Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFRalpha3-RET receptor complex

The glial cell line-derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFRalpha1-GFRalpha4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFRalpha3-RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFRalpha1-RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRalpha1-RET) and NTN (GFRalpha2-RET), Artemin has a preferred receptor (GFRalpha3-RET) but that alternative receptor interactions also occur.     

Genomic structure of the gene for the SH2 and pleckstrin homology domain-containing protein GRB10 and evaluation of its role in Hirschsprung disease

Hirschsprung disease (HSCR), or congenital aganglionic megacolon, is the most frequent cause of congenital bowel obstruction. Germline mutations in the RET receptor tyrosine kinase have been shown to cause HSCR. Mice that carry null alleles for RET or for its ligand, glial cell line-derived neurotrophic factor (GDNF), both exhibit complete intestinal aganglionosis and renal defects. Recently, the Src homology 2 (SH2) domain-containing protein Grb10 has been shown to interact with RET in vitro and in vivo, early in development. We have confirmed the map location of GRB10 on human chromosome 7, isolated human BACs containing the gene, elucidated its genomic structure, isolated a highly polymorphic microsatellite marker adjacent to exon 14 and scanned the gene for mutations in a large panel of HSCR patients. No evidence of linkage was detected in HSCR kindreds and no mutations were found in patients. These data suggest that while GRB10 may be important for signal transduction in developing embryos, it does not play an obvious role in HSCR.     

Various mechanisms cause RET-mediated signaling defects in Hirschsprung's disease

Hirschsprung's disease (HSCR) is a common congenital malformation characterized by the absence of intramural ganglion cells of the hindgut. Recently, mutations of the RET tyrosine kinase receptor have been identified in 50 and 15-20% of familial and sporadic HSCR, respectively. These mutations include deletion, insertion, frameshift, nonsense, and missense mutations dispersed throughout the RET coding sequence. To investigate their effects on RET function, seven HSCR missense mutations were introduced into either a 1114-amino acid wild-type RET isoform (RET51) or a constitutively activated form of RET51 (RET-MEN 2A). Here, we report that one mutation affecting the extracytoplasmic cadherin domain (R231H) and two mutations located in the tyrosine kinase domain (K907E, E921K) impaired the biological activity of RET-MEN 2A when tested in Rat1 fibroblasts and pheochromocytoma PC12 cells. However, the mechanisms resulting in RET inactivation differed since the receptor bearing R231H extracellular mutation resulted in an absent RET protein at the cell surface while the E921K mutation located within the catalytic domain abolished its enzymatic activity. In contrast, three mutations mapping into the intracytoplasmic domain neither modified the transforming capacity of RET-MEN 2A nor stimulated the catalytic activity of RET in our ligand-independent system (S767R, P1039L, M1064T). Finally, the C609W HSCR mutation exerts a dual effect on RET since it leads to a decrease of the receptor at the cell surface and converted RET51 into a constitutively activated kinase due to the formation of disulfide-linked homodimers. Taken together, our data show that allelic heterogeneity at the RET locus in HSCR is associated with various molecular mechanisms responsible for RET dysfunction.     

Glial cell line-derived neurotrophic factor/neurturin-induced differentiation and its enhancement by retinoic acid in primary human neuroblastomas expressing c-Ret, GFR alpha-1, and GFR alpha-2

Neuroblastomas often undergo spontaneous differentiation and/or regression in vivo, which is at least partly regulated by the signals through neurotrophins and their receptors. Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN), have been found to mediate their signals by binding to a heterotetrameric complex of c-Ret tyrosine kinase receptors and glycosylphosphatidylinositol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiation in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment with 5 microM all-trans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancement of neurite outgrowth induced by treatment with both GDNF and all-trans-retinoic acid was significantly correlated with younger age (less than 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single copy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0062). To examine the expression levels of GFR alpha-1, we cloned a short form of the human GFR alpha-1 gene with a 15-bp deletion by screening a human adult substantia nigra cDNA library. Many primary neuroblastomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their ligands, GDNF and NTN, suggesting the presence of a paracrine or autocrine signaling system within the tumor tissue. The effect of NTN on primary culture cells of neuroblastoma was similar to that of GDNF. These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.     

