Glial cell line derived neurotrophic factor (GDNF) signals through a multicomponent receptor complex consisting of RET receptor tyrosine kinase and a member of GDNF family receptor a (GFRa). Recently, it was shown that tyrosine 1062 in RET represents a binding site for SHC adaptor proteins and is crucial for both RAS/mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways. In the present study, we characterized how these two pathways diverge from tyrosine 1062, using human neuroblastoma and primitive neuroectodermal tumor cell lines expressing RET at high levels. In response to GDNF stimulation, SHC bound to GAB1 and GRB2 adaptor proteins as well as RET, and SHC and GAB1 were highly phosphorylated on tyrosine. The complex formation consisting of SHC, GAB1 and GRB2 was almost abolished by replacement of tyrosine 1062 in RET with phenylalanine. Tyrosine-phosphorylated GAB1 was also associated with p85 subunit of PI3-K, resulting in PI3-K and AKT activation, whereas SHC-GRB2-SOS complex was responsible for the RAS/ERK signaling pathway. These results suggested that the RAS and PI3-K pathways activated by GDNF bifurcate mainly through SHC bound to tyrosine 1062 in RET. Furthermore, using luciferase reporter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-kB in GDNF-treated cells, respectively. Oncogene (2000) 19, 4469 ± 4475.

The Ret receptor tyrosine kinase plays a crucial role in the development of the enteric nervous system and the kidney. Tyrosine 1062 in Ret represents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for the activation of intracellular signaling pathways, such as the RAS/ERK, phosphatidylinositol 3-kinase/AKT, and Jun-associated N-terminal kinase pathways. To investigate the importance of tyrosine 1062 for organogenesis in vivo, knock-in mice in which tyrosine 1062 in Ret was replaced with phenylalanine were generated. Although homozygous knock-in mice were born normally, they died by day 27 after birth and showed growth retardation. The development of the enteric nervous system was severely impaired in homozygous mutant mice, about 40% of which lacked enteric neurons in the whole intestinal tract, as observed in Ret-deficient mice. The rest of the mutant mice developed enteric neurons in the intestine to various extents, although the size and number of ganglion cells were significantly reduced. Unlike Ret-deficient mice, a small kidney developed in all knock-in mice, accompanying a slight histological change. The reduction of kidney size was due to a decrease of ureteric bud branching during embryogenesis. Thus, these findings demonstrated that the signal via tyrosine 1062 plays an important role in histogenesis of the enteric nervous system and nephrogenesis.

SNT/FRS2 is a lipid anchored docking protein that contains an amino-terminal myristylation signal, followed by a phosphotyrosine-binding (PTB) domain and a carboxy-terminal region with multiple tyrosine residues. Here we show that the SNT/FRS2 PTB domain binds to RET receptor tyrosine kinase activated by glial cell linederived neurotrophic factor (GDNF) or multiple endocrine neoplasia (MEN) 2 mutations. Analyses by site directed-mutagenesis revealed that it binds to tyrosine 1062 in RET that is also known to be a binding site for the SHC adaptor protein. Whereas SHC bound to RET was associated with GRB2 and GAB1 proteins, SNT/ FRS2 was associated with GRB2 only, suggesting that SNT/FRS2 is involved mainly in the activation of the RAS/mitogen activated protein kinase (MAPK) pathway but not the phosphatidylinositol 3-kinase (PI3-K)/AKT pathway. In addition, phosphorylated SNT/FRS2 appeared to directly complex with SHP-2 tyrosine phosphatase. These results suggest that tyrosine 1062 in RET provides a site for the interaction of multiple signaling molecules and that the balance of SHC and SNT/FRS2 binding may a ect the nature of the intracellular signaling for cell proliferation, di erentiation and survival induced by activated RET. Oncogene (2001) 20, 1929 ± 1938

