RET Isoforms Research Articles

Differential interaction of Enigma protein with the two RET isoforms

The receptor tyrosine kinase RET, with a known role in embryonic development and in human pathologies, is alternatively spliced to yield at least two functional isoforms, which differ only in their carboxyl terminal. Enigma protein is a member of the PDZ–LIM family and is known to interact with the short isoform of RET/PTC2, a chimeric oncoprotein isolated from papillary thyroid carcinoma. Here, we show that Enigma also interacts in intact cells with the short isoform of RET-wt and of its pathologic mutants associated to MEN2 syndromes, RET-C634R and RET-M918T. In contrast, Enigma binds all the corresponding RET long isoforms very poorly and colocalizes with short but not long RET/PTC2 isoforms. The RET docking tyrosine for Enigma is the last but one before the divergence between the two isoforms and we demonstrated that short-isoform-specific amino acid residues +2 to +4 to this tyrosine are required for the interaction of RET/PTC2 with Enigma.

Differential activities of the RET tyrosine kinase receptor isoforms during mammalian embryogenesis

The RET receptor tyrosine kinase has a critical role in kidney organogenesis and the development of the enteric nervous system. Two major isoforms, RET9 and RET51, differ in the amino acid sequence of the C-terminal tail as a result of alternative splicing. To determine the roles of these isoforms in vivo, we used targeted mutagenesis to generate mice that express either RET9 or RET51. Monoisoformic RET9 mice, which lack RET51, are viable and appear normal. In contrast, monoisoformic RET51 animals, which lack RET9, have kidney hypodysplasia and lack enteric ganglia from the colon. To study the differential activities of the two RET isoforms further, we generated transgenic mice expressing ligand-dependent and constitutively active forms of RET9 or RET51 under the control of the Hoxb7 regulatory sequences. Such RET9 transgenes are capable of rescuing the kidney agenesis in RET-deficient mice or causing kidney hypodysplasia in wild-type animals. In contrast, similar RET51 transgenes fail to rescue the kidney agenesis or cause hypodysplasia. Our findings show that RET9 and RET51 have different signaling properties in vivo and define specific temporal and spatial requirements of c-Ret function during renal development and histogenesis of the enteric nervous system.

C-cell and thyroid epithelial tumours and altered follicular development in transgenic mice expressing the long isoform of MEN 2A RET.

Gain-of-function mutations in the gene encoding the receptor tyrosine kinase RET have been identified as the aetiological factor for multiple endocrine neoplasia type 2A (MEN2A). MEN2A is a dominantly-inherited cancer predisposition syndrome characterized by medullary thyroid carcinoma, a tumour of the calcitonin-producing thyroid C-cells. There are three isoforms of RET: RET9, RET43 and RET51, and although in vitro evidence suggests they vary in cellular transformation activities, little is known about their function in tumorigenesis in vivo. To address this, we used RET51 cDNA to construct mice in which the most frequent MEN2A mutation, Cys-634-Arg, was expressed under the control of the human calcitonin promoter (CT-2A mice). These mice developed C-cell tumours resembling human MTC and follicular tumours resembling human papillary thyroid carcinoma (PTC) depending on the founder line examined. One founder line developed compound MTC/PTC at low frequency (8%) and pancreatic cystadenocarcinoma. CT-2A mice also displayed a developmental defect in thyroid follicular structure, in which much of the thyroid was occupied by large irregular cystic follicles thought to be derived from the ultimobranchial body, a developmental precursor of the thyroid gland. The CT-2A mice will provide a suitable model to further study the effects of the MEN 2A RET mutation in vivo.