Familial medullary thyroid carcinoma: not a distinct entity? Genotype-phenotype correlation in a large family

BACKGROUND: Multiple endocrine neoplasia type 2A (MEN 2A) is a hereditary syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Familial MTC (FMTC) is characterized by MTC only. Both MEN 2A and FMTC are caused by germline mutations of the RET proto-oncogene. PURPOSE: To assess genotype/phenotype correlations, large families have to be examined periodically over a long period using an extensive screening program. PATIENTS AND METHODS: Since 1973, we screened a large family with hereditary C cell carcinoma for MTC, pheochromocytoma, and parathyroid disease by clinical tests and imaging methods. A germline codon Cys618 to Ser mutation in the RET proto-oncogene was recently identified in this family. The disease phenotype associated with this mutation was compared with that of Cys634 mutations in some other large MEN 2A families. RESULTS: The distinct course of disease in the family described here is similar to that in other FMTC families and MEN 2A families with a Cys618 mutation of the RET gene, but clearly different from that in families with a Cys634 mutation. The frequency of pheochromocytomas and parathyroid disease is clearly lower, whereas cure rates and life expectancy are higher. However, in families with a Cys618 mutation, pheochromocytoma and parathyroid disease do occur. CONCLUSION: In FMTC families with cysteine codon mutations of the RET proto-oncogene, screening for other endocrinopathies is mandatory, since these may not be MTC-only families. Therefore, we suggest that MEN 2A families should not be subclassified into MEN 2A and FMTC, but rather according to their specific mutation in the RET protein (i.e., for this family MEN 2A RET C618S).     

Mutational analysis of the endothelin-B receptor gene in Japanese Hirschsprung's disease

BACKGROUND/PURPOSE: Hirschsprung's disease (HD, HSCR) is one of the most common diseases in the field of pediatric surgery. It is well known that the aganglionic bowel is primarily a causative factor of dismotility of distal narrow segment. Recent studies have shown that mutations in endothelin-B receptor (EDNRB), endothelin-3, RET, glial cell line-derived neurotrophic factor (GDNF) genes are responsible for the occurrence of congenital aganglionosis. Here, the authors describe two new mutations of the EDNRB gene in Japanese patients with HD. RESULTS: One patient had a heterozygous point mutation at the splice donor site of intron 3, leading to premature termination of translation of EDNRB mRNA. Another patient has a heterozygous missense mutation (N1041) in exon 1, but the same mutation was found in two of 50 normal individuals, so the mutation may be a noncausative polymorphism of the EDNRB gene. CONCLUSION: These results provide further evidence that a spectrum of different mutations within the EDNRB gene are responsible for HD.     

Neurturin shares receptors and signal transduction pathways with glial cell line-derived neurotrophic factor in sympathetic neurons

Neurturin (NTN) is a neurotrophic factor that shares homology with glial cell line-derived neurotrophic factor (GDNF). Recently, a receptor complex has been identified for GDNF that includes the Ret tyrosine kinase receptor and a glycosylphosphatidylinositol-linked protein termed "GDNFRalpha." However, differences in the phenotype of Ret and GDNF knockout animals suggest that Ret has at least one additional ligand. In this report, we demonstrate that NTN induces Ret phosphorylation in primary cultures of rat superior cervical ganglion (SCG) neurons. NTN also caused Ret phosphorylation in fibroblasts that were transfected stably with Ret and GDNFRalpha but not in cells expressing Ret alone. A glycosylphosphatidylinositol-linked protein also was important for NTN and GDNF signaling in SCG neurons; phosphatidylinositol-specific phospholipase C treatment of SCG cultures reduced the ability of NTN to phosphorylate Ret and the ability of NTN or GDNF to activate the mitogen-activated protein kinase pathway. NTN and GDNF also caused sustained activation of Ret and the mitogen-activated protein kinase pathway in SCG neurons. Finally, both NTN and GDNF activated the phosphatidylinositol 3-kinase pathway in SCG neurons, which may be important for the ability of NTN and GDNF to promote neuronal survival. These data indicate that NTN is a physiologically relevant ligand for the Ret receptor and suggest that NTN may have a critical role in the development of many neuronal populations.     

