scholarly journals E3-13.7 Integral Membrane Proteins Encoded by Human Adenoviruses Alter Epidermal Growth Factor Receptor Trafficking by Interacting Directly with Receptors in Early Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3559-3572 ◽  
Author(s):  
Denise Crooks ◽  
Song Jae Kil ◽  
J. Michael McCaffery ◽  
Cathleen Carlin
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Viral Protein ◽  
Egf Receptor ◽  
Receptor Trafficking ◽  
Model Systems ◽  
Egf Receptors ◽  
Down Regulation ◽  
Human Adenoviruses ◽  
Early Endosomes

Animal cell viruses provide valuable model systems for studying many normal cellular processes, including membrane protein sorting. The focus of this study is an integral membrane protein encoded by the E3 transcription region of human adenoviruses called E3-13.7, which diverts recycling EGF receptors to lysosomes without increasing the rate of receptor internalization or intrinsic receptor tyrosine kinase activity. Although E3-13.7 can be found on the plasma membrane when it is overexpressed, its effect on EGF receptor trafficking suggests that the plasma membrane is not its primary site of action. Using cell fractionation and immunocytochemical experimental approaches, we now report that the viral protein is located predominantly in early endosomes and limiting membranes of endosome-to-lysosome transport intermediates called multivesicular endosomes. We also demonstrate that E3-13.7 physically associates with EGF receptors undergoing E3-13.7–mediated down-regulation in early endosomes. Receptor–viral protein complexes then dissociate, and EGF receptors proceed to lysosomes, where they are degraded, while E3-13.7 is retained in endosomes. We conclude that E3-13.7 is a resident early endocytic protein independent of EGF receptor expression, because it has identical intracellular localization in mouse cells lacking endogenous receptors and cells expressing a human cytomegalovirus-driven receptor cDNA. Finally, we demonstrate that EGF receptor residues 675–697 are required for E3-13.7–mediated down-regulation. Interestingly, this sequence includes a known EGF receptor leucine-based lysosomal sorting signal used during ligand-induced trafficking, which is also conserved in the viral protein. E3-13.7, therefore, provides a novel model system for determining the molecular basis of selective membrane protein transport in the endocytic pathway. Our studies also suggest new paradigms for understanding EGF receptor sorting in endosomes and adenovirus pathogenesis.

scholarly journals The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR

2016 ◽  
Vol 215 (4) ◽  
pp. 543-558 ◽  
Author(s):  
Sandra Scharaw ◽  
Murat Iskar ◽  
Alessandro Ori ◽  
Gaelle Boncompain ◽  
Vibor Laketa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Egf Receptor ◽  
Regulatory Mechanism ◽  
Transcriptional Regulator ◽  
Transport Efficiency ◽  
Early Endosomes ◽  
Epidermal Growth ◽  
Partial Degradation ◽  
Stimulation Of

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.

scholarly journals Endocytic Down-Regulation of ErbB2 Is Stimulated by Cleavage of Its C-Terminus

2007 ◽  
Vol 18 (9) ◽  
pp. 3656-3666 ◽  
Author(s):  
Mads Lerdrup ◽  
Silas Bruun ◽  
Michael V. Grandal ◽  
Kirstine Roepstorff ◽  
Malene M. Kristensen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Retention Mechanism ◽  
Full Length ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
Down Regulation ◽  
C Terminus ◽  
Early Endosomes

High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2.

scholarly journals Structurally related class I and class II receptor protein tyrosine kinases are down-regulated by the same E3 protein coded for by human group C adenoviruses.

1993 ◽  
Vol 120 (5) ◽  
pp. 1271-1279 ◽  
Author(s):  
E Kuivinen ◽  
B L Hoffman ◽  
P A Hoffman ◽  
C R Carlin
Keyword(s):  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Class Ii ◽  
Class I ◽  
Receptor Protein ◽  
Egf Receptors ◽  
Class Iii ◽  
Human Group ◽  
Down Regulation ◽  
In Cells

