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1.
Congenital aganglionic megacolon, commonly known as Hirschsprung disease (HSCR), is the most frequent cause of congenital bowel obstruction. Germline mutations in theRETreceptor tyrosine kinase have been shown to cause HSCR. Knockout mice forRETand for its ligand, glial cell line-derived neurotrophic factor (GDNF), exhibit both complete intestinal aganglionosis and renal defects. Recently, GDNF and GFRA1 (GDNF family receptor, also known as GDNFR-α), its GPI-linked coreceptor, were demonstrated to be components of a functional ligand for RET. Moreover,GDNFhas been implicated in rare cases of HSCR. We have mappedGFRA1to human chromosome 10q25, isolated human and mouse genomic clones, determined the gene's intron–exon boundaries, isolated a highly polymorphic microsatellite marker adjacent to exon 7, and scanned forGFRA1mutations in a large panel of HSCR patients. No evidence of linkage was detected in HSCR kindreds, and no sequence variants were found to be in significant excess in patients. These data suggest thatGFRA1's role in enteric neurogenesis in humans remains to be elucidated and that RET signaling in the gut may take place via alternate pathways, such as the recently described GDNF-related molecule neurturin and its GFRA1-like coreceptor, GFRA2.  相似文献   

2.
Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) alpha subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFRalpha1, neurturin via GFRalpha2, artemin via GFRalpha3, whereas the mammalian GFRalpha receptor for persephin (PSPN) is unknown. Here we characterize the human GFRalpha4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRalpha4 lack the first Cys-rich domain characteristic of other GFRalpha receptors. Unlabeled PSPN displaces (125)I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRalpha4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFRalpha4 may restrict the MEN2 syndrome to these cells.  相似文献   

3.
Four members of the glial cell line-derived neurotrophic factor family have been identified (GDNF, neurturin, persephin, and enovin/artemin). They bind to a specific membrane-anchored GDNF family receptor as follows: GFRalpha-1 for GDNF, GFRalpha-2 for neurturin, GFRalpha-3 for enovin/artemin, and (chicken) GFRalpha-4 for persephin. Subsequent signaling occurs through activation of a common transmembrane tyrosine kinase, cRET. GFRalpha-4, the coreceptor for persephin, was previously identified in chicken only. We describe the cloning and characterization of a mammalian persephin receptor GFRalpha-4. The novel GFRalpha receptor is substantially different in sequence from all known GFRalphas, including chicken GFRalpha-4, and lacks the first cysteine-rich domain present in all previously characterized GFRalphas. At least two different GFRalpha-4 splice variants exist in rat tissues, differing at their respective COOH termini. GFRalpha-4 mRNA is expressed at low levels in different brain areas in the adult as well as in some peripheral tissues including testis and heart. Recombinant rat GFRalpha-4 variants were expressed in mammalian cells and shown to be at least partially secreted from the cells. Persephin binds specifically and with high affinity (K(D) = 6 nm) to the rat GFRalpha-4 receptor, but no cRET activation could be demonstrated. Although the newly characterized mammalian GFRalpha-4 receptor is structurally divergent from previously characterized GFRalpha family members, we suggest that it is a mammalian orthologue of the chicken persephin receptor. This discovery will allow a more detailed investigation of the biological targets of persephin action and its potential involvement in diseases of the nervous system.  相似文献   

4.
5.
Molecular analysis of congenital central hypoventilation syndrome   总被引:7,自引:0,他引:7  
Congenital central hypoventilation syndrome (CCHS or Ondines curse; OMIM 209880) is a disorder characterized by an idiopathic failure of the automatic control of breathing. CCHS is frequently complicated with neurocristopathies such as Hirschsprungs disease (HSCR). The genes involved in the RET-GDNF signaling and/or EDN3-EDNRB signaling pathways have been analyzed as candidates for CCHS; however, only a few patients have mutations of the RET, EDN3, and GDNF genes. Recently, mutations of the PHOX2B gene, especially polyalanine expansions, have been detected in two thirds of patients. We studied the RET, GDNF, GFRA1, PHOX2A, PHOX2B, HASH-1, EDN1, EDN3, EDNRB, and BDNF genes in seven patients with isolated CCHS and three patients with HSCR. We detected polyalanine expansions and a novel frameshift mutation of the PHOX2B gene in four patients and one patient, respectively. We also found several mutations of the RET, GFRA1, PHOX2A, and HASH-1 genes in patients with or without mutations of the PHOX2B gene. Our study confirmed the prominent role of mutations in the PHOX2B gene in the pathogenesis of CCHS. Mutations of the RET, GFRA1, PHOX2A, and HASH-1 genes may also be involved in the pathogenesis of CCHS. To make clear the pathogenesis of CCHS, the analysis of more cases and further candidates concerned with the development of the autonomic nervous system is required.  相似文献   