Mutation analysis of glial cell line-derived neurotrophic factor, a ligand for an RET/coreceptor complex, in multiple endocrine neoplasia type 2 and sporadic neuroendocrine tumors

Causative germline missense mutations in the RET proto-oncogene have been associated with over 92% of families with the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). MEN 2A is characterized primarily by medullary thyroid carcinoma (MTC) and pheochromocytoma, both tumors of neural crest origin. Parathyroid hyperplasia or adenoma is also seen in MEN 2A, but rarely in MEN 2B, which has additional stigmata, including a marfanoid habitus, mucosal neuromas, and ganglioneuromatosis of the gastrointestinal tract. In familial MTC, MTC is the only lesion present. Somatic RET mutations have also been identified in a subset of sporadic MTCs, pheochromocytomas, and rarely, small cell lung cancer, but not in sporadic parathyroid hyperplasias/adenomas or other neuroendocrine tumors. Glial cell line-derived neurotrophic factor (GDNF) and its receptor molecule GDNFR-alpha, have recently been identified as members of the RET ligand binding complex. Therefore, the genes encoding both GDNF and GDNFR-alpha are excellent candidates for a role in the pathogenesis of those MEN 2 families and sporadic neuroendocrine tumors without RET mutations. No mutations were found in the coding region of GDNF in DNA samples from 9 RET mutation negative MEN 2 individuals (comprising 6 distinct families), 12 sporadic MTCs, 17 sporadic cases of parathyroid adenoma, and 10 small cell lung cancer cell lines. Therefore, we find no evidence that mutation within the coding regions of GDNF plays a role in the genesis of MEN 2 and sporadic neuroendocrine tumors.     

Glial cell line-derived neurotrophic factor differentially stimulates ret mutants associated with the multiple endocrine neoplasia type 2 syndromes and Hirschsprung's disease

Ret is a receptor tyrosine kinase involved in several neoplastic and developmental diseases affecting the thyroid gland and tissues of neuroectodermal origin. Different ret mutations are associated with different disease phenotypes. Gain-of-function of ret is caused by gene rearrangements in thyroid papillary carcinomas and by point mutations in multiple endocrine neoplasia (MEN) type 2A syndrome (MEN2A), in familial medullary thyroid carcinoma (FMTC), and in the more severe MEN2B syndrome. Conversely, Hirschsprung's disease (HSCR) is associated with loss of function of ret. Recently, it has been shown that glial cell line-derived neurotrophic factor (GDNF), by binding to the accessory molecule GDNFR-alpha, acts as a functional ligand of Ret and stimulates its tyrosine kinase and biological activity. To ascertain whether the biological effects of ret mutations are modulated by GDNF, we have investigated the responsiveness to GDNF of ret mutants in cell lines coexpressing GDNFR-alpha and MEN2A-, MEN2B-, FMTC-, or HSCR-associated ret mutants. Here, we show that triggering of GDNF affected only ret/MEN2B, i.e. it stimulated ret/MEN2B mitogenic and kinase activities, as well as its ability to phosphorylate Shc, a bona fide Ret substrate. In contrast, ret mutants associated with MEN2A or FMTC (carrying Cys634 or Cys620 mutations) were unresponsive to GDNF. HSCR mutations, by affecting either the extracellular or the intracellular Ret domain, impaired responsiveness to GDNF. These data suggest that the phenotype of human diseases caused by ret mutations can be differentially influenced by GDNF.     

Neurturin exerts potent actions on survival and function of midbrain dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) exhibits potent effects on survival and function of midbrain dopaminergic (DA) neurons in a variety of models. Although other growth factors expressed in the vicinity of developing DA neurons have been reported to support survival of DA neurons in vitro, to date none of these factors duplicate the potent and selective actions of GDNF in vivo. We report here that neurturin (NTN), a homolog of GDNF, is expressed in the nigrostriatal system, and that NTN exerts potent effects on survival and function of midbrain DA neurons. Our findings indicate that NTN mRNA is sequentially expressed in the ventral midbrain and striatum during development and that NTN exhibits survival-promoting actions on both developing and mature DA neurons. In vitro, NTN supports survival of embryonic DA neurons, and in vivo, direct injection of NTN into the substantia nigra protects mature DA neurons from cell death induced by 6-OHDA. Furthermore, administration of NTN into the striatum of intact adult animals induces behavioral and biochemical changes associated with functional upregulation of nigral DA neurons. The similarity in potency and efficacy of NTN and GDNF on DA neurons in several paradigms stands in contrast to the differential distribution of the receptor components GDNF Family Receptor alpha1 (GFRalpha1) and GFRalpha2 within the ventral mesencephalon. These results suggest that NTN is an endogenous trophic factor for midbrain DA neurons and point to the possibility that GDNF and NTN may exert redundant trophic influences on nigral DA neurons acting via a receptor complex that includes GFRalpha1.     

