A two-tiered mechanism of EGFR inhibition by RALT/MIG6 via kinase suppression and receptor degradation

Abstract

Signaling by epidermal growth factor receptor (EGFR) must be controlled tightly because aberrant EGFR activity may cause cell transformation. Receptor-associated late transducer (RALT) is a feedback inhibitor of EGFR whose genetic ablation in the mouse causes phenotypes due to EGFR-driven excess cell proliferation. RALT inhibits EGFR catalytic activation by docking onto EGFR kinase domain. We report here an additional mechanism of EGFR suppression mediated by RALT, demonstrating that RALT-bound EGF receptors undergo endocytosis and eventual degradation into lysosomes. Moreover, RALT rescues the endocytic deficit of EGFR mutants unable to undergo either endocytosis (Dc214) or degradation (Y1045F) and mediates endocytosis via a domain distinct from that responsible for EGFR catalytic suppression. Consistent with providing a scaffolding function for endocytic proteins, RALT drives EGFR endocytosis by binding to AP-2 and Intersectins. These data suggest a model in which binding of RALT to EGFR integrates suppression of EGFR kinase with receptor endocytosis and degradation, leading to durable repression of EGFR signaling.

Figure 1.

Kinase inhibition by RALT, but not by AG1478, is associated to normal rates of EGFR endocytosis and down-regulation. (A) Expression of EGFR and MYC-RALT was reconstituted in NR6 fibroblasts, which lack endogenous EGFR expression. Control and RALT-overexpressing NR6-EGFR cells were rendered quiescent and then stimulated with 100 ng/ml EGF (±3 µM AG1478) for the indicated time. Lysates were immunoblotted as indicated. (B) Control (±3 µM AG1478) and MYC-RALT–expressing NR6-EGFR cells were assayed for [¹²⁵I]-EGF uptake (1 ng/ml at 37°C for the indicated time). The graph reports the average results ± SD from four independent experiments. (C) Control (±3 µM AG1478) and MYC-RALT–expressing NR6-EGFR cells were made quiescent, incubated at 37°C with 100 ng/ml EGF for the indicated time, and processed to determine residual [¹²⁵I]-EGF binding at the cell surface (percentage of binding at time 0). Results were averaged from three independent experiments ± SD. (D) EGFR degradation. Control and MYC-RALT–expressing NR6-EGFR cells were rendered quiescent and then stimulated with 100 ng/ml EGF for the indicated time (hours). Lysates were immunoblotted with the indicated antibodies. Two different exposures of the same anti-EGFR autoradiograph are shown. (E) Quiescent MYC-RALT–expressing NR6-EGFR cells were incubated with 20 ng/ml EGF on ice for 30 min. After EGF wash-out, cells were chased at 37°C in EGF-free medium for either 10 min (for EGFR/EEA1 and EGFR/RALT colocalization) or 30 min (for EGFR/LAMP-1 colocalization). Nuclei were stained with Hoechst 33258 (blue). Bar, 5 µm.

Figure 2.

RALT rescues the endocytic deficit of EGFR Dc214. (A) Quiescent control and MYC-RALT–expressing NR6-EGFR Dc214 cells were either left untreated or stimulated with 20 ng/ml EGF for 10 min at 37°C. Cells were fixed and stained with anti-EGFR mAb 108 (red) and Hoechst 33258 (blue). (B) MYC-RALT–expressing NR6-EGFR Dc214 cells were rendered quiescent and then incubated with 20 ng/ml EGF on ice for 30 min. After EGF wash-out, cells were chased at 37°C in EGF-free medium for 10 min, fixed, and stained for EGFR, EEA1, and MYC-RALT immunodetection. Nuclei were stained with Hoechst 33258 (blue). (C) NR6-EGFR Dc214 cells were transfected with either control or RALT-specific siRNAs. Cells were made quiescent by serum deprivation and then challenged with 10% newborn calf serum (NCS) for 3 h to induce RALT protein expression. After serum wash-out, cells were left untreated or stimulated with 20 ng/ml EGF for 10 min at 37°C before being processed for anti-EGFR staining (red). Nuclei were stained with Hoechst 33258 (blue). Sister cultures were lysed to monitor the efficiency of RALT KD by immunoblot (top). Nontransfected NR6-EGFR Dc214 cells (±NCS stimulation) were included as additional immunoblotting controls. β-Actin was used as loading control. (D) Control and RALT siRNA-transfected NR6 Dc214 cells were made quiescent and then stimulated with NCS as in C. After serum wash-out, cells were incubated with 1 ng/ml [¹²⁵I]-EGF for 8 min at 37°C. Nontransfected cells (±NCS stimulation) were included as additional control. Columns report the average of three independent experiments ± SD. Bars: (A and C) 20 µm; (B) 5 µm.

