Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane (original) (raw)
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Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling
PLoS ONE, 2011
Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3) and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrinindependent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms.
Global Snapshot of the Influence of Endocytosis upon EGF Receptor Signaling Output
Journal of Proteome Research, 2012
Trafficking of activated receptors may dictate the signaling output through the exposure to a changing palette of substrates and effectors. Here, we have used the acute application of a chemical inhibitor of dynamin activity, Dynasore, to inhibit internalization of activated EGF receptors together with quantitative mass spectrometry. This has generated a global snapshot of phosphorylation associated changes, which are contingent upon the endosomal trafficking of the activated EGF receptor. Using a SILAC approach, we have been able to quantitate >500 proteins in pTyr immunoprecipitation experiments and close to 800 individual phosphopeptides through affinity based enrichment strategies. This study provides >2 orders of magnitude increase in the coverage of potential EGF effectors than hitherto assessed in the context of endocytosis. There is a strong positive correlation between EGF responsiveness and sensitivity to Dynasore, with ∼40% of EGF responses being significantly changed by endocytic inhibition. Proteins which are functionally linked to endosomal sorting are strongly influenced by receptor entry, suggesting that the activated receptor can govern its fate by influencing endosomal dynamics. However, the majority of EGF-responsive enzymes which we quantify, do not exhibit this property. Hence, our results provide many examples of key signaling proteins that are impervious to EGFR receptor endocytosis but nevertheless confirm the broad principle of endocytosis influence upon the network response.
Control of EGF Receptor Signaling by Clathrin-Mediated Endocytosis
Science, 1996
14. The suckling mouse model (10) was used to quantify the STa-induced dianhea in vivo. STa (50 rig) was dissolved in 0.5 ml of isotonic PBS and injected intragastrically in 3-to 4-day-old mice. After a 2-hour incubation at 25"C, the whole intestine without stomach was carefully removed and weighed. The g/c ratio was calculated as the ratio of gut weight to remaining carcass weight. To evaluate the response to CT, we used the sealed mouse model [S. H. Richardson, J. C. Giles, K. S. Kruger, Infect. Immun. 43, 482 (1984)l. Data are expressed as mean -t SEM of five mice for each treatment. P c 0.05 versus control.
Traffic, 2011
The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.
Developmental Cell, 2008
Clathrin-mediated endocytosis (CME) is the major pathway of epidermal growth factor receptor (EGFR) internalization. It is commonly believed that CME mediates long-term attenuation of EGFR signaling by targeting the receptor for degradation. However, the EGFR can also be internalized through (a) clathrin-independent pathway(s), and it remains unclear why distinct mechanisms of internalization have evolved. Here, we report that EGFRs internalized via CME are not targeted for degradation, but instead are recycled to the cell surface. By contrast, clathrin-independent internalization preferentially commits the receptor to degradation. This finding has profound implications for signaling, as by skewing EGFR fate toward recycling rather than degradation, CME prolongs the duration of signaling. Our data show that CME determines the longevity of some EGFR-activated signaling pathways and that EGF-dependent biological responses, such as DNA synthesis, absolutely require CME. Thus, CME of the EGFR unexpectedly has a greater impact on receptor signaling than on receptor degradation.
Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells
Experimental Cell Research, 2008
The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads can stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.
Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif
Journal of Cell Science, 2013
EGFR signaling is attenuated by endocytosis and degradation of receptor/ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple posttranslational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization mot...
Inhibitors of clathrin-dependent endocytosis enhance TGF signaling and responses
Journal of Cell Science, 2009
Clathrin-dependent endocytosis is believed to be involved in TGFβ-stimulated cellular responses, but the subcellular locus at which TGFβ induces signaling remains unclear. Here, we demonstrate that inhibitors of clathrin-dependent endocytosis, which are known to arrest the progression of endocytosis at coated-pit stages, inhibit internalization of cell-surface-bound TGFβ and promote colocalization and accumulation of TβR-I and SARA at the plasma membrane. These inhibitors enhance TGFβ-induced signaling and cellular responses (Smad2 phosphorylation/nuclear localization and expression of PAI-1). Dynasore, a newly identified inhibitor of dynamin GTPase activity, is one of the most potent inhibitors among those tested and, furthermore, is a potent enhancer of TGFβ. Dynasore ameliorates atherosclerosis in the aortic endothelium of hypercholesterolemic ApoE-null mice by counteracting the suppressed TGFβ responsiveness caused by the hypercholesterolemia, presumably acting through its effec...