Receptor tyrosine kinases (RTKs) are grouped into subcategories based on shared sequence and structural features. Human group C adenoviruses down-regulate EGF receptors, which are members of the class I family of RTKs, during the early stages of infection. Adenovirus appears to utilize a nonsaturable intracellular pathway since it causes EGF-R down-regulation even in cells that significantly overexpress EGF-R. Adenovirus-induced down-regulation is mediated by a small hydrophobic molecule coded for by the E3 early transcription region that has recently been localized to plasma membrane. Here we examine intracellular trafficking of other RTKs in adenovirus-infected cells, to better understand the molecular basis for the action of the E3 protein. Although p185c-neu, which is a class I RTK closely related to the EGF receptor, is down-regulated in cells expressing physiological concentrations of this molecule, it is not down-regulated in tumor cell lines that significantly overexpress p185c-neu. Cell surface receptors for insulin and IGF1, which are class II RTKs, are also reduced in cells expressing the E3 protein, although to a slightly lesser extent than the EGF receptor. Moreover, whereas EGF receptors are degraded between 3- and 9-h postinfection, insulin and IGF1 receptors are degraded between 6- and 12-h postinfection under identical conditions. In contrast to the class I and class II RTKs, there is no difference in the expression of the class III receptors for PDGF and aFGF in cells infected with a virus with an intact E3 region versus a virus mutant with an internal deletion in the relevant E3 gene. These results suggest that the E3 protein provides an internalization and degradative sorting signal for some class I and class II RTKs, although down-regulation of class II RTKs is somewhat less efficient. Molecular recognition of class I and class II RTKs during adenovirus infection may not be due strictly to amino acid structure, however, since EGF-R but not p185c-neu is down-regulated in cells where it is significantly overexpressed.

scholarly journals Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin

1997 ◽  
Vol 136 (4) ◽  
pp. 811-821 ◽  
Author(s):  
Sanne van Delft ◽  
Christopher Schumacher ◽  
Willem Hage ◽  
Arie J. Verkleij ◽  
Paul M.P. van Bergen en Henegouwen
Keyword(s):  
Egf Receptor ◽  
Critical Role ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Endocytic Pathway ◽  
Egf Receptors ◽  
Coated Pits ◽  
Early Endosomes ◽  
Endosomal Membranes ◽  
Cell Fractions

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.

scholarly journals Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains

1999 ◽  
Vol 337 (3) ◽  
pp. 591-597 ◽  
Author(s):  
Mark G. WAUGH ◽  
Durward LAWSON ◽  
J. Justin HSUAN
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Cell Fractionation ◽  
Membrane Domains ◽  
Egf Receptors ◽  
Cell Extracts ◽  
Epidermal Growth

Increasing evidence for the organization of cell-surface proteins and lipids into different detergent-insoluble rafts led us to investigate epidermal growth factor (EGF) receptor activation in the plasma membranes of A431 carcinoma cells, using a combination of cell fractionation and immunoprecipitation techniques. Density-gradient centrifugation of sodium carbonate cell extracts revealed that the vast majority of both stimulated and unstimulated EGF receptors were concentrated in a caveolin-rich light membrane (CLM) fraction, with the biochemical characteristics of detergent-insoluble glycolipid-rich domains (DIGs). However, ultrastructural analysis of the CLM fraction revealed that it contained a heterogeneous collection of vesicles, some with sizes greater than that expected for individual caveolae. Experiments with detergent-solubilized cells and isolated CLMs indicated that, in contrast with caveolin, EGF receptors were unlikely to be localized to DIG domains. Furthermore, immunoisolation of caveolin from CLMs revealed that EGF receptor activation occurs in a compartment distinct from caveolae. Similarly, using an anti-(EGF receptor) antibody, the bulk of the cellular caveolin was not co-immunoprecipitated from CLMs, thereby confirming that these two proteins reside in separate membrane domains. The deduction that caveolar signalling and EGF receptor activation occur in separable rafts argues for a multiplicity of signal transduction compartments within the plasma membrane. In addition, by demonstrating that EGF receptor activation is compartmentalized within low-density, non-caveolar regions of the plasma membrane, it is also shown that the co-localization of proteins in a CLM fraction is insufficient to prove caveolar localization.

scholarly journals Role of the Sec61 Translocon in EGF Receptor Trafficking to the Nucleus and Gene Expression

