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

Johannessen, Lene E.; Pedersen, Nina Marie; Pedersen, Ketil W.; Madshus, Inger Helene; Stang, Espen

doi:10.1128/mcb.26.2.389-401.2006

Cited by 110 publications

(127 citation statements)

References 43 publications

Supporting

Mentioning

112

Contrasting

Unclassified

Order By: Relevance

“…Furthermore, the essential role of Grb2 and Cbl proteins in clathrin-mediated internalization of EGFR has been previously demonstrated (10,11,(22)(23)(24)(25)(26). To test whether internalization of the 15KR and 16KR/2RK mutants used the same proteins, Grb2 and Cbl proteins (c-Cbl and Cbl-b) were knocked down by specific siRNA as described in refs.…”

Section: Internalization Of Egfr Lysine Mutants Requires Clathrin Grmentioning

confidence: 99%

EGF receptor ubiquitination is not necessary for its internalization

Huang¹,

Goh

Sorkin

2007

Proc. Natl. Acad. Sci. U.S.A.

171

165

View full text Add to dashboard Cite

Ubiquitination of the EGF receptor (EGFR) has been implicated in EGF-induced receptor internalization, lysosomal degradation, and down-regulation. Mutation of EGFR ubiquitination sites identified by mass spectrometry yielded receptor mutants that are weakly ubiquitinated and not down-regulated by EGF. However, these EGFR mutants were normally internalized. To examine whether this internalization was mediated by the residual ubiquitination, systematic mutagenesis of lysine residues in the kinase domain of the EGFR was performed to generate a receptor mutant that is not ubiquitinated. Mutations of a number of lysines inhibited kinase activity of the EGFR, thus leading to the inhibition of receptor internalization. However, a mutant lacking 15 lysine residues (15KR), which was negligibly ubiquitinated and normally phosphorylated, was internalized at a rate similar to that of the wild-type EGFR. As in the case of the wild-type EGFR, internalization of the 15KR mutant depended on the presence of clathrin, Grb2 adaptor, and Cbl ubiquitin ligase. These data imply that EGFR ubiquitination is not necessary for its internalization by clathrincoated pits. Interestingly, the reconstitution of two major ubiquitination sites in the 16KR receptor mutant, which had impaired kinase activity and slow internalization kinetics, resulted in a partial rescue of ubiquitination and a complete rescue of receptor internalization. This result suggests that ubiquitination of the kinase-impaired receptor can mediate its internalization by the clathrin pathway. Altogether these data emphasize the robustness of the EGFR internalization process, which can be controlled by multiple kinase-and ubiquitination-dependent and -independent mechanisms. endocytosis ͉ clathrin ͉ ubiquitin A ctivation of the EGF receptor (EGFR) by EGF or other ligands at the cell surface triggers several signaling cascades leading to a cell-specific biological response (1). Activated EGFR also is rapidly endocytosed by clathrin-coated pits (2). After internalization, ligand-receptor complexes traffic through a series of endosomal compartments and are either returned back to the plasma membrane or degraded in lysosomes. The accelerated internalization and efficient sorting of activated EGFR to lysosomes result in the dramatic down-regulation of the receptor and serve as a negative-feedback regulation of receptor signaling (3). At the same time, internalized EGFR continues to signal from endosomes, and this endosomal signaling is thought to play an important role in determining the duration, intensity, and specificity of signaling processes (2).Endocytosis and intracellular sorting of EGFR have been the most popular experimental models to study pathways and mechanisms of endocytic trafficking that are specific to signaling receptors. However, despite extensive research for more than two decades, the mechanisms of internalization and degradation of EGFR, as well as other receptor tyrosine kinases, are not well understood. Our understanding of the molecular interactions lead...

show abstract

Section: Internalization Of Egfr Lysine Mutants Requires Clathrin Grmentioning

confidence: 99%

EGF receptor ubiquitination is not necessary for its internalization

Huang¹,

Goh

Sorkin

2007

Proc. Natl. Acad. Sci. U.S.A.

171

165

View full text Add to dashboard Cite

show abstract

“…Clearly without EGF stimulation, EGFRs were more mobile on the membrane. Dimerization of EGFRs was believed to the cause of reduced mobility for EGF-stimulated EGFRs (50,63), which also induced endocytosis (64). Although untreated EGFRs were more mobile, their time proportions in Brownian and confined diffusion were similar to those of EGF-stimulated EGFRs (Fig.…”

Section: Distributions Of the Dynamic Parameters Of Egfrmentioning

confidence: 89%

“…Internalization of EGFR was initiated by replacing the media with DMEM containing 10 ng/ml EGF (recombinant human epidermal growth factor, PHG0311L, Thermo Fisher Scientific) (64). As membrane-bound EGFRs were typically internalized within 30 min upon EGF stimulation (78), 2-4 EGFR trajectories (duration ranged from 2-10 min) were typically obtained from each well.…”

Section: Fluorescent Probe Labeling To Egfrsmentioning

confidence: 99%

See 1 more Smart Citation

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

Liu

Perillo

Liu

et al. 2016

Biophysical Journal

View full text Add to dashboard Cite

Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using singleparticle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

show abstract

“…Furthermore, some have reported that downregulation of the 2 subunit of AP-2 did not affect EGFR internalization (Motley et al, 2003), whereas others have found that 2 is important for EGFR downregulation (Huang et al, 2004). There has also been contradiction regarding the importance of the 2 subunit of AP-2 in internalization of the EGFR (Hinrichsen et al, 2003;Motley et al, 2003;Huang et al, 2004;Johannessen et al, 2006). Receptors are believed to be recruited into preexisting clathrin-coated pits.…”

Section: Yxx ) and The Dileucine Based (Consensus Motifs [De]xxxl[li]mentioning

confidence: 99%

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Grøvdal

Stang

Sorkin

et al. 2004

Experimental Cell Research

Self Cite

160

143

View full text Add to dashboard Cite

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

Cited by 110 publications

References 43 publications

EGF receptor ubiquitination is not necessary for its internalization

EGF receptor ubiquitination is not necessary for its internalization

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Contact Info

Product

Resources

About