C-terminal heparin-binding domain of fibronectin regulates integrin-mediated cell spreading but not the activation of mitogen-activated protein kinase

2001 ◽  
Vol 360 (1) ◽  
pp. 239-245 ◽  
Author(s):  
Jungyean KIM ◽  
Innoc HAN ◽  
Yeonhee KIM ◽  
Seungin KIM ◽  
Eok-Soo OH
Keyword(s):  
Cell Spreading ◽  
Binding Domain ◽  
Mitogen Activated Protein Kinase ◽  
Heparin Binding ◽  
Cell Binding ◽  
Rat Embryo ◽  
Mapk Activation ◽  
Mitogen Activated Protein ◽  
Heparin Binding Domain ◽  
Cell Binding Domain

Fibronectin (FN) stimulates multiple signalling events including mitogen-activated protein kinase (MAPK) activation. During cell spreading, both the cell-binding domain and the C-terminal heparin-binding domain (HepII) of FN co-operatively regulate cytoskeleton organization. However, in comparison with the large number of studies on the functions of cell-binding domain, there is little information about the role of HepII. We therefore investigated the effect of HepII on integrin-mediated cell spreading and adhesion on FN and MAPK activation. In contrast with cells on FN substrates, rat embryo fibroblasts on FN120, which lacks HepII, were less spread, had weaker adhesion to FN and failed to form focal adhesions and actin stress fibres. Phosphotyrosine was present in the focal contacts of rat embryo fibroblasts on FN within 30min but was absent from cells on FN120. Overall, tyrosine phosphorylation was much less in cell lysates from cells on FN120, with decreased phosphorylation of focal adhesion kinase (‘pp125FAK’) on tyrosine-397, implying additional regulation of tyrosine phosphorylation by HepII. Nevertheless, adhesion-mediated MAPK activity was similar in cells on FN and on FN120. Furthermore, cells spread on FN and on FN120 substrates showed similar MAPK activation in response to treatment with epidermal growth factor and with platelet-derived growth factor. Consistently, overexpression of syndecan-4, which binds to HepII, enhanced cell spreading and adhesion on FN but did not affect integrin-mediated MAPK activation. We therefore conclude that both HepII and syndecan-4 regulate integrin-mediated cell spreading but not MAPK activation.

scholarly journals Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein.

1991 ◽  
Vol 2 (11) ◽  
pp. 951-964 ◽  
Author(s):  
J L Guan ◽  
J E Trevithick ◽  
R O Hynes
Keyword(s):  
Tyrosine Phosphorylation ◽  
Concanavalin A ◽  
Cell Spreading ◽  
Binding Domain ◽  
Heparin Binding ◽  
Cell Binding ◽  
Heparin Binding Domain ◽  
Coated Surfaces ◽  
Cell Binding Domain ◽  
In Cells

We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment.

scholarly journals Fibronectin Facilitates Mycobacterium tuberculosis Attachment to Murine Alveolar Macrophages

2002 ◽  
Vol 70 (3) ◽  
pp. 1287-1292 ◽  
Author(s):  
Rajamouli Pasula ◽  
Paul Wisniowski ◽  
William J. Martin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Alveolar Macrophages ◽  
Pulmonary Infection ◽  
Polyclonal Antibodies ◽  
Binding Domain ◽  
Heparin Binding ◽  
Cell Binding ◽  
Adhesive Protein ◽  
Heparin Binding Domain ◽  
Cell Binding Domain

