scholarly journals A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells

2003 ◽  
Vol 371 (2) ◽  
pp. 565-571 ◽  
Author(s):  
José V. MOYANO ◽  
Alfredo MAQUEDA ◽  
Juan P. ALBAR ◽  
Angeles GARCIA-PARDO
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Melanoma Cells ◽  
Small Gtpase ◽  
Binding Domain ◽  
Heparin Binding ◽  
Chondroitin Sulphate Proteoglycans ◽  
Heparin Binding Domain

Cell adhesion to fibronectin results in formation of actin stress fibres and focal adhesions. In fibroblasts, this response requires two co-operative signals provided by interactions of the RGD sequence with α5β1 integrin and the heparin-binding domain II (Hep II) domain with syndecan-4. Within Hep II, this activity was mapped to repeat III13 and to the peptide FN-C/H-V(WQPPRARITGY, repeat III14). We previously described that the synthetic heparin-binding peptide/III5 (HBP/III5) (WTPPRAQITGYRLTVGLTRR, repeat III5) binds heparin and mediates cell adhesion via chondroitin sulphate proteoglycans. We have now studied whether HBP/III5 co-operates with α5β1 and drives a full cytoskeletal response in melanoma cells. SKMEL-178 cells attached and spread on the RGD-containing FNIII7–FNIII10 (FNIII7–10) fragment, but did not form stress fibres or focal adhesions. Co-immobilization of HBP/III5 with FNIII7–10 or adding soluble HBP/III5 to cells prespread on FNIII7–10, effectively induced these structures. Cell transfection with dominant-negative N19RhoA, a member of the small GTPase family, abolished the HBP/III5 effect. Both chondroitinase and heparitinase diminished focal adhesions, indicating that both types of proteoglycans bound HBP/III5 in melanoma cells. We have mapped the active sequence of HBP/III5 to YRLTVGLTRR, which is a novel sequence in fibronectin with focal-adhesion-promoting activity. The last two arginine (R) residues of this sequence are required for activity, since their replacement by alanine completely abrogated the HBP/III5 cytoskeletal effect. Moreover, this sequence is also active in the context of large fibronectin fragments. Our results establish that the Hep III region provides co-operative signals to α5β1 for the progression of the cytoskeletal response and that these include activation of RhoA.

scholarly journals Thrombospondin modulates focal adhesions in endothelial cells.

1989 ◽  
Vol 109 (3) ◽  
pp. 1309-1319 ◽  
Author(s):  
J E Murphy-Ullrich ◽  
M Höök
Keyword(s):  
Endothelial Cells ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Heparin Binding ◽  
Focal Adhesion Formation ◽  
Heparin Binding Domain ◽  
Adhesion Plaques ◽  
Number Of Cells

We examined the effects of thrombospondin (TSP) in the substrate adhesion of bovine aortic endothelial cells. The protein was tested both as a substrate for cell adhesion and as a modulator of the later stages of the cell adhesive process. TSP substrates supported the attachment of some BAE cells, but not cell spreading or the formation of focal adhesion plaques. In contrast, cells seeded on fibrinogen or fibronectin substrates were able to complete the adhesive process, as indicated by the formation of focal adhesion plaques. Incubation of cells in suspension with soluble TSP before or at the time of seeding onto fibronectin substrates resulted in an inhibition of focal adhesion formation. Furthermore, the addition of TSP to fully adherent cells in situ or prespread on fibronectin substrates caused a reduction in the number of cells, which were positive for focal adhesions, although there was no significant effect on cell spreading. In a dose-dependent manner, TSP reduced the number of cells with adhesion plaques to approximately 60% of control levels. The distribution of remaining adhesion plaques in TSP-treated cells was also altered: plaques were primarily limited to the periphery of cells and were not present in the central cell body, as in control cells treated with BSA. The observed effects were specific for TSP and were not observed with platelet factor 4, beta-thromboglobulin, or fibronectin. The TSP-mediated loss of adhesion plaques was neutralized by the addition of heparin, fucoidan, other heparin-binding proteins, and by a monoclonal antibody to the heparin binding domain of TSP, but not by antibodies to the core or carboxy-terminal regions of TSP. The interaction of the heparin-binding domain of TSP with cell-associated heparan sulfate appears to be an important mechanistic component for this activity of TSP. These data indicate that TSP may have a role in destabilizing cell adhesion through prevention of focal adhesion formation and by loss of preformed focal adhesions.

scholarly journals A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation.

