The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations aecting one of ®ve cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and aected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disul®de homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent ®broblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These ®ndings 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 eects.Keywords: MEN 2; Hirschsprung disease; RET receptor; tyrosine kinase IntroductionMutations in the RET proto-oncogene, which encodes a transmembrane protein tyrosine kinase, cause two inherited neural crest disorders: multiple endocrine neoplasia type 2 (MEN 2) and Hirschsprung disease (HSCR). Recent studies have provided evidence that RET is a component of a multi-subunit complex which acts as a receptor for the Glial cell-line Derived Neurotrophic Factor (GDNF) and for neurturin, two homologous members of the transforming growth factor b (TGF-b) family (reviewed in Lindsay and Yancopoulos, 1996. For references see Milbrandt et al., 1998). GDNF and neurturin-binding require accessory receptors called GFRa-1 and GFRa-2 that associate with RET and are anchored to the plasma membrane via a glycosyl ± phosphatidylinositol linkage (reviewed in Lindsay and Yancopoulos, 1996. For references see Milbrandt et al., 1998). Mice homozygous for a disruptive mutation either in the RET or the GDNF gene show renal agenesis or dysgenesis of the kidneys and lack the intestinal nervous system (reviewed in Lindsay and Yancopoulos, 1996). These results indicate that RET and GDNF play a critical role during renal organogenesis and are required for the development of a subset of vagal neural crest cells.MEN 2 is a f...