The c-ret proto-oncogene encodes a transmembrane tyrosine kinase that contains a cadherin-like structure in the extracellular domain (9,10,19,22,23). Its expression was detected at high levels in the peripheral nervous systems such as the enteric and autonomic nervous systems as well as in the excretory system during embryogenesis (1a, 15, 25). In addition, it is expressed preferentially in human tumors such as neuroblastoma, pheochromocytoma, and thyroid medullary carcinoma (8,18,24). Since the peripheral nervous systems and tumors mentioned above derive from neural crest cells, the physiological function of the c-ret proto-oncogene appears related to their normal growth and differentiation.Recent studies revealed that germ line mutations in the c-ret proto-oncogene are associated with the development of four different neural crest disorders (neurocristopathies): multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma, and Hirschsprung's disease (2,4,5,7,13,16). MEN 2A and MEN 2B are autosomal dominant cancer syndromes characterized by the development of medullary thyroid carcinoma and pheochromocytoma. MEN 2B is distinguished from MEN 2A by a more complex phenotype including mucosal neuroma, hyperganglionosis of the gastrointestinal tract, and marfanoid habitus. MEN 2A and familial medullary thyroid carcinoma mutations always involve cysteine residues present in the extracellular domain of the c-ret proto-oncogene (4, 12, 13). These cysteine residues are conserved in both human and mouse c-ret proto-oncogenes, suggesting that they are important for normal conformation of the c-Ret protein (9,22,23). On the other hand, a single point mutation in exon 16 of the tyrosine kinase domain has been found in 95% of patients with MEN 2B (6). This difference of the mutation sites may account for different phenotypes of MEN 2A and MEN 2B. Alternatively, it is possible that the diverse phenotypes observed in MEN 2A and MEN 2B are due to mutations in other modifier genes.Hirschsprung's disease is a developmental disorder of the enteric nervous system, inherited in an autosomal dominant manner with incomplete penetrance and variant expressivity. Several mutations have been found in different domains of the c-ret proto-oncogene, including the extracellular and tyrosine kinase domains (5, 16). Since mice homozygous for c-ret disruption showed phenotypes similar to Hirschsprung's disease (20), it is likely that the abnormalities observed in Hirschsprung's disease are caused by inactivation of the c-Ret function. On the other hand, MEN 2A and MEN 2B mutations might represent gain-of-function mutations.To elucidate the mechanism of development of MEN 2A syndrome, we introduced MEN 2A mutations in the extracellular domain of the c-ret proto-oncogene and analyzed their functions. Biochemical analysis of the Ret protein with MEN 2A mutations indicated that it is activated by ligand-independent dimerization on the cell surface. In addition, we showed that a mutation in a putative Ca 2ϩ -binding site of the ...