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.     

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.     

Mutation of the RET ligand, neurturin, supports multigenic inheritance in Hirschsprung disease

Hirschsprung disease (HSCR) is a frequent neurocristopathy characterized by the absence of submucosal and myenteric plexuses in a variable length of the gastrointestinal tract. Pedigrees and segregation analyses suggested the involvement of one or several dominant genes with low penetrance in HSCR. Considering that RET and glial cell line-derived neurotrophic factor (GDNF) mutations have been reported in the disease, we regarded the other RET ligand, neurturin (NTN), as an attractive candidate gene, especially as it shares large homologies with GDNF. Here, we report on the finding of a heterozygous missense NTN mutation in a large non-consanguineous family including four children affected with a severe aganglionosis phenotype extending up to the small intestine. Interestingly, it appears that the NTN mutation reported here is not sufficient to cause HSCR, and this multiplex family also segregates a RET mutation. This cascade of independent and additive genetic events fits well with the multigenic pattern of inheritance expected in HSCR, and further support the role of RET ligands in development of the enteric nervous system.     

Full activation of MEN2B mutant RET by an additional MEN2A mutation or by ligand GDNF stimulation

Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).     

Germline mutation of the RET proto-oncogene in children with total intestinal aganglionosis

To clarify the pathogenesis of total intestinal aganglionosis, an extremely severe from of neural crest-derived cell migration disorder of the gut, the authors studied possible germline mutations of the RET proto-oncogene (10q11.2) in five pedigrees at high risk for congenital aganglionosis. All five patients analyzed were boys, and one had a family history of Hirschsprung's disease. Genomic DNA was extracted from lymphoblastoid cell lines established from patients and their relatives. Polymerase chain reaction (PCR) products, which were amplified using specific primers (RET; exon 1 approximately 20), were electrophoresed to analyze the single-strand conformational polymorphism (SSCP) patterns. DNA sequences were determined in pedigrees showing abnormal SSCP bands. Among the five patients, three germline mutations were found in the receptor tyrosine kinase domain (exon 15; codons 884, 897, and 904). Amino acid substitutions of the Ret protein were predicted based on the mutated nucleotide changes. Phenotypic variations of congenital aganglionosis may depend on the RET mutation pattern and other genetic or environmental determinants. In our series of patients, male sexuality and germline mutation of the RET tyrosine kinase domain were the most likely factors contributing to this form of Hirschsprung'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.     

Only the substitution of methionine 918 with a threonine and not with other residues activates RET transforming potential

Specific point-mutations of the RET receptor tyrosine kinase protooncogene are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). MEN2B is caused by the substitution of methionine 918 by a threonine in the tyrosine kinase (TK) domain of RET. This mutation converts RET into a dominant transforming oncogene. We have substituted Met918 with four different residues and found that RET acquired transforming activity only when Met918 was substituted with a threonine. However, also when serine and valine, but not leucine or phenylalanine, were inserted in position 918, the RET TK function was activated and induced, especially in the case of the RET(918Ser), immmediate-early response genes. We conclude that the preservation of Met918 is critical for the control of RET kinase. However, only when a threonine residue is present in position 918, does RET efficiently couple with a transforming pathway.     

Mutations in the ligand-binding domain of the kit receptor: an uncommon site in human piebaldism

Heterozygous mutations in the gene for the Kit transmembrane receptor have been identified recently in human piebaldism and mouse "dominant spotting." Interestingly, all of the 14 known missense mutations that cause depigmentation in these species map to the tyrosine kinase domain of the receptor, whereas none have involved the extracellular ligand-binding domain. In an attempt to detect these uncommon mutations, we screened the nine exons encoding the extracellular portion of Kit for single-strand conformation polymorphisms (SSCP) in eight piebald subjects previously reported to be negative for kinase mutations. Four of these eight kindreds proved to carry novel mutations. The first mutation, found in two apparently unrelated probands with mild piebaldism and English ancestry, substitutes an arginine for a highly conserved cysteine at codon 136. This substitution disrupts a putative disulfide bond required for formation of the second Ig-like (D2) loop of the Kit ligand-binding domain. The second mutation, detected in a piebald kindred characterized by unusually limited depigmentation, substitutes a threonine for an alanine at codon 178, a site just proximal to conserved cysteines at codons 183 and 186. The third mutation, occurring in a kindred with more extensive depigmentation, is a novel four-base insertion in exon 2 that results in a proximal frameshift and premature termination. The data strongly suggest that piebaldism can result from missense mutations in the Kit ligand-binding domain, although the resulting phenotype may be milder than that observed for null or kinase mutations. The apparent clustering of these uncommon mutations at or near the conserved cysteines for the D2 Ig-like loop further suggests a critical role for this region in Kit receptor function.     

