Assessing the effect of RET Transmembrane Domain Mutations in receptor self-association capability using the in vivo TOXCAT System

Abstract

Mutations of c-RET proto-oncogene with a unique localization within the human transmembrane receptor represent a challenge for contemporary molecular oncology techniques. RET transmembrane domain (TMD)-driven dimerization of the receptor leads to its permanent activation that eventually results in the development of medullary thyroid neoplasia. In this study, we describe the employment of the TOXCAT system which enables to investigate mutation-induced alterations in the strength of RET TMD dimerization in vivo. We suggest an improvement of the method by adding reporter gene quantification at the mRNA levels as a support to the commonly used reporter protein level. We have investigated possible changes in RET TMD dimerization in case of two germline RET TMD mutations found in in several individual cases and MEN2 families worldwide, p.Ala641Ser and p.Ser649Leu. According to our results, substitution of Ser-649 residue by leucine, found as a result of germline mutation, caused a significant decrease of RET TMD self-association in comparison to RET wild-type transmembrane domain. The impaired ability of self-association suggests a novel, yet unknown mechanism of tyrosine kinase domain activation, possibly independent of RET homodimerization.