ABSTRACT Mycobacterium tuberculosis remains a major cause of pulmonary infection worldwide. Attachment of M. tuberculosis organisms to alveolar macrophages (AMs) represents the earliest phase of primary infection in pulmonary tuberculosis. In this study fibronectin (Fn), an adhesive protein, is shown to bind M. tuberculosis organisms and facilitates attachment of M. tuberculosis to murine AMs. A monoclonal antibody (MAb) specific to the heparin binding domain (HBD) of Fn decreases 125I-Fn binding to M. tuberculosis; whereas MAbs specific to either the cell binding domain (CBD) or the gelatin binding domain (GBD) have no effect on Fn binding to M. tuberculosis. In the presence of exogenous Fn (10 μg/ml) M. tuberculosis attachment to AMs increased significantly from control levels (means ± standard errors of the means) of 11.5% ± 1.1% to 44.2% ± 4.2% (P < 0.05). Fn-enhanced attachment was significantly decreased from 44.2% ± 4.2% to 10.8% ± 1.2% (P < 0.05) in the presence of anti-Fn polyclonal antibodies. The attachment is also inhibited in the presence of MAbs specific for the HBD and CBD, whereas MAbs specific to GBD did not affect the attachment. Further, an Fn cell binding peptide, Arg-Gly-Asp-Ser (RGDS), decreased the attachment from 44.2% ± 4.2% to 15.3% ± 1.2% (P < 0.05), whereas addition of a control peptide, Arg-Gly-Glu-Ser (RGES) did not affect the attachment (40.5% ± 1.8%). These results suggest that Fn-mediated attachment of M. tuberculosis can occur through the binding of Fn to the AM via the CBD and to M. tuberculosis organisms via the HBD.

Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

2001 ◽  
Vol 356 (2) ◽  
pp. 531-537 ◽  
Author(s):  
Jinsook JEONG ◽  
Innoc HAN ◽  
Yangmi LIM ◽  
Jungyean KIM ◽  
Ilseon PARK ◽  
...  
Keyword(s):  
Cell Survival ◽  
Binding Domain ◽  
Heparin Binding ◽  
Cell Binding ◽  
Rat Embryo ◽  
Nuclear Condensation ◽  
Embryo Fibroblasts ◽  
Induced Apoptosis ◽  
Domain Peptide ◽  
Cell Binding Domain

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.

Phosphatidylserine (PS)-Positive Erythrocytes Bind to Both Immobilized and Soluble Thrombospondin-1 (TSP-1) Via Its Heparin Binding Domain.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1568-1568
Author(s):  
Yamaja B.N. Setty ◽  
Suhita Gayen Betal ◽  
Surekha Kulkarni ◽  
Marie J. Stuart
Keyword(s):  
Cell Adhesion ◽  
Binding Domain ◽  
Red Cells ◽  
Binding Motif ◽  
Dependent Manner ◽  
Red Cell ◽  
Heparin Binding ◽  
Cell Binding ◽  
Erythrocyte Adhesion ◽  
Heparin Binding Domain

Abstract Phosphatidylserine (PS)-dependent erythrocyte adhesion to both cultured endothelial cells and the components of sub-endothelial matrix appears to be mediated in part via thrombospondin-1 (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP has not been identified. Since a cell-binding domain for anionic heparin is located at the amino-terminal domain of TSP, we hypothesized that anionic PS-positive (PS+ve) red cells bind to this domain. In a recent preliminary study, using a flow adhesion system and PS+ve erythrocytes (prepared by treating control AA red cells with A23187), we have demonstrated that heparin inhibited PS+ve erythrocyte adhesion to immobilized TSP in a concentration-dependent manner with 58 to 77% inhibition noted at concentrations between 1 and 50 U/ml (n=9, P<0.001). Other anionic polysaccharides including high molecular weight dextran sulfate and chondroitin sulfate A also inhibited PS+ve erythrocyte adhesion to immobilized TSP with the magnitude of the inhibitory effects comparable to heparin. These results suggested that the heparin-binding domain on TSP may be involved in PS-mediated red cell adhesion to immobilized TSP. We have extended these studies to characterize the PS-binding site on TSP using monoclonal antibodies directed against specific cell-binding domains on the molecule and also using specific TSP peptides. We demonstrate that pre-incubation of immobilized TSP with an antibody directed against the heparin-binding domain on TSP (TSP-Ab9, Lab Vision) blocked PS-mediated red cell adhesion to immobilized TSP (80% inhibition compared to an isotype-matched negative control antibody, n=7, P<0.001), whereas an antibody that recognizes the collagen-binding domain on TSP (TSP-Ab4) did not affect this process. In addition, incubation of PS+ve erythrocytes with a TSP peptide containing the specific heparin-binding motif, KKTRG, inhibited PS-mediated red cell adhesion by 55% (P<0.001, n=6), whereas a peptide lacking the binding motif had no effect. Since protein confirmation of immobilized TSP appears to be different from that of soluble TSP, we next investigated whether soluble TSP, like immobilized TSP, also interacted with PS+ve erythrocytes. Erythrocytes containing 8 to 10% PS+ve cells were incubated in the absence or the presence of increasing concentrations of soluble TSP (0.1 to 10 μg/ml), and then analyzed by flow cytometry for surface bound TSP using both adhesion blocking (TSP-Ab9) and non-blocking (TSP-Ab4) anti-TSP antibodies. We demonstrate that soluble TSP binds to PS+ve erythrocytes in a concentration-dependent manner with 3 to 11% TSP-positive (TSP+ve) red cells measured at soluble TSP concentrations between 1 to 10 μg/ml (n=4). In addition, TSP binding could be detected only with the non-adhesion blocking antibody TSP-Ab4, which recognizes the collagen-binding domain on TSP. The adhesion blocking antibody TSP-Ab9 that interacts with the heparin binding domain, failed to detect any TSP+ve red cells. No TSP+ve erythrocytes were detected when PS-negative control red cells were evaluated in binding assays. In parallel adhesion experiments, soluble TSP inhibited PS+ve erythrocyte adhesion to immobilized TSP at concentrations at which significant TSP binding to erythrocytes occurred (43% and 44% inhibition at 5 and 10 μg of soluble TSP per ml, n=4). These results conclusively demonstrate that PS-positive erythrocytes interact with both immobilized and fluid phase TSP through the heparin-binding domain, and that heparin blocks this interaction.

