scholarly journals Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

2004 ◽  
Vol 15 (8) ◽  
pp. 3591-3604 ◽  
Author(s):  
Espen Stang ◽  
Frøydis D. Blystad ◽  
Maja Kazazic ◽  
Vibeke Bertelsen ◽  
Tonje Brodahl ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Protein S ◽  
Egf Receptors ◽  
Ubiquitinated Protein ◽  
Coated Pits ◽  
Independent Manner ◽  
Sh3 Domains

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

scholarly journals Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits

2006 ◽  
Vol 26 (2) ◽  
pp. 389-401 ◽  
Author(s):  
Lene E. Johannessen ◽  
Nina Marie Pedersen ◽  
Ketil Winther Pedersen ◽  
Inger Helene Madshus ◽  
Espen Stang
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Mitogen Activated Protein Kinase ◽  
Coated Pits ◽  
Α Subunit ◽  
Sh3 Domains ◽  
Epidermal Growth

ABSTRACT In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the μ2 or α subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the α subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the α subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.

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 Disabled Is a Putative Adaptor Protein That Functions during Signaling by the Sevenless Receptor Tyrosine Kinase

1998 ◽  
Vol 18 (8) ◽  
pp. 4844-4854 ◽  
Author(s):  
Ngocdiep Le ◽  
Michael A. Simon
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathway ◽  
Receptor Tyrosine Kinase ◽  
Biochemical Analysis ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Sh3 Domains ◽  
Sevenless Receptor Tyrosine Kinase ◽  
Ptb Domain

ABSTRACT DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by thesevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.

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.

Mutually dependent localization of megalin and Dab2 in the renal proximal tubule

2005 ◽  
Vol 289 (3) ◽  
pp. F569-F576 ◽  
Author(s):  
J. Nagai ◽  
E. I. Christensen ◽  
S. M. Morris ◽  
T. E. Willnow ◽  
J. A. Cooper ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Proximal Tubule ◽  
Binding Protein ◽  
Adaptor Protein ◽  
Renal Proximal Tubule ◽  
Retinol Binding Protein ◽  
Coated Pits ◽  
Receptor Associated Protein ◽  
Protein Levels

Disabled-2 (Dab2) is a cytoplasmic adaptor protein that binds to the cytoplasmic tail of the multiligand endocytic receptor megalin, abundantly expressed in renal proximal tubules. Deletion of Dab2 induces a urinary increase in specific plasma proteins such as vitamin D binding protein and retinol binding protein (Morris SM, Tallquist MD, Rock CO, and Cooper JA. EMBO J 21: 1555–1564, 2002). However, the subcellular localization of Dab2 in the renal proximal tubule and its function have not been fully elucidated yet. Here, we report the characterization of Dab2 in the renal proximal tubule. Immunohistocytochemistry revealed colocalization with megalin in coated pits and vesicles but not in dense apical tubules and the brush border. Kidney-specific megalin knockout almost abolished Dab2 staining, indicating that Dab2 subcellular localization requires megalin in the proximal tubule. Reciprocally, knockout of Dab2 led to a redistribution of megalin from endosomes to microvilli. In addition, there was an overall decrease in levels of megalin protein observed by immunoblotting but no decrease in clathrin or α-adaptin protein levels or in megalin mRNA. In rat yolk sac epithelial BN16 cells, Dab2 was present apically and colocalized with megalin. Introduction of anti-Dab2 antibody into BN16 cells decreased the internalization of 125I-labeled receptor-associated protein, substantiating the role of Dab2 in megalin-mediated endocytosis. The present study shows that Dab2 is localized in the apical endocytic apparatus of the renal proximal tubule and that this localization requires megalin. Furthermore, the study suggests that the urinary loss of megalin ligands observed in Dab2 knockout mice is caused by suboptimal trafficking of megalin, leading to decreased megalin levels.

Effect of cortisol acetate on wool quality in sheep selected for divergent staple strength

2002 ◽  
Vol 53 (2) ◽  
pp. 183 ◽  
Author(s):  
A. C. Schlink ◽  
P. C. Wynn ◽  
J. M. Lea ◽  
J. R. Briegel ◽  
N. R. Adams
Keyword(s):  
Egf Receptor ◽  
Fibre Diameter ◽  
Egf Receptors ◽  
Wool Growth ◽  
Receptor Concentration ◽  
Epidermal Growth ◽  
The Mean ◽  
Staple Strength ◽  
The Impact

These studies utilised cortisol treatment to clarify the role of stress in reducing staple strength (SS). The first study established the impact of the duration of exposure and nutritional status on SS, wool parameters, and epidermal growth factor (EGF) receptors. Sheep (n = 42) were fed at 0.75 or 1.5 times maintenance for 62 days and then administered with 120 mg cortisol acetate/day for 0, 3, or 12 days. SS was reduced significantly (P < 0.05) only in the group fed below maintenance and treated with cortisol for 12 days. In the sheep fed below maintenance, the mean SS was 31.6, 32.7, and 21.5 N/ktex for groups treated for 0, 3, and 12 days of cortisol, respectively. Cortisol administration in these sheep also increased the rate of fibre shedding (P < 0.001) but did not affect their mean fibre diameter. The shed fibres had a finer diameter than those that were not (P < 0.001). The concentration of EGF receptors was not affected by cortisol administration, although submaintenance feeding (P < 0.05) significantly reduced EGF receptor concentration. The second experiment examined whether some sheep were predisposed to reduce SS in response to cortisol. Sheep (n = 42) of low and high prior SS were fed to maintain liveweight for 84 days and then administered with 0 or 120 mg cortisol/day for 12 days. Cortisol administration significantly reduced SS (P < 0.001) and wool growth (P < 0.001), and increased the rate of fibre shedding (P < 0.001) but did not affect fibre diameter. SS history did not affect the response to cortisol administration. We conclude that cortisol reduced SS by increasing fibre shedding, without decreasing fibre diameter. Treatment was only effective in sheep fed at or below maintenance, and required between 3 and 12 days of exposure to cortisol. This is longer than would be observed in many stressful situations in the field.

