The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 1251_ labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.

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The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in neural crest as well as kidney development. Genetic studies have demonstrated that germ line mutations in the ret oncogene are the direct cause of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease. However, despite the large body of genetic and biological evidence suggesting the importance of RET in development and neoplastic processes, the signal transduction mechanisms of RET remain unknown. To begin to understand the molecular mechanisms of the disease states caused by mutations in RET, the patterns of autophosphorylation of the wild-type RET and the MEN mutants were studied using site-directed mutagenesis and phosphopeptide mapping. Among the 6 autophosphorylation sites found in the wild-type RET receptor, the MEN2B mutant lacked phosphorylation at Tyr-1096, leading to decreased Grb2 binding, while simultaneously creating a new phosphorylation site. These changes in autophosphorylation suggest that the MEN2B mutation may result in the more aggressive MEN2B phenotype by altering the receptor's signaling capabilities.The c-ret proto-oncogene is a member of the receptor tyrosine kinase superfamily and consists of an extracellular ligand binding domain with a cysteine-rich region, a single transmembrane domain, and an intracellular kinase domain (1). Studies involving transgenic mice lacking the RET receptor have demonstrated a critical function for this receptor in kidney and enteric nervous system development (2).Mutations in the ret gene result in several forms of human disease including familial medullary thyroid carcinoma (FMTC), 1 multiple endocrine neoplasia types , and Hirschsprung's disease (3-6). FMTC is a familial cancer syndrome characterized by medullary thyroid carcinoma while MEN2A variably expresses pheochromocytomas, hyperparathyroidism and rarely Hirschsprung's disease in addition to medullary thyroid carcinoma (7,8). These cancerous states arise by substitution of a cysteine residue in the extracellular domain of RET resulting in ligand independent activation of the RET receptor (9, 10). MEN2B displays all the characteristics of MEN2A but is additionally characterized by a more rapid disease progression, skeletal abnormalities, ganglioneuromas of the intestinal tract, and mucosal neuromas (7). In MEN2B, a Met 3 Thr substitution occurs at position 918 in the kinase domain (5). Finally, Hirschsprung's disease is a loss of function mutation characterized by decreased parasympathetic innervation of the lower intestine (10).It is now well established that the phosphotyrosine residues of the receptor tyrosine kinases can serve as docking sites for molecules containing SH2 domains (11) or PTB domains (12). In order to elucidate potential RET signaling pathways, we initiated efforts to map the phosphorylation sites of the wildtype, MEN2A, and MEN2B RET receptors. Both the wild-type RET and the MEN2A mutant were autophosphorylated at identical ...

Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing phosphotyrosine, dsPTPs can hydrolyze phosphoserine/threonine-containing substrates and have been shown to dephosphorylate activated MAPK. We have identified a novel dsPTP, rVH6, from rat hippocampus. rVH6 contains the conserved dsPTP active site sequence, VXVHCX 2 GX 2 RSX 5 AY(L/I)M, and exhibits phosphatase activity against activated MAPK. In PC12 cells, rVH6 mRNA is induced during nerve growth factor-mediated differentiation but not during insulin or epidermal growth factor mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly expressed in proliferating cells and declines rapidly during differentiation. rVH6 expression correlates with the inability of fibroblast growth factor to stimulate MAPK activity in proliferating but not in differentiating MM14 cells. rVH6 protein localizes to the cytoplasm and is the first dsPTP to be localized outside the nucleus. This novel subcellular localization may expose rVH6 to potential substrates that differ from nuclear dsPTPs substrates.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta family of growth factors, was first identified by its ability to promote the survival of midbrain dopaminergic neurons in culture. We demonstrate that GDNF treatment of several neuroblastoma cell lines leads to dose-dependent tyrosine phosphorylation of the RET receptor and that other transforming growth factor-beta family members are not able to activate the RET receptor. GDNF treatment of neuroblastoma cells also results in increased transcription of an Elk luciferase reporter gene, suggesting that GDNF activates the mitogen-activated protein kinase signal transduction pathway.