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

Mostafavi‐Pour, Zohreh; Askari, Janet A.; Whittard, John D.; Humphries, Martin J.

doi:10.1016/s0945-053x(00)00131-1

Cited by 63 publications

(60 citation statements)

References 40 publications

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“…Several extracellular matrix proteins, including fibronectin, collagen, thrombospondin, laminin, and tenascin, interact with cell surface proteoglycans and are thought to modulate cell adhesion through this interaction (2,21,33,45,50,82). In the present study, we have shown that vitronectin also supports proteoglycan dependent cell adhesion.…”

Section: Discussionsupporting

confidence: 56%

Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

Wilkins-Port

Sanderson²,

Tominna-Sebald³

et al. 2003

Cell Communication & Adhesion

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During the process of tissue remodeling, vitronectin (Vn) is deposited in the extracellular matrix where it plays a key role in the regulation of pericellular proteolysis and cell motility. In previous studies we have shown that extracellular levels of vitronectin are controlled by receptormediated endocytosis and that this process is dependent upon vitronectin binding to sulfated proteoglycans. We have now identified vitronectin's 12 amino acid "basic domain" which is contained within the larger 40 amino acid heparin binding domain, as a syndecan binding site. Recombinant vitronectins representing wild type vitronectin (rVn) and vitronectin with the basic domain deleted (rVn 347-358) were prepared in a baculoviral expression system. The rVn as well as a glutathione S-transferase (GST) fusion protein, consisting of vitronectin's 40 amino acid heparin binding domain (GST-VnHBD), exhibited dose dependent binding to HT-1080 cell surfaces, which was attenuated following deletion of the basic domain. In addition, GST-VnHBD supported both HT-1080 and dermal fibroblast cell adhesion, which was also dependent upon the basic domain. Similarly, ARH-77 cells transfected with syndecans -1, -2, or -4, but not Glypican-1, adhered to GST-VnHBD coated wells, while adhesion of these same cells was lost following deletion of the basic domain. HT-1080 cells were unable to degrade rVn 347-358. Degradation of rVn 347-358 was completely recovered in the presence of GST-VnHBD but not in the presence of GST-VnHBD 347-358. These results indicate that turnover of soluble vitronectin requires ligation of vitronectin's basic domain and that this binding event can work in trans to regulate vitronectin degradation.

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Section: Discussionsupporting

confidence: 56%

Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

Wilkins-Port

Sanderson²,

Tominna-Sebald³

et al. 2003

Cell Communication & Adhesion

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“…A rabbit polyclonal antibody against a peptide in rat LTBP1 was also used, which recognizes mouse and rat but not human LTBP1 (18). Fibronectin antibodies included a mouse monoclonal antibody (IgM) against cellular fibronectin (Sigma), a mouse monoclonal antibody (IgG) against the human fibronectin ED-A domain (Harlan), a mouse monoclonal antibody IgG (39B6) directed against type II repeats 12-14 of human fibronectin (30), and a rabbit polyclonal antibody against fibronectin purified from human platelets (Neomarkers, Freemont, CA).…”

Section: Methodsmentioning

confidence: 99%

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Dallas

Sivakumar

Jones

et al. 2005

Journal of Biological Chemistry

278

233

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Latent transforming growth factor-␤-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGF␤ in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-␤ (TGF␤) 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 TGF␤ 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 ␣5␤1 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␤ via LTBP1 interactions.

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“…Briefly, the TG-FN matrix was prepared by incubating 20 g/ml gplTG in a 96-well plate precoated with 5 g/ml FN as optimized previously (7,22). In order to block the heparin binding sites within FN (30) or TG2 molecules (31), 300 g/ml heparin in 50 mM Tris-HCl, pH 7.4, was used before and after the immobilization of gplTG as described previously (22). In certain experiments, the cells were pretreated with specific antibodies, inhibitors, or peptides for 30 min to 1 h. For inhibition of ␣4 integrinmediated cell adhesion, cells were pretreated with anti-␣4 integrin antibody 9C10 for 1 h in the presence of a 100 g/ml concentration of either GRADSP or GRGDTP peptide.…”

Section: Establishment Of the Tg2-transfected Mef (Tg2-mef) Cell Linementioning

confidence: 99%

RGD-independent Cell Adhesion via a Tissue Transglutaminase-Fibronectin Matrix Promotes Fibronectin Fibril Deposition and Requires Syndecan-4/2 and α5β1 Integrin Co-signaling

Wang

Collighan

Gross

et al. 2010

Journal of Biological Chemistry

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Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, weshow that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and ␤1 integrin co-signaling pathway. By using ␣5 null cells, ␤1 integrin functional blocking antibody, and a ␣5␤1 integrin targeting peptide A5-1, we demonstrate that the ␣5 and ␤1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKC␣ is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains. Fibronectin (FN)3 mediates the cell adhesion process by interacting with cell surface receptors through its different cell binding sites. It is essential to embryogenesis and found associated with the extracellular matrix in large quantities during wound healing and angiogenesis. The amino acid sequence Arg-Gly-Asp (RGD) found within FN and many other matrix proteins is the most widely occurring cell-adhesive motif (1). It is the most important recognition site for about half of all known integrins, such as ␣5␤1, ␣V␤1, ␣V␤3, and ␣V␤5 (2). However, this cell adhesion process can easily be inhibited by the presence of competitive peptides containing the RGD sequence, often leading to apoptosis (anoikis) (3, 4). Such peptides are commonly released during matrix remodeling, a process that is essential to both wound healing and angiogenesis (5-7). Of the integrins, ␣5␤1 integrin is probably the major cell surface integrin that interacts with the RGDcell binding site on FN, initiating the cell adhesion process (8), whereas the binding of ␣v␤3 integrin to the RGD domain can also support cell adhesion on FN (9). In addition to the RGDcell binding domains, the heparin binding sites within FN have also been reported to be involved in cell adhesion because, although cell binding to the RGD-cell binding domains of FN can initiate the cell adhesion process, it is not sufficient to fully support actin cytoskeleton formation, an observation tha...

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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

Cited by 63 publications

References 40 publications

Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

RGD-independent Cell Adhesion via a Tissue Transglutaminase-Fibronectin Matrix Promotes Fibronectin Fibril Deposition and Requires Syndecan-4/2 and α5β1 Integrin Co-signaling

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