scholarly journals Activation of Distinct α5β1-mediated Signaling Pathways by Fibronectin's Cell Adhesion and Matrix Assembly Domains

1998 ◽  
Vol 141 (1) ◽  
pp. 241-253 ◽  
Author(s):  
Denise C. Hocking ◽  
Jane Sottile ◽  
Paula J. McKeown-Longo
Keyword(s):  
Cell Adhesion ◽  
Amino Terminus ◽  
Type I ◽  
New Paradigm ◽  
Matrix Assembly ◽  
Rgd Peptides ◽  
Surface Receptors ◽  
Amino Terminal ◽  
Dynamic Cell ◽  
Α5β1 Integrin

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α5β1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α5β1 integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α5β1 integrin, suggesting that these two domains of fibronectin cannot bind to the α5β1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α5β1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.

scholarly journals A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

1996 ◽  
Vol 133 (2) ◽  
pp. 431-444 ◽  
Author(s):  
D C Hocking ◽  
R K Smith ◽  
P J McKeown-Longo
Keyword(s):  
Binding Site ◽  
Wound Repair ◽  
Solid Phase ◽  
Focal Adhesions ◽  
Amino Terminus ◽  
Matrix Assembly ◽  
Amino Terminal ◽  
Fibronectin Matrix ◽  
Beta 1 Integrin ◽  
Rgd Site

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

Fibronectin Distribution in Human Bone Marrow Stroma: Matrix Assembly and Tumor Cell Adhesion via α5β1 Integrin

1997 ◽  
Vol 230 (1) ◽  
pp. 111-120 ◽  
Author(s):  
D. Van der Velde-Zimmermann ◽  
M.A.M. Verdaasdonk ◽  
L.H.P.M. Rademakers ◽  
R.A. De Weger ◽  
J.G. Van den Tweel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Tumor Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Bone Marrow Stroma ◽  
Matrix Assembly ◽  
Tumor Cell Adhesion ◽  
Marrow Stroma ◽  
Α5β1 Integrin

A novel role for alpha 3 beta 1 integrins in extracellular matrix assembly

1995 ◽  
Vol 108 (6) ◽  
pp. 2511-2523 ◽  
Author(s):  
C. Wu ◽  
A.E. Chung ◽  
J.A. McDonald
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Binding Activity ◽  
Integrin Subunit ◽  
Pericellular Matrix ◽  
Matrix Assembly ◽  
Amino Terminal ◽  
Beta 1 Integrin ◽  
Fibronectin Deposition

To study the biological role of alpha 3 beta 1 integrins in cell adhesion, migration, and in the deposition of extracellular matrix, we stably expressed the human alpha 3 integrin subunit in the alpha 4, alpha 5 integrin deficient CHO cell line B2. The expression of alpha 3 beta 1 integrins enhanced cell adhesion on entactin (also known as nidogen), but not on fibronectin. Using recombinant GST-fusion proteins that span the entire length of the entactin molecule, we located cell adhesive activity to the G2 domain of entactin. These results suggest that the alpha 3 beta 1 integrin functions as an adhesion receptor interacting with the G2 domain of entactin. On the other hand, the expression of alpha 3 beta 1 integrins did not confer the ability to migrate on entactin. Strikingly, the expression of alpha 3 beta 1 dramatically increased the deposition of entactin and fibronectin into the pericellular matrix. This was accompanied by increased binding activity of the 29 kDa amino-terminal domain of fibronectin. Thus, similar to alpha 5 beta 1 integrins, alpha 3 beta 1 integrins can play an important role in modulating the assembly of pericellular matrices. However, unlike fibronectin deposition supported by alpha 5 beta 1, alpha 3 beta 1 supported fibronectin deposition into pericellular matrix was not inhibited by antibodies binding to the RGD containing cell adhesion domain of fibronectin, demonstrating that the two processes are mechanistically distinct. The role of alpha 3 beta 1 in pericellular matrix assembly potentially implicates this receptor in the assembly and/or recognition of entactin-containing pericellular matrices, an observation consistent with its apparent role in the renal glomerulus of the mammalian kidney.

scholarly journals Evidence for an Activation Domain at the Amino Terminus of Simian Immunodeficiency Virus Vpx

2009 ◽  
Vol 84 (3) ◽  
pp. 1387-1396 ◽  
Author(s):  
Thomas Gramberg ◽  
Nicole Sunseri ◽  
Nathaniel R. Landau
Keyword(s):  
Dendritic Cells ◽  
Simian Immunodeficiency Virus ◽  
Structural Similarity ◽  
Amino Terminus ◽  
Type I ◽  
Activation Domain ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Viruslike Particles ◽  
Hiv 1