Mutations in the extracellular domain cause RET loss of function by a dominant negative mechanism

The RET proto-oncogene encodes a tyrosine kinase receptor expressed in neuroectoderm-derived cells. Mutations in specific regions of the gene are responsible for the tumor syndromes multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B), while mutations along the entire gene are involved in a developmental disorder of the gastrointestinal tract, Hirschsprung's disease (HSCR disease). Two mutants in the extracellular domain of RET, one associated with HSCR disease and one carrying a flag epitope, were analyzed to investigate the impact of the mutations on RET function. Both mutants were impeded in their maturation, resulting in the lack of the 170-kDa mature form and the accumulation of the 150-kDa immature form in the endoplasmic reticulum. Although not exposed on the cell surface, the 150-kDa species formed dimers and aggregates; this was more pronounced in a double mutant bearing a MEN 2A mutation. Tyrosine phosphorylation and the transactivation potential were drastically reduced in single and double mutants. Finally, in cotransfection experiments both mutants exerted a dominant negative effect over protoRET and RET2A through the formation of a heteromeric complex that prevents their maturation and function. These results suggest that HSCR mutations in the extracellular region cause RET loss of function through a dominant negative mechanism.     

Neurturin protects dopaminergic neurons following medial forebrain bundle axotomy

To determine whether neurturin (NTN), a recently identified homologue of glial cell line-derived neurotrophic factor (GDNF), is able to preserve tyrosine hydroxylase immunoreactivity (TH-IR) in a rat model of Parkinson's disease, polymer encapsulated cells genetically engineered to release NTN were implanted near the substantia nigra 1 week before a unilateral medial forebrain bundle axotomy. Animals were allowed to survive for 1 week post-axotomy. Upon sacrifice, animals that received a NTN capsule had a significantly higher percentage of TH-IR (lesioned side vs non-lesioned side) than animals that had received a capsule containing non-transfected parent cells. However, in contrast to GDNF, no reduction of turning was observed upon amphetamine rotation with NTN. Nevertheless, these results suggest that NTN might have a therapeutic value for the treatment of Parkinson's disease.     

Novel mutations of the endothelin B receptor gene in patients with Hirschsprung's disease and their characterization

Hirschsprung's disease (HSCR) is a congenital intestinal disease, characterized by the absence of ganglion cells in the distal portion of the intestinal tract. Recently, three susceptibility genes have been identified in HSCR, namely the RET protooncogene, the endothelin B (ETB) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3). To investigate whether mutations in EDNRB could be related with HSCR in non-inbred populations in Japan, we examined alterations of the gene in 31 isolated patients. Three novel mutations were detected as follows: two transversions, A to T and C to A at nucleotides 311 (N104I) and 1170 (S390R), respectively, and a transition, T to C at nucleotide 325 (C109R). To analyze functions of these mutant receptors, they were expressed in Chinese hamster ovary cells. S390R mutation did not change the binding affinities but caused the decreases in the ligand-induced increment of intracellular calcium and in the inhibition of adenylyl cyclase activity, showing the impairment of the intracellular signaling. C109R receptors were proved to be localized near the nuclei as an unusual 44-kDa protein with the extremely low affinity to endothelin-1 (ET-1) and not to be translocated into the plasma membrane. On the other hand, N104I receptors showed almost the same binding affinities and functional properties as those of the wild type. Therefore, we conclude that S390R and C109R mutations could cause HSCR but that N104I mutation might be polymorphous.     

