scholarly journals Tissue Transglutaminase Is an Integrin-Binding Adhesion Coreceptor for Fibronectin

2000 ◽  
Vol 148 (4) ◽  
pp. 825-838 ◽  
Author(s):  
Sergey S. Akimov ◽  
Dmitry Krylov ◽  
Laurie F. Fleischman ◽  
Alexey M. Belkin
Keyword(s):  
Cell Surface ◽  
Focal Adhesion ◽  
Factor Xiii ◽  
Tissue Transglutaminase ◽  
Focal Adhesions ◽  
Cross Linking ◽  
Binding Motifs ◽  
High Affinity ◽  
Β2 Integrins

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of β1 and β3 subfamilies, but not with β2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

scholarly journals ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

2018 ◽  
Vol 19 (10) ◽  
pp. 3089 ◽  
Author(s):  
Marie Hlavničková ◽  
Milan Kuchař ◽  
Radim Osička ◽  
Lucie Vaňková ◽  
Hana Petroková ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clinical Manifestations ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interleukin 17 ◽  
Normal Human Skin ◽  
High Affinity ◽  
Th 17 ◽  
Surface Receptor

Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists.

Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats

1999 ◽  
Vol 112 (24) ◽  
pp. 4589-4599 ◽  
Author(s):  
F. Li ◽  
Y. Zhang ◽  
C. Wu
Keyword(s):  
Focal Adhesion ◽  
Critical Role ◽  
Focal Adhesions ◽  
Subcellular Compartment ◽  
Cell Matrix ◽  
Integrin Binding Site ◽  
Integrin Linked Kinase ◽  
Cell Cell

Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. In this study, we show that ILK is a constituent of cell-matrix focal adhesions. ILK was recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK was absent in E-cadherin-mediated cell-cell adherens junctions. In previous studies, we have identified PINCH, a protein consisting of five LIM domains, as an ILK binding protein. We demonstrate in this study that the ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeats domain, which is capable of interacting with PINCH in vitro, could also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex was markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, was unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, as an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site failed to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function.

Aberrant fibrin formation and cross-linking of fibrinogen Nieuwegein, a variant with a shortened Aα-chain, alters endothelial capillary tube formation

Blood ◽  
2001 ◽  
Vol 97 (4) ◽  
pp. 973-980 ◽  
Author(s):  
Annemie Collen ◽  
Annemarie Maas ◽  
Teake Kooistra ◽  
Florea Lupu ◽  
Jos Grimbergen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Capillary Tube ◽  
Stop Codon ◽  
Tissue Transglutaminase ◽  
Tube Formation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Molecular Abnormalities ◽  
Fibrin Network

Abstract A congenital dysfibrinogenemia, fibrinogenNieuwegein, was discovered in a young man without any thromboembolic complications or bleeding. A homozygous insertion of a single nucleotide (C) in codon Aα 453 (Pro) introduced a stop codon at position 454, which resulted in the deletion of the carboxyl-terminal segment Aα 454-610. The ensuing unpaired cysteine at Aα 442 generated fibrinogen-albumin complexes of different molecular weights. The molecular abnormalities of fibrinogenNieuwegein led to a delayed clotting and a fibrin network with a low turbidity. Electron microscopy confirmed that thin fibrin bundles were organized in a fine network. The use of fibrinogenNieuwegein-derived fibrin (fibrinNieuwegein) in an in vitro angiogenesis model resulted in a strong reduction of tube formation. The ingrowth of human microvascular endothelial cells (hMVEC) was independent of αvβ3, indicating that the reduced ingrowth is not due to the absence of the RGD-adhesion site at position Aα 572-574. Rather, the altered structure of fibrinNieuwegeinis the cause, since partial normalization of the fibrin network by lowering the pH during polymerization resulted in an increased tube formation. Whereas factor XIIIa further decreased the ingrowth of hMVEC in fibrinNieuwegein, tissue transglutaminase (TG), which is released in areas of vessel injury, did not. This is in line with the absence of the cross-linking site for TG in the α-chains of fibrinogenNieuwegein. In conclusion, this newly discovered congenital dysfibrinogenemia has a delayed clotting time and leads to the formation of an altered fibrin structure, which could not be cross-linked by TG and which is less supportive for ingrowth of endothelial cells.

scholarly journals Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms

2000 ◽  
Vol 348 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Madeleine TOUTANT ◽  
Jeanne-Marie STUDLER ◽  
Ferran BURGAYA ◽  
Alicia COSTA ◽  
Pascal EZAN ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Src Family Kinases ◽  
Critical Residue ◽  
Green Fluorescent ◽  
And Function

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues (‘boxes’ 6 and 7), located on either side of Tyr397, which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr397, a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr397 independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr397 in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr397. They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

scholarly journals Restructuring of Focal Adhesion Plaques by Pi 3-Kinase

2000 ◽  
Vol 150 (3) ◽  
pp. 627-642 ◽  
Author(s):  
Jeffrey A. Greenwood ◽  
Anne B. Theibert ◽  
Glenn D. Prestwich ◽  
Joanne E. Murphy-Ullrich
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Adhesive Strength ◽  
Focal Adhesions ◽  
Lipid Product ◽  
Phosphoinositide 3 Kinase ◽  
Triton X ◽  
Adhesion Plaque ◽  
Adhesion Complex