2007 ◽  
Vol 18 (3) ◽  
pp. 1064-1072 ◽  
Author(s):  
Hong-Jun Liao ◽  
Graham Carpenter
Keyword(s):  
Gene Expression ◽  
Endoplasmic Reticulum ◽  
Growth Factor ◽  
Egf Receptor ◽  
Transmembrane Proteins ◽  
Receptor Trafficking ◽  
Cyclin D ◽  
Egf Receptors ◽  
Epidermal Growth

The epidermal growth factor (EGF)-dependent trafficking of the intact EGF receptor to the nucleus and its requirement for growth factor induction of cyclin D and other genes has been reported. Unresolved is the mechanism by which this or other transmembrane proteins are excised from a lipid bilayer before nuclear translocalization. We report that, after the addition of EGF, the cell surface EGF receptor is trafficked to the endoplasmic reticulum (ER) where it associates with Sec61β, a component of the Sec61 translocon, and is retrotranslocated from the ER to the cytoplasm. Abrogation of Sec61β expression prevents EGF-dependent localization of EGF receptors to the nucleus and expression of cyclin D. This indicates that EGF receptors are trafficked from the ER to the nucleus by a novel pathway that involves the Sec61 translocon.

scholarly journals Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric Gαs Protein

2004 ◽  
Vol 15 (12) ◽  
pp. 5538-5550 ◽  
Author(s):  
Bin Zheng ◽  
Christine Lavoie ◽  
Ting-Dong Tang ◽  
Phuong Ma ◽  
Timo Meerloo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Membrane Trafficking ◽  
Egf Receptor ◽  
Egf Receptors ◽  
Protein Subunit ◽  
Cooperative Effects ◽  
Knock Down ◽  
Early Endosomes ◽  
Epidermal Growth

Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Gαs) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Gαs expression by RNA interference (RNAi) delays receptor degradation. We also show that Gαs and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Gαs coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Gαs colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Gαs on endosomes. Furthermore, knock-down of both Hrs and Gαs by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Gαs and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Gαs in EGF receptor degradation and provide mechanistic insights into the function of Gαs in endocytic sorting.

scholarly journals A Novel Type III Endosome Transmembrane Protein, TEMP

Cells ◽  
2012 ◽  
Vol 1 (4) ◽  
pp. 1029-1044 ◽  
Author(s):  
Rajith N. Aturaliya ◽  
Markus C. Kerr ◽  
Rohan D. Teasdale
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Subcellular Localisation ◽  
Cell Periphery ◽  
Recycling Endosomes ◽  
Type Iii ◽  
Extracellular Region ◽  
Early Endosomes

As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP's plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport.

A novel action of epidermal growth factor in rat granulosa cells: its potentiation of gonadotrophin action

1995 ◽  
Vol 15 (3) ◽  
pp. 283-291 ◽  
Author(s):  
M-A Hattori ◽  
E Yoshino ◽  
Y Shinohara ◽  
R Horiuchi ◽  
I Kojima
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Egf Receptors ◽  
Down Regulation ◽  
Control Value ◽  
Progesterone Synthesis ◽  
Affinity Labelling ◽  
Epidermal Growth

ABSTRACT It is well known that epidermal growth factor (EGF) induces down-regulation of LH receptors and desensitization to gonadotrophin stimulation in gonadal cells, including granulosa cells. In a previous study we showed that EGF receptor levels in rat granulosa cells were increased up to fourfold after 96 h of culture with human GH in the presence of FSH, and the present study has evaluated the action of EGF on these cells. The induced EGF receptors were identical in size to the pre-existing receptors as assessed by affinity labelling with 125I-EGF. After 48 h in culture, various amounts of EGF (0·5–10 ng) were added and the cells were cultured for a further 48 h. The addition of EGF caused down-regulation of LH receptors in cells expressing high levels of EGF receptors. However, this down-regulation was less than that in control cells. After the cells were washed, cAMP synthesis in response to human chorionic gonadotrophin (hCG) increased by two to three times the control value and this increase was closely correlated with an increase in EGF receptor content. However, stimulation with cholera toxin or forskolin showed no such augmentation, indicating that it may not be due to quantitative alterations in G proteins and their effector systems. Induction of EGF potentiation required long-term exposure to EGF, for at least more than 24 h. In addition, progesterone synthesis was sensitive to stimulation with lower doses of hCG. These findings indicate that the activation of hGH-induced EGF receptors may potentiate gonadotrophin action in granulosa cells.

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