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PubMed2003

Ret protein expression in adrenal medullary hyperplasia and pheochromocytoma.

Ret is a developmentally regulated tyrosine kinase involved in formation and maintenance of the nervous system. Ret mutations predisposing to pheochromocytomas and medullary thyroid carcinomas occur in multiple endocrine neoplasia (MEN) syndromes 2A and 2B. Biochemical studies have demonstrated overexpression of Ret mRNA and protein in pheochromocytomas compared to normal adrenal medulla. However, the cellular distribution of Ret in the normal human adrenal and in hyperplastic lesions that antecede pheochromocytomas are unclear. The present investigation was undertaken to resolve the histological distribution of Ret in the normal human adrenal, in pheochromocytomas evolving from adrenal medullary hyperplasia in MEN2A and in sporadic pheochromocytomas. Ret expression was studied by immunohistochemistry using both a polyclonal and a monoclonal antibody, with confirmation by immunoblotting of representative cases. Only occasional cells stained for Ret in the normal adrenal, consistent with the distribution in adult adrenals of other species. Heterogeneous, progressively increased Ret expression was observed during the evolution of pheochromocytomas. In both normal and neoplastic adrenal, the most intense immunoreactivity was observed in cells with neuron-like features. Our finding that Ret is not expressed at high levels in the early stages of disease suggests that elucidation of mechanisms that regulate Ret expression is required for understanding the pathobiology of MEN2A. The association of high-level Ret expression with neuronal morphology suggests that the variable overexpression of Ret in pheochromocytomas might in part be an epiphenomenon, reflecting the known phenotypic plasticity of these tumors.

Adrenal Gland Neoplasms↗
PubMed2006

[Expressions of wildtype-RET and RET/PTC rearrangements in sporadic adult papillary thyroid carcinoma and their clinicopathologic correlation].

OBJECTIVE: To evaluate the expressions of wildtype-RET (WT-RET) and RET/PTC in sporadic adult papillary thyroid carcinoma and to investigate their clinicopathologic correlation. METHODS: Sixty-six papillary thyroid carcinomas (PTC) and thirty-six control cases with frozen and paraffin-embedded tissues were analyzed for the expressions of WT-RET and oncogene RET/PTC1 or RET/PTC3 by nested RT-PCR. RESULTS: (1) 62 percent (41/66) of PTC patients were above 40 years of age. Thirty-eight percent (25/66) of the tumors showed lymphocytic thyroiditis. Lymph node and distant metastasis were seen in 59% (39/66) and 7.6% (5/66) respectively. (2) Forty-five cases (68.1%) of PTCs expressed RET tyrosine kinase domain (RET-TK). Simultaneous expressions of RET-BP and TK were seen in nineteen PTCs (28.8 %). One of eight adenomas (12.5 %) expressed wild-type RET (WT-RET). (3) Fourteen PTCs (21.2%) expressed RET/PTC, including five cases expressing RET/PTC1 and nine cases expressing RET/PTC3. Six cases (9%) expressed both RET/PTC and WT-RET. (4) Statistic analysis did not show any correlation between the expression of WT-RET or RET/PTC and clinicopathologic parameters. CONCLUSIONS: The expression of RET/PTC was specific to PTC. However, its prevalence was low and, therefore, of limited diagnostic utility. The expression patterns of WT-RET in PTC and adenoma suggest that there are different molecular mechanisms in activating RET proto-oncogene in thyroid tumors.

Adenoma↗
PubMed1995

Spatial and temporal expression of the ret proto-oncogene product in embryonic, infant and adult rat tissues.

