A novel RGD-independent fibronectin assembly pathway initiated by alpha4beta1 integrin binding to the alternatively spliced V region

2000 ◽  
Vol 113 (8) ◽  
pp. 1491-1498 ◽  
Author(s):  
J.L. Sechler ◽  
A.M. Cumiskey ◽  
D.M. Gazzola ◽  
J.E. Schwarzbauer
Keyword(s):  
Integrin Receptors ◽  
Matrix Assembly ◽  
Rgd Sequence ◽  
Assembly Pathway ◽  
B Lymphoma Cells ◽  
Pathway Activation ◽  
Exogenous Agents ◽  
Alternatively Spliced ◽  
V Region ◽  
Alpha4beta1 Integrin

Fibronectin (FN) matrix assembly is a multi-step process that involves binding to integrin receptors, FN-FN interactions and connections to the actin cytoskeleton. Ultimately, FN is converted into stable matrix fibrils that are detergent-insoluble. RGD-binding integrins such as alpha5beta1 play a major role in the assembly of fibrillar FN. Here we show that alpha4beta1 binding to the alternatively spliced V (IIICS) region of FN initiates an alternative assembly pathway. Activation of alpha4beta1 with exogenous agents such as Mn(2+) or a beta1-stimulatory antibody TS2/16 was sufficient to induce initiation of FN fibrillogenesis by Ramos B lymphoma cells and by CHO(B2)alpha4 cells. Using recombinant FNs lacking specific sequences, we show that assembly is independent of the RGD sequence but requires the V25/CS-1 segment. Previously, we have characterized an activated recombinant FN (FN III(1–7)) that rapidly forms detergent-insoluble multimers upon binding to alpha5beta1 integrin. Alpha4beta1 also formed FNdeltaIII(1–7) multimers without the aid of exogenous stimulants, suggesting that an activated form of FN can override the need for activation of the integrin. In contrast to assembly by alpha5beta1, actin filaments remained largely cortical and no change in cell growth rate was observed with alpha4beta1-mediated assembly. These results show that binding sites on FN other than the RGD sequence/synergy site and distant from the cell binding domain can promote FN assembly. Thus, there appear to be multiple, integrin-specific mechanisms for assembly of FN matrix.

scholarly journals Altered rate of fibronectin matrix assembly by deletion of the first type III repeats.

1996 ◽  
Vol 134 (2) ◽  
pp. 573-583 ◽  
Author(s):  
J L Sechler ◽  
Y Takada ◽  
J E Schwarzbauer
Keyword(s):  
Early Stage ◽  
Expression System ◽  
Integrin Receptors ◽  
Matrix Assembly ◽  
Type Iii ◽  
Amino Terminal ◽  
Rgd Sequence ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression

The assembly of fibronectin (FN) into a fibrillar matrix is a complex stepwise process that involves binding to integrin receptors as well as interactions between FN molecules. To follow the progression of matrix formation and determine the stages during which specific domains function, we have developed cell lines that lack an endogenous FN matrix but will form fibrils when provided with exogenous FN. Recombinant FNs (recFN) containing deletions of either the RGD cell-binding sequence (RGD-) or the first type III repeats (FN delta III1-7) including the III1 FN binding site were generated with the baculovirus insect cell expression system. After addition to cells, recFN matrix assembly was monitored by indirect immunofluorescence and by insolubility in the detergent deoxycholate (DOC). In the absence of any native FN, FN delta III1-7 was assembled into fibrils and was converted into DOC-insoluble matrix. This process could be inhibited by the amino-terminal 70 kD fragment of FN, showing that FN delta III1-7 follows an assembly pathway similar to FN. The progression of FN delta III1-7 assembly differed from native FN in that the recFN became DOC-insoluble more quickly. In contrast, RGD- recFNs were not formed into fibrils except when added in combination with native FN. These results show that the RGD sequence is essential for the initiation step but fibrils can form independently of the III1-7 modules. The altered rate of FN delta III1-7 assembly suggests that one function of the missing repeats might be to modulate an early stage of matrix formation.

scholarly journals The alternatively spliced V region contributes to the differential incorporation of plasma and cellular fibronectins into fibrin clots.