Role of the Heparin-Binding Domain of Chimeric Peptides Derived from Fibronectin in Cell Spreading and Motility

1995 ◽  
Vol 217 (1) ◽  
pp. 169-179 ◽  
Author(s):  
Junya Yoneda ◽  
Ikuo Saiki ◽  
Yu Igarashi ◽  
Hideo Kobayashi ◽  
Hideki Fujii ◽  
...  
Keyword(s):  
Cell Spreading ◽  
Binding Domain ◽  
Heparin Binding ◽  
Heparin Binding Domain ◽  
Chimeric Peptides

scholarly journals 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.

1991 ◽  
Vol 114 (6) ◽  
pp. 1295-1305 ◽  
Author(s):  
T Nagai ◽  
N Yamakawa ◽  
S Aota ◽  
S S Yamada ◽  
S K Akiyama ◽  
...  
Keyword(s):  
Cell Spreading ◽  
Binding Domain ◽  
Central Cell ◽  
Site Directed Mutagenesis ◽  
Cell Binding ◽  
Matrix Assembly ◽  
Fibronectin Receptor ◽  
Molar Basis ◽  
Rgd Site ◽  
Cell Binding Domain

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.

scholarly journals Platelet interactions with fibronectin: divalent cation-independent platelet adhesion to the gelatin-binding domain of fibronectin

Blood ◽  
1993 ◽  
Vol 81 (7) ◽  
pp. 1778-1786
Author(s):  
KJ Winters ◽  
JJ Walsh ◽  
BG Rubin ◽  
SA Santoro
Keyword(s):  
Divalent Cation ◽  
Disulfide Bonds ◽  
Platelet Adhesion ◽  
Divalent Cations ◽  
Binding Domain ◽  
Heparin Binding ◽  
Cell Binding ◽  
Binding Domains ◽  
Platelet Spreading ◽  
Cell Binding Domain

Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation- independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin- binding domain of FN by a novel divalent cation-independent mechanism.

