Both clathrin-positive and -negative coats are involved in endosomal sorting of the EGF receptor

Sorting of endocytosed EGF receptor (EGFR) to internal vesicles of multivesicular bodies (MVBs) depends on sustained activation and ubiquitination of the EGFR. Ubiquitination of EGFR is mediated by the ubiquitin ligase Cbl, being recruited to the EGFR both directly and indirectly through association with Grb2. Endosomal sorting of ubiquitinated proteins further depends on interaction with ubiquitin binding adaptors like Hrs. Hrs localizes to flat, clathrin-coated domains on the limiting membrane of endosomes. In the present study, we have investigated the localization of EGFR, Cbl and Grb2 with respect to coated and non-coated domains of the endosomal membrane and to vesicles within MVBs. Both EGFR, Grb2, and Cbl were concentrated in coated domains of the limiting membrane before translocation to inner vesicles of MVBs. While almost all Hrs was in clathrin-positive coats, EGFR and Grb2 in coated domains only partially colocalized with Hrs and clathrin. The extent of colocalization of EGFR and Grb2 with Hrs and clathrin varied with time of incubation with EGF. These results demonstrate that both clathrin-positive and clathrin-negative electron dense coats exist on endosomes and are involved in endosomal sorting of the EGFR.     

Recruitment of Uev1B to Hrs-containing endosomes and its effect on endosomal trafficking

Endocytosis of signaling receptors, such as epidermal growth factor receptor (EGFR), tightly controls the signal transduction process triggered by ligand activation of these receptors. To identify new regulators of the endocytic trafficking of EGFR an RNA interference screen was performed for genes involved in ubiquitin conjugation and down-regulation of EGFR. The screen revealed that small interfering RNAs (siRNAs) that target the conserved ubiquitin-binding domain Uev1 increased down-regulation of EGFR. Since these siRNAs simultaneously targeted multiple genes containing a Uev1 domain, we analyzed the role of these gene products by overexpressing individual Uev1-related proteins. This analysis revealed that overexpression of Uev1A (UBE2V1) has no effect on the degradation of EGFR:EGF complexes. In contrast, overexpression of Uev1B (TMEM189-UBE2V1 isoform 2) slowed the degradation of EGF:receptor complexes. The Uev1B protein was found to strongly colocalize and associate with ubiquitin and Hrs in endosomes. Moreover, overexpression of Uev1B abrogated the ability of Hrs to colocalize with EGFR. The B-domain of Uev1B, and not the UEV-domain, was mainly responsible for the observed phenotypes suggesting the presence of a novel endosomal targeting sequence within the B-domain. Together, the data show that elevated levels of Uev1B protein in cells lead to decreased efficiency of endosomal sorting by associating with ubiquitinated proteins and Hrs.     

SCF acts in endosomal sorting of the GH receptor

The ubiquitin ligase SCF^TrCP is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF^TrCP is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF^TrCP is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis

Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of ¹²⁵I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl–CIN85–endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.     

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.     

Regulation of EGF-induced ERK/MAPK activation and EGFR internalization by G protein-coupled receptor kinase 2

G protein-coupled receptor kinases （GRKs） mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors （GPCRs）. We investigate the role of GRK2 on epidermal growth factor （EGF） receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase （ERK/MAPK） activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5fold （P〈0.05） at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2,suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled δ5 opioid receptor internalization by approximately 40% （P〈0.01）. Overall,these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.     