Conservation of RET proto-oncogene splicing variants and implications for RET isoform function

The RET proto-oncogene encodes a receptor tyrosine kinase required for development of the kidney and neural crest-derived cell types. Alternative splicing of the 3′ exons of human RET results in three protein isoforms with distinct C-termini: RET9, RET51, and RET43. These RET isoforms show differential binding to downstream adapter molecules, suggesting they may have distinct signaling functions. We have characterized Ret 3′ sequences in mouse and investigated alternative splicing of this region. We found that the organization of Ret 3′ sequences is very similar to human RET. The mouse locus also has alternatively spliced C-terminal coding regions, and the sequences corresponding to RET9 and RET51 are highly conserved in both position and sequence with the human locus. Further, we compared the predicted C-terminal amino acids of RET9 and RET51 in seven vertebrate species, and found that they are well conserved. We have identified sequence encoding a putative ret43 isoform in mouse, however the predicted amino acid sequence showed low homology to human RET43. Our data suggest that RET isoforms are evolutionarily highly conserved over a broad range of species, which may indicate that each isoform has a distinct role in normal RET function.

Signaling through Ras Is Essential for retOncogene-induced Cell Differentiation in PC12 Cells

Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret-transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade.

CDNA sequence and genomic structure of the rat RET proto-oncogene.

The RET proto-oncogene, a member of the Receptor Tyrosine Kinase family, plays a crucial role during the development of the excretory system and the enteric nervous system, as demonstrated by in vivo animal studies and by its involvement in the pathogenesis of several human neurocristopathies like Hirschsprung disease and Multiple Endocrine Neoplasia type 2. Using a multistep RT-PCR approach we have isolated and sequenced the cDNA of the whole rat RET proto-oncogene, reporting the deduced amino acid sequence in comparison with the human and mouse counterparts. Moreover, two different isoforms (RET9 and RET51) have been confirmed in the rat, while a third RET isoform demonstrated in human (RET43) has not resulted to be conserved in this species. Finally, we have determined the genomic structure of the rat RET proto-oncogene comparing the exon-intron boundaries and intron sizes with the known structure of the human homologous gene. Our findings will facilitate the molecular study of appropriate rat models of RET related human diseases.

Grb2 binding to the different isoforms of Ret tyrosine kinase.

The RET proto-oncogene encodes two isoforms of a receptor tyrosine kinase which plays a role in neural crest and kidney development. Ret ligands have been recently identified as the neuron survival factor GDNF (Glial-Derived Neurotrophic Factor) and Neurturin. Somatic rearrangements of RET, designated RET/PTCs, have been frequently detected in papillary thyroid carcinomas. In addition, distinct germ-line mutations of RET gene have been associated with the inherited cancer syndromes MEN (Multiple Endocrine Neoplasia) 2A, 2B and FMTC (Familial Medullar Thyroid Carcinomas) as well as with the congenital megacolon or Hirschsprung's disease, thus enlightening a significant role of this receptor gene in diverse human pathologic conditions. In this study, by performing classical inhibition experiments using synthetic phosphopeptides and by site-directed mutagenesis of the putative docking site, we have determined that for Grb2 the latter is provided by the tyrosine 620 of Ret/ptc2 long isoform (corresponding to Tyr 1096 on proto-Ret). However, in intact cells, the interaction of Grb2 with the two short and long Ret isoforms expressed separately is of similar strength, thus suggesting that Ret short isoform interaction with Grb2 could be mediated not only by Shc but also by a molecule that binds preferentially to this isoform. This possibility is supported by the evidence that the mutant Ret/ptc2Y620F long isoform displays a weak coimmunoprecipitation with Grb2 and that this mutant, lacking the docking site for Grb2 but owing all the others phosphotyrosines, surprisingly displays a reduced transforming activity compared to that of the two WTs oncogenes. We thus conclude that in intact cells both Ret isoforms bind to Grb2, although with different modalities. In addition, the present results are in agreement with the possibility that different signal transduction pathways are associated with the two isoforms of Ret.

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.

