Proteins and mechanisms involved in endosomal sorting of the epidermal growth factor receptor

Signaling from growth factor receptors is tightly regulated. Failure of this regulation can lead to overstimulation of growth, proliferation and differentiation, and is often observed in cancer. The epidermal growth factor receptor (EGFR) is desensitized by internalization and degradation in lysosomes. Internalization takes place rapidly upon ligand induced activation, whereas degradation depends on retained ligand binding and subsequent sorting to inner vesicles on multivesicular bodies (MVBs). Using electron-microscopy, confocal immunofluorescence microscopy and standard molecular biology techniques, we have investigated endocytosis and endosomal sorting of EGFR. Endocytosis of the activated EGFR is believed to depend on ubiquitination of the receptor. Ubiquitination depends on the ubiquitin ligase Cbl, and we show that the direct interaction between EGFR and Cbl at the phosphorylated tyrosine residue 1045 (pY1045) of the EGFR is sufficient for endocytosis of the EGFR, but not sufficient for sorting to MVBs and degradation. On endosomes, different membrane domains are involved in sorting of the endocytosed EGFR, and we found that two different electron dense coats are involved, one coat containing clathrin and Hrs, and another coat devoid of clathrin and Hrs. Hrs is involved in sorting of ubiquitinated receptors for degradation, and we further found that the ubiquitination induced by direct interaction between EGFR and Cbl at the pY1045 of the EGFR is necessary for co-localization between the EGFR and Hrs. Finally, we found the non-receptor tyrosine kinase Ack1, which is often over-expressed in cancer, to be involved in EGFR endocytosis, most likely at the level of endosomal sorting. Ack1 colocalized with the EGFR on early endosomes and overexpression of Ack1 retained the EGFR on the limiting membrane of early endosomes and inhibited sorting of the EGFR to inner vesicles of MVBs

Molecular constituents of the extracellular matrix in rat liver mounting a hepatic progenitor cell response for tissue repair

BACKGROUND: Tissue repair in the adult mammalian liver occurs in two distinct processes, referred to as the first and second tiers of defense. We undertook to characterize the changes in molecular constituents of the extracellular matrix when hepatic progenitor cells (HPCs) respond in a second tier of defense to liver injury. RESULTS: We used transcriptional profiling on rat livers responding by a first tier (surgical removal of 70% of the liver mass (PHx protocol)) and a second tier (70% hepatectomy combined with exposure to 2-acetylaminofluorene (AAF/PHx protocol)) of defense to liver injury and compared the transcriptional signatures in untreated rat liver (control) with those from livers of day 1, day 5 and day 9 post hepatectomy in both protocols. Numerous transcripts encoding specific subunits of collagens, laminins, integrins, and various other extracellular matrix structural components were differentially up- or down-modulated (P < 0.01). The levels of a number of transcripts were significantly up-modulated, mainly in the second tier of defense (Agrn, Bgn, Fbn1, Col4a1, Col8a1, Col9a3, Lama5, Lamb1, Lamb2, Itga4, Igtb2, Itgb4, Itgb6, Nid2), and their signal intensities showed a strong or very strong correlation with Krt1-19, a well-established marker of a ductular/HPC reaction. Furthermore, a significant up-modulation and very strong correlation between the transcriptional profiles of Krt1-19 and St14 encoding matriptase, a component of a novel protease system, was found in the second tier of defense. Real-time PCR confirmed the modulation of St14 transcript levels and strong correlation to Krt-19 and also showed a significant up-modulation and strong correlation to Spint1 encoding HAI-1, a cognate inhibitor of matriptase. Immunodetection and three-dimensional reconstructions showed that laminin, Collagen1a1, agrin and nidogen1 surrounded bile ducts, proliferating cholangiocytes, and HPCs in ductular reactions regardless of the nature of defense. Similarly, matriptase and HAI-1 were expressed in cholangiocytes regardless of the tier of defense, but in the second tier of defense, a subpopulation of HPCs in ductular reactions co-expressed HAI-1 and the fetal hepatocyte marker Dlk1. CONCLUSION: Transcriptional profiling and immunodetection, including three-dimensional reconstruction, generated a detailed overview of the extracellular matrix constituents expressed in a second tier of defense to liver injury

Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

<div><p>The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.</p> </div

EGFR internalization after caveolin1 knockdown.

