The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein

Abstract

Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations affecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disulfide-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This finding specifically correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion affecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is affected by mutation type.

Introduction

Medullary thyroid carcinoma (MTC) is a malignant tumor arising from calcitonin-secreting parafollicular C cells. MTCs may occur sporadically or as a component of the familial cancer syndrome known as multiple endocrine neoplasia type 2 (MEN 2A). Germline mutations within the RET proto-oncogene, coding for a receptor tyrosine kinase, can cause the MEN 2A, MEN 2B, and FMTC syndromes. The majority of germline mutations reportedly associated with MEN 2A and FMTC lie within exons 10 and 11 of RET. These mutations specifically occur at codons 609, 611, 618, 620 and 634 and universally result in the substitution of a cysteine residue (Mulligan et al., 1993; Donis-Keller et al., 1993). A single point mutation at codon 918, causing the substitution of a methionine (ATG) with a threonine (ACG), is associated with both MEN 2B and sporadic MTC (Eng et al., 1994, 1995b; Carlson et al., 1994; Hofstra et al., 1994; Muragaki et al., 1995; Blaugrund et al., 1994). Approximately 80% of sporadic MTCs are positive for this specific RET codon 918 mutation (Eng et al., 1995b, 1996). In contrast, two other relatively rare somatic mutations, at codons 768 (exon 13) and 883 (exon 15), are detected in approximately 10% of sporadic MTCs (Bolino et al., 1995; Eng et al., 1995a; Komminoth et al., 1995). We have recently described two additional interstitial deletions affecting exons 10 and 11 (Romei et al., 1996; Ceccherini et al., 1997). These novel somatic mutations, found in two different patients, were heterozygous in frame deletions of 48 (cod 592 – 607) and 6 bp (cod 632 – 633) respectively, yet did not directly involve any cysteine residues. Other similar codon deletions have been observed in sporadic MTCs, namely a 3 bp deletion (cod 633) (Hofstra et al., 1996), and a 27 bp deletion comprising exon 10 (Kalinin and Frilling, 1998). Of interest, a duplication of 12 bp between codon Cys634 and Arg635, resulting in the insertion of four amino acids including a cysteine, has been described as a novel MEN 2A family germline mutation (Hoppner and Ritter, 1997). It has been demonstrated that missense mutations involving any of the cysteine residues 609, 611, 618, 620 and 634, typical of the MEN 2A and FMTC phenotypes yet rarely identified in sporadic MTCs, can promote ligand independent Ret dimerization leading to constitutive enzymatic activation. It is further evident that the substitution of one of these five crucial cysteines leads to both ligand independent dimerization and receptor phosphorylation. We have recently demonstrated in an in vitro system that the deletion of the codons 632 – 633 more effectively activated the RET gene with respect to the Cys634Arg missense mutation. This specifically correlated to the clinical severity of the corresponding tumor, characterized by an unusual aggressive progression with both multiple and recurrent metastases (Ceccherini et al., 1997).

In this paper, we addressed the problem of the molecular mechanism underlying this oncogenic activation. We examined the possibility that these deletions act in a similar way to Cys point mutations. Phosphorylated dimers of the deleted Ret receptor are detected when immunoprecipitates are separated under non-reducing conditions. Therefore, like Cys point mutations, this deletion affecting two Cys-proximal residues is capable of activating the transforming ability of Ret by promoting ligand-independent receptor dimerization.

Furthermore, we have studied the biogenesis of RetCys634Arg and Retdel1632 – 633 and found that the latter to be more slowly processed to the cell surface. A large fraction of Retdel632 – 633 intermediates and a significantly smaller fraction of RetCys634Arg, is retained within the endoplasmic reticulum where it dimerizes and transduces mitogenic signals.

Results

Effect of Δ6 deletion mutation on dimerization and autophosphorylation of Ret receptor

Unlabeled lysates from Retdel632 – 633 (RetΔ6), RetCys634Arg (Ret2A) and Ret wild-type (Retwt) expressing cell lines were immunoprecipitated with an anti-Ret antibody. Immunocomplexes were tested for kinase activity in the presence of [γ-32P]ATP. A significant amount of Ret autophosphorylation was seen in the cell lines expressing the RetΔ6 and Ret2A constructs but not in the wild-type (data not shown). This was demonstrated by the labeling of two proteins corresponding to the Ret isoforms of approximately 140 and 160 kDa. Therefore, even in the absence of ligands, both RetΔ6 and Ret2A can be phosphorylated.