Immunohistochemical analysis with the anti-Ret antibody was performed to investigate the expression of the c-ret proto-oncogene product (c-Ret protein) in embryonic, infant and adult rat tissues. During embryogenesis, the c-Ret expression became detectable by day 11.5 in the developing peripheral and central nervous systems as well as in the excretory system. In the peripheral nervous system of the trunk, it was expressed at high levels in the enteric neuroblasts and the autonomic and dorsal root ganglia. c-Ret positive cells appeared in the mesenchyme around the foregut and the dorsal aorta at day 11.5 and formed the myenteric plexus of the whole embryonic gut and the sympathetic trunk at later stages respectively. Examination of the cranial region revealed that the c-Ret protein was expressed in neural crest cells migrating from rhombomere 4 at day 11.5 and then became positive in the facial, glossopharyngeal and vagus cranial ganglia at day 12.5-13.5. After day 16.5 of gestation, the c-Ret expression was also observed in the trigeminal ganglion. In the central nervous system, the c-Ret protein was expressed in the neuroepithelial cells of the ventral neural tube (day 11.5-14.5), the motor neurons of the spinal cord (day 18.5) as well as in the embryonic neuroretina (day 18.5). In addition to the nervous system, the c-Ret expression was detected in the nephric duct (day 11.5), the ureteric bud (day 13.5) and the collecting ducts of the kidney (day 16.5). After birth, neurons in the nervous systems mentioned above continued to express the c-Ret protein at variable levels while no c-Ret expression was observed in the kidney of adult rats. Furthermore, the c-Ret expression was found in the acinar cells of the salivary gland, the epithelial cells of the thymus and the follicular dendritic cells of the spleen and lymph node in infant and adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1996

Molecular heterogeneity of RET loss of function in Hirschsprung's disease.

The RET proto-oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non-neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non-neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprung's disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild-type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972-->Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32-->Leu) and HSCR393 (Phe393-->Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.

3T3 Cells↗
PubMed2005

High prevalence of c-RET expression in papillary thyroid carcinomas from the Korean population.

BACKGROUND: Activation of the RET proto-oncogene, located on the long arms of chromosome 10, contributes to the development of thyroid cancers in two different ways. First, somatic rearrangements of RET with variable activation genes are frequently found in papillary thyroid carcinomas. Second, germ-line point mutations are responsible for the development of medullary thyroid carcinomas and multiple endocrine neoplasia type 2 (MEN 2). There are several conflicting reports on the influences of RET expression and RET/PTC rearrangements on the clinical outcome of thyroid cancers. Therefore, the wild-type RET gene expression and RET/PTC-1, RET/PTC-2, RET/PTC-3 rearrangements were examined in thyroid carcinomas and other thyroid diseases. MATERIALS AND METHODS: Thirty-six papillary thyroid carcinomas (PTCs), 8 follicular thyroid carcinomas (FTCs), 4 anaplastic thyroid carcinomas (ATC), 7 follicular adenomas (FAs), 23 hyperplasias, 6 normal thyroid tissues, and 39 normal portions from each tumor were included in this study. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses were used to identify the RET gene and RET/PTC rearrangements. RESULTS: From the RT-PCR analysis, 68.9% of the PTCs, a single case of FTC, and 22.2% of the hyperplasias expressed the RET gene. No RET gene expression was observed in ATCs, FAs, or normal thyroid tissues. One RET/PTC-1 and one RET/PTC-2 rearrangement were detected in the PTCs. No RET/PTC-3 rearrangement was detected in any specimen. The immunohistochemical results revealed that 66.7% of PTCs, 28.6% of FAs, and 18.2% of hyperplastic thyroid tissue specimens showed high levels of RET protein expression. Neither the normal thyroid tissues nor the FTCs and ATC, showed high levels of RET protein expression. The two methods are agreed in PTC and hyperplastic nodules, but not in FA and FTC. CONCLUSION: PTCs among Koreans rarely showed RET/PTC rearrangements, but commonly showed increased RET gene expression. Compared to earlier reports indicating that the expression of the RET gene was limited to PTCs, the RET gene was also expressed in hyperplasias in this study.

Adenocarcinoma, Papillary↗
PubMed1997

Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs.

Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to signal by binding to GDNF receptor-alpha (GDNFR-alpha), after which the GDNF-GDNFR-alpha associates with and activates the tyrosine kinase receptor Ret. We have localized Ret messenger RNA (mRNA) in the developing and adult rodent and compared with to the expression of GDNF and GDNFR-alpha mRNA. Ret mRNA is strongly expressed in dopamine neurons and alpha-motor neurons as well as in thalamus, ruber and occluomotor nuclei, the habenular complex, septum, cerebellum, and brain stem nuclei. Ret mRNA was also found in several sensory systems, in ganglia, and in nonneuronal tissues such as teeth and vibrissae. Very strong Ret mRNA signals are present in kidney and the gastrointestinal tract, where Ret and GDNF mRNA expression patterns are precisely complementary. The presence of Ret protein was confirmed in adult dopamine neurons using immunohistochemistry. GDNFR-alpha mRNA was strongly expressed in the developing and adult dopamine neurons. It was also found in neurons in deep layers of cortex cerebri, in hippocampus, septum, the dentate gyrus, tectum, and the developing spinal cord. In the kidney and the gastrointestinal tract, GDNFR-alpha mRNA and Ret mRNA distribution overlapped. Dorsal root ganglia, cranial ganglia, and developing peripheral nerves were also positive. GDNFR-alpha was additionally found in sensory areas and in developing teeth. Sensory areas included inner ear, eye, olfactory epithelium, and the vomeronasal organ, as well as developing tongue papillae. The temporospatial pattern of expression of GDNFR-alpha mRNA did not always match that of Ret mRNA. For instance, GDNFR-alpha mRNA was also found in the developing ventral striatum, including the olfactory tubercle, and in hippocampus. These areas seemed devoid of Ret mRNA, suggesting that GDNFR-alpha might also have functions unrelated to Ret.

Animals↗
PubMed2005

Up-regulation of ret by reserpine in the adult rat adrenal medulla.

The receptor tyrosine kinase, ret, is activated by glial cell line-derived neurotrophic factor, neurturin and related ligands that bind to glycosylphosphatidylinositol-tailed receptors GFRalpha1-4. Ret expression is developmentally regulated and detectable only at very low levels in adult adrenal medulla. However, mutations of ret that cause constitutive activation or alter signal transduction give rise to adrenal medullary hyperplasia and pheochromocytomas in humans with hereditary multiple endocrine neoplasia (MEN) syndromes 2A and 2B and in animal models. These discordant observations pose the conundrum of how a molecule barely detectable in the adult adrenal can contribute to development of adrenal medullary pathology that typically occurs in adults. We recently reported that depolarization and phorbol esters that activate protein kinase C act synergistically with neurturin to up-regulate ret protein and mRNA expression in adult rat chromaffin cell cultures. Those findings suggested that ret expression in vivo is not static and might be regulated in part by neurally derived signals. We show here that the anti-hypertensive agent reserpine, which is known to cause a reflex increase in trans-synaptic stimulation of chromaffin cells, increases expression of ret mRNA and protein in adult rat adrenal medullary tissue in vivo. Elevated ret protein levels are detectable both by immunoblots and immunohistochemistry, which shows immunoreactive ret in chromaffin cells and neurons after reserpine administration. The finding that ret expression is subject to up-regulation by environmental signals in vivo suggests that epigenetic factors might influence the development of adrenal medullary disease by affecting the expression of ret. It is known that long-term administration of reserpine leads to the development of adrenal medullary hyperplasia and pheochromocytomas in rats. Our findings suggest potential utility of the rat model for studying the roles of ret in the adrenal medulla and the mechanisms of its involvement in MEN 2 and other pheochromocytoma syndromes.

Adrenal Medulla↗
PubMed2003

Development of adrenal chromaffin cells is largely normal in mice lacking the receptor tyrosine kinase c-Ret.

c-Ret encodes a receptor tyrosine kinase that is essential for normal development of the kidney as well as enteric and sympathetic neurons. Since sympathetic neurons and neuroendocrine chromaffin cells originate from a common progenitor cell, we have examined the relevance of c-Ret for the development of adrenal chromaffin cells by analyzing mouse mutants lacking c-Ret. Adrenal chromaffin cells express c-Ret mRNA at embryonic day (E) 12.5 and 13.5, yet levels of expression decline at later embryonic and postnatal ages. Adrenal medullae of c-Ret deficient mice show normal numbers of tyrosine hydroxylase (TH)-immunoreactive cells at E13.5 and at birth. Ultrastructurally, adrenal chromaffin cells of c-Ret(-/-) mice appear unaltered: chromaffin cells develop typical secretory chromaffin granules, the morphological hallmark of chromaffin cells, and synaptic terminals appear normal. However, adrenaline levels and numbers of chromaffin cells immunoreactive for the adrenaline synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) are reduced by about 30% in c-Ret-deficient mice arguing for a direct or indirect role of c-Ret in the regulation of PNMT. Thus, despite expression of c-Ret, adrenal chromaffin cells develop largely normal in mice lacking c-Ret. We therefore conclude that sympathetic neurons and neuroendocrine chromaffin cells profoundly differ in their requirement for c-Ret signaling during development.