ABSTRACT Vpx and Vpr are related lentiviral accessory proteins that enhance virus replication in macrophages and dendritic cells. Both proteins are packaged into virions and mediate their effects in the target cell through an interaction with an E3 ubiquitin ligase that contains DCAF1 and DDB1. When introduced into primary macrophages and dendritic cells in viruslike particles, Vpx can enhance the efficiency of a subsequent infection. Here, we confirm the ability of Vpx to enhance simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) infection of macrophages up to 100-fold by using single-cycle reporter viruses and by pretreatment of the cells with Vpx-containing viruslike particles. Vpx was also active in differentiated THP-1 cells but not in other cell lines. Induction of an antiviral state in macrophages with type I interferon significantly magnified the effect of Vpx on HIV-1 infection, suggesting that Vpx helps the virus to overcome an inducible intracellular restriction. Quantitative PCR quantitation of SIV and HIV-1 reverse transcripts in newly infected macrophages showed that the block was at an early step in reverse transcription. In spite of its structural similarity, Vpr was inactive. This difference allowed us to map the functional domains of Vpx with a panel of Vpr/Vpx chimeras. Analysis of the chimeras demonstrated that the amino-terminal domain of Vpx is important for the enhancement of infection. Fine mapping of the region indicated that amino acids at positions 9, 12, and 15 to 17 were required. Although the mutants failed to enhance infection, they retained their ability to interact with DCAF1. These findings suggest that the Vpx amino terminus contains an activation domain that serves as the binding site for a cellular restriction factor.

scholarly journals Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maisel J. Caliva ◽  
Won Seok Yang ◽  
Shirley Young-Robbins ◽  
Ming Zhou ◽  
Hana Yoon ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cellular Migration ◽  
Endosomal Trafficking ◽  
Matrix Assembly ◽  
Transmembrane Receptors ◽  
Surface Receptors ◽  
Proximity Ligation ◽  
Assembly Cell ◽  
Proteomics Approach ◽  
Α5β1 Integrin

AbstractEndosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility.

scholarly journals Identification of the fibronectin sequences required for assembly of a fibrillar matrix.

1991 ◽  
Vol 113 (6) ◽  
pp. 1463-1473 ◽  
Author(s):  
J E Schwarzbauer
Keyword(s):  
Cell Surface ◽  
Disulfide Bonds ◽  
Mammalian Cells ◽  
Retroviral Vectors ◽  
Binding Activity ◽  
Type I ◽  
Matrix Assembly ◽  
Surface Receptors ◽  
Matrix Fraction ◽  
Carboxy Terminal

During extracellular matrix assembly, fibronectin (FN) binds to cell surface receptors and initiates fibrillogenesis. As described in this report, matrix assembly has been dissected using recombinant FN polypeptides (recFNs) expressed in mammalian cells via retroviral vectors. RecFNs were assayed for incorporation into the detergent-insoluble cell matrix fraction and for formation of fibrils at the cell surface as detected by indirect immunofluorescence. Biochemical and immunocytochemical data are presented defining the minimum domain requirements for FN fibrillogenesis. The smallest functional recFN is half the size of native FN and contains intact amino- and carboxy-terminal regions with a large internal deletion spanning the collagen binding domain and the first seven type III repeats. Five type I repeats at the amino terminus are required for assembly and have FN binding activity. The dimer structure mediated by the carboxy-terminal interchain disulfide bonds is also essential. Surprisingly, recFNs lacking the RGDS cell binding site formed a significant fibrillar matrix. Therefore, FN-FN interactions and dimeric structure appear to be the major determinants of fibrillogenesis.

scholarly journals Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin.

1993 ◽  
Vol 121 (2) ◽  
pp. 469-477 ◽  
Author(s):  
J M Sipes ◽  
N Guo ◽  
E Nègre ◽  
T Vogel ◽  
H C Krutzsch ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Type I Collagen ◽  
Selective Inhibitor ◽  
Type I ◽  
Surface Receptors ◽  
Extracellular Matrix Components ◽  
Flanking Sequences ◽  
Fibronectin Binding

The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin-binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix.

scholarly journals Inhibition of binding of fibronectin to matrix assembly sites by anti-integrin (alpha 5 beta 1) antibodies.

1990 ◽  
Vol 111 (2) ◽  
pp. 699-708 ◽  
Author(s):  
F J Fogerty ◽  
S K Akiyama ◽  
K M Yamada ◽  
D F Mosher
Keyword(s):  
Cell Adhesion ◽  
Direct Interaction ◽  
Fibroblast Cell ◽  
Inhibitory Antibody ◽  
Matrix Assembly ◽  
Fibronectin Receptor ◽  
Amino Terminal ◽  
Fab Fragments ◽  
Cell Layers ◽  
Fibronectin Binding

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.

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