To examine whether increased kinase activity correlated with increases in covalent dimer formation, we analysed immunoprecipitated RetΔ6, Ret2A and Retwt proteins by SDS – PAGE under both reducing and non-reducing conditions. As shown in Figure 1A, Ret immunoprecipitates separated under non-reducing conditions revealed the presence of Ret dimers in cell lines expressing mutant receptors. Stable dimer formation was not detected in a cell line expressing the wild-type Ret receptor. Furthermore, RetΔ6 and Ret2A dimers were shown to be strongly phosphorylated (Figure 1B) by anti-phosphotyrosine immunoblotting. Two high molecular bands corresponding to the dimerized form of the mature (160 kDa) and the partially glycosylated precursor (140 kDa) of the mutated receptor, appeared phosphorylated. Duplicate immunoprecipitates were subjected to immunoblot analysis for Ret after separation under reducing conditions to determine whether receptor dimerization required disulfide bonding (Figure 1C). Results revealed that under reducing conditions, only monomeric forms of Ret were detected. These results provide evidence that the Ret Glu632-Leu633 deletion promotes disulfide-linked dimer formation. Ret proteins were further analysed under non-reducing conditions by 125I-labeled protein-A binding (data not shown). Densitometric analysis of bands corresponding to dimers or monomers of the two mutant receptors revealed that RetΔ6 protein exhibits a greater ability to dimerize in comparison to Ret2A protein. The molar ratio of dimers to monomers for RetΔ6 and Ret2A was 0.95 and 0.70 respectively. In contrast, the Retwt control ratio was 0.05. Results from in vitro kinase assays, and from both reducing and non-reducing immunoblots demonstrated that the Δ6 deletion mutation is a gain-of-function mutation. This results in the constitutive activation of Ret, thus allowing for ligand-independent signal transduction.

Figure 1
figure1

Dimerization and tyrosine phosphorylation of Ret receptors. Lysates from NIH3T3 cells transfected with the RetΔ6 (lanes Δ6), Ret2A (lanes 2A) and Retwt (lanes wt) constructs were immunoprecipitated (IP) with anti-Ret antiserum (αRet) and the resulting complexes were separated by SDS – PAGE (6%) under non-reducing (A and B) or reducing conditions (C). Western blot filters were developed (BLOT) with anti-Ret (αRet, A and C) or anti-phosphotyrosine (αPtyr, B) antiserum. The positions of the Ret oligomers and monomers are indicated

Effect of 2-mercaptoethanol treatment on Ret mutants transforming activity

Reducing agents have previously been used to interfere with the signaling from activated forms of both the thrombopoietin and NEU receptor (Alexander et al., 1995; Siegel and Muller, 1996). We examined whether the addition of 2-mercaptoethanol (2-ME) could interfere with the ability of the RetΔ6 deletion mutant or the Ret2A point mutant to transform NIH3T3 cells. Addition of increasing concentrations of 2-ME (Table 1) failed to affect the transforming activity of the RetΔ6 construct. In contrast, Ret2A transforming activity decreased by 84% at the maximal dose (500 μM) of 2-ME. Toxic effects due to 2-ME at 500 μM were not evident as demonstrated by the unaffected ability of RetΔ6 and HRAS in transforming NIH3T3 cells. This data demonstrates that RetΔ6 has a greater resistance to a reducing agent in comparison with Ret2A. This specifically correlates to a stronger disulfide bond formation in the RetΔ6 mutant.

Table 1 2-mercaptoethanol effect on the transforming activity of Ret mutants

Biosynthesis and glycan processing of RetΔ6 and Ret2A proteins and effects of 2-mercaptoethanol treatment