Adrenal Glands↗
PubMed1997

Distinct biological properties of two RET isoforms activated by MEN 2A and MEN 2B mutations.

Germline mutations of the RET proto-oncogene, which codes for a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). MEN 2 mutations have been shown to result in RET oncogenic activation. The RET gene encodes several isoforms whose biological properties, when altered by MEN 2 mutations, have not been thoroughly addressed yet. In this study, we have introduced a MEN 2A mutation (Cys634-->Arg) and the unique MEN 2B mutation (Met918-->Thr) in two RET isoforms of 1114 and 1072 amino acids which differ in the carboxy-terminus part. Herein, we report that each RET isoform activated by MEN 2A or MEN 2B mutation was transforming in fibroblasts and induced neuronal differentiation of pheochromocytoma PC12 cells. However, among the different RET-MEN 2 mutants, the long RET isoform activated by the MEN 2B mutation stimulated the most prominent neurite outgrowth in PC12 cells, while the short RET isoform counterpart elicited a very weak differentiation effect in PC12 cells. We further demonstrate that the morphological changes of PC12 cells caused by constitutively activated RET oncoproteins involved the engagement of a Ras-dependent pathway. These findings provide evidence that the biological properties of RET-MEN 2 mutants depend on the interplay between the RET isoforms and the nature of the activating MEN 2 mutation.

Animals↗
PubMed1998

A 5'-CG-3'-rich region in the promoter of the transcriptionally frequently silenced RET protooncogene lacks methylated cytidine residues.

In a large proportion of familial and sporadic cases of Hirschsprung disease (HSCR) mutations in the RET (rearranged during transfection) protooncogene have been described. We have investigated the structure of the RET gene promoter and have analysed a region of approximately 1000 nucleotides in its promoter and 5'-upstream segments for the occurrence of 5-methyldeoxycytidine (5-mC) residues by using the bisulfite protocol of the genomic sequencing method. With an estimated sensitivity of about 93% of this technique, not a single 5-mC residue could be detected in the control region of a gene that seems to be silenced or exhibit low activity in many adult tissues. In these experiments, the DNAs of peripheral white blood cells (PWBC) from four healthy individuals, from seven patients with familial HSCR, as well as DNAs from different human tissues and from a human embryonic kidney (HEK) cell line have been included. In a DNA segment starting 790 nucleotides upstream of the transcriptional start site of the RET gene, a few 5-mC residues have been identified. This region possibly constitutes the transition site from an unmethylated promoter to a more extensively methylated region in the human genome. The data presented are remarkable in that a highly 5'-CG-3'-enriched segment of the human genome with 49 5'-CG-3' dinucleotide pairs in 400 bp within the putative promoter region is completely devoid of 5-mC residues, although this control region is not actively transcribed in most adult human tissues. By hybridization of a PCR-amplified RET protooncogene cDNA probe harboring exons 9-15 to a membrane (Clontech) containing poly-A selected RNAs from 50 different human tissues, weak RET protooncogene expression in many of the neural cell derived tissues has been detected. RNAs extracted from many other human tissues do not share sequence homologies to this 32P-labeled probe. Mechanisms other than DNA methylation obviously play the crucial role in the inactivation of the RET gene promoter in these tissues. We have also demonstrated by the in vitro premethylation of a RET promoter-chloramphenicol acetyltransferase (CAT) gene construct and transient transfection experiments into neuroblastoma cells that the transcriptional activity of the RET promoter is decreased by HpaII (5'-CCGG-3') methylation and abolished by SssI (5'-CG-3') methylation. Hence, the RET promoter region is sensitive to this regulatory signal. However in vivo, DNA methylation of the promoter region seems not to be the predominant regulatory mechanism for the RET protooncogene. Possibly, in adults the RET gene can be occasionally activated.

