Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction.

An important question regarding autocrine transformation by v-sis is whether intracellularly activated PDGF receptors are sufficient to transform cells or whether activated receptor-ligand complexes are required at the cell surface. We have addressed this question by inhibiting cell surface transport of a membrane-anchored v-sis protein utilizing the ER retention signal of the adenoviral transmembrane protein E3/19K. A v-sis fusion protein containing this signal was retained within the cell and not transported to the cell surface as confirmed by immunofluorescent localization experiments. Also, proteolytic maturation of this protein was suppressed, indicating inefficient transport to post-Golgi compartments of the secretory pathway. When compared with v-sis proteins lacking a functional retention signal, the ER-retained protein showed a diminished ability to transform NIH 3T3 cells, as measured by the number and size of foci formed. In newly established cell lines, the ER-retained protein did not down-regulate PDGF receptors. However, continued passage of these cells selected for a fully transformed phenotype exhibiting downregulated PDGF receptors and proteolytically processed v-sis protein. These results indicate that productive autocrine interactions occur in a post-ER compartment of the secretory pathway. Transport of v-sis protein beyond the Golgi correlated with acquisition of the transformed phenotype. Furthermore, suramin treatment reversed transformation and upregulated the expression of cell surface PDGF receptors, suggesting an important role for receptor-ligand complexes localized to the cell surface.

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Mechanisms of Signaling through Urokinase Receptor and the Cellular Response

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615847 ◽

1999 ◽

Vol 82 (08) ◽

pp. 305-311 ◽

Cited By ~ 31

Author(s):

Yuri Koshelnick ◽

Monika Ehart ◽

Hannes Stockinger ◽

Bernd Binder

Keyword(s):

Cell Surface ◽

Proteolytic Activity ◽

Tumor Invasion ◽

Cellular Response ◽

Transmembrane Protein ◽

Urokinase Receptor ◽

Biological Processes ◽

In Vivo Models ◽

Proteolytic Activation ◽

Core Proteins

IntroductionThe urokinase-urokinase receptor (u-PA-u-PAR) system seems to play a crucial role in a number of biological processes, including local fibrinolysis, tumor invasion, angiogenesis, neointima and atherosclerotic plaque formation, inflammation, and matrix remodeling during wound healing and development.1-6 Binding of urokinase to its specific receptor provides cells with a localized proteolytic potential. It stimulates conversion of cell surface-bound plasminogen into active plasmin, which, in turn, is required for proteolytic degradation of basement membrane components, including fibronectin, collagen, laminin, and proteoglycan core proteins.7 Moreover, plasmin activates other matrix-degrading enzymes, such as matrix metalloproteinases.8 Overexpression of u-PA/u-PAR correlates with tumor invasion and metastasis formation,9-13 while reduction of cell-surface bound u-PA and inhibition of u-PAR expression leads to a significant decrease of invasive and metastatic activity.14 Specific antagonists that suppress binding of u-PA to u-PAR have been shown to inhibit cell-surface plasminogen activation, tumor growth, and angiogenesis both in vitro and in vivo models.15,16 Independently of its proteolytic activity, u-PA is implicated in many biological processes that seem to require u-PAR-mediated intracellular signal transduction, such as proliferation, chemotactic movement and adhesion, migration, and differentiation.17 Data obtained in the late 1980s indicated that u-PA not only provides cells with local proteolytic activity, but might also be capable of transducing signals to the cell.18-22 At that time, however, the u-PAR has just been isolated, cloned, and identified as a glycosylphosphatidylinositol (GPI)-linked protein and not a transmembrane protein. Signaling via the u-PAR was, therefore, regarded as being unlikely, and the effects of u-PA on cell proliferation18-22 were thought to be mediated by proteolytic activation of latent growth factors. The assumption of direct signaling via u-PAR was, in fact, considered controversial, until about 10 years later when a physical association between u-PAR and signaling proteins was found.23 From this report on, several proteins associated with u-PAR have been identified. Now, u-PAR seems to be part of a large “signalosome” associated and interacting with several proteins on both the outside and inside of the cell.