It has previously been demonstrated that proteins exposing reactive thiols are often retained within the endoplasmic reticulum (ER) (Isidoro et al., 1996). This protein quality control system has been termed thiol-mediated retention. This mechanism is believed to monitor the oxidation status of one or more cysteines contained in protein sequences. Since RetΔ6 and Ret2A mutations affect critical cysteine residues, we studied both the biosynthesis and relative kinetics of dimerization by following the fate of metabolically labeled mutant receptors in NIH3T3 cells. Sub-confluent cultures were labeled with 0.35 mCi/ml 35S-methionine-cysteine for 15 min and subsequently chased for varying times. Under reducing conditions, newly synthesized protein for both mutant receptors appeared in the 140 kDa form immediately after the pulse (Figure 2). The amount of Ret2A p140 protein increased significantly within the first hour of chase and gradually decreased after 2 h. The mature p160 form appeared after 1 h of chase. In contrast, RetΔ6 p140 reached a maximum intensity only after 2 h of chase, the same chase time at which the mature p160 form became evident. Since the 140 kDa is the endoglycosidase H sensitive form (data not shown and (Asai et al., 1995)) whilst the 160 kDa is resistant, we deduced the longer persistence of the RetΔ6 140 kDa precursor. This indicated that RetΔ6 has a less efficient ER processing compared to the Ret2A precursor. The amount of the mature 160 kDa Ret2A protein was similar to that of the precursor after 2 h of chase, with a maximum reached after 4 h. In contrast, the amount of the 160 kDa RetΔ6 protein increased slowly, yet always remained less than the 140 kDa form. To explore the mechanism of dimerization, features of the labeled RetΔ6 and Ret2A proteins were also followed as a function of time by non-reducing SDS – PAGE (Figure 2). Both the precursor and mature forms of RetΔ6 and Ret2A were capable of dimerization, each with individual specific kinetics (Figure 2). The formation of RetΔ6 homodimers in both the p140 and p160 forms was observed from 2 h. In contrast, after 1 h of chase in the Ret2A mutant, a consistent amount of p140 homodimers and only a small fraction of p160 homodimers were observed. Furthermore, p160 homodimers were observed almost exclusively after longer chase times. Analysis of phosphorylation levels in all RetΔ6 and Ret2A proteins indicated that most of the precursors were significantly phosphorylated, irrespective of whether in the monomeric or dimeric form (Figures 3B and 1B and data not shown). This is due to the fact that phosphorylation associated to dimer forms is intermolecular. Therefore, SDS – PAGE under reducing conditions produces phosphorylated monomers.

Figure 2
figure2

Pulse-chase labeling of RetΔ6 and Ret2A proteins in reducing and non-reducing conditions. Autoradiograms of metabolically labeled radioactive RetΔ6 and Ret2A proteins derived from transfectants pulsed for 15 min and harvested immediately (lanes marked 0) or after the indicated chase time (lanes marked 1 – 6). Cell extracts were immunoprecipitated with anti-Ret antiserum and the complexes were separated by SDS – PAGE under either reducing or non-reducing conditions. Gels were exposed to X-ray film for 4 – 5 days. Numbers are molecular weights in kDa

Figure 3
figure3

Coimmunoprecipitation of Shc phosphorylation proteins with both the precursor and the mature forms of RetΔ6 and Ret2A receptors. Lysates from NIH3T3 cells (lane NIH) or from NIH3T3 cell lines expressing the RetΔ6 (lane Δ6) and Ret2A (lane 2A) proteins were immunoprecipitated (IP) with anti-Shc (αShc, A and B) or anti-Ret (αRet, C and D) antiserum. The resulting complexes were electrophoresed on a 7.5% SDS – PAGE under reducing conditions. Proteins were transferred to nitrocellulose and subjected to immunoblot analysis (BLOT) with anti-phosphotyrosine (αPtyr) or antiRet (αRet) antiserum. RetΔ6 and Ret2A expressing cell lines were serum starved for 48 h. Numbers are molecular weights in kDa and correspond to two of the three different isoforms of Shc (p52 and p46) and to Ret (p160 and p140), respectively

Co-immunoprecipitation of SHC phosphorylated proteins with both the precursor and mature forms of the RetΔ6 and Ret2A receptors

RetΔ6 proteins are characterized by slower maturation kinetics yet display stronger oncogenic activity in comparison to Ret2A proteins. With the aim to explain this fact, we speculated that both the p140 precursor and p160 mature forms of Ret, when phosphorylated and dimerized, are able to transduce mitogenic activity in spite of their different localization; the endoplasmic reticulum or surface membrane respectively. To address this, we investigated whether the precursor forms of RetΔ6 and Ret2A were able to bind the Shc adapter protein. This protein is localized to the membrane of the rough endoplasmic reticulum and has been previously demonstrated to bind Ret/PTCs, Ret2A and Ret2B (a mutant Ret receptor carrying an M918T mutation) oncoproteins (Lotti et al., 1996). Cell lysates from RetΔ6 and Ret2A transfected NIH3T3 cells were immunoprecipitated with anti-Shc or anti-Ret antibodies and subsequently blotted with anti-Ptyr antibodies (Figure 3). Both the precursor and mature forms of Ret were co-immunoprecipitated with Shc phosphorylated proteins. The band density of both RetΔ6 and Ret2A co-immunoprecipitated or not with Shc protein and probed with anti-Ptyr were checked by densitometric analysis. The observed affinity of Shc for the precursor and mature forms of both the mutated receptors is similar. Considering the sum of the relative amounts, it was calculated that 40% of RetΔ6 and 20% of Ret2A were co-immunoprecipitated with Shc phosphorylated proteins. It is thus possible to conclude from this data that the mitogenic signal transduced by Shc does not depend on maturation or localization of the mutated receptor, but possibly influenced by the specific type of activating mutation.

