Subcellular localization and trafficking of the RET receptor tyrosine kinase: implications for signalling and disease
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The RET proto-oncogene encodes a receptor tyrosine kinase (RTK) that is widely expressed in neuroendocrine tissues and is essential for embryonic development of the kidney and enteric nervous system. Mutations leading to constitutive activation of the RET protein underlie various tumours of endocrine tissues. Conversely, loss-of-function mutations of RET lead to Hirschsprung disease, a congenital disorder characterized by a loss of enteric neurons throughout the colon and small intestine. Intracellular trafficking of RTKs through multiple cellular compartments has been shown to impact on downstream signalling. To date, the intracellular trafficking of RET has not been investigated. Here, we show that RET is rapidly internalized after activation and that trafficking to cytoplasmic endosomes plays an important role in downstream signalling. RET is alternatively spliced into multiple isoforms that are co-expressed in cells; therefore, we further investigated RET internalization in an isoform-specific context. This study revealed a number of differences between RET isoforms including differences in sub-cellular localization pre-activation, rate of internalization, and ability to recycle to the plasma membrane. Differential trafficking of RET isoforms alter their downstream signalling properties, providing an additional mechanism to explain the distinct contributions of RET isoforms to cellular processes. Finally, we investigated the impact of altered sub-cellular localization in the context of thyroid carcinoma. Activation of RET has been implicated in a number of thyroid tumours that differ in their inherent oncogenicity. We observed that altered subcellular localization of oncogenic forms of RET, RET/PTCs, enhance their oncogenicity. Interestingly, RET/PTC tumours are indolent and rarely metastasize compared to other RET-mediated forms of cancer. Further investigation revealed that RET/PTC oncogenes are expressed off relatively weak promoters, resulting in quantitatively less RET/PTC oncoprotein expression in these tumours compared to mutant RET expression in more aggressive cancers. Together, our results represent the first in-depth study of the trafficking properties of RET and indicate the importance of proper sub-cellular localization and trafficking in the maintenance of normal cell metabolism.