with immobilized anti-CD3 antibody to promote proliferation of total human peripheral blood lymphocytes (HPBL) in the absence of exogenous IL-2. Proliferation was inhibited by both the alpha 5 beta 1 and alpha 4 beta 1 recognition peptides. ARG-GLY-ASP (RGD), and GLU-ILE-LEU-ASP-VAL-PRO-SER-THR (EILDVPST), respectively. Expression of CD25 (IL-2 receptor) was significantly higher on cells cultured on anti-CD3 and FN, indicative of T-cell activation. Additionally, cells cultured on immobilized anti-CD3 and FN for 3 days showed increased adhesion to FN and increased forward light scatter/side scatter profile. Synthesis of both IL-1 and to a lesser extent IL-2 was elevated in supernatants from cultures containing both anti-CD3 and FN. These data are consistent with published reports which demonstrate that ECM proteins can act as costimulants of lymphocyte proliferation. Finally, our results show that cells cultured on anti-CD3 antibody and FN have an activated phenotype and that cytokines may be involved in this process.     

Guidance of Mesenchymal Stem Cells on Fibronectin Structured Hydrogel Films

Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.     

Fibronectin and heparin binding domains of latent TGF-beta binding protein (LTBP)-4 mediate matrix targeting and cell adhesion

Latent transforming growth factor (TGF)-β binding proteins are extracellular matrix (ECM) proteins involved in the regulation of TGF-β sequestration and activation. In this study, we have identified binding domains in LTBP-4, which mediate matrix targeting and cell adhesion. LTBP-4 was found to possess heparin binding activity, especially in its N-terminal region. The C-terminal domain of LTBP-4 supported fibroblast adhesion, a property reduced by soluble heparin. In addition, we found that LTBP-4 binds directly to fibronectin (FN), which was indispensable for the matrix assembly of LTBP-4. The FN binding sites were also located in the N-terminal region. Interestingly, heparin was able to reduce the binding of LTBP-4 to FN. In fibroblast cultures, LTBP-4 colocalized first with FN and subsequently with fibrillin-1, pointing to a role for FN in the early assembly of LTBP-4. In FN −/− fibroblasts, LTBP-mediated ECM targeting was disturbed, resulting in increased TGF-β activity. These results revealed new molecular interactions which are evidently important for the ECM targeting, but which also are evidence of novel functions for LTBP-4 as an adhesion molecule.