6.
The glial cell line-derived neurotrophic factor (GDNF) family coreceptor alpha1 (GFRalpha1) is a critical component of the RET receptor kinase signal-transducing complex. The activity of this multicomponent receptor is stimulated by the glial cell line-derived neurotrophic factor (GDNF) and is involved in neuronal cells survival and kidney development. GFRalpha1 pre-mRNA is alternatively spliced and produces two isoforms: GFRalpha1a, which includes the exon 5; and GFRalpha1b, which excludes it. Here we show that the Gfralpha1a isoform is predominantly expressed in neuronal tissues and in PC12 cells differentiated toward a neuronal phenotype. GFRalpha1 splicing is also regulated during kidney development, GFRalpha1a is the minor isoform before birth and then rapidly becomes the major form after birth. We established cell lines expressing either GFRalpha1 isoforms and demonstrated that the GFRalpha1b isoform binds GDNF more efficiently than GFRalpha1a. Consistently, GFRalpha1b promotes a stronger RET phosphorylation than GFRalpha1a. These results indicate that specific inclusion of the GFRalpha1 exon 5 in neuronal tissues or during kidney development may alter the binding properties of GDNF to GFRalpha1, and thus could constitute an additional regulatory mechanism of the RET signaling pathway.  相似文献   

7.
In animal models, kidney formation is known to be controlled by the proteins RET, GDNF, and GFRA1; however, no human studies to date have shown an association between abnormal kidney development and mutation of these genes. We hypothesized that stillborn fetuses with congenital renal agenesis or severe dysplasia would possess mutations in RET, GDNF, or GFRA1. We assayed for mutations in these genes in 33 stillborn fetuses that had bilateral or unilateral renal agenesis (29 subjects) or severe congenital renal dysplasia (4 subjects). Mutations in RET were found in 7 of 19 fetuses with bilateral renal agenesis (37%) and 2 of 10 fetuses (20%) with unilateral agenesis. In two fetuses, there were two different RET mutations found, and a total of ten different sequence variations were identified. We also investigated whether these mutations affected RET activation; in each case, RET phosphorylation was either absent or constitutively activated. A GNDF mutation was identified in only one fetus with unilateral agenesis; this subject also had two RET mutations. No GFRA1 mutations were seen in any fetuses. These data suggest that in humans, mutations in RET and GDNF may contribute significantly to abnormal kidney development.  相似文献   

8.
We have cloned a partial cDNA of chicken glial cell line-derived neurotrophic factor (GDNF) and systematically examined its expression pattern as well as that of GDNF-binding components (GDNF family receptor alpha-1 and 2: GFRalpha-1 and 2) and a common signal transduction receptor (c-ret protooncogene: RET) during very early developmental stages. In addition, we also examined the expression pattern of an apparent avian-specific binding component, GFRalpha-4. The cloned chicken cDNA for GDNF had approximately 80% homology to mammalian counterparts. The expression of GDNF mRNA occurred in many spatially and temporally discrete regions such as the intermediate mesoderm, the floor plate of the spinal cord, pharyngeal endoderm contacting the epibranchial placodes, distal ganglia of cranial nerves, subpopulations of mesenchyme cells in the craniofacial region, and in the mesodermal wall of the digestive tract. Both a GDNF receptor signal transduction component (RET) and a binding component (GFRalpha-1 or GFRalpha-2) were independently expressed in nearby interacting tissues such as the somites, peripheral and central nervous system, and mesenchyme cells in the craniofacial region. These observations suggest that possible combinations of novel unidentified receptors acting with RET or with GFRalphas may mediate GDNF-derived signals and indicate that GDNF or other family members may have previously unidentified actions in early organogenesis in the chick embryo.  相似文献   