1992 ◽  
Vol 119 (4) ◽  
pp. 923-933 ◽  
Author(s):  
C L Wilson ◽  
J E Schwarzbauer
Keyword(s):  
Blood Clot ◽  
Coagulation Factor ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Asymmetric Distribution ◽  
Alternatively Spliced ◽  
V Region ◽  
Fibrin Clots

During blood clot formation in vivo, plasma fibronectin (pFN) is cross-linked to fibrin by coagulation factor XIIIa. Cellular FN (cFN), which localizes to connective tissue, is distinguished from pFN by the inclusion of alternatively spliced segments. To determine if these two FNs are functionally equivalent in blood clotting, the cross-linking of rat pFN and cFN to fibrin was compared in an in vitro clotting assay. Fibrinogen and FN were incubated at physiological ratios in the presence of thrombin and factor XIIIa. Cross-linking of FN to fibrin was monitored by SDS-PAGE and immunoblotting. Over 24 h, cFN was incorporated at a significantly slower rate than pFN and was not completely cross-linked to fibrin at a temperature that favors this interaction (0 degrees C). This difference was observed with purified fibrinogens from human, rat, and bovine and with rat plasma and was maintained even after incubation of pFN with rat fibroblasts for several days. Using the same assay, purified recombinant V(+)-V0 and V(+)-V+ FN dimers resembling pFN and cFN, respectively, showed a similar difference in cross-linking kinetics. These results suggest that the asymmetric distribution of the V region among pFN dimers plays a role in regulating its incorporation into blood clots. In fibrin clots, cFN was converted into a set of cross-linked intermediates distinct from those of pFN. For example, while pFN was initially cross-linked into a pFN-fibrin alpha heterodimer, this product was not a major intermediate in clots formed with cFN. This finding, in conjunction with evidence for the formation of factor XIIIa-catalyzed cFN-cFN cross-links, indicated that cFN molecules interact with each other, and with fibrin, differently from pFN. Together, these results show an important functional distinction between pFN and cFN.

scholarly journals Inhibition of nonsense-mediated decay rescues functional p53β/γ isoforms in MDM2-amplified cancers

2020 ◽  
Author(s):  
Jayanthi P. Gudikote ◽  
Tina Cascone ◽  
Alissa Poteete ◽  
Piyada Sitthideatphaiboon ◽  
Qiuyu Wu ◽  
...  
Keyword(s):  
Cancer Cells ◽  
P53 Gene ◽  
Hpv Infection ◽  
P53 Mutations ◽  
Nonsense Mediated Decay ◽  
Pathway Activation ◽  
Alternatively Spliced ◽  
Exon 9 ◽  
Truncated Isoforms ◽  
E6 Protein

ABSTRACTCommon mechanisms for p53 loss in cancer include expression of MDM2 or the human papilloma virus (HPV)-encoded E6 protein which both mediate degradation of wild-type (WT) p53 (p53α). Here, we show that two alternatively-spliced, functional, truncated isoforms of p53 (p53β and p53γ, containing exons 1-9 of the p53 gene) can be markedly upregulated by pharmacologic or genetic inhibition of nonsense mediated decay (NMD), a regulator of aberrant mRNA stability. These isoforms lack the MDM2 binding domain and hence have reduced susceptibility to MDM2-mediated degradation. In MDM2-overexpressing cells bearing wildtype TP53 gene, NMD blockade increased p53β/γ expression and p53 pathway activation, enhanced radiosensitivity, and inhibited tumor growth. A similar pattern was observed in HPV+ cancer cells and in cancer cells with p53 mutations downstream of exon 9. These results identify a novel therapeutic strategy for restoration of p53 function in tumors rendered p53 deficient through MDM2 overexpression, HPV infection, or certain p53 mutations.

scholarly journals Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells.

1987 ◽  
Vol 7 (12) ◽  
pp. 4297-4307 ◽  
Author(s):  
P A Norton ◽  
R O Hynes
Keyword(s):  
Alternative Splicing ◽  
Mrna Level ◽  
Receptor Expression ◽  
Transcriptional Level ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Oncogenic Transformation ◽  
Rous Sarcoma ◽  
Alternatively Spliced ◽  
V Region

To study the alternative splicing of fibronectin during embryogenesis and oncogenic transformation, we isolated cDNA clones of chicken fibronectin. The partial amino acid sequence deduced from sequencing of these clones showed that, overall, chicken fibronectin is approximately 80% identical with mammalian fibronectins. However, two of the three known regions of alternative splicing differed from this average. The V region was significantly more divergent, and RNA from embryonic chicken liver showed a pattern of V exon splicing which was distinct from that seen in human or rat fibronectins. In contrast, the EIIIB segment was very highly conserved (96%). As in mammals, this segment and another (EIIIA) were alternatively spliced in a cell-type-specific fashion. EIIIA+ and EIIIB+ species were almost absent in liver but predominated in total embryo RNA at all times from 2.5 to 11 days postfertilization. We also examined the possible contributions of fibronectin splicing and integrin receptor expression to the loss of fibronectin on oncogenic transformation. We detected little change in fibronectin splicing, other than a slight increase in representation of EIIIB+ species in fibroblasts after transformation by Rous sarcoma virus. It was also established that the overall reduction in fibronectin mRNA level observed after transformation was not accompanied by a decrease in integrin mRNA levels, indicating that fibronectin and integrin receptors are not coordinately regulated at the transcriptional level.