Characterization of Ret-Shc-Grb2 Complex Induced by GDNF, MEN 2A, and MEN 2B Mutations

We analyzed the intracellular signalling pathway through Ret activated by glial-cell-line-derived neurotrophic factor (GDNF), multiple endocrine neoplasia (MEN) 2A and 2B mutations. The results showed that all of them induce a signal transducing complex consisting of Ret, Shc, and Grb2 proteins. In addition, GDNF clearly activated a Ras-MAPK pathway in human neuroblastoma cells. Ret is expressed mainly as two isoforms that differ in the carboxy-terminal sequence: a long isoform (1114 amino acids) and a short isoform (1072 amino acids). The long isoform contains the consensus sequence for binding of the Shc PTB domain but not of its SH2 domain, whereas the short isoform has the consensus sequences for binding of both domains.In vitrobinding assay revealed that the long isoform of the MEN2A-Ret protein and both isoforms of the MEN2B-Ret protein bound preferentially to the Shc PTB domain. On the other hand, the short isoform of MEN2A-Ret bound to the PTB and SH2 domains. In neuroblastoma cells expressing both isoforms of Ret, its activation by GDNF also resulted in the binding of both domains. GDNF and MEN 2A mutations activate Ret by inducing its dimerization, whereas the MEN 2B mutation increases Ret catalytic activity without dimerization. Our results thus suggest that Ret dimerization might be required for binding of the Shc SH2 domain to the short isoform.

Oncogenic activation of RET by two distinct FMTC mutations affecting the tyrosine kinase domain.

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are two dominantly inherited disorders caused by germline mutations of the RET proto-oncogene. The RET gene codes for a receptor tyrosine kinase. The majority of MEN2A and FMTC mutations are clustered in the extra-cellular cysteine-rich domain and result in constitutive activation of the tyrosine kinase through the formation of disulfide-bonded RET homodimers. Recently, two novel point mutations have been identified in the germline of five distinct FMTC families. Both mutations occur within the catalytic domain of the RET kinase and lead to the substitution of either glutamic acid 768 or valine 804 by an aspartic acid and a leucine respectively. We have introduced each FMTC mutation in two RET isoforms: RET51 the long isoform (1114 aa) and RET9 the short isoform (1072 aa) which differ in the C-terminal region of the protein. The RET51 isoform carrying either E768D or V804L mutation was autophosphorylated, displayed a transforming activity upon expression in Rat1 fibroblasts and induced neuronal differentiation of PC12 cells. However, the transforming capacity of these RET51-FMTC mutants was found to be severalfold less potent compared to the same isoform carrying either the MEN2A mutation (C634R) or the MEN2B mutation (M918T). In contrast, RET9 containing mutations E768D or V804L was not autophosphorylated, exhibited a poor oncogenic potential in fibroblasts and did not promote neuritic outgrowth upon expression in PC12 cells. Overall, these findings demonstrate that mutations E768D and V804L are gain-of-function mutations that confer to the long RET isoform the capacity to exert a biological effect, although these mutations are more weakly activating than the MEN2A and MEN2B mutations. These results may provide a biochemical basis as to why the phenotypic consequences of these mutations are restricted to thyroid C-cells.

Identification of Shc docking site on Ret tyrosine kinase.

The RET proto-oncogene encodes two isoforms of a receptor type tyrosine kinase which plays a role in neural crest and kidney development. Distinct germ-line mutations of RET have been associated with the inherited cancer syndromes MEN2A, MEN2B and FMTC as well as with the congenital disorder Hirschsprung disease (HSCR), whereas somatic rearrangements (RET/PTCs) have been frequently detected in the papillary thyroid carcinoma. Despite these findings, suggesting a relevant role for RET product in development and neoplastic processes, little is known about the signalling triggered by this receptor. In this study, we have demonstrated that the transducing adaptor molecule Shc is recruited and activated by both Ret isoforms and by the rearranged cytoplasmatic Ret/ptc2 oncoproteins as well as by the membrane bound receptor activated by MEN2A or by MEN2B associated mutations. Moreover, our analysis has identified the Ret tyrosine residue and the Shc domains involved in the interaction. In fact, here we show that both the phosphotyrosine binding domains of Shc, PTB and SH2, interact with Ret/ptc2 in vitro. However, PTB domain binds 20 folds higher amount of Ret/ptc2 than SH2. The putative binding site for either SH2 and PTB domains has been identified as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this finding, by using RET/PTC2-Y586F mutant, we have demonstrated that this tyrosine residue, the last amino acid but one before the divergence of the two Ret isoforms, is the docking site for Shc.

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.