Figure 3.

Relocation onto EGFR is required for RALT-mediated endocytosis. (A and B) Quiescent NR6-EGFR Dc214 cells expressing the indicated MYC-RALT alleles were assayed for [¹²⁵I]-EGF uptake (1 ng/ml at 37°C for the indicated time). The graphs report average results ± SD from four (A) and three (B) independent experiments. (C and D) NR6-EGFR Dc214 cells expressing the indicated MYC-RALT alleles were made quiescent and then assayed for [¹²⁵I]-EGF uptake (1 ng/ml for 8 min at 37°C) either before or after a 3-h stimulation with 10% NCS to induce RALT expression. Results were averaged ± SD from three independent experiments. Comparison between endogenous and ectopic RALT proteins indicates that the latter are largely overexpressed (see Fig. S2 A), thus allowing the assessment of their potential dominant-negative activity over endogenous RALT.

Figure 4.

Identification of the RALT endocytic domain. (A) Schematic representation of chimeras generated by fusing the indicated fragments of RALT to the C-terminal end of EGFR_1–682. (B) The indicated EGFR_1–682-RALT chimeras (referred to as ER, see panel A) were expressed in NR6 cells. Quiescent cells were assayed for [¹²⁵I]-EGF uptake (3 ng/ml for 5 min at 37°C). Columns report average results ± SD from four independent experiments. (C) Quiescent NR6 cells expressing either wt EGFR or ER_144–323 were allowed to bind mAb 108 (3 µg/ml) for 30 min on ice either in the absence or presence of EGF (20 ng/ml). After mAb wash-out, cells were shifted to 37°C in mAb/EGF-free medium for 10 min. After fixation, cells were stained with anti–mouse IgG to visualize mAb 108 (red) and Hoechst 33258 (blue). (D) Quiescent NR6-ER_144–323 cells were incubated for 30 min on ice with 3 µg/ml mAb 108. After mAb wash out, cells were chased for 10 min at 37°C in serum-free medium before being processed for mAb 108 and EEA1 immuno-imaging. Nuclei were stained with Hoechst 33258 (blue). Bars: (C) 20 µm; (D) 5 µm.

Figure 5.

RALT drives degradation of EGFR Dc214 and EGFR Y1045F. (A) EGFR Dc214 down-regulation. Control and MYC-RALT–expressing NR6-EGFR Dc214 cells were made quiescent and assayed for receptor down-regulation as in Fig. 1 C. Graphs indicate the average results ± SD from three independent experiments. (B) [¹²⁵I]-EGF degradation. Quiescent control and MYC-RALT–expressing NR6-EGFR Dc214 cells were allowed to bind [¹²⁵I]-EGF on ice (15 ng/ml for 45 min). After washing, cells were shifted to 37°C in EGF-free medium for the indicated time. Degraded [¹²⁵I]-EGF was measured as the fraction of TCA-soluble radioactivity (i.e., the sum of TCA-soluble cpm measured in cell lysates and extracellular medium) over bound [¹²⁵I]-EGF. Results were averaged (±SD) from three independent experiments. Note that under these conditions EGF stimulation induces significant RALT expression in control NR6-EGFR Dc214 cells by 90 min (not depicted), which likely explains why in control cells [¹²⁵I]-EGF degradation increases above background past the 1.5-h time point. (C) Quiescent MYC-RALT–expressing NR6-EGFR Dc214 cells were incubated with EGF on ice as in Fig. 2 B, washed, chased in EGF-free medium for 30 min at 37°C, and processed for EGFR, LAMP-1, and MYC-RALT immuno-imaging. Nuclei were stained by Hoechst 33258. (D) Top: control and MYC-RALT–expressing NR6-EGFR Y1045F cells were made quiescent and then stimulated with 100 ng/ml EGF for the indicated time. Where indicated, chloroquine was added to the culture medium throughout the EGF incubation time. Lysates were immunoblotted with the indicated antibodies. Two different exposures of the same anti-EGFR autoradiograph are shown. Bottom: sister plates were stimulated with 100 ng/ml EGF for 5 min at 37°C to document the extent of catalytic suppression of EGFR Y1045F by MYC-RALT. Blots were probed as indicated; AKT was used as loading control. (E) NR6 fibroblasts expressing either wtEGFR or EGFR Y1045F in the absence (Ctrl) or presence of ectopic MYC-RALT (RALT) were made quiescent and either left untreated or incubated with 20 ng/ml EGF for the indicated time (min). Anti-EGFR immunoprecipitates and total cell lysates were immunoblotted as indicated. Bar, 5 µm.