PKC-δ binds to E-cadherin and mediates EGF-induced cell scattering

EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinomas but the underlying mechanisms are not completely understood. Using human tumor epithelial cells overexpressing EGFR we demonstrated that EGF-induced cell scattering was mediated by protein kinase C-delta (PKC-δ). PKC-δ knockdown by siRNA significantly inhibited EGF-induced internalization of E-cadherin into the cytoplasm and blocked cell scattering. EGF phosphorylated PKC-δ at Y311 and ectopic expression of the mutant Y311F prevented PKC-δ binding to E-cadherin and EGF-induced cell scattering. Moreover, depletion of Src using siRNA decreased EGF-induced phosphorylation of PKC-δ at Y311 and blocked scattering. Finally, EGF reduced expression of the tight junction protein, occludin, and this effect was also mediated by PKC-δ through Src. In summary, PKC-δ mediated the effects of EGF on adherens and tight junctions thereby playing an important role in cell-cell adhesion with possible wider implications in tumor metastasis or epithelial-to-mesenchymal transition.     

Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of (125)I-EGF internalization, whereas internalization of (125)I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized (125)I-EGF was increased, while degradation of (125)I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.     

EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells

Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Met^flx/flx and Met^−/− oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-β)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Met^flx/flx cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Met^flx/flx and Met^−/− oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in normal, untransformed oval cells, c-Met and EGFR represent critical molecular players to control proliferation and survival that function independent of one another.     

Transactivation of epidermal growth factor receptor by insulin-like growth factor 1 requires basal hydrogen peroxide

Insulin-like growth factor (IGF-1) plays an important role in prostate cancer development. Recent studies suggest that IGF-1 has mitogenic action through epidermal growth factor receptor (EGFR). However, the mechanism remains largely unknown. Here, we demonstrated in prostate cancer DU145 cells that IGF-1 induced EGFR transactivation, leading to ERK activation. Matrix metalloproteinase-mediated shedding of heparin-binding EGF is involved in this process. Antioxidants and catalase inhibited IGF-1-stimulated EGFR phosphorylation, indicating that H(2)O(2) is required for EGFR activation. However, exogenous H(2)O(2) did not activate EGFR or IGF-1R in DU145 cells. IGF-1 did not induced production of H(2)O(2) in DU145 cells. Our results suggest that transactivation of EGFR by IGF-1 requires basal intracellular H(2)O(2) in DU145 cells.     

E3B1, a human homologue of the mouse gene product Abi-1, sensitizes activation of Rap1 in response to epidermal growth factor

E3B1, a human homologue of the mouse gene product Abi-1, has been implicated in growth-factor-mediated regulation of the small GTPases p21Ras and Rac. E3b1 is a regulator of Rac because it can form a complex with Sos-1 and eps8, and such a Sos-1-e3B1-eps8 complex serves as a guanine nucleotide exchange factor for Rac. In the present study, we found that overexpression of e3B1 in NIH3T3/EGFR cells sensitized EGF-induced activation of Rac1, whereas it had no impact on EGF-induced activation of p21Ras. Remarkably, we found that EGF-induced activation of the p21Ras-related GTPase Rap1 was also sensitized in NIH3T3/EGFR-e3B1 cells. Thus, in NIH3T3/EGFR-e3B1 cells, maximal EGF-induced activation of Rap1 occurs with a dose of EGF much lower than in NIH3T3/EGFR cells. We also report that overexpression of e3B1 in NIH3T3/EGFR cells renders EGF-induced activation of Rap1 completely dependent on Src tyrosine kinases but not on c-Abl. However, EGF-induced tyrosine phosphorylation of the Rap GEF C3G occurred regardless of whether e3B1 was overexpressed or not, and this did not involve Src tyrosine kinases. Accordingly, we propose that overexpression of e3B1 in NIH3T3/EGFR cells leads to mobilization of Src tyrosine kinases that participate in EGF-induced activation of Rap1 and inhibition of cell proliferation.     

