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. 2012 Mar 20;109(12):4419-24.
doi: 10.1073/pnas.1200164109. Epub 2012 Feb 27.

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

Leiliane P Sousa  1 , Irit Lax, Hongying Shen, Shawn M Ferguson, Pietro De Camilli, Joseph Schlessinger
Affiliations

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

Leiliane P Sousa et al. Proc Natl Acad Sci U S A. .

Abstract

The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Ligand binding characteristics of EGFR are not influenced by dynamin depletion. (A). Binding experiments were performed using a single concentration of 125I-labeled EGF in the presence of increasing concentration on unlabeled native EGF. The graphs depict competition experiments of 125I-labeled EGF binding to control (blue squares) or DKO (red circles) fibroblasts in the presence of increasing concentration of native EGF. Curve fitting to binding data shown by lines and bars indicate standard deviation values. (B). Immunoblotting analysis of total cell lysates of control or DKO fibroblasts with anti-dynamin antibodies. Immunobloting with anti-AKT antibodies was used as loading control. (C). Dissociation constants (Kd) and numbers of low and high affinity EGFR binding sites on control and DKO fibroblasts were determined using Scatchard analysis of quantitative 125I-EGF binding experiments to these cells. Similar results were obtained in three separate 125I-EGF binding experiments.
Fig. 2.
Fig. 2.
Endocytosis of EGFR is strongly impaired in dynamin depleted cells. Quantitative ligand internalization experiments using 1.5 ng/mL (A–D) or 20 ng/mL (E–H) of 125I-EGF to control (A, E) and DKO (B, F) fibroblasts are shown. Surface bound 125I-EGF (blue), internalized 125I-EGF (red) and degraded 125I-EGF (green) are shown. The TCA precipitatable radioactivity representing intact released or recycled 125I-EGF molecules are not included in the figures. The ratio of the amount of 125I EGF internalized vs. surface bound 125I-EGF molecules are shown in (C) and (G) for control (solid line) and DKO (dashed line) fibroblasts. Each data point is the average value of duplicate results. Data are presented as mean ± SD, as indicated by the bars. Also shown immunobloting analyzes with anti-dynamin antibodies or anti-EGFR antibodies to reveal dynamin or EGFR expression respectively, in control or DKO fibroblasts. Similar results were obtained in three different experiments.
Fig. 3.
Fig. 3.
Ligand induced degradation of EGFR is compromised in dynamin depleted cells. Serum starved control or DKO fibroblasts were pretreated with 10 μM of cycloheximide for 1 h followed by stimulation with 10 ng/mL (A) or 100 ng/mL (B) of EGF for indicated times. Equal amounts of cell lysates were subjected to immunobloting with anti-EGFR antibodies and as controls with anti-dynamin or anti-AKT antibodies. Arrow points to an EGFR degradation product that is detected in EGF stimulated WT cells and not in dynamin depleted cells. Similar results were obtained in three different experiments.
Fig. 4.
Fig. 4.
Enhanced EGF induced autophosphorylation and ubiquitination of EGFR and altered pattern of phosphorylation of Shc isoforms in dynamin depleted cells. Serum starved control or DKO fibroblasts were stimulated with 1.5 ng/mL (A, D), 5 ng/mL (B, E) or 100 ng/mL (C, F) of EGF for different times. Equal amounts of cell lysates were subjected to immunoprecipitation with either anti-EGFR or anti-Shc antibodies. (A–C) The anti-EGFR immunoprecipitates were immunobloted with anti-phosphotyrosine (pY) and reprobed with anti-ubiquitin (UB) antibodies. Enhanced tyrosine phosphorylation of EGFR is marked with asterisks. (D–E) Anti-Shc immunoprecipitates were subjected to immunobloting with anti-pY antibodies (asterisk marks the presence of an nonspecific protein band observed in control cells) and reprobed using anti-Shc or anti-Grb2 antibodies. The efficiency of tamoxifen induced dynamin depletion as well as the levels of EGFR expression in control and DKO fibroblasts were determined by immunobloting with anti-dynamin or anti-EGFR antibodies, respectively (not shown for clarity). Similar results were obtained in three different experiments.
Fig. 5.
Fig. 5.
Similar stimulation of MAP kinase and AKT responses in dynamin depleted cells. Serum starved control or DKO fibroblasts were stimulated with 1.5 ng/mL (A), 5 ng/mL (B), or 100 ng/mL (C) of EGF. Cells were collected at the indicated time points and equal amounts of lysates were subjected to immunobloting with anti-AKT or anti-ERK antibodies. Membranes were reprobed with anti-pAKT or anti-pERK antibodies to monitor their enzymatic activities. EGFR and dynamin levels were monitored by inmunoblotting with anti-EGFR or anti-dynamin antibodies respectively. Similar results were obtained in three different experiments.
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