Analysis of RET proto-oncogene abnormalities in patients with MEN 2A, MEN 2B, familial or sporadic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) may occur either as a sporadic or familial (FMTC) disease. Multiple endocrine neoplasia (MEN) type 2, inherited as an autosomal dominant disease, is characterized by coexistence of MTC with other endocrine neoplasia. Activating mutations of the RET proto-oncogene, involving the somatic or the germinal cell lineage, are found in both inherited and acquired forms. In this study, RET mutations were screened in 47 individuals either affected by MTC or belonging to families with hereditary MTC. Exons 10, 11, 13, 14, 15 and 16 of the RET gene were amplified by polymerase chain reaction and examined by DNA sequence and/or restriction enzyme analysis to detect mutations in purified amplicons. Six MEN 2A families with a germline mutation at codon 634, one FMTC family carrying a mutation at codon 618 and two MEN 2B families with a mutation at codon 918 were identified. In affected members of a MEN 2A family no known RET mutations were observed. Besides, we identified a germline mutation in a patient with apparently sporadic MTC and in two out of three sons, indicating the presence of a sporadic misclassified familial disease. In all of the families examined we were able to distinguish the affected vs unaffected (not at risk) members. A somatic mutation of codon 918 was detected in three out of ten patients with apparently sporadic MTC.     

Complementary and overlapping expression of glial cell line-derived neurotrophic factor (GDNF), c-ret proto-oncogene, and GDNF receptor-alpha indicates multiple mechanisms of trophic actions in the adult rat CNS

Glial cell line-derived neurotrophic factor (GDNF), the most potent trophic factor yet described for both dopaminergic neurons of the substantia nigra and spinal motorneurons, has recently been shown to signal through a multireceptor complex composed of a novel glycosylphosphatidylinositol-anchored GDNF receptor-alpha (GDNFR-alpha) and the receptor tyrosine kinase product of the c-ret proto-oncogene (RET). Despite its importance, the individual expression patterns and the relationships between domains of expression of the different components of this trophic system are not understood. We show here by in situ hybridization that GDNF mRNA is expressed in the normal adult rat brain in several targets of substantia nigra neurons, including striatum, nucleus accumbens, thalamic nuclei, olfactory tubercle, hippocampus, cerebellum, and cingulate cortex as well as in the internal granular cell layer of the olfactory bulb. Within the basal ganglia we observe a pronounced segregation of regions expressing GDNF from those expressing GDNF receptors, suggesting that within these structures GDNF is functioning in its anticipated role as a target-derived trophic factor. In addition, the expression of GDNF and both GDNF receptors within the cerebellum, hippocampus, and olfactory bulb may indicate a paracrine mode of action. Importantly, we also see expression of RET mRNA in cellular populations within the cerebellum and the glomerular layer of the olfactory bulb, as well as in the subthalamic nucleus, which lack GDNFR-alpha expression, indicating that RET functions either independently of GDNFR-alpha or with GDNFR-alpha presented in trans. Conversely, GDNFR-alpha is widely expressed in many regions in which RET expression is absent, suggesting that GDNFR-alpha may associate with additional signaling receptors. Finally, RET and GDNFR-alpha show distinct patterns of regulated expression in the brain after kainic acid stimulation and in the sciatic nerve after nerve transection. Taken together these findings indicate that GDNF, RET, and GDNFR-alpha utilize multiple mechanisms to comprise physiologically relevant trophic circuits for different neuronal populations.     

Neurturin responsiveness requires a GPI-linked receptor and the Ret receptor tyrosine kinase

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor (GDNF). Both factors promote the survival of a variety of neurons, and GDNF is required for the development of the enteric nervous system and kidney. GDNF signals through a receptor complex consisting of the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor termed GDNFR-alpha. Here we report the cloning of a new GPI-linked receptor termed NTNR-alpha that is homologous with GDNFR-alpha and is widely expressed in the nervous system and other tissues. By using microinjection to introduce expression plasmids into neurons, we show that coexpression of NTNR-alpha with Ret confers a survival response to neurturin but not GDNF, and that coexpression of GDNFR-alpha with Ret confers a survival response to GDNF but not neurturin. Our findings indicate that GDNF and neurturin promote neuronal survival by signalling through similar multicomponent receptors that consist of a common receptor tyrosine kinase and a member of a GPI-linked family of receptors that determines ligand specificity.