Control of epidermal growth factor receptor endocytosis by receptor dimerization, rather than receptor kinase activation

Abstract

Given that ligand binding is essential for the rapid internalization of epidermal growth factor receptor (EGFR), the events induced by ligand binding probably contribute to the regulation of EGFR internalization. These events include receptor dimerization, activation of intrinsic tyrosine kinase activity and autophosphorylation. Whereas the initial results are controversial regarding the role of EGFR kinase activity in EGFR internalization, more recent data suggest that EGFR kinase activation is essential for EGFR internalization. However, we have shown here that inhibition of EGFR kinase activation by mutation or by chemical inhibitors did not block EGF-induced EGFR internalization. Instead, proper EGFR dimerization is necessary and sufficient to stimulate EGFR internalization. We conclude that EGFR internalization is controlled by EGFR dimerization, rather than EGFR kinase activation. Our results also define a new role for EGFR dimerization: by itself it can drive EGFR internalization, independent of its role in the activation of EGFR kinase.

Figure 1

Internalization of kinase-dead epidermal growth factor receptor K721A. 293T cells (A–D) or Chinese hamster ovary (CHO) cells (E) were transiently transfected with epidermal growth factor receptor (EGFR)–YFP (yellow fluorescent protein) or K721A–YFP. (A) Immunoblotting. After EGF (100 ng/ml) stimulation, 293T cells were lysed and immunoblotted with indicated antibodies. (B) Intrinsic fluorescence. The cells were stimulated with Texas red (TR)-conjugated EGF (TR–EGF; 100 ng/ml), and the internalization of both EGFR and EGF was viewed by intrinsic fluorescence. (C) Indirect immunofluorescence. After EGF stimulation, the localization of EGFR (green) and phospho-tyrosine (pTyr; red) was shown by indirect immunofluorescence. (D) Quantitative analysis of EGFR–YFP and K721A–YFP internalization by flow cytometry. Data are the mean of at least three experiments performed in triplicate. (E) Indirect immunofluorescence analysis of the internalization of EGFR–YFP and K721A–YFP in CHO cells. CHO cells were transiently transfected with EGFR–YFP or K721A–YFP. After EGF (100 ng/ml) stimulation, the localization of EGFR (green) and pTyr (red) or EEA1 (red) was shown by indirect immunofluorescence. Scale bars, 20 μm.

Figure 2

Internalization of epidermal growth factor receptor after treatment with PD158780. (A) BT20 cells were treated with PD158780 (100 nM) for 15 min and then stimulated with epidermal growth factor (EGF) (100 ng/ml) at 37°C for the indicated time. EGF receptor (EGFR; green) and phospho-tyrosine (pTyr; red) or phospho-EGFR (pEGFR; red) localization was determined by double indirect immunofluorescence. (B) BT20 cells were treated with PD158780 (100 nM) for 15 min and then stimulated with EGF (100 ng/ml) at 37°C for the indicated time. EGFR (green) and EEA1 (red) localization was determined by double indirect immunofluorescence. (C) Quantitative analysis of the effects of PD158780 on EGFR internalization in BT20 cells by flow cytometry. Data are the means of at least three experiments performed in triplicate. Scale bars, 20 μm.

Figure 3

Epidermal growth factor-induced receptor dimerization in the inhibition of EGFR kinase. (A) EGF-induced dimerization of K721A–YFP (yellow fluorescent protein). 293T cells were transiently transfected with EGFR–YFP or K721A–YFP. After EGF stimulation for 15 min, 293T cells were crosslinked with bis-(sulphosuccinimidyl suberate) (BS³). The cell lysates were immunoblotted with an anti-EGFR antibody. (B) EGF-induced EGFR dimerization in BT20 cells treated with either PD158780 or AG1478. BT20 cells were treated with either PD158780 or AG1478, and then stimulated with EGF for 15 min. The cells were crosslinked with bis-(sulphosuccinimidyl suberate) (BS³) and the cell lysates were immunoblotted with an anti-EGFR antibody.

Figure 4

Epidermal growth factor receptor internalization after non-ligand-induced dimerization. (A) Schematic representation of full-length epidermal growth factor receptor (EGFR) fused with FKBP (EGFR–FKBP). C terminus, carboxyl terminus; TM, transmembrane. (B) EGFR–FKBP was expressed in 293T cells by transient transfection. After treatment with either AP20187 (100 nM) or EGF (100 ng/ml), the proteins were crosslinked with disuccinimidyl suberate (DSS). The cells were then lysed and the protein samples were immunoblotted with anti-EGFR and anti-pEGFR antibodies. (C) EGFR–FKBP was expressed in 293T cells by transient transfection. After treatment with AP20187 (100 nM) and/or AG1478 (0.5 μM), the cells were lysed and the protein samples were immunoblotted with anti-phospho-EGFR (pEGFR) antibodies. (D) Double indirect immunofluorescence. EGFR–FKBP was expressed in 293T cells by transient transfection. After treatment with EGF (100 ng/ml), AP20187 (100 nM) and AG1478 (0.5 μM), the localization of EGFR (green) and phospho-tyrosine (pTyr; red) was shown by double indirect immunofluorescence. Scale bar, 20 μm.

Figure 5

Inhibition of epidermal growth factor receptor internalization by blocking EGFR dimerization. The EGFR dimerization loop (amino acids 244–259) was deleted and the deletion mutant was tagged with enhanced yellow fluorescent protein (EYFP; ΔCR1–YFP). ΔCR1–YFP or EGFR–YFP was expressed in Cos7 cells by transient transfection and the cells were treated with EGF (100 ng/ml) or Texas red (TR)-conjugated EGF (TR–EGF; 100 ng/ml). (A) The cells were lysed and the protein samples were immunoblotted with indicated antibodies. (B) The cells were crosslinked with bis-(sulphosuccinimidyl suberate) (BS³) and lysed. The protein samples were immunoblotted with an anti-green fluorescent protein (GFP) antibody. (C) The localization of ΔCR1–YFP (green), EGFR–YFP (green) and TR–EGF (red) was shown by intrinsic fluorescence. Scale bar, 20 μm.

Figure 6

Inhibition of epidermal growth factor-induced internalization of ΔCR1 in BaF/3 cells stably expressing ΔCR1. BaF/3 cells stably expressing epidermal growth factor receptor (EGFR) or ΔCR1 were stimulated with EGF (100 ng/ml) for 15 min. (A) The cells were lysed and the protein samples were immunoblotted with anti-EGFR and anti-phospho-EGFR (pEGFR) antibodies. (B) The cells were crosslinked with bis-(sulphosuccinimidyl suberate) (BS³) and lysed. The protein samples were immunoblotted with anti-EGFR and anti-pEGFR antibodies. (C) Quantitative analysis of EGFR internalization in BaF/3 cells by flow cytometry. BaF/3 cells stably expressing EGFR or ΔCR1 were stimulated with EGF (100 ng/ml) for the indicated time and the internalization of ΔCR1 was analysed by flow cytometry. WT, wild type.