Discussion

We have demonstrated that deletion of the residues Glu632-Leu633 of Ret induces stable receptor dimer formation in the absence of ligand. This suggests that constitutive dimerization and autophosphorylation of the receptor protein underlies the potent transforming activity of this Ret mutant. MEN 2A mutations destroy Cys residues, thus indicating that their mechanism of action involves the creation of ligand-independent dimers most likely through aberrant intermolecular disulfide-bond formation. Whilst not directly involving Cys residues, the Δ6 mutation also induces such dimerization. This deletion mutation may indirectly affect neighboring Cys residues by disrupting the normal and most likely intramolecular cysteine pairing, which occurs in the non-activated receptor. This thus leaves the possibility that at least one unpaired cysteine residue may participate in an intermolecular binding event with another altered receptor, resulting in a disulfide-bonded receptor dimer leading to aberrant receptor activation. In fact, a similar mechanism has been proposed to account for the oncogenic activation in Neu deletion mutants. Deletion of conserved cysteine residues in the juxtatransmembrane region of Neu resulted in elevated transforming activities compared to that of the C647S point mutation form (Siege and Muller, 1996). One potential explanation for this observation is that the spatial arrangement between the remaining cysteine residues may play a crucial role in mediating receptor dimerization. For example, certain deletions may result in a free cysteine being exposed at the surface of the receptor, thus making it more accessible to intermolecular disulfide bond formation.

Interestingly, the transforming activity of Ret2A receptor is decreased following the addition of a reducing agent. In contrast, the transforming activity of the RetΔ6 deletion mutant is not significantly impaired at the same non toxic concentration of 2-ME. This may be explained by the formation of more stable RetΔ6 disulfide-linked dimers, thus generating a more efficient covalent dimerization. Alternatively, it is possible that the loss of residues near cysteine 634 results in an unfolding of the cysteine rich domain that may lead to a less compact and more flexible conformation. Such an unfolding could increase the probability of receptor interaction on the plasma membrane, leading to the tyrosine kinase activation of a greater number of mutant receptors compared to the Ret2A type receptor. Finally, the lower sensitivity of the Δ6 mutant to reducing agents could also be explained by its particular molecular equilibrium shift from monomeric to dimeric/oligomeric forms.

Taken together, these observations suggest that both the number and spatial arrangement of cysteine residues may induce a ligand independent dimerization.

More generally, it is possible that the loss, gain or mutation or residues within the cysteine-rich region of Ret has the capacity to induce stabilization of the dimeric conformation through intermolecular disulfide bonds.

Biosynthesis studies of RetΔ6 and Ret2A proteins have shown a slower rate of maturation for RetΔ6 protein due to precursor retention in the endoplasmic reticulum. However, both RetΔ6 and Ret2A show intracellular retention in comparison with the Retwt protein, which reaches the membrane within a 30 min chase time (data not shown). This is expected since, as a rule, only proteins that have attained a proper three-dimensional structure are secreted or expressed at the cell surface. Since RetΔ6 and Ret2A are mutated near or within Cys, their folding and assembly could potentially be delayed by intracellular retention for a longer time with respect to Retwt.

