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Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTK) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumour pheochromocytoma (PCC) can be caused by activating mutations of the RET receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumour suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization, including membrane protein diffusability and protein complex assembly, and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

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Multiple Endocrine Neoplasia type 2 (MEN2) is a cancer syndrome characterized by medullary thyroid carcinoma (MTC) and adrenal tumors. MEN2 is caused by activating point mutations of the REarranged during Transfection (RET) receptor tyrosine kinase, a protein essential for normal cell proliferation, migration, and differentiation in multiple tissues. MEN2 RET mutations can result in constitutive receptor dimerization (MEN2A) or in loss of receptor autoinhibition (MEN2B), which are associated with distinct disease courses and oncogenic potential. However, the specific cellular mechanisms contributing to these differences have not been well characterized. We have previously shown that RET maturation, cell surface localization and trafficking through the endolysosomal system modulate RET signaling and contribute to regulation of normal RET functions. Here, we have explored the contributions of these processes to MEN2A and MEN2B RET mutant activity. In preliminary studies using immunofluorescence (IF) microscopy and MTC cell lines endogenously expressing MEN2A (2ARET) or MEN2B (2BRET) RET mutant forms, we showed that RET mutants do not accumulate in pre-membrane compartments (ER, Golgi) at significant levels. Both 2ARET and 2BRET mutant receptors matured and reached the cell membrane efficiently. Using IF and cell surface protein labeling methods, we demonstrated that wildtype RET, 2ARET and 2BRET forms had differential patterns of plasma membrane distribution in the absence of ligand stimulation. Further, constitutive stimulation of wildtype RET receptors could not recapitulate the pattern observed for MEN2 RET mutants. We showed that, in the absence of ligand, 2ARET and 2BRET, but not wildtype RET, were constitutively phosphorylated, activated downstream signaling pathways and could localize to multiple endosomal compartments. We also showed different extents of protein turnover in 2ARET and 2BRET, which is important for sustained signals and suggests distinct endosomal sorting mechanisms for these receptors. Together, our data indicate that constitutive activation of MEN2 RET mutant receptors is not the only mechanism contributing to aberrant RET function in MEN2. Our results suggest that receptor localization at the membrane and trafficking through endosomal compartments is mutation-specific and may modulate RET signals that contribute to its roles in MEN2 oncogenicity. Citation Format: Eduardo Reyes-Alvarez, Brandy D. Hyndman, Lois M. Mulligan. Effects of MEN2 mutations on RET receptor localization and function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2426.