The Carboxy-Terminal Cell-Binding Domain of Thrombospondin Is Essential for Sickle Red Blood Cell Adhesion

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 302-309 ◽  
Author(s):  
Cheryl A. Hillery ◽  
J. Paul Scott ◽  
Ming C. Du
Keyword(s):  
Cell Adhesion ◽  
Matrix Protein ◽  
Binding Domain ◽  
Terminal Cell ◽  
Heparin Binding ◽  
Cell Binding ◽  
Adhesive Site ◽  
Carboxy Terminal ◽  
Cell Binding Domain

Sickle red blood cells (SS-RBCs) have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. TSP has at least four domains that mediate cell adhesion. The goal of this study was to map the site(s) on TSP that binds SS-RBCs. Purified TSP proteolytic fragments containing either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (&lt;10% adhesion). However, a 140-kD thermolysin TSP fragment, containing the carboxy-terminal cell-binding domain in addition to the type 1, 2, and 3 repeats fully supported the adhesion of SS-RBCs (126% ± 25% adhesion). Two cell-binding domain adhesive peptides, 4N1K (KRFYVVMWKK) and 7N3 (FIRVVMYEGKK), failed to either inhibit or support SS-RBC adhesion to TSP. In addition, monoclonal antibody C6.7, which blocks platelet and melanoma cell adhesion to the cell-binding domain, did not inhibit SS-RBC adhesion to TSP. These data suggest that a novel adhesive site within the cell binding domain of TSP promotes the adhesion of sickle RBCs to TSP. Furthermore, soluble TSP did not bind SS-RBCs as detected by flow cytometry, nor inhibit SS-RBC adhesion to immobilized TSP under conditions of flow, indicating that the adhesive site on TSP that recognizes SS-RBCs is exposed only after TSP binds to a matrix. We conclude that the intact carboxy-terminal cell-binding domain of TSP is essential for the adhesion of sickle RBCs under flow conditions. This study also provides evidence for a unique adhesive site within the cell-binding domain that is exposed after TSP binds to a matrix.

scholarly journals Topographic localization of the heparin-binding domain of the neural cell adhesion molecule N-CAM.

1986 ◽  
Vol 103 (5) ◽  
pp. 1739-1744 ◽  
Author(s):  
G J Cole ◽  
A Loewy ◽  
N V Cross ◽  
R Akeson ◽  
L Glaser
Keyword(s):  
Cell Adhesion Molecule ◽  
Neural Cell Adhesion Molecule ◽  
Binding Domain ◽  
Heparin Binding ◽  
Cell Binding ◽  
Terminal Sequence ◽  
Amino Terminal ◽  
Neural Cell Adhesion ◽  
Heparin Binding Domain ◽  
Amino Terminal Sequence

Previous studies have reported that the cell-binding region of the neural cell adhesion molecule (N-CAM) resides in a 65,000-D amino-terminal fragment designated Frl (Cunningham, B. A., S. Hoffman, U. Rutishauser, J. J. Hemperly, and G. M. Edelman, 1983, Proc. Natl. Acad. Sci. USA, 80:3116-3120). We have reported the presence of two functional domains in N-CAM, each identified by a specific mAb, that are required for cell-cell or cell-substratum adhesion (Cole, G. J., and L. Glaser, 1986, J. Cell Biol., 102:403-412). One of these domains is a heparin (heparan sulfate)-binding domain. In the present study we have determined the topographic localization of the heparin-binding fragment from N-CAM, which has been identified by our laboratory. The B1A3 mAb recognizes a 25,000-D heparin-binding fragment derived from chicken N-CAM, and also binds to a 65,000-D fragment, presumably Frl, produced by digestion of N-CAM with Staphylococcus aureus V8 protease. Amino-terminal sequence analysis of the isolated 25,000-D heparin-binding domain of N-CAM yielded the sequence: Leu-Gln-Val-Asp-Ile-Val-Pro-Ser-Gln-Gly. This sequence is identical to the previously reported amino-terminal sequence for murine and bovine N-CAM. Thus, the 25,000-D polypeptide fragment is the amino-terminal region of the N-CAM molecule. We have also shown that the B1A3 mAb recognizes not only chicken N-CAM but also rat and mouse N-CAM, indicating that the heparin-binding domain of N-CAM is evolutionarily conserved among different N-CAM forms. Additional peptide-mapping studies indicate that the second cell-binding site of N-CAM is located in a polypeptide region at least 65,000 D from the amino-terminal region. We conclude that the adhesion domains on N-CAM identified by these antibodies are physically distinct, and that the previously identified cell-binding domain on Frl is the heparin-binding domain.

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