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

Julie A Gosney,Daniel W Wilkey,Michael L Merchant,Brian P Ceresa

doi:10.1074/jbc.ra117.000632

Julie A Gosney, Daniel W Wilkey + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra117.000632

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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.

Highlights

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling
To determine the time point at which the EGFR was maximally localized to the early endosome, we used indirect immunofluorescence probing for the early endosome marker, EEA1 (21) following Alexa 647–EGF treatment (Fig. 1)
After 30 min of EGF treatment, there is a decrease in EGF and EEA1 costaining, which is consistent with reports that the EGF1⁄7EGFR complex is trafficked out of the early endosome 30 – 60 min after EGF stimulation (23, 24)

Summary

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Developmental biology, tissue homeostasis, and wound healing (1). In addition, overexpression and activating mutations of EGFR have been associated with many cancers including brain, lung, breast, pancreatic, and colon cancers (2). Alterations in regulatory mechanisms are associated with cell transformation (6) and have been targeted to promote receptor activity to enhance wound healing (7). Depending on multiple factors such as the activating ligand (9), cell type, and receptor density, the early endosome readies the receptor for its ultimate cellular destination, be it the plasma membrane (10), lysosome (11), endoplasmic reticulum (12), or nucleus (13). This process governs how long the ligand1⁄7receptor complex is active, the localization of the receptor, and its proximity to downstream effectors. There are implications in understanding the contribution of the endocytic pathway in EGFR signaling under physiological and pathological conditions, as well as for the signaling of other cell surface receptors

Results

Discussion

Experimental procedures