Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2

Abstract

The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers.RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes.RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish 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.

A specific Ret receptor isoform is required for pioneer axon outgrowth and growth cone dynamics

“…Because of pair to pair variability, we compare ret hu2846 mutant embryos to sibling controls within clutches from individual pair matings. To test if a particular Ret isoform was required for pLLg axon outgrowth, we injected ret hu2846 mutant and sibling embryos from individual clutches with in vitro transcribed human Ret isoform mRNA tagged with mCherry, either RET9mcherry or RET51-mcherry 18 , at the one-cell stage and assayed at 3 dpf for axon length, measuring the shortest pLLg axon bundle in the embryo as a conservative measure of axon extension rescue ( Fig. 2C,D).…”

“…Live imaging of RET51/Jip3 retrograde co-transport as well as the disruption of RET51 retrograde axonal transport and pRet localization followed by loss of Jip3, suggests Ret51 retrograde transport may be required for this signaling role. In other cell types, Ret51 is much more rapidly internalized from the plasma membrane and differentially ubiquitylated compared to Ret9 18,40 . In mouse sympathetic neuron culture, Ret51 is more rapidly degraded in axon terminals compared to Ret9 following GDNF treatment 16 .…”

“…Neurod5kb:mCherry plasmid was generated by recombining the neurod5kb promoter 33 p5e entry vector into the pDEST394 vector in the Gateway system by previously described methods 47 . Human RET9-mCherry and RET51-mCherry constructs in the EGFP-N1 vector were obtained from the Mulligan lab 18 and digested with HindIII, NotI, and SphI to release the whole tagged construct and digest the EGFP-N1 vector backbone. Released RET fusion construct cassettes were ligated into HindIII/NotI-digested Gateway pME-MCS vector and subsequently recombined with the neurod5kb or cmv/sp6 promoter p5e entry vectors to yield neurod5kb:RET9/51-mCherry or cmv/sp6:RET9/51-mCherry in the pDEST394 destination vector 47 .…”

“…In sympathetic neuron culture, RET9 degrades more slowly than RET51, which is thought to allow retrograde trafficking of RET9 and ultimately promote neuronal survival 16 . In human epithelial cell lines, RET9 is transcribed at much higher levels than RET51, but RET51 is comparatively more frequently localized at the plasma membrane, recycled to the plasma membrane after endocytosis, and more rapidly internalized into endosomes after GDNF treatment 17,18 . Thus, Ret9 and Ret51 have notably different properties and responses to ligand binding even within the same cell.…”

Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization

“…As a result, RET9 and RET51 recruit distinct signalling and regulatory protein complexes (Tsui-Pierchala et al, 2002). We have previously shown that RET9 and RET51 differ in maturation, subcellular localization and protein trafficking after ligand stimulation (Crupi et al, 2015a;Richardson et al, 2012), suggesting that isoform-specific regulatory mechanisms also contribute to functional differences. In response to ligand, RET receptors at the cell surface are internalized into the endosomal network via clathrin-coated pits (CCPs) (Crupi et al, 2015a;Richardson et al, 2006).…”