Adult↗
PubMed2006

Self-association of the transmembrane domain of RET underlies oncogenic activation by MEN2A mutations.

In patients with medullary thyroid carcinoma (MTC) and type 2A multiple endocrine neoplasia (MEN2A), mutations of cysteine residues in the extracellular juxtamembrane region of the RET receptor tyrosine kinase cause the formation of covalent receptor dimers linked by intermolecular disulfide bonds between unpaired cysteines, followed by oncogenic activation of the RET kinase. The close proximity to the plasma membrane of the affected cysteine residues prompted us to investigate the possible role of the transmembrane (TM) domain of RET (RET-TM) in receptor-receptor interactions underlying dimer formation. Strong self-association of the RET-TM was observed in a biological membrane. Mutagenesis studies indicated the involvement of the evolutionary conserved residues Ser-649 and Ser-653 in RET-TM oligomerization. Unexpectedly, RET-TM interactions were also abrogated in the A639G/A641R double mutant, first identified in a sporadic case of MTC. In agreement with this, no transforming activity could be detected in full-length RET carrying the A639G and A641R mutations, which remained fully responsive to glial cell-line-derived neurotrophic factor (GDNF) stimulation. When introduced in the context of C634R - a cysteine replacement that is prevalent in MEN2A cases - the A639G/A641R mutations significantly reduced dimer formation and transforming activity in this otherwise highly oncogenic RET variant. These data suggest that a strong propensity to self-association in the RET-TM underlies - and may be required for - dimer formation and oncogenic activation of juxtamembrane cysteine mutants of RET, and explains the close proximity to the plasma membrane of cysteine residues implicated in MEN2A and MTC syndromes.

Alanine↗
PubMed2000

Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway.

The RET gene codes for a receptor tyrosine kinase that plays a crucial role during the development of both the enteric nervous system and the kidney. Germ line missense mutations at one of six codons specifying extracytoplasmic cysteines are responsible for two related cancer disorders as follows: multiple endocrine neoplasia type2A (MEN2A) and familial medullary thyroid carcinoma (FMTC). MEN2A and FMTC mutations result in a constitutive catalytic activity and as a consequence convert RET into a dominantly acting transforming gene. Although it has been shown that RET-MEN2 mutants activate several transduction pathways, their respective contribution to the neoplastic phenotype remains poorly understood. Over the past few years, it has become increasingly clear that the transforming ability of several viral and cellular oncoproteins depends on their capacity to activate phosphatidylinositol 3-kinase (PI3K). We now report that RET carrying a representative MEN2A mutation at Cys-634 (termed RET-MEN2A) activates PI3K and its downstream effector, the serine/threonine kinase AKT/protein kinase B. Previous studies have demonstrated that mutation of Tyr-1062, which is the intracellular docking site for Shc and Enigma on RET, abolishes the RET-MEN2A transforming activity. We provide evidence that mutation of Tyr-1062 abrogates the binding of the p85 regulatory subunit of PI3K to RET-MEN2A and the subsequent stimulation of the PI3K/AKT pathway. Furthermore, infection of rat fibroblasts with a retrovirus expressing a dominant-interfering form of PI3K suppresses RET-MEN2A-dependent transformation, whereas overexpression of AKT enhances the RET-MEN2A oncogenic potential. In summary, these data are consistent with the notion that RET-mediated cell-transforming effect is critically dependent on the activation of the PI3K/AKT pathway.

Animals↗
PubMed2004

RET proto-oncogene mutations are restricted to codon 634 and 618 in Korean families with multiple endocrine neoplasia 2A.