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of α-actinin and vinculin from the focal adhesion complex to the Triton X-100–soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P3 binds to α-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of α-actinin with the integrin β subunit, and that PtdIns (3,4,5)-P3 could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P3, has important implications for the regulation of cellular adhesive strength and motility.

scholarly journals Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

2016 ◽  
Vol 27 (9) ◽  
pp. 1431-1441 ◽  
Author(s):  
Aniqua Rahman ◽  
Shawn P. Carey ◽  
Casey M. Kraning-Rush ◽  
Zachary E. Goldblatt ◽  
Francois Bordeleau ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Focal Adhesions ◽  
Three Dimensions ◽  
Collagen Matrices ◽  
Migration Direction ◽  
Unidirectional Motion

During metastasis, cells can use proteolytic activity to form tube-like “microtracks” within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro three-dimensional (3D) micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Because focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on two-dimensional (2D) substrates and in 3D uniform collagen matrices, as indicated by reduced speed, shorter net displacement, and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for focal adhesion kinase (FAK) activation in three dimensions, as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks but not on 2D substrates, and, accordingly, FAK inhibition halts cell migration in 3D microtracks. Together these data indicate that vinculin plays a key role in polarization during migration.

Functional analysis of fibrin γ-chain cross-linking by activated factor XIII: determination of a cross-linking pattern that maximizes clot stiffness

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 902-907 ◽  
Author(s):  
Kristina F. Standeven ◽  
Angela M. Carter ◽  
Peter J. Grant ◽  
John W. Weisel ◽  
Irina Chernysh ◽  
...  
Keyword(s):  
Factor Xiii ◽  
Tissue Transglutaminase ◽  
Coagulation Factor ◽  
Fold Increase ◽  
Cross Linking ◽  
Cross Links ◽  
Α Chain ◽  
And Function ◽  
Hybrid Cross

Abstract Activated coagulation factor XIII (FXIIIa) cross-links the γ-chains of fibrin early in clot formation. Cross-linking of the α-chains occurs more slowly, leading to high molecular weight multimer formations that can also contain γ-chains. To study the contribution of FXIIIa-induced γ-chain cross-linking on fibrin structure and function, we created 2 recombinant fibrinogens (γQ398N/Q399N/K406R and γK406R) that modify the γ-chain cross-linking process. In γK406R, γ-dimer cross-links were absent, but FXIIIa produced a cross-linking pattern similar to that observed in tissue transglutaminase cross-linked fibrin(ogen) with mainly α-γ cross-links. In Q398N/Q399N/K406R, cross-links with any γ-chain involvement were completely absent, and only α-chain cross-linking occurred. Upon cross-linking, recombinant normal fibrin yielded a 3.5-fold increase in stiffness, compared with a 2.5-fold increase by α-chain cross-linking alone (γQ398N/Q399N/K406R). γK406R fibrin showed a 1.5-fold increase in stiffness after cross-linking. No major differences in clot morphology, polymerization, and lysis rates were observed, although fiber diameter was slightly lower in cross-linked normal fibrin relative to the variants. Our results show that γ-chain cross-linking contributes significantly to clot stiffness, in particular through γ-dimer formation; α-γ hybrid cross-links had the smallest impact on clot stiffness.

A new anti-idiotype antibody capable of binding rituximab on the surface of lymphoma cells

Blood ◽  
2004 ◽  
Vol 104 (8) ◽  
pp. 2540-2542 ◽  
Author(s):  
Mark S. Cragg ◽  
Mike B. Bayne ◽  
Alison L. Tutt ◽  
Ruth R. French ◽  
Stephen Beers ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Immunoglobulin G ◽  
Human Immunoglobulin ◽  
Lymphoma Cells ◽  
Fine Specificity ◽  
High Affinity ◽  
Anti Cd20

Abstract The chimeric anti-CD20 monoclonal antibody (mAb), rituximab, is an established part of the management of many non-Hodgkin lymphomas. The in vivo action of rituximab remains elusive, and this partially reflects a lack of highly specific reagents to detect rituximab binding at the cell surface. Here we report a new high-affinity mAb (MB2A4) with fine specificity for the idiotype of rituximab. It is able to detect rituximab in vitro, in the presence of high levels of human immunoglobulin G (IgG), in the serum of patients receiving rituximab therapy, and, surprisingly, when rituximab is bound to CD20 on the cell surface. We propose that the anti–idiotype (Id) binds to rituximab molecules bound univalently at the cell surface, facilitated by the relatively high off-rate of rituximab. This reagent provides new insights into the binding of rituximab at the cell surface and demonstrates a mode of binding that could be exploited for the surface detection of other mAbs with clinical and biologic applications.

Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin

1994 ◽  
Vol 107 (2) ◽  
pp. 709-717 ◽  
Author(s):  
C.K. Wood ◽  
C.E. Turner ◽  
P. Jackson ◽  
D.R. Critchley
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Binding Site ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Terminal Region ◽  
Nih3t3 Cells ◽  
Cell Biol

Paxillin and vinculin are cytoskeletal proteins that colocalise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059–1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383–2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881–1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881–978. Transfection experiments using cDNAs encoding chick vinculin residues 398–1066 and 398–1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398–1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979–1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.

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