Proteomics reveals novel protein associations with early endosomes in an epidermal growth factor-dependent manner

Abstract

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. EGFRs on the cell surface become activated upon EGF binding and have an increased rate of endocytosis. Once in the cytoplasm, the EGF·EGFR complex is trafficked to the lysosome for degradation, and signaling is terminated. During trafficking, the EGFR kinase domain remains active, and the internalized EGFR can continue signaling to downstream effectors. Although effector activity varies based on the EGFR's endocytic location, it is not clear how this occurs. In an effort to identify proteins that uniquely associate with the internalized, liganded EGFR in the early endosome, we developed an early endosome isolation strategy to analyze their protein composition. Post-nuclear supernatant from HeLa cells stimulated with and without EGF were separated on an isotonic 17% Percoll gradient. The gradient was fractionated, and early endosomal fractions were pooled and immunoisolated with an EEA1 mAb. The isolated endosomes were validated by immunoblot using antibodies against organelle-specific marker proteins and transmission EM. These early endosomes were also subjected to LC-MS/MS for proteomic analysis. Five proteins were detected in endosomes in a ligand-dependent manner: EGFR, RUFY1, STOML2, PTPN23, and CCDC51. Knockdown of RUFY1 or PTPN23 by RNAi indicated that both proteins play a role in EGFR trafficking. These experiments indicate that endocytic trafficking of activated EGFR changes the protein composition, membrane trafficking, and signaling potential of the early endosome.

Keywords: endosome; epidermal growth factor receptor (EGFR); membrane trafficking; proteomics; receptor endocytosis.

Figure 1.

EGF colocalization with EEA1-positive vesicles. Serum-starved HeLa cells were pulse-chased with Alexa 647–EGF (10 ng/ml) for 0, 5, 15, and 30 min. The cells were fixed and processed for indirect immunofluorescence using an EEA1 antibody and fluorescently labeled secondary antibody (goat anti-rabbit Alexa 488). Scale bar, 20 μm. A, images are representative of three independent experiments. B, the extent of colocalization between EGF and EEA1 was measured as described under “Experimental procedures.” The data are plotted as the percentages of colocalization for each time point (four images were taken per time point, i.e. each data point measured one image). Three independent experiments are represented with three distinct bars. Approximately 300 cells total were analyzed per time point per condition, per experiment. Scale bars, 20 μm. The images were quantified using ImageJ software. A Pearson's correlation was calculated for each of the three experiments comparing EGF fluorescence to total EEA1 fluorescence: r = 0.8790, r = 0.9608, and r = 0.9659.

Figure 2.

Total and phosphorylated EGFR colocalize with early endosomal markers following isotonic Percoll gradient fractionation. A, PNS was prepared from HeLa cells treated with and without EGF (10 ng/ml) for 15 min. PNS was resolved on a 17% isotonic Percoll gradient, fractionated, and resolved by 7.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunoblotted for phosphorylated (Tyr(P)-1068, pY1068) and total EGFR as well as the following marker proteins: EEA1 (early endosomes), TfnR (early and recycling endosomes), LAMP2 (late endosomes and lysosomes), Na/K-ATPase (plasma membrane), and Calnexin (endoplasmic reticulum). The immunoblots are representative of three independent experiments. B, relative intensity of the immunoblots in A. Circles on the x axis represent density bead migration (R_f ∼0.93 = 1.109 g/ml, ∼0.91 = 1.070 g/ml, ∼0.89 = 1.057 g/ml, ∼0.59 = 1.049 g/ml, and ∼0.20 = 1.042 g/ml).

Figure 3.

Schematic of the Percoll gradient purification protocol.

Figure 4.

Affinity purification of early endosomes from Percoll gradient fractions. A, PNS from HeLa cells treated with or without EGF (10 ng/ml) were separated on a 17% isotonic Percoll gradient. Fractions containing early endosome markers were immunoisolated using an EEA1 antibody as outlined under “Experimental procedures.” Samples were loaded by percentage of total sample volume, and proteins were resolved on a 7.5% SDS-PAGE. E, elution; PT, pass-through; PF, pooled fractions (Percoll gradient fractions with R_f values of ∼0.25–0.10). The percentages of sample total are noted above each lane. Membranes were immunoblotted for EEA1, total EGFR, phospho-EGFR (Tyr(P)-1068, pY1068), TfnR, LAMP2, Na/K-ATPase, and Calnexin. The data are representative of three independent experiments. B, quantifications are shown as percentages of the total IP sample (i.e. elution + pass-through = 100%). The data are plotted ± S.D. C, electron micrograph of immunoisolated early endosomes. A representative micrograph of Dynabeads and early endosomes (30,000×). Scale bar, 200 nm. The diameters of 651 individual endosomes were measured using ImageJ software. Correcting with the Fullman equation, the mean diameter of the endosomes was calculated to be 68.63 nm. A histogram of endosome size is inset in C. Arrows indicate endosomes.

Figure 5.

Immunoblot validation of candidate proteins from immunoisolated early endosomes. HeLa cells treated with ±10 ng/ml EGF were subjected to Percoll gradient fractionation and EEA1-targeted immunoisolation as described under “Experimental procedures.” A, Percoll gradient fractions were resolved on a 10% SDS-PAGE and immunoblotted for EEA1, PTPN23, RUFY1, or STOML2. B, quantification of the immunoblots in A.

Figure 6.

RUFY1, STOML2, and PTPN23 colocalize with early endosomes and internalized EGF. A–C, HeLa cells were pulse-labeled with 10 ng/ml Alexa Fluor 647–EGF ligand (Invitrogen) for 0, 5, 10, 15, 20 and 30 min, followed by fixation in 4% paraformaldehyde. The cells were permeabilized and immunostained for EEA1 and either RUFY1, STOML2, or PTPN23 and visualized using either a goat anti-rabbit Alexa 568 or goat anti-mouse Alexa 488, respectively. Images are representative of 0-, 5-, 15-, and 30-min time points from three independent experiments. The extent of colocalization between EGF and each candidate protein or EEA1 and each candidate protein was measured as described under “Experimental procedures.” The data are plotted as the percentages of colocalization for each time point. Approximately 300 cells were analyzed per time point per condition, per experiment. Scale bar, 20 μm. The images were quantified using ImageJ software.

Figure 7.

EGF and EGFR colocalization with EEA1 in siCON, RUFY1 KD, and PTPN23 KD cells. A–C, HeLa cells were incubated with siCON, RUFY1, or PTPN23 siRNA for 72 h prior to serum starving. The serum-starved cells were pulse-labeled with 10 ng/ml Alexa Fluor 647–EGF ligand (Invitrogen) for 0, 15, 30, 60, and 120 min, followed by fixation in 4% paraformaldehyde. The cells were permeabilized and immunostained for EEA1 and EGFR and visualized using either a goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 568, respectively. Images are representative of time points from three independent experiments. The extent of colocalization between EGF or EGFR and EEA1 was measured as described under “Experimental procedures.” The data are plotted as the percentages of colocalization for each time point. Approximately 300 cells were analyzed per time point per condition, per experiment. Scale bars, 20 μm. Images were quantified using ImageJ software. D, a representative immunoblot from each knockdown experiment, probing for PTPN23, RUFY1, and α-tubulin. For each knockdown experiment, the samples were loaded in multiple protein concentrations (20, 10, and 5 μg), and the percentage of knockdown was calculated. Only experiments with a knockdown efficiency of >90% were used.