Efficient poly A trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos

We have created a deletion mutant of the insulin-like growth factor type 1 receptor (IGF-1 R) which lacks the 36 amino acids (aa) immediately N-terminal to the transmembrane domain (Delta870-905 IGF-1 R). This region has been reported to have a negative effect on the transforming potential of an avian sarcoma virus gag-IGF-1 R fusion protein. We have sought to determine whether this region plays a similar role in the intact IGF-1 R. Analysis of the tyrosine kinase activity of the Delta870-905 IGF-1 R shows that the mutant receptor is autophosphorylated without IGF-1 stimulation, indicating that the tyrosine kinase domain is constitutively active. In addition, processing of the receptor is decreased, resulting in accumulation of a high molecular weight proreceptor containing both alpha and beta-subunits. A well-characterized substrate of the IGF-1 R, IRS-1, is constitutively phosphorylated by the Delta870-905 IGF-1 R and phosphoinositide (PI) 3-kinase activity, which is normally activated by the phosphorylation of IRS-1 following IGF-1 stimulation, is increased even in the absence of IGF-1. A second intracellular signal pathway normally activated by IGF-1, the MAP kinase pathway, showed no increase in activity in the absence of IGF-1. The Delta870-905 IGF-1 R promoted cell proliferation only in the presence of IGF-1. We conclude that this deletion increases the basal activity of the IGF-1 receptor tyrosine kinase and activates PI 3-kinase, but is unable to stimulate MAP kinase in the absence of ligand. These results confirm those seen in the gag-IGF-1 R fusion protein and indicate that aa 870-905 exert a negative effect on the tyrosine kinase domain of the beta-subunit of the IGF-1 R.     

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.     

Identification of the major autophosphorylation sites of Nyk/Mer, an NCAM-related receptor tyrosine kinase

Nyk/Mer receptor tyrosine kinase is a new member of the Ufo/Axl tyrosine kinase family and is characterized by its neural cell adhesion molecule-like extracellular domain. By using a vaccinia virus expression system to express a constitutively activated form of Nyk, we identified the major sites of Nyk autophosphorylation in tryptic peptide IY749SGDY753Y754R. Tyr-749, Tyr-753, and Tyr-754 in this peptide lie in the activation loop of the kinase domain. We also studied a series of Nyk mutants in which the three tyrosine residues were replaced individually, in pairs, or all together by phenylalanine. Single mutations of Tyr-749 or Tyr-753 to phenylalanine reduced Nyk kinase activity toward exogenous substrate to 39 or 10% of that of the wild type Nyk, respectively, whereas the Tyr-754 mutant is completely inactive. All of the double and triple Tyr-Phe mutants reduced Nyk kinase activity to a level below the background. Similar results were obtained when Nyk autophosphorylation levels were examined. Our studies suggest that full activity of Nyk/Mer kinase requires phosphorylation of all three tyrosine residues in the kinase domain (Tyr-749, Tyr-753, and Tyr-754) and that Nyk kinase activity is modulated by the level of autophosphorylation in the kinase domain. Given the highly conserved nature of this region among the Ufo/Axl receptor family members, the information presented in this report may provide insight to the biochemical properties of other members of this family.     

Molecular and structural characterization of five novel mutations in the Bruton's tyrosine kinase gene from patients with X-linked agammaglobulinemia

BACKGROUND: The Btk (Bruton's tyrosine kinase) gene has been shown to be mutated in the human immunodeficiency disease, XLA (X-linked agammaglobulinemia). Btk is a member of the Tec family of cytosolic protein tyrosine kinases with distinct functional domains PH, TH, SH3, SH2, and kinase. Mutations have been observed in each of the Btk subdomains in XLA. We have analyzed the Btk gene in six XLA patients from five unrelated families. MATERIALS AND METHODS: DNA was prepared from the patients peripheral blood. The Btk exons including the junctional sequences were analyzed by single-strand conformation polymorphism (SSCP) followed by direct nucleotide sequencing after PCR-amplification. For structural analysis, the missense mutations were introduced into three-dimensional models of the PH and kinase domains of Btk and the outcome was predicted based on the knowledge of the protein function. RESULTS: Five novel mutations and two novel polymorphisms, all of which resulted from single-base alterations, were identified. Three of the five mutations were in the PH domain and two were in the kinase domain of Btk. Three of these mutations were of the missense type, two of which altered the same codon in the PH domain; the third one was located in the kinase domain. The fourth mutation was a point deletion in the PH domain causing a frameshift followed by premature termination. The fifth mutation was a splice donor-site mutation within the kinase domain which could result in an exon skipping. In four of the five instances, mothers of the patients were shown to be obligate carriers. In one instance, a sibling sister was identified as a heterozygote establishing her as a carrier. CONCLUSIONS: Functional consequences of the mutations causing frameshifts and altered splicing can be inferred directly. Functional consequences of the missense mutations were interpreted by 3-dimensional structural modeling of Btk domains. It is proposed that the two PH domain mutations will interfere with membrane localization while the kinase domain mutation will interfere with the enzymatic function of Btk. This study provides further insight into the role of Btk in XLA.     