<p>A: Cells treated with siRNA were incubated with 10 nM ligand for 15 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing caveolin siRNA treated cells to mock treatment for each ligand was performed using two-way ANOVA with Bonferroni posttest. ns = non significant. B: Test of the caveolin1 knockdown and EGFR levels. Cells were lysed in RIPA buffer and resolved on SDS-PAGE and western blotting. Actin is used as a loading control.</p

EGFR internalization after filipin treatment.

<p>A: Cells were incubated with or without 1 µg/ml filipin for 1 hour, and then treated with 10 nM ligand for 15 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing filipin to control treatment for each ligand was performed using two-way ANOVA with Bonferroni posttest. ns = non significant. B: Cells were incubated with or without 1 µg/ml filipin for 1 hour, and then allowed to bind fluorescently labeled cholera toxin on ice for 30 minutes. The cholera toxin solution was removed and the cells were allowed to internalize the cholera toxin for 1 hour at 37°C. After uptake the cells were washed and cholera toxin uptake was determined using flow cytometry. Statistical analysis comparing filipin to control treatment was performed using t-test. *** = p<0.001. C: Test of EGFR levels. Cells were lysed in RIPA buffer and resolved by SDS-PAGE and western blotting. Actin is used as a loading control.</p

EGFR degradation after knockdown with CHC siRNA and ligand stimulation.

<p>Cells were incubated with 10 nM of the indicated ligand at 37°C for different time periods. Cells were lysed and the amount of EGFR determined by ELISA. Data points represent mean +/− SEM. A: EGF, B: TGF- α, C: BTC. Statistical analysis comparing degradation in unstimulated cells to ligand-treated and ligand+CHC siRNA-treated cells was performed using two-way ANOVA with Bonferroni posttest. * = p<0.05, ** = p<0.01, *** = p<0.001, ns = non significant.</p

EGFR co-localization with EEA1 after clathrin knockdown.

<p>A: Cells treated with siRNA were incubated with 10 nM ligand for 15 minutes at 37°C. The cells were fixed and labeled for EGFR and EEA1. B: Quantitative analysis of the amount of EGFR co-localizing with EEA1 in an average of 32–51 cells for each ligand+SEM. Statistical analysis comparing each column to their relevant unstimulated control (normalized to 1) was performed using one-way ANOVA with Bonferroni posttest. ** = p<0.01, *** = p<0.001, ns = non significant.</p

EGFR internalization after clathrin knockdown.

<p>A: Cells treated with siRNA were incubated with 3.22 nM ligand for 15 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing each column to their relevant unstimulated control (normalized to 100) was performed using one-way ANOVA with Bonferroni posttest. *** = p<0.001, ns = non significant. B: Knockdown cells were incubated with 10 nM (EGF, TGF-α, HB-EGF, BTC) or 100 nM (AR, EPI) ligand for 15 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing each column to their relevant unstimulated control (normalized to 100) was performed using one-way ANOVA with Bonferroni posttest. * = p<0.05, ** = p<0.01, *** = p<0.001, ns = non significant. C: Knockdown cells were incubated with 10 nM ligand for 5 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing CHC to mock-treatment for each ligand was performed using two-way ANOVA with Bonferroni posttest. * = p<0.05, ** = p<0.01, ns = non significant. D: Test of the clathrin knockdown and EGFR levels. Cells were lysed in RIPA buffer and resolved by SDS-PAGE and western blotting. Actin is used as a loading control.</p