scholarly journals Defining the RND-binding residues of AcrA

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Ilyas Alav ◽  
Ricardo Torres ◽  
Vassiliy Bavro ◽  
Jessica Blair
Keyword(s):  
Western Blotting ◽  
Efflux Pump ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Phenotypic Effect ◽  
Resistance Nodulation Division ◽  
Binding Residues ◽  
Outer Membrane Channel

The resistance-nodulation-division (RND) family of efflux pumps confer clinically relevant antibiotic resistance in Gram-negative bacteria, such as Salmonella enterica. RND pumps, including AcrB, are organized as tri-partite systems, consisting of an inner membrane RND pump, a periplasmic adaptor protein (PAP) and an outer membrane channel. Previously, inactivation of the PAPs AcrA and AcrE in S. enterica has been shown to significantly increase susceptibility to antimicrobials and reduce virulence. Therefore, PAPs are seen as attractive targets for the development of efflux pump inhibitors. However, the role of PAPs in the assembly of tri-partite pumps and the residues involved in PAP-RND pump binding is poorly understood. In this study, point mutations in the predicted RND binding residues of AcrA were generated by site-directed mutagenesis. The point mutants were characterised phenotypically through ethidium bromide efflux assays and antimicrobial susceptibility testing. Furthermore, Western blotting was used to verify that the phenotypic effect of the point mutations was not due to destabilisation of the AcrA protein. Point mutations in certain residues, such as G58, F292, R294 and G363 were found to significantly impair efflux activity and increase susceptibility to various antibiotics and dyes, suggesting an important role for these AcrA residues in RND pump binding. Western blotting confirmed that these point mutants were stable and exhibited similar expression levels to the wild-type. These residues could be important targets for the design and development of PAP inhibitors to restore the activity of existing antibiotics and reduce virulence of Salmonella.

scholarly journals CIN85 Associates with Multiple Effectors Controlling Intracellular Trafficking of Epidermal Growth Factor Receptors

2004 ◽  
Vol 15 (7) ◽  
pp. 3155-3166 ◽  
Author(s):  
Katarzyna Kowanetz ◽  
Koraljka Husnjak ◽  
Daniela Höller ◽  
Marcin Kowanetz ◽  
Philippe Soubeyran ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Adaptor Protein ◽  
Phosphatidyl Inositol ◽  
Adaptor Proteins ◽  
Egf Receptors ◽  
Molecular Scaffold ◽  
Epidermal Growth ◽  
Inositol Phosphatases

CIN85 is a multidomain adaptor protein involved in Cbl-mediated down-regulation of epidermal growth factor (EGF) receptors. CIN85 src homology 3 domains specifically bind to a proline-arginine (PxxxPR) motif in Cbl, and this association seems to be important for EGF receptor endocytosis. Here, we report identification of novel CIN85 effectors, all containing one or more PxxxPR motifs, that are indispensable for their mutual interactions. These effectors include phosphatidyl-inositol phosphatases SHIP-1 and synaptojanin 2B1, Arf GTPase-activating proteins ASAP1 and ARAP3, adaptor proteins Hip1R and STAP1, and a Rho exchange factor, p115Rho GEF. Acting as a molecular scaffold, CIN85 clusters its effectors and recruits them to high-molecular-weight complexes in cytosolic extracts of cells. Further characterization of CIN85 binding to ASAP1 revealed that formation of the complex is independent on cell stimulation. Overexpression of ASAP1 increased EGF receptor recycling, whereas ASAP1 containing mutated PxxxPR motif failed to promote this event. We propose that CIN85 functions as a scaffold molecule that binds to numerous endocytic accessory proteins, thus controlling distinct steps in trafficking of EGF receptors along the endocytic and recycling pathways.

scholarly journals Role of the ubiquitin ligase ITCH in clathrin-mediated endocytosis of the epidermal growth factor receptor

2021 ◽  
Author(s):  
Riham Ayoubi ◽  
Peter S. McPherson ◽  
Annie Angers
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ubiquitin Ligase ◽  
Point Mutations ◽  
Growth Factor Receptor ◽  
Coated Pits ◽  
Epidermal Growth ◽  
First Time

AbstractOnce activated by ligand, epidermal growth factor receptor (EGFR) is endocytosed in clathrin-coated pits. ITCH is an E3 ubiquitin ligase that interacts with and ubiquitinates several proteins involved in clathrin-mediated endocytosis (CME) including endophilin. To further investigate the function of ITCH in EGFR endocytosis, the internalization of fluorescent EGF was measured in ITCH-/- HeLa cells. In the absence of ITCH, there was a significant decrease in the CME of EGF. Rescue experiments using wild-type ITCH confirmed the importance of the protein for normal EGF uptake. ITCH point mutations that disrupt the interaction of ITCH with endophilin failed to rescue the defects in EGFR uptake, as did a non-catalytic form of ITCH. ITCH-/- cells also displayed a delay in the rate of phospho-EGFR degradation as well as prolonged ERK1/2 signaling. Our study uncovers a pathway regulating EGFR trafficking and reveals for the first time that the protein ITCH is required for CME of EGFR.

Sign in / Sign up

Export Citation Format

Share Document