9.
The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) (GDNF, neurturin, artemin, and persephin) are critical regulators of neurodevelopment and support the survival of midbrain dopaminergic and spinal motor neurons in vitro and in animal disease models making them attractive therapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affinity binding component (GDNF-family receptor alpha-component (GFRalpha1-GFRalpha4)) and the receptor tyrosine kinase RET. To begin characterization of GFL receptor specificity at the molecular level, we performed comprehensive homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from persephin (PSPN) (which cannot bind or activate GFRalpha1.RET or GFRalpha2.RET) identified sites along the second finger of GDNF critical for activating the GFRalpha1.RET and GFRalpha2.RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRalpha1. RET, but additional determinants are required for interaction with the other GFRalphas. This difference in the molecular basis of GFL-GFRalpha specificity allowed the production of GFRalpha1. RET-specific agonists and provides a foundation for understanding of GFL-GFRalpha.RET signaling at the molecular level.  相似文献   

10.
Using bioinformatic tools, mutagenesis, and binding studies, we have investigated the structural organization of the extracellular region of the RET receptor tyrosine kinase, a functional receptor for glial cell line-derived neurotrophic factor (GDNF). Multiple sequence alignments of seven vertebrate sequences and one invertebrate RET sequence delineated four distinct N-terminal domains, each of about 110 residues, containing many of the consensus motifs of the cadherin fold. Based on these alignments and the crystal structures of epithelial and neural cadherins, we have generated molecular models of each of the four cadherin-like domains in the extracellular region of human RET. The modeled structures represent realistic models from both energetic and geometrical points of view and are consistent with previous observations gathered from biochemical analyses of the effects of Hirschsprung's disease mutations affecting the folding and stability of the RET molecule, as well as our own site-directed mutagenesis studies of RET cadherin-like domain 1. We have also investigated the role of Ca(2+) in ligand binding by RET and found that Ca(2+) ions are required for RET binding to GDNF but not for GDNF binding to the GFRalpha1 co-receptor. In agreement with these results, RET, but not GFRalpha1, was found to bind Ca(2+) directly. Our results indicate that the overall architecture of the extracellular region of RET is more closely related to cadherins than previously thought. The models of the cadherin-like domains of human RET represent valuable tools with which to guide future site-directed mutagenesis studies aimed at identifying residues involved in ligand binding and receptor activation.  相似文献   

11.
Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial).Here we present the screening for RVs in the RET CDS and intron/exon boundaries of 601 Chinese HSCR patients, the largest number of patients ever reported. We identified 61 different heterozygous RVs (50 novel) distributed among 100 patients (16.64%). Those include 14 silent, 29 missense, 5 nonsense, 4 frame-shifts, and one in-frame amino-acid deletion in the CDS, two splice-site deletions, 4 nucleotide substitutions and a 22-bp deletion in the intron/exon boundaries and 1 single-nucleotide substitution in the 5' untranslated region. Exonic variants were mainly clustered in RET the extracellular domain. RET RVs were more frequent among patients with the most severe phenotype (24% vs. 15% in short-HSCR). Phasing RVs with the RET HSCR-associated haplotype suggests that RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants were found in 250 Chinese controls.  相似文献   

12.
Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related neurotrophic factors that have both been shown to prevent the degeneration of dopaminergic neurons in vitro and in vivo. NTN and GDNF are thought to bind with different affinities to the GDNF family receptor alpha-2 (GFRalpha2), and can activate the same multi-component receptor system consisting of GFRalpha2, receptor tyrosine kinase Ret (RET) and NCAM. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that regulate gene expression through translational repression or RNA degradation. miRNAs have diverse functions, including regulating differentiation, proliferation and apoptosis in several organisms. It is currently unknown whether GDNF and NTN regulate the expression of miRNAs through activation of the same multi-component receptor system. Using quantitative real-time PCR, we measured the expression of some miRNA precursors in human BE(2)-C cells that express GFRalpha2 but not GFRalpha1. GDNF and NTN differentially regulate the expression of distinct miRNA precursors through the activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2). This study showed that the expression of distinct miRNA precursors is differentially regulated by specific ligands through the activation of GFRalpha2.  相似文献   