Epitope Mapping of Inhibitory Monoclonal Antibodies to Human von Willebrand Factor by Using Recombinant cDNA Libraries

1994 ◽  
Vol 71 (06) ◽  
pp. 788-792 ◽  
Author(s):  
Geneviéve Piétu ◽  
Anne-Sophie Ribba ◽  
Ghislaine Chérel ◽  
Virginie Siguret ◽  
Bernadette Obert ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Recombinant Expression ◽  
Cdna Libraries ◽  
Integrin Receptors ◽  
Binding Domains ◽  
Rgd Sequence ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTwo recombinant expression libraries containing small (300-600 base pairs) cDNA fragments of von Willebrand Factor (vWF) were screened in order to map the epitope of monoclonal antibodies (MAbs) to vWF. Among eleven MAbs tested, seven were effectively mapped. The epitopes of MAbs 418 and 522, which inhibit the binding of vWF to Factor VIII (FVIII), were localized between Leu 2 and Arg 53 and between Glu 35 and He 81 of the vWF subunit respectively, within the N-terminal trypsin fragment called SpIII-T4 [amino acids (aa) 1-272] which contains a binding domain for FVIII. The epitope of MAb 710, which inhibits the binding of vWF to glycoprotein lb (GPIb), was identified between Ser 593 and Ser 678 on the tryptic 52/48 kDa fragment (aa 449-728) which contains binding domains for GPIb, collagen, heparin, sulfatides and subendothclium extracellular matrices. The epitope of MAb 723, which does not interfere with any known function of vWF, was localized between Ser 523 and Gly 588. The epitopes of MAb 505 and MAb 400, which inhibit the binding of v WF to collagen, were identified between Leu 927 and Arg 1114 within the SPI fragment (aa 911-1365) corresponding to the central part of the vWF subunit. The epitope of MAb 9, which inhibits the binding of vWF to GPIIb/IIIa, was identified in the C-terminal part of the vWF subunit between Gin 1704 and Asp 1746, the latter being the third aa of the RGD sequence common to adhesive proteins and serving as a recognition site for integrin receptors.

scholarly journals Structural mechanism of laminin recognition by integrin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Takao Arimori ◽  
Naoyuki Miyazaki ◽  
Emiko Mihara ◽  
Mamoru Takizawa ◽  
Yukimasa Taniguchi ◽  
...  
Keyword(s):  
Metal Ion ◽  
Electrostatic Interactions ◽  
High Mobility ◽  
Laminin Receptor ◽  
Integrin Receptors ◽  
Unique Role ◽  
Structural Mechanism ◽  
Cryo Electron Microscopy ◽  
Alternatively Spliced ◽  
Anchor Points

AbstractRecognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6β1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin β1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the β-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.

scholarly journals Multiple functions for the catenin family member plakoglobin in cadherin-dependent adhesion, fibronectin matrix assembly and Xenopus gastrulation movements

2018 ◽  
Author(s):  
Glen D. Hirsh ◽  
Bette J. Dzamba ◽  
Pooja R. Sonavane ◽  
David R. Shook ◽  
Claire M. Allen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Actin Binding ◽  
Convergent Extension ◽  
Integrin Receptors ◽  
Matrix Assembly ◽  
Cell Matrix ◽  
Fibronectin Matrix ◽  
Cytoplasmic Tails ◽  
Fibronectin Assembly

AbstractShaping an embryo requires tissue-scale cell rearrangements known as morphogenetic events. These force-dependent processes require cells to adhere to their neighbors, through cadherin-catenin complexes, and to their extracellular matrix substrates, through integrin-based focal contacts. Integrin receptors are not only important for attachment to the extracellular matrix, but also for its fibrillar assembly. Fibrillogenesis requires actomyosin contractility, regulated in part by cadherin-catenin complexes. One such catenin, plakoglobin, mediates the attachment of actin stress fibers to cadherin cytoplasmic tails through its interactions with actin-binding proteins. In Xenopus gastrulae, plakoglobin has been identified as an essential member in the force-induced collective migration of the mesendoderm tissue. In the current study, we have further characterized the role of plakoglobin in two additional morphogenetic processes, epiboly and convergent extension. Plakoglobin-deficient tadpoles are 40% shorter and gastrulae contain notochords that are 60% wider than stage-matched controls, indicating convergent extension defects. The radially intercalating ectoderm of morphant animal caps is nearly twice as thick as controls. Furthermore, morphant embryos exhibit a failure to assemble a fibronectin matrix at the notochord-somite-boundary or along the blastocoel roof. The loss of the fibronectin matrix, while not due to changes in overall patterning, is a result of a failure to assemble the soluble dimers into long fibrils. The force of attachment to a cadherin or fibronectin substrate is reduced in plakoglobin morphants, indicating defects in adhesion to both cadherin and fibronectin. These data suggest that plakoglobin regulates morphogenesis and fibronectin assembly through cell-cell and cell-matrix adhesion.

scholarly journals Selective secretion of alternatively spliced fibronectin variants.

