Date on Master's Thesis/Doctoral Dissertation

8-2018

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Ceresa, Brian

Committee Co-Chair (if applicable)

Hood, Joshua

Committee Member

Hood, Joshua

Committee Member

Beverly, Levi

Committee Member

Merchant, Michael

Committee Member

Clark, Geoffrey

Author's Keywords

EGFR; endocytosis; signaling; trafficking; proteomics

Abstract

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. When activated by a ligand at the plasma membrane, EGFR dimerizes with another ErbB family receptor, leading to kinase domain activation and transphosphorylation of C-terminus tyrosine residues. These phosphotyrosines act as crucial regulators of EGFR signaling as effector proteins dock to the receptor at these sites. The receptor undergoes clathrin-mediated endocytosis into early endosomes, where it can then be trafficked to a lysosome for degradation. However, the kinase domain of EGFR retains its activity during trafficking, suggesting that EGFR can continue to elicit signaling cascades after internalization. Unfortunately, there is no consensus as to how EGFR spatial regulation affects its signaling or interaction with downstream effectors. We hypothesize that EGFR localization in early endosomes permits unique interactions with downstream effectors. In an effort to identify proteins that uniquely associate with the internalized EGFR, we have developed a strategy for isolating early endosomes and analyzed the protein make-up of these compartments. HeLa cells were stimulated with and without EGF, and the post-nuclear supernatant (PNS) was loaded onto a 17% Percoll gradient which separates endosomes based on density. The gradient was fractionated, and fractions containing early endosomes were pooled and immunoisolated with an EEA1 monoclonal antibody. The morphology of isolated compartments was monitored using transmission electron microscopy. Endosomes were also subjected to liquid chromatography/tandem mass spectrometry (LC-MS/MS) for proteomic analysis. The isolation method precipitates early endosome marker proteins, but not marker proteins specific to other organelles. Electron microscopy revealed that the isolated vesicles are intact and have an average diameter of 68.63 ± 26.74nm. More than 900 proteins were isolated with the early endosome, and five proteins were detected in endosomes in a ligand-dependent manner: EGFR, RUFY1, STOML2, PTPN23, and CCDC51. RNAi was used to knock down RUFY1 and PTPN23 in HeLa cells to monitor changes in the trafficking of EGFR. Knock-down studies revealed that loss of PTPN23 leads to endocytic accumulation of EGFR and decreased degradation of the ligand:receptor complex. Loss of RUFY1 resulted in a significant increase in cell growth as well as a resistance to cell death. We have developed a rapid and high-throughput isolation technique to collect early endosomes from HeLa cells that can be analyzed by LC-MS/MS to detect a distinct proteome. The purification protocol yields a highly enriched population of early endosomes, as evidenced by immunoblot and LC-MS/MS analyses. The isolated vesicles are also intact and exhibit morphology and size distribution similar to early endosomes. These data provide evidence that endocytic trafficking of the activated EGFR changes the protein composition and signaling potential of the early endosome.

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