Vps22/EAP30 in ESCRT-II mediates endosomal sorting of growth factor and chemokine receptors destined for lysosomal degradation

The ubiquitin-binding protein Hrs and endosomal sorting complex required for transport (ESCRT)-I and ESCRT-III are involved in sorting endocytosed and ubiquitinated receptors to lysosomes for degradation and efficient termination of signaling. In this study, we have investigated the role of the ESCRT-II subunit Vps22/EAP30 in degradative protein sorting of ubiquitinated receptors. Vps22 transiently expressed in HeLa cells was detected in endosomes containing endocytosed epidermal growth factor receptors (EGFRs) as well as Hrs and ESCRT-I and ESCRT-III. Depletion of Vps22 by small interfering RNA, which was accompanied by decreased levels of other ESCRT-II subunits, greatly reduced degradation of EGFR and its ligand EGF as well as the chemokine receptor CXCR4. EGFR accumulated on the limiting membranes of early endosomes and aberrantly small multivesicular bodies in Vps22-depleted cells. Phosphorylation and nuclear translocation of extracellular-signal-regulated kinase1/2 downstream of the EGF-activated receptor were sustained by depletion of Hrs or the ESCRT-I subunit Tsg101. In contrast, this was not the case when Vps22 was depleted. These results indicate an important role for Vps22 in ligand-induced EGFR and CXCR4 turnover and suggest that termination of EGF signaling occurs prior to ESCRT-II engagement.     

Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation

The ubiquitin ligase Cbl mediates ubiquitination of activated receptor tyrosine kinases (RTKs) and interacts with endocytic scaffold complexes, including CIN85/endophilins, to facilitate RTK endocytosis and degradation. Several mechanisms regulate the functions of Cbl to ensure the fine-tuning of RTK signalling and cellular homeostasis. One regulatory mechanism involves the binding of Cbl to Sprouty2, which sequesters Cbl away from activated epidermal growth factor receptors (EGFRs). Here, we show that Sprouty2 associates with CIN85 and acts at the interface between Cbl and CIN85 to inhibit EGFR downregulation. The CIN85 SH3 domains A and C bind specifically to proline-arginine motifs present in Sprouty2. Intact association between Sprouty2, Cbl and CIN85 is required for inhibition of EGFR endocytosis as well as EGF-induced differentiation of PC12 cells. Moreover, Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation, providing a molecular explanation for functional differences between Sprouty isoforms. Sprouty2 therefore acts as an inducible inhibitor of EGFR downregulation by targeting both the Cbl and CIN85 pathways.     

In vivo inhibition of epidermal growth factor receptor autophosphorylation prevents receptor internalization

The question whether epidermal growth factor (EGF)-induced receptor endocytosis requires the prior autophosphorylation via the EGF receptor (EGFR) kinase domain has been a matter of long-standing debate. In the airway epithelial cell line NCI-H292, the EGFR kinase domain inhibitor BIBW 2948 BS was found to inhibit both autophosphorylation and subsequent internalization of the endogenous EGFR with similar IC₅₀ values. Applying an ex vivo EGFR internalization assay in a clinical study, the in vivo effect of inhalatively administered BIBW 2948 BS was determined directly at the targeted receptor in airway tissues from COPD patients. In these experiments, the in vivo inhibition of the EGFR kinase domain prevented the EGF-induced internalization of EGFR.     

Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of (125)I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized (125)I-225 mAb is recycled to the surface much more efficiently than internalized (125)I-EGF. Also, we found that internalization of (125)I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidenced by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

Activation of tyrosine kinase of EGFR induces Gbetagamma-dependent GRK-EGFR complex formation

This study demonstrated that activation of tyrosine kinase of epidermal growth factor receptor (EGFR) induces its association with G protein-coupled receptor kinase 2 (GRK2). Immunoprecipitation experiments showed that EGF stimulation increased GRK2 binding to EGFR complex in HEK293 cells coexpressing EGFR and GRK2. The EGF-induced GRK2-EGFR complex formation was greatly reduced by perturbation of EGFR and Src tyrosine kinase activity. Furthermore, studies with GRK2 mutants showed that neither catalytic activity nor the N-terminal domain of GRK2 was required for EGF-induced GRK2-EGFR complex formation. However, overexpression of Gbetagamma scavengers blocked EGF-induced formation of GRK2-EGFR complex.