Melanocortin receptor 1 (MC1R) mutations and coat color in pigs

The melanocortin receptor 1 (MC1R) plays a central role in regulation of eumelanin (black/brown) and phaeomelanin (red/yellow) synthesis within the mammalian melanocyte and is encoded by the classical Extension (E) coat color locus. Sequence analysis of MC1R from seven porcine breeds revealed a total of four allelic variants corresponding to five different E alleles. The European wild boar possessed a unique MC1R allele that we believe is required for the expression of a wild-type coat color. Two different MC1R alleles were associated with the dominant black color in pigs. MC1R*2 was found in European Large Black and Chinese Meishan pigs and exhibited two missense mutations compared with the wild-type sequence. Comparative data strongly suggest that one of these, L99P, may form a constitutively active receptor. MC1R*3 was associated with the black color in the Hampshire breed and involved a single missense mutation D121N. This same MC1R variant was also associated with EP, which results in black spots on a white or red background. Two different missense mutations were identified in recessive red (e/e) animals. One of these, A240T, occurs at a highly conserved position, making it a strong candidate for disruption of receptor function.     

RET protooncogene mutational analysis in multiple endocrine neoplasia syndrome type 2B: case report and review of the literature

Multiple endocrine neoplasia, type 2B (MEN 2B), is a phenotypic variant of a group of autosomal-dominant neurocristopathies. MEN 2B is associated with medullary thyroid carcinoma and pheochromocytoma with oral, ocular, and alimentary submucosal ganglioneuromas and Marfanoid body features. Approximately 50% of cases are thought to be spontaneous mutations. The RET protooncogene (RET) is a 21-exon gene encoding a tyrosine kinase receptor. A codon 918 germ line mutation, which converts a highly conserved methionine to a threonine in the intracellular tyrosine kinase portion of this receptor of RET, has been identified in 95% of patients with MEN 2B. This mutation is easily detected by a direct deoxyribonucleic acid sequencing or restriction enzyme (Fok 1) analysis of amplified polymerase chain reaction products. The RET gene is normally expressed in the oral and gastrointestinal submucosal neural ganglia, and the codon 918 mutation is thought to cause neuromas by virtue of its transforming activity in these ganglia. Identifying clinical features of MEN 2B in an 11-year-old boy by an oral pathologist led to confirmation by mutational analysis. Before genetic testing was available, the patient, and at a later date his mother, underwent thyroidectomies based solely on biochemical testing. Results indicated the patient had the codon 918 mutation, whereas his phenotypically normal mother, father, and older brother had normal RET analyses. Studies in families have demonstrated that the mutant allele is derived from the father with possible acquisition during spermatogenesis. We believe the mother of our affected patient to be normal; the absence of phenotypic features of MEN 2B and a normal genotype suggest her calcitonin abnormalities and minimal evidence for C-cell hyperplasia were inconsequential. Molecular analysis for RET abnormalities will likely supplant biochemical methods of diagnosis in patients with MEN 2B.     

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.     

Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas

The MET protooncogene encodes a transmembrane tyrosine kinase identified as the receptor of a polypeptide known as hepatocyte growth factor/scatter factor. We performed PCR-based single-strand conformational polymorphism and sequencing analysis of the tyrosine kinase domain of the MET gene (exon 15-19) in 75 primary liver cancers. Three missense mutations were detected exclusively in 10 childhood hepatocellular carcinomas (HCCs), while no mutations were detected in 16 adult HCCs, 21 cholangiocarcinomas, or 28 hepatoblastomas. The extremely short incubation period from hepatitis B virus infection to the genesis of childhood HCC as compared with the adult HCC suggests that there may be an additional mechanism that accelerates the carcinogenesis of childhood HCC. Our results indicate that mutations of the tyrosine kinase domain of the MET gene may be involved in the acceleration of the carcinogenesis in childhood HCC.     

The physical map of the human RET proto-oncogene

The RET proto-oncogene, a transmembrane tyrosine kinase receptor, is involved in the development of at least five different disease phenotypes. RET is activated through somatic rearrangements in a number of cases of papillary thyroid carcinoma while germ-line point mutations are associated with three inherited cancer syndromes MEN 2A, MEN 2B and FMTC. Moreover, point mutations or heterozygous deletions of RET are found in the dominant form of Hirschsprung disease or congenital colonic aganglionosis. We cloned the entire RET genomic sequence in a contig of cosmids encompassing 150 kb, from the CA repeat sTCL-2 to the region upstream the RET promoter, and established the position of the 20 exons of the RET gene with respect to a detailed restriction map based on eight endonucleases. A new highly polymorphic CA repeat sequence was identified within intron 5 of RET (RET-INT5). Finally the orientation of RET on chromosome 10q11.2 made it possible to orientate three other genes rearranged with RET in papillary thyroid carcinomas, namely H4/D10S170 on 10q21, R1 alpha on 17q23 and RFG2/Ele1 on 10q11.2.