MOLECULAR MECHANISMS OF DEVELOPMENT OF MULTIPLE ENDOCRINE NEOPLASIA-2BY RET MUTATIONS

Biological and biochemical effects of multiple endocrine neoplasia typ e 2A (MEN-2A), type 2B (MEN-2B) and familial medullary thyroid carcino ma (FMTC) mutations on Ret function were investigated by transfection of NIH 3T3 cells, All mutations examined conferred transforming activi ty on Ret at variable levels. Cysteine mutations detected in MEN-2A an d FMTC induced disulphide-linked homodimers of Ret on the cell surface , leading to activation of its intrinsic tyrosine kinase. Of these cys teine-mutated proteins. Ret with a codon 634 mutation had the highest transforming activity. The activity of Ret with a codon 609, 611, 618 or 620 mutation was approximately three-to five-fold lower than that o f Bet with a codon 634 mutation, The first four mutations impaired the Ret cell surface-expression or its correct maturation, resulting in t he low transforming activity, On the other hand, the MEN-2B mutation a ppeared to activate Ret by an intramolecular mechanism without dimeriz ation. In addition, we investigated the role of tyrosines present in t he intracellular domain for the transforming activity of the mutant Re t proteins. As a result, we found that tyrosine 905 is essential for t he transforming activity of the MEN-2A-Ret mutant protein whereas tyro sines 864 and 952 are critical for that of the MEN-2B-Ret mutant prote in. Moreover, it turned out that tyrosine 1062 regulates the activity of both MEN-2A-Ret and MEN-2B-Ret and represents a binding site for th e Shc adaptor protein.