1989 ◽  
Vol 109 (6) ◽  
pp. 3445-3453 ◽  
Author(s):  
J E Schwarzbauer ◽  
C S Spencer ◽  
C L Wilson
Keyword(s):  
Protein Interactions ◽  
Time Course ◽  
Deletion Mapping ◽  
Protein Protein Interactions ◽  
Dimer Formation ◽  
Alternatively Spliced ◽  
Selective Retention ◽  
V Region

We demonstrate that the alternatively spliced variable (V) region of fibronectin (FN) is required for secretion of FN dimers during biosynthesis. Alternative splicing of the V segment of the rat FN transcript generates three subunit variants (V120, V95, V0) that differ by the inclusion or omission of an additional 120 or 95 amino acids. We are exploring the functions of this segment by expressing variant cDNAs in normal and transformed fibroblasts. Like FN itself, the cDNA-encoded polypeptides (deminectins [DNs]) containing the V120 or V95 segment are efficiently secreted as disulfide-bonded homodimers. However, few homodimers of DNs lacking this region, V0 DNs, are secreted. V0 homodimers do form inside the cell, as demonstrated by biosynthetic analyses of dimer formation and secretion using pulse-chase and time course experiments, but these dimers seldom reach the cell surface and are probably degraded intracellularly. Coexpression of V0 and V120 subunits results in intracellular formation of three types of dimers, V0-V0, V0-V120, and V120-V120, but only the V120-containing dimers are secreted. This selective retention of V0 homodimers indicates that the V region is required for formation and secretion of native FN dimers. In an analogous in vivo situation, we show that plasma FN also lacks V0-V0 dimers and consists of V0-V+ and V+-V+ combinations. Dissection of V region sequences by deletion mapping localizes the major site involved in DN dimer secretion to an 18-amino acid segment within V95. In addition, high levels of dimer secretion can be restored by insertion of V into a heterologous site 10 kD COOH terminal to its normal location. We discuss the potential role of intracellular protein-protein interactions in FN dimer formation.

Melatonin provokes cell death in human B-lymphoma cells by mitochondrial-dependent apoptotic pathway activation

2005 ◽  
Vol 39 (4) ◽  
pp. 425-431 ◽  
Author(s):  
Oriana Trubiani ◽  
Rina Recchioni ◽  
Fausto Moroni ◽  
Jacopo Pizzicannella ◽  
Sergio Caputi ◽  
...  
Keyword(s):  
Cell Death ◽  
Lymphoma Cells ◽  
Apoptotic Pathway ◽  
B Lymphoma ◽  
B Lymphoma Cells ◽  
Pathway Activation

scholarly journals Modulatory Roles for Integrin Activation and the Synergy Site of Fibronectin during Matrix Assembly

1997 ◽  
Vol 8 (12) ◽  
pp. 2563-2573 ◽  
Author(s):  
Jan L. Sechler ◽  
Siobhan A. Corbett ◽  
Jean E. Schwarzbauer
Keyword(s):  
De Novo ◽  
Slow Phase ◽  
Lag Phase ◽  
Integrin Receptors ◽  
Cell Binding ◽  
Matrix Assembly ◽  
Prolonged Incubation ◽  
Absolute Requirement ◽  
Binding Sequence

Initiation of fibronectin (FN) matrix assembly is dependent on specific interactions between FN and cell surface integrin receptors. Here, we show that de novo FN matrix assembly exhibits a slow phase during initiation of fibrillogenesis followed by a more rapid growth phase. Mn2+, which acts by enhancing integrin function, increased the rate of FN fibril growth, but only after the initial lag phase. The RGD cell-binding sequence in type III repeat 10 is an absolute requirement for initiation by α5β1 integrin. To investigate the role of the cell-binding synergy site in the adjacent repeat III9, a full-length recombinant FN containing a synergy mutation, FN(syn−), was tested for its ability to form fibrils. Mutation of this site drastically reduced FN assembly by CHOα5 cells. Only sparse short fibrils were formed even after prolonged incubation, indicating that FN(syn−) is defective in progression of the assembly process. These results show that the synergy site is essential for α5β1-mediated accumulation of a FN matrix. However, the incorporation of FN(syn−) into fibrils and the deoxycholate-insoluble matrix could be stimulated by Mn2+. Therefore, exogenous activation of integrin receptors can overcome the requirement for FN’s synergy site as well as modulate the rate of FN matrix formation.

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