Figure 6.

RALT-mediated endocytosis is clathrin- and AP-2–dependent. (A) MYC-RALT–expressing HeLa cells were made quiescent and then incubated with 20 ng/ml EGF for 5 min at 37°C before MYC-RALT and CHC immunostaining. (B) NR6-EGFR Dc214 RALT cells were transfected with control and CHC-specific siRNAs. Cells were rendered quiescent and assayed for [¹²⁵I]-EGF (1 ng/ml for 6 min at 37°C) and [¹²⁵I]-Tf (0.5 µg/ml for 4 min at 37°C) uptake. Results were averaged ± SD from four independent experiments. The immunoblot documents the typical efficiency of CHC KD; β-actin was used as loading control. (C) CHO cells were transfected with either GFP or dominant-negative (DN) DYN2 K44A-GFP vectors along with either pcDNA3-EGFR + pCS2MT (left) or pcDNA3-EGFR Dc214 + pCS2MT-RALT (right). 2 d after transfection, cells were made quiescent and stimulated with 20 ng/ml EGF for 10 min at 37°C before being processed for anti-EGFR staining. Nuclei were stained with Hoechst 33258 (blue). (D) Quiescent NR6-EGFR Dc214 cells were incubated for 30 min at 37°C with either vehicle or 80 µM dynasore before being assayed for [¹²⁵I]-EGF uptake (± dynasore) for 6 min at 37°C. Results were averaged ± SD from three independent experiments. (E) NR6-EGFR Dc214 cells were transfected with control and two different μ2-specific siRNAs. Cells were made quiescent and assayed for [¹²⁵I]-EGF uptake (1 ng/ml for the indicated time at 37°C). Results were averaged ± SD from three independent experiments. The immunoblot documents μ2 KD, which causes also concomitant reduction of the β2 subunit, as reported previously (Johannessen et al., 2006); CHC was used as loading control. Bars, (A) 10 µm; (inset) 5 µm; (C) 20 µm.

Figure 7.

ITSN recruitment by RALT is necessary for RALT-mediated endocytosis. (A) NR6-EGFR Dc214 cells were transfected with either control or ITSN-specific siRNAs. Cells were made quiescent before being tested for [¹²⁵I]-EGF uptake (1 ng/ml for the indicated time at 37°C). An experiment representative of three independent experiments is shown. ITSN KD efficiency was documented by immunoblot (ITSN1, bottom) and RT-PCR (ITSN2, top; note that our anti-ITSN2 antibody does not react against the mouse protein). GAPDH was used as RT-PCR reaction control. (B and C) HEK 293 cells were transfected with either empty vector (Ctrl) or vectors directing the expression of the indicated EGFR_1–682-RALT chimeras. Lysates were subjected to immunoprecipitation with anti-EGFR mAb 108 (B) and anti-ITSN2 antiserum (C). Total cell lysates (TCL, 5% of input lysate in IPs) and IPs were immunoblotted as indicated. (D) HEK 293 cells were cotransfected with vectors encoding HA-tagged 5SH3 X. laevis ITSN and the indicated MYC-RALT alleles. Cell lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-MYC 9E10 mAb. (E) EGFR Dc214 cells expressing comparable amounts of either wt or MYC-RALT 4Ala (see immunoblot) were made quiescent and assayed for [¹²⁵I]-EGF uptake (1 ng/ml at 37°C). Columns report the endocytic rate constants K_e averaged from three independent experiments (0.131 ± 0.006 for wtRALT and 0.099 ± 0.013 for RALT 4Ala, P = 0.039).