Differential Trafficking of RET Receptor Tyrosine Kinase Isoforms

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.