Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines

The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system, Germli ne mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634 ) located in the juxtamembrane domain of the RET receptor are responsible f or the vast majority of two cancer-prone disorders, multiple endocrine neop lasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). The se mutations lead to the replacement of a cysteine by an alternate amino ac id, Mutations of the RET gene are also the underlying genetic cause of Hirs chsprung disease (HSCR), a congenital aganglionosis of the hindgut, In a fr action of kindreds, MEN 2A cosegregate with HSCR and affected individuals c arry a single mutation at codons 609, 618 or 620, To examine the consequenc es of cysteine substitution on RET function, we have introduced a Cys to Ar g mutation into the wild-type RET at either codons 609, 618, 620, 630 or 63 4, We now report that each mutation induces a constitutive catalytic activi ty due to the aberrant disulfide homodimerization of RET, However, mutation s 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocyt oma PC12 cells, Biochemical analysis revealed that mutations 618 and 620, a nd to a lesser extent mutation 609, result in a marked reduction of the lev el of RET at the cell surface and as a consequence decrease the amount of R ET covalent dimer, These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.