Differential Trafficking of RET Receptor Tyrosine Kinase Isoforms
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Authors
Crupi, Mathieu
Date
Type
thesis
Language
eng
Keyword
RET Proto-Oncogene , Receptor Tyrosine Kinase , RET Isoforms , Internalization , AP2M1 , Clathrin , Ubiquitination , CBL , NEDD4 , GRB2 , SHANK2 , GRB10 , Recycling , GGA3 , ARF6 , Migration , Invasion , Total Internal Reflection Fluorescence Microscopy , Cell Surface Biotinylation , Protein Interactions
Alternative Title
Abstract
The RET receptor tyrosine kinase is implicated in both normal development and cancer.
RET is expressed as two protein isoforms, RET9 and RET51, which differ in the number of their
C-terminal amino acids. RET isoforms are activated at the cell surface in response to glial cell
line-derived neurotropic factor ligand stimulation but the specific mechanisms of RET trafficking
remain to be elucidated. Here, we demonstrated that interactions with the AP2 complex promote
RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have
distinct internalization kinetics that may contribute to differences in their biological functions.
We also showed that RET9 and RET51 differ in their abilities to recruit E3-ubiquitin ligase
complexes. RET51, but not RET9, interacts with, and is ubiquitinated by CBL, which is recruited
through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by
CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitination requires
interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the
NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitination is required for RET9
localization to clathrin coated pits and subsequent internalization. Our data establish differences
in the mechanisms of RET9 and RET51 ubiquitination and internalization that may influence the
strength and duration of RET isoform signals and cellular outputs. Finally, we investigated the
ability of RET51 to recycling back to the plasma membrane. We observed that isoform-specific
interactions of RET51 with GGA3 and ARF6 promote RAB11 recycling, as well as cell motility,
migration and invasion through activation of AKT signalling. Together, our findings provide
novel insight into the trafficking and signalling pathways of RET that may in future be targeted in
cancer therapy.
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.