Identification of the germline mutation in the RET proto-oncogene is important for the diagnosis of hereditary medullary thyroid carcinoma (MTC). Hereditary forms account for approximately 25%-30% of all cases of MTC. The objective of this study was to evaluate the prevalence of the RET mutation and the genotype-phenotype relation in Korean patients with MTC. Genomic DNAs were obtained from 33 patients with MTC (M:F = 10:23, 39.8 +/- 12.0 years) who underwent total thyroidectomy between 1997 and 2003 at the Samsung Medical Center. Exons 10, 11, 13, 14, 15 and 16 of the RET proto-oncogene were amplified with specific primers using polymerase chain reaction (PCR). Sequence analysis was performed on the polymerase chain reaction (PCR) product using an automatic sequence analyzer. Nine of the 33 patients (M:F = 3:6, 33.3 +/- 10.0 years) were identified as having RET mutations. Six patients had multiple endocrine neoplasia (MEN) 2A and one had familial medullary thyroid carcinoma (FMTC). The remaining two patients were thought to have sporadic MTC. Five of the patients with MEN 2A had RET mutations in codon 634 of exon 11 (3 patients, C634Y; 2 patients, C634R) and the other patient with MEN 2A had a RET mutation in codon 618 of exon 10 (C618R). The patient with FMTC had a mutation in codon 634 (C634W). The two patients with sporadic MTC had RET mutations in codon 634 (1 patient, C634Y; 1 patient, C634S). We were not able to identify any genotype-phenotype relations because of the limited number of patients. Twenty-seven percent (9/33) of the patients with MTC in this study had RET mutations. Taking other studies into account, 77% (10/13) of Korean families with MEN 2A, including 7 other families in three reports from Korea, had RET mutations in codon 634 (5 families, C634Y; 4 families, C634R; 1 family, C634W), and 23% (3/13) had RET mutations in codon 618 (2 families, C618R; 1 family, C618S). RET proto-oncogene mutations were restricted to codon 634 and 618 in Korean families with MEN 2A.

Adult↗
PubMed1999

Two distinct mutations of the RET receptor causing Hirschsprung's disease impair the binding of signalling effectors to a multifunctional docking site.

The RET gene codes for a transmembrane tyrosine kinase which is a subunit of a multimeric complex that acts as a receptor for four structurally related molecules: the glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin and persephin. Germline mutations of RET cause a dominantly inherited dysgenesis of the enteric nervous system known as Hirschsprung's disease (HSCR; aganglionosis megacolon). The majority of HSCR mutations results either in a reduction of dosage of the RET protein or in the loss of RET function. Two novel distinct mutations of RET that led either to the deletion of codon 1059 (denoted Delta1059) or to the substitution of a Pro for Leu1061 have been identified in five HSCR families. In one large pedigree, two children born from asymptomatic consanguineous parents presented a severe form of HSCR and were found to carry the mutation at codon 1061 in the homozygous state. A tyrosine residue at position 1062 is an intracytoplasmic docking site that enables RET to recruit several signalling molecules, including the Shc adaptor protein. We now report that both HSCR mutations impair the fixation of Shc to RET and consequently prevent its phosphorylation. In addition, quantitative analysis in PC12 cells reveals that mutation Delta1059 inactivates the ability of RET to transduce a downstream signal whereas mutation L1061P only partially inhibits the signalling of RET. Finally, we provide evidence that these effects are partly mediated via the disruption of the RET/Shc interaction. Collectively, these results demonstrate that HSCR can be ascribed to mutations of RET which interfere with the binding of transduction effectors, such as Shc, and further provide a biochemical explanation for the phenotype of patients carrying a homozygous mutation at codon 1061. Finally, these data indicate that Y1062 is a multifunctional docking site that confers to RET the capacity to engage downstream signalling pathways which exert a crucial role during enteric neurogenesis.

3T3 Cells↗
PubMed2002

Molecular mechanisms of RET activation in human cancer.