Several mutations were identi®ed in the kinase domain of the RET proto-oncogene in patients with multiple endocrine neoplasia (MEN) 2B, familial medullary thyroid carcinoma (FMTC) or sporadic medullary thyroid carcinoma. We introduced seven mutations (glutamic acid 768?aspartic acid (E768D), valine 804?leucine (V804L), alanine 883?phenylalanine (A883F), serine 891?alanine (S891A), methionine 918 ?threonine (M918T), alanine 919?proline (A919P) and E768D/A919P) into the short and long isoforms of RET cDNA and transfected the mutant cDNAs into NIH3T3 cells. The transforming activity of the long isoform of Ret with each mutation was much higher that that of its short isoform. Based on the levels of the transforming activity, these mutant RET genes were classi®ed into two groups; a group with high transforming activity (A883F, M918T and E768D/A919P) and a group with low transforming activity (E768D, V804L, S891A and A919P) (designated high group and low group). Interestingly, the level of transforming activity correlated with clinical phenotypes; high group Ret with the A883F or M918T mutation and low group Ret with the E768D, V804L or S891A mutation were associated with the development of MEN 2B and FMTC, respectively. In addition, we found that substitution of phenylalanine for tyrosine 905 present in the kinase domain abolished both transforming and autophosphorylation activities of low group Ret whereas it did not a ect the activities of high group Ret. (cysteines 609, 611, 618, 620, 630 and 634) in the extracellular domain of Ret (Mulligan et al., 1993(Mulligan et al., , 1994 Donis-Keller et al., 1993;Lips et al., 1994; Eng et al., 1996), whereas a point mutation (methionine 918?threonine, M918T) in the tyrosine kinase domain was detected in MEN 2B (Hofstra et al., 1994; Carlson et al., 1994). We and others demonstrated that MEN 2A, MEN 2B and FMTC mutations represent gain-of-function mutations (Santoro et al

RET is a tyrosine kinase receptor that plays an important role in the development of the enteric nervous system and the kidney (1, 2). It has been demonstrated that members of the glial cell line-derived neurotrophic factor (GDNF) 1 family including GDNF, neurturin, artemin, and persephin represent the RET ligands. The RET activation by these neurotrophic factors are mediated by their binding to glycosylphosphatidylinositol-anchored co-receptors termed GDNF family receptor ␣ 1-4 (GFR␣ 1-4) (1, 2). GDNF, neurturin, artemin, and persephin use GFR␣ 1, GFR␣ 2, GFR␣ 3, and GFR␣ 4 as the preferred receptors, respectively, and play specific roles in vivo through these preferred ligand-receptor complex formation. For example, Gdnf-or Gfr␣1-deficient mice had the phenotype quite similar to that of Ret-deficient mice, exhibiting the defects of enteric neurons as well as renal agenesis or dysgenesis (3-8). In Nrtn-or Gfra2-deficient mice, the parasympathetic innervation was markedly reduced in the lachrymal and submandibular glands and the intestine (9, 10). Gfr␣3-deficient mice showed severe defect of the superior cervical ganglion (11).RET mutations are responsible for development of several human diseases including Hirschsprung's disease, multiple endocrine neoplasia (MEN) type 2A and 2B, familial medullary thyroid carcinoma (FMTC), and papillary thyroid carcinoma (PTC) (1, 2, 12). Loss-of-function mutations of RET lead to the development of Hirschsprung's disease (13, 14), a malformation characterized by the absence of autonomous enteric neurons. On the other hand, gain-of-function mutations of RET contribute to the development of human neoplastic diseases including MEN 2A, MEN 2B, FMTC, and PTC (12,15,16). MEN 2A, MEN 2B, and FMTC are caused by germ-line point mutations of RET, and PTC is caused by its somatic rearrangement. MEN 2A and MEN 2B share the clinical feature of medullary thyroid carcinoma (MTC) and pheochromocytoma, and FMTC is characterized by the development of MTC alone. In addition, ϳ10 -30% of MEN 2A patients develop parathyroid hyperplasia, whereas MEN 2B patients show a more complex phenotype including ganglioneuromatosis of the gastrointestinal tract, mucosal neuroma, and marfanoid habitus. MTCs developed in MEN 2B appear more aggressive than those in MEN 2A and FMTC.Activated RET recruits a variety of signaling molecules including Grb2, Grb7, Grb10, Shc, Enigma, SNT/Frs2, Dok, insulin receptor substrate-1 (IRS-1), and phospholipase C␥ (17-29). As is the case for other receptor tyrosine kinases, phosphorylated tyrosine residues in the kinase domain and carboxyl-terminal tail of RET represent docking sites for these molecules. For example, phosphorylated tyrosines 905, 1015, and 1096 were identified as docking sites for Grb7/Grb10, phospholipase C␥, and Grb2, respectively (17,20,28,29). Intriguingly, several signaling molecules including Shc, Enigma, SNT/ Frs2, Dok, and IRS-1 bind to phosphorylated tyrosine 1062 (18, 19, 21-23, 25, 26) through which the Ras/Erk, PI3-K/Akt, p38MAPK, c-Jun amino-term...