1993 ◽  
Vol 4 (6) ◽  
pp. 605-613 ◽  
Author(s):  
A Woods ◽  
J B McCarthy ◽  
L T Furcht ◽  
J R Couchman
Keyword(s):  
Cell Surface ◽  
Focal Adhesion ◽  
Cell Attachment ◽  
Adhesion Formation ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Fiber Formation ◽  
Heparin Binding ◽  
Focal Adhesion Formation ◽  
Heparin Binding Domain

Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process of focal adhesion and stress fiber formation requires additional interactions. Heparin-binding fragments of fibronectin can provide this signal. The COOH-terminal heparin-binding domain of fibronectin contains five separate heparin-binding amino acid sequences. We show here that all five sequences, as synthetic peptides coupled to ovalbumin, can support cell attachment. Only three of these sequences can promote focal adhesion formation when presented as multicopy complexes, and only one of these (WQPPRARI) retains this activity as free peptide. The major activity of this peptide resides in the sequence PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition with chondroitin sulfate does not.

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.

Cell adhesion to laminin 1 or 5 induces isoform-specific clustering of integrins and other focal adhesion components

1998 ◽  
Vol 111 (6) ◽  
pp. 793-802 ◽  
Author(s):  
D. Dogic ◽  
P. Rousselle ◽  
M. Aumailley
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Specific Expression ◽  
Normal Human Skin ◽  
Laminin 5 ◽  
Cell Shapes ◽  
Alpha3beta1 Integrin ◽  
Laminin 1

Laminin 1 (alpha1beta1gamma1) and laminin 5 (alpha3beta3gamma2) induce cell adhesion with different involvement of integrins: both are ligands for the alpha6beta1 integrin, while alpha3beta1 integrin has affinity for laminin 5 only. These two laminin isoforms therefore provide good models to investigate whether alpha3beta1 and alpha6beta1 integrins play different roles in signal transduction and in focal adhesion formation. Laminin 1 or 5 induced adhesion of normal human skin fibroblasts to a similar extent but promoted different overall cell shapes. On laminin 1 the fibroblasts formed mainly filopodia-like structures, while on laminin 5 they developed lamellipodias. Staining of fibrillar actin with fluorescein-phalloidin revealed a similar organisation of the actin cytoskeleton on both substrates. However, integrin subunits and several cytoskeletal linker proteins, including vinculin, talin, and paxillin, showed an isoform-specific arrangement into focal adhesions. On laminin 1 they were recruited into thick and short aggregates localized at the termini of actin stress fibers, while on laminin 5 they appeared as dots or streaks clustered on a long portion of actin microfilaments. To test whether the differing affinity of laminin 1 or 5 for alpha3beta1 integrin would explain the formation of morphologically different focal adhesions, cells were seeded on laminin 1 under conditions in which alpha3beta1 integrins were occupied by a function-blocking antibody. This resulted in the formation of focal adhesions similar to that observed on laminin 5, where the integrin is occupied by its natural ligand. These results provide the first evidence for a cross-talk between alpha3beta1 and alpha6beta1 integrins and indicate that occupancy of alpha3beta1 integrins results in a trans-dominant regulation of alpha6beta1 integrin clustering and of focal adhesions. It suggests that recruitment of integrins and cytoskeletal linker proteins are laminin isoform-specific and that tissue specific expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.

scholarly journals Platelet thrombospondin mediates attachment and spreading of human melanoma cells.

1987 ◽  
Vol 104 (1) ◽  
pp. 131-139 ◽  
Author(s):  
D D Roberts ◽  
J A Sherwood ◽  
V Ginsburg
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Attachment ◽  
Melanoma Cells ◽  
Binding Domain ◽  
Heparin Binding ◽  
Amino Terminal ◽  
Sulfated Fucan ◽  
Heparin Binding Domain ◽  
Carboxyl Terminal ◽  
Inhibition Studies

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.

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