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Interaction of Pestiviral E1 and E2 Sequences in Dimer Formation and Intracellular Retention

International Journal of Molecular Sciences ◽

10.3390/ijms22147285 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7285

Author(s):

Yu Mu ◽

Birke Andrea Tews ◽

Christine Luttermann ◽

Gregor Meyers

Keyword(s):

Life Cycle ◽

Cell Surface ◽

Double Mutant ◽

Essential Role ◽

Infectious Clone ◽

Intracellular Localization ◽

Dimer Formation ◽

Covalent Linkage ◽

Crucial Step ◽

Retention Signal

Pestiviruses contain three envelope proteins: Erns, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely. E1 also establishes disulfide linked heterodimers with E2, which are crucial for the recovery of infectious viruses. Co-expression analyses with the HA-tagged E1 wt/E1 mutants and E2 wt/E2 mutants demonstrated that C123 in E1 and C295 in E2 are the critical sites for E1/E2 heterodimer formation. Introduction of mutations preventing E1/E2 heterodimer formation into the full-length infectious clone of BVDV CP7 prevented the recovery of infectious viruses, proving that C123 in E1 and C295 in E2 play an essential role in the BVDV life cycle, and further support the conclusion that heterodimer formation is the crucial step. Interestingly, we found that the retention signal of E1 is mandatory for intracellular localization of the heterodimer, so that absence of the E1 retention signal directs the heterodimer to the cell surface even though the E2 retention signal is still present. The covalent linkage between E1 and E2 plays an essential role for this process.

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Induction of β3-Integrin Gene Expression by Sustained Activation of the Ras-Regulated Raf–MEK–Extracellular Signal-Regulated Kinase Signaling Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.21.9.3192-3205.2001 ◽

2001 ◽

Vol 21 (9) ◽

pp. 3192-3205 ◽

Cited By ~ 94

Author(s):

Douglas Woods ◽

Holly Cherwinski ◽

Eleni Venetsanakos ◽

Arun Bhat ◽

Stephan Gysin ◽

...

Keyword(s):

Cell Surface ◽

Signaling Pathway ◽

Erk Signaling ◽

Integrin Expression ◽

3T3 Cells ◽

Extracellular Signal ◽

Integrin Gene ◽

Β3 Integrin ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf–MEK–extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of α6- and β3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface α6- and β3-integrin expression. Induced β3-integrin was expressed on the cell surface as a heterodimer with αv-integrin; however, the overall level of αv-integrin expression was not altered by Ras or Raf. Raf-induced β3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce β3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated β3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, β3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on β3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface αvβ3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

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Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility

Journal of Cell Science ◽

10.1242/jcs.113.18.3227 ◽

2000 ◽

Vol 113 (18) ◽

pp. 3227-3240 ◽

Cited By ~ 3

Author(s):

P.U. Le ◽

N. Benlimame ◽

A. Lagana ◽

A. Raz ◽

I.R. Nabi

Keyword(s):

Cell Surface ◽

Cell Motility ◽

Endocytic Pathway ◽

Limiting Factor ◽

Autocrine Motility Factor ◽

Endocytic Compartment ◽

Motility Factor ◽

Microtubule Disruption ◽

Nih 3T3 ◽

Fibronectin Fibrils

Autocrine motility factor receptor (AMF-R) is internalized via a clathrin-independent pathway to smooth endoplasmic reticulum tubules. This endocytic pathway is shown here to be inhibited by methyl-(beta)-cyclodextrin (m(beta)CD) implicating caveolae or caveolae-like structures in AMF internalization to smooth ER. AMF-R is also internalized via a clathrin-dependent pathway to a transferrin receptor-negative, LAMP-1/lgpA-negative endocytic compartment identified by electron microscopy as a multivesicular body (MVB). Endocytosed AMF recycles to cell surface fibrillar structures which colocalize with fibronectin; AMF-R recycling is inhibited at 20 degrees C, which blocks endocytosis past the early endosome, but not by m(beta)CD demonstrating that AMF-R recycling to fibronectin fibrils is mediated by clathrin-dependent endocytosis to MVBs. Microtubule disruption with nocodazole did not affect delivery of bAMF to cell surface fibrils indicating that recycling bAMF traverses the MVB but not a later endocytic compartment. Plating NIH-3T3 cells on an AMF coated substrate did not specifically affect cell adhesion but prevented bAMF delivery to cell surface fibronectin fibrils and reduced cell motility. AMF-R internalization and recycling via the clathrin-mediated pathway are therefore rate-limiting for cell motility. This recycling pathway to the site of deposition of fibronectin may be implicated in the de novo formation of cellular attachments or the remodeling of the extracellular matrix during cell movement.