Mutations that produce oncogenes with dominant gain of function target receptor protein tyrosine kinases (PTKs) in cancer and confer uncontrolled proliferation, impaired differentiation, or unrestrained survival to the cancer cell. However, insufficient PTK signaling may be responsible for developmental diseases. Gain of function of the RET receptor PTK is associated with human cancer. At the germline level, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN2A, MEN2B, and FMTC). Mutations of extracellular cysteines are found in MEN2A patients, and a Met918Thr mutation is responsible for most MEN2B cases. At the somatic level, gene rearrangements juxtaposing the tyrosine kinase domain of RET to heterologous gene partners are found in papillary carcinomas of the thyroid. These rearrangements generate the chimeric RET/PTC oncogenes. Both MEN2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, the RET downstream signaling events. A multidocking site of the C-tail of RET is essential for both mitogenic and survival RET signaling. Such a site is involved in the recruitment of several intracellular molecules, such as the Shc, FRS2, IRS1, Gab1/2, and Enigma. The different activating mutations not only potentiate the enzymatic activity of the RET kinase but also may alter qualitatively RET signaling properties by: (1) altering RET autophosphorylation (in the case of the MEN2B mutation), (2) modifying the subcellular distribution of the active kinase, and (3) providing the active kinase with a scaffold for novel protein-protein interactions (as in the case of RET/PTC oncoproteins). This review describes the molecular mechanisms by which the different genetic alterations cause the conversion of RET into a dominant transforming oncogene.

Carcinoma, Papillary↗
PubMed2002

The neuron-specific Rai (ShcC) adaptor protein inhibits apoptosis by coupling Ret to the phosphatidylinositol 3-kinase/Akt signaling pathway.

Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals.

Adaptor Proteins, Signal Transducing↗
PubMed2004

RET exon 11 (G691S) polymorphism is significantly more frequent in sporadic medullary thyroid carcinoma than in the general population.

The RET protooncogene is constitutively activated by point mutations in hereditary medullary thyroid carcinomas (MTCs). RET somatic point mutations have also been reported in 40-50% of sporadic MTCs. Several single nucleotide polymorphisms of the RET gene have been described in the general population as well as in patients with MTC. These allelic variants do not seem to confer any transforming activity to the tyrosine kinase domain of the RET gene. Because the exon 11 RET polymorphism determines an important aminoacidic variation (G691S), we studied its frequency in 212 subjects, 106 sporadic MTC patients and 106 normal age-, sex-, race-, and geographic origin-matched controls. In 46 cases of sporadic MTCs, we also studied the cosegregation of somatic RET gene mutation and G691S polymorphism as well as the linkage of the polymorphism with RET germline mutation in 60 members of eight multiple endocrine neoplasia type 2 families. The influence of this polymorphism on the RET gene transcription has also been studied. In parallel we analyzed the frequencies of another three neutral polymorphisms (L769L, S836S, S904S). We found a statistically significant (P = 0.029) higher allelic frequency of G691S polymorphism in MTCs (27.83%) than that found in normal controls (18.86%), at variance with the three neutral polymorphisms whose frequencies were not different in patients and controls. With this study we excluded the influence of the G691S polymorphism on RET mRNA expression, the development of the somatic RET mutation, the linkage with the germline RET mutation, the younger onset of the MTCs, and the clinical outcome of the disease. A putative role of the G691S polymorphism as genetic modifier in the normal subjects remains to be established.

Carcinoma, Medullary↗
PubMed2006

RET is dispensable for maintenance of midbrain dopaminergic neurons in adult mice.

Glial cell-line derived neurotrophic factor (GDNF)-mediated RET tyrosine kinase signaling is implicated in the survival of several PNS and CNS neuronal populations that are important in the pathogenesis of several disorders including Parkinson's disease and drug addiction. However, it has been difficult to study these processes and the physiological importance of this pathway in adult mice because of the neonatal lethality of Gdnf and Ret null mice. We report successful creation of RET conditional reporter mice to investigate postnatal physiologic roles of RET and monitor the fate of RET-expressing cell types. To delete RET specifically in dopaminergic neurons and determine the physiologic requirement of RET in the maintenance of substantia nigra compacta (SNC) and ventral tegmental area (VTA), we bred the RET conditional mice with mice that specifically express Cre from the dopamine transporter (Dat) locus. A detailed morphometric and biochemical analysis including dopaminergic neuron number and size in SNC and VTA, and fiber density in the striatum and nucleus accumbens, and dopamine levels indicate that RET is not required for providing global trophic support to midbrain dopaminergic neurons in adult mice. Furthermore, RET deficiency in these neurons does not cause major sensorimotor abnormalities. Hence our results support the idea that RET signaling is not critical for the normal physiology of the SNC and VTA in adult mice.

Animals↗