Accumulation of misfolded immature Ret2A and RetΔ6 proteins in the intracellular vesicular compartment and its homodimerization due to aberrant intermolecular disulfide bonds have important consequences on receptor functions in the downstream mitogenic program. In fact, a very large portion of RetΔ6 and Ret2A precursors strongly bind phosphorylated Shc proteins, thus indicating their ability to induce this mitogenic signal pathway. This finding may also aid in explaining why the RetΔ6 protein is more oncogenic, even when less efficiently processed than Ret2A mutant; an obvious paradox. The possibility that intracellular Ret precursors still trigger a mitogenic activity is extremely important also in the context of the effects of cysteine mutation in positions 609, 618 and 620. There is evidence suggesting that these cysteine mutations induce constitutive catalytic activity due to aberrant disulfide homodimerization of Ret, but in the same time are responsible for a decrease in the amount of mature RET protein expressed at the cell surface (Chappuis-Flament et al., 1998). Since 20 – 30% of families with mutations C618R or C620R (Mulligan et al., 1994) present a combination of HSRC and MEN 2A, it has been hypothesized that these cysteine mutations exert a dual impact on RET. In particular, these mutations can be activating or deactivating depending on the tissue in which RET is expressed. Thus, RET carrying either the C618R or C620R mutation, due to constitutive dimerization, exhibits a transforming potential responsible for MEN2A and in the same time, due to scarce localization at the surface membrane, an inability to promote a survival of enteric neurons. Our data well supports this model, indicating that not exclusively the membrane can trigger mitogenic signals. This reinforces the concept that an efficient membrane localization of Ret receptors is very important for triggering of neuronal differentiation signals.

Materials and methods

Cell culture and transfection

Mouse NIH3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum. Transfection experiments were performed in NIH3T3 cells by calcium phosphate precipitation as previously described (Bongarzone et al., 1993). Plasmids carrying Retwt, RetΔ6 and Ret2A inserts have been described in detail previously (Ceccherini et al., 1997). The HRAS oncogene was mutated at its 12th codon by a G→T transversion. Transformation foci were selected in DMEM plus 5% calf serum. The reducing agent 2-ME was added when cells had formed a monolayer. This monolayer was maintained for 14 days in DMEM supplemented with 5% bovine calf serum.

Immunoprecipitation and Western blot analysis

Protein samples were prepared as previously described (Borrello et al., 1996) and immunoprecipitated with the specified antisera: affinity purified anti-Ret polyclonal antiserum (Borrello et al., 1996) and rabbit polyclonal anti-Shc antiserum (Upstate Biotechnology Incorporated). Immunoprecipitates were resolved by electrophoresis on 7.5 or 8.5% SDS polyacrylamide gels (PAGE). Proteins were transferred to nitrocellulose filters, blocked with 5% bovine serum albumin (BSA) or 0.5% gelatin in Tris-buffered saline (TBS) pH 7.6 and immunoblotted with the same anti-Ret antiserum described above or with the monoclonal antiserum anti-phosphotyrosine (anti-Ptyr, Upstate Biotechnology Incorporated). Immunoreactive bands were visualized using horseradish peroxidase conjugated anti-rabbit or anti-mouse antisera and ECL detection reagents (Amersham) or using 125I-labeled protein A (Amersham) followed by autoradiography. When 125I-labeled protein A was used, filters were exposed to storage phosphor screen films (Molecular Dynamics) in order to quantify the counts per minute (c.p.m.) associated with the Ret-specific bands reacting with anti-Ret or anti-Shc with a Phosphorimager. For Western blotting under non-reducing conditions, 2-mercaptoethanol was excluded.

Metabolic labeling and immunoprecipitation

Prior to labeling, subconfluent cells were incubated with methionine-free DMEM for 1 h. Metabolically labeling was performed with 370 μCi/mL 35S-Methionine-Cysteine (DuPont/NEN) for 15 min. The labeling medium was then removed and replaced with completed DMEM medium for the duration of the chase period. Further processing, extraction, and immunoprecipitation of labeled Ret was carried out as above with the following modifications: after incubation, bound immune complexes (Protein A/G-Plus-Agarose beads, Santa Cruz Biotechnology) were washed extensively (seven times) with lysis buffer (0.15 mM NaCl/0.05 mM Tris-HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/0.1% SDS), either supplemented with or without 1% Triton and 1 M NaCl and on a shaking platform, eluted, and subjected to SDS – PAGE fractionation and autoradiography. Densities of radioactive bands on X-ray films were estimated using a Phosphorimager.

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Acknowledgements

This work was partially supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Italiana per la Ricerca sul Cancro (FIRC), CNR (Biotecnologie) No. 97.01258.PF49, Project BIOMED2 No. BMH4-CT97-2157 and Istituto Superiore di Sanita’. The authors thank Dr. R Sitia for critical comments and suggestions on the manuscript. We also thank Cristina Mazzadi for secretarial help.

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Correspondence to Italia Bongarzone.

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Bongarzone, I., Vigano, E., Alberti, L. et al. The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein. Oncogene 18, 4833–4838 (1999). https://doi.org/10.1038/sj.onc.1202848

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Keywords

  • RET
  • oncogenic activation
  • dimerization

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