The Y606C RET mutation causes a receptor gain of function

Summary Context Medullary thyroid carcinoma (MTC) is the most common feature of multiple endocrine neoplasia type 2A (MEN2A) and occurs in almost all patients affected by germline RET mutations. Objective We identified and characterized an activating germline RET point mutation (G>A substitution leading to the heterozygous missense mutation Y606C in exon 10), in a 58‐year‐old female affected by MTC. Design The RET/Y606C and RET/C620Y, obtained by site‐directed mutagenesis, as well as the RET/wild‐type (wt) were cloned in an expression vector and transiently transfected in NIH3T3 fibroblasts. In vitro cell model was used to evaluate the effect of Y606C mutation on the RET downstream signalling pathways through Western blot analysis. Results We found that the cysteine insertion, due to the Y606C mutation, results in increased receptor dimerization, which is accompanied by an increased tyrosine phosphorylation of the Y905 residue in the RET/Y606C, demonstrating that the Y606C mutation is associated with constitutive receptor activation. As RET activation results in an intracellular signalling cascade involving extracellular signal‐regulated kinases (ERKs), we investigated ERK activity in our transfected cells. Results demonstrated a significant increase in ERK2 phosphorylation in the RET/Y606C vs. the RET/wt and RET/C620Y transfected cells, suggesting an up‐regulation of RET signalling. Conclusions All these findings demonstrate that the Y606C mutation is associated with RET constitutive activation and thus has to be considered of pathogenetic relevance in the development of MTC.