13.
Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.  相似文献   

14.
15.
The GDNF family ligands (GFLs: GDNF, neurturin, persephin, and artemin) signal through RET and a gly-cosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRalpha1-alpha4) that binds ligand with high affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRalpha1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRalpha1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.  相似文献   

16.
Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.  相似文献   

17.
The GDNF family ligands signal through a receptor complex composed of a ligand binding subunit, GFRalpha, and a signaling subunit, the RET tyrosine kinase. GFRalphas are expressed not only in RET-expressing cells, but also in cells lacking RET. A body of evidence suggests that RET-independent GFRalphas are important for (1) modulation of RET signaling in a non-cell-autonomous fashion (trans-signaling) and (2) regulation of NCAM function. To address the physiological significance of these roles, we generated mice specifically lacking RET-independent GFRalpha1. These mice exhibited no deficits in regions where trans-signaling has been implicated in vitro, including enteric neurons, motor neurons, kidney, and regenerating nerves. Furthermore, no abnormalities were found in the olfactory bulb, which requires proper NCAM function for its formation and is putatively a site of GDNF-GFRalpha-NCAM signaling. Thus RET-independent GFRalpha1 is dispensable for organogenesis and nerve regeneration in vivo, indicating that trans-signaling and GFRalpha-dependent NCAM signaling play a minor role physiologically.  相似文献   

18.
Hirschsprung disease (HSCR) is a common genetic disorder characterized by intestinal obstruction secondary to enteric aganglionosis. HSCR demonstrates a complex pattern of inheritance, with the RET proto-oncogene acting as a major gene and with several additional susceptibility loci related to the Ret-signaling pathway or to other developmental programs of neural crest cells. To test how the HSCR phenotype may be affected by the presence of genetic variants, we investigated the role of a single-nucleotide polymorphism (SNP), 2508C-->T (S836S), in exon 14 of the RET gene, characterized by low frequency among patients with HSCR and overrepresentation in individuals affected by sporadic medullary thyroid carcinoma. Typing of several different markers across the RET gene demonstrated that a whole conserved haplotype displayed anomalous distribution and nonrandom segregation in families with HSCR. We provide genetic evidence about a protective role of this low-penetrant haplotype in the pathogenesis of HSCR and demonstrate a possible functional effect linked to RET messenger RNA expression.  相似文献   

19.
Wang  Gang  Wang  Hefeng  Zhang  Lijuan  Guo  Feng  Wu  Xiangyu  Liu  Yang 《Molecular and cellular biochemistry》2021,476(5):2061-2073
Molecular and Cellular Biochemistry - Studies have reported that miR-195-5p plays a role in the Hirschsprung disease (HSCR). Our previous work found GDNF family receptor alpha 4 (GFRA4) is also...  相似文献   

20.
Pozas E  Ibáñez CF 《Neuron》2005,45(5):701-713
Cortical GABAergic neurons are generated in the ventral telencephalon and migrate dorsally into the cortex following a tangential path. GDNF signaling via GFRalpha1 was found to promote the differentiation of ventral precursors into GABAergic cells, enhancing their neuronal morphology and motility. GDNF stimulated axonal growth in cortical GABAergic neurons and acted as a potent chemoattractant of GABAergic cells. These effects required GFRalpha1 but neither RET nor NCAM, the two transmembrane signaling receptors known for GDNF. Mutant mice lacking GDNF or GFRalpha1, but neither RET nor NCAM, showed reduced numbers of GABAergic cells in the cerebral cortex and hippocampus. We conclude that one of the normal functions of GDNF signaling via GFRalpha1 in the developing brain is to promote the differentiation and migration of cortical GABAergic neurons. The lack of involvement of RET or NCAM in these processes suggests the existence of additional transmembrane effectors for GDNF.  相似文献   

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