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Modulation of the transport of a lysosomal enzyme by PDGF.

The Journal of Cell Biology ◽

10.1083/jcb.110.2.319 ◽

1990 ◽

Vol 110 (2) ◽

pp. 319-326 ◽

Cited By ~ 46

Author(s):

E M Prence ◽

J M Dong ◽

G G Sahagian

Keyword(s):

Secretory Pathway ◽

Cathepsin L ◽

Control Level ◽

3T3 Cells ◽

Lysosomal Protease ◽

Affinity Ligand ◽

The Media ◽

Mouse Cells ◽

Nih 3T3 ◽

The Relationship

The major excreted protein (MEP) of transformed mouse fibroblasts is the lysosomal protease, cathepsin L. MEP is also secreted by untransformed mouse cells in response to growth factors and tumor promoters, and is thought to play a role in cell growth and transformation. To determine the relationship between MEP synthesis and MEP secretion, we have examined these events in PDGF-treated NIH 3T3 cells. PDGF enhanced MEP synthesis and caused the diversion of MEP from the lysosomal delivery pathway to a secretory pathway. These two effects were found to be regulated independently at various times after growth factor addition. Short PDGF treatments (0.5 or 1 h) resulted in quantitative secretion of MEP although synthesis was near the control level. High levels of both synthesis and secretion occurred between 2 and 14 h of PDGF treatment. Between 18 and 30 h, the amount of secreted MEP returned to the low control level even though synthesis remained elevated. The secretion was specific for MEP; other lysosomal enzymes were not found in the media from PDGF-treated cells. PDGF-induced secretion of MEP was inhibited 84% by cycloheximide, suggesting that protein synthesis is required to elicit this effect. PDGF also caused a time-dependent increase in mannose 6-phosphate (Man-6-P) receptor-mediated endocytosis. These data support a model in which PDGF alters the distribution of Man-6-P receptors such that the Golgi concentration of receptors becomes limiting, thereby causing the selective secretion of the low affinity ligand, MEP.

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HIV-2 transmembrane protein GP36 binds to human cell surface proteins and inhibits lymphocyte proliferation

Journal of Cancer Research and Clinical Oncology ◽

10.1007/bf02559889 ◽

1995 ◽

Vol 121 (S1) ◽

pp. S34-S34

Author(s):

Y. -H. Chen ◽

A. Christiansen ◽

G. Böck ◽

M. P. Dierich

Keyword(s):

Cell Surface ◽

Human Cell ◽

Lymphocyte Proliferation ◽

Transmembrane Protein ◽

Surface Proteins ◽

Cell Surface Proteins

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Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport.

The Journal of Cell Biology ◽

10.1083/jcb.118.2.285 ◽

1992 ◽

Vol 118 (2) ◽

pp. 285-299 ◽

Cited By ~ 93

Author(s):

H Liu ◽

A Bretscher

Keyword(s):

Cell Surface ◽

Secretory Pathway ◽

Synthetic Lethality ◽

Vesicular Transport ◽

Yeast Cells ◽

Carboxypeptidase Y ◽

Apparent Loss ◽

Abnormal Accumulation ◽

Late Step ◽

Actin Cables

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.

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Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region

International Journal of Molecular Sciences ◽

10.3390/ijms221910207 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10207

Author(s):

Julien Vitry ◽

Guillaume Paré ◽

Andréa Murru ◽

Xavier Charest-Morin ◽

Halim Maaroufi ◽

...

Keyword(s):

Cell Surface ◽

Cell Activation ◽

Secretory Pathway ◽

Surface Expression ◽

Site Directed Mutagenesis ◽

Cell Surface Expression ◽

Dependent Manner ◽

Inhibitory Receptor ◽

Lectin Receptors ◽

Stalk Domain

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

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