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Endocrinology Vol. 139, No. 8 3613-3619
Copyright © 1998 by The Endocrine Society

ARTICLES

Glial Cell Line-Derived Neurotrophic Factor Differentially Stimulates Ret Mutants Associated with the Multiple Endocrine Neoplasia Type 2 Syndromes and Hirschsprung’s Disease1

Francesca Carlomagno2, Rosa Marina Melillo2, Roberta Visconti, Giuliana Salvatore, Gabriella De Vita, Gelsy Lupoli, Yanbin Yu, Shuqian Jing, Giancarlo Vecchio3, Alfredo Fusco and Massimo Santoro

Centro di Endocrinologia ed Oncologia Sperimentale del CNR (F.C., R.M.M., R.V., G.S., G.D.V., G.L., G.V., M.S.), c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltá di Medicina e Chirurgia, Universitá di Napoli "Federico II", 80131 Naples, Italy; Amgen (Y.Y., S.J.), Thousand Oaks, California 91320-1789; and Dipartimento di Medicina Sperimentale e Clinica (A.F.), Facoltá di Medicina e Chirurgia di Catanzaro, Universitá di Reggio Calabria, 88100 Catanzaro, Italy

Address all correspondence and requests for reprints to: Massimo Santoro, Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale Delle Ricerche, via S. Pansini 5, 80131 Napoli, Italy. E-mail: masantor@unina.it or afusco{at}synapsis.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ret gene encodes a tyrosine-kinase receptor, Ret (1), which has transforming potential. Somatic rearrangements of ret cause its oncogenic activation in papillary thyroid carcinomas (ret/PTC oncogenes) (2). Mutations of ret are associated with human neurochristopathies (diseases affecting tissues derived from the neural crest), including multiple endocrine neoplasia (MEN) type 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung’s disease (HSCR). Substitutions of extracellular cysteines of ret cause most MEN2A and FMTC cases (3, 4), and a specific mutation of an intracellular methionine (M918) cause most MEN2B cases (5, 6). Tumors characterizing MEN2B are more severe and have an earlier onset age, with respect to the other diseases; moreover, the M918T (MEN2B) mutation is frequent also in sporadic medullary thyroid carcinomas (5, 6, 7). The residue more frequently mutated in MEN2A is C634; other cysteines, frequently C620, are mutated in most FMTC cases (8). Whereas the substitution of extracellular cysteines leads to a constitutive dimerization of the receptor in MEN2A/FMTC (9–12; and Chappuis-Flament, S., A. Pasini, G. De Vita, A. Fusco, S. Lyonnet, G. Lenoir, M. Santoro, and M. Billaud, manuscript submitted), the M918T mutation, associated with MEN2B, causes activation of the kinase function of Ret and a change of its substrate specificity (9, 14). Finally, HSCR is the consequence of the absence of autonomic ganglion cells within intestinal parasympathetic plexuses, which results in functional obstruction and megacolon. Heterogeneous ret mutations characterize HSCR (15, 16). When HSCR mutations were cloned in active ret isoforms (ret/PTC and ret/MEN2 mutants), they caused a loss of function of ret (17, 18).

It recently has been reported that the glial cell line-derived neurotrophic factor (GDNF) acts as a functional ligand for Ret. GDNF induces Ret tyrosine phosphorylation, and survival and proliferation of Ret-expressing cells (19, 20, 21, 22). Moreover, mice with targeted disruption of the GDNF gene show a phenotype similar to that of ret knock-out mice, including megacolon (23, 24). The mechanism by which GDNF stimulates Ret has been elucidated: GDNF interacts with GDNFR-{alpha}, a glycosyl phosphatidylinositol (GPI)-linked cell surface receptor, which, in turn, mediates Ret activation (19, 20). More recently, another GPI-linked protein, NTNR-{alpha}, has been cloned, which mediates Ret stimulation by a GDNF-related neurotrophin named neurturin or NTN (25, 26).

So far, the effects of MEN2 and of HSCR mutations on ret function have been tested only under ligand-free conditions. However, in vivo, the presence of the ligand probably affects the activity of ret mutants in tissues targeted by these diseases. We have studied the responsiveness of HSCR-associated and of oncogenic Ret mutants to ligand stimulation. Because Ret and GDNFR-{alpha} coexpression is required for high affinity binding of GDNF (and, consequently, for full biological activity of the receptor), we cotransfected NIH 3T3 cells with GDNFR-{alpha} and with constructs encoding wild-type Ret, Ret/Cys634 (more frequently associated with MEN2A), Ret/Cys620 (more frequently associated with FMTC), Ret/Met918 (MEN2B), and Ret/HSCR972 and Ret/HSCR32 (HSCR). GDNF stimulated tyrosine phosphorylation of the wild-type receptor, and this was followed by a mitogenic response. Two phenotypes were identified among the Ret mutants analyzed: Ret/Cys634, Ret/Cys620, Ret/HSCR972 and Ret/HSCR32 were unresponsive to GDNF, whereas Ret/Met918 was responsive to GDNF triggering.

These data demonstrate that HSCR mutants are unresponsive to the physiological Ret ligand. Moreover, our data on the activated Ret mutants show that GDNF influences the biological behavior of the Ret/Met918 mutant but not of the MEN2A- and FMTC-associated Ret mutants and (consistent with the clinical aggressiveness of the MEN2B syndrome) indicate that, when triggered by the ligand, Ret/Met918 is a very potent oncoprotein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Molecular constructs
The molecular constructs used in this study are described elsewhere: the Long Terminal Repeat-based ret expression vector (9), vectors expressing ret/Cys634 (C634Y), ret/Met918 (M918T) (9), ret/Cys620 (C620Y) (11), ret/HSCR972 (R972G) and ret/HSCR32 (S32L) (18), and the expression vector for GDNFR-{alpha} (pSJA45-GDNFR-{alpha}), which carries G418-resistance (19).

Cells and transfection experiments
NIH 3T3 cells were grown in DMEM supplemented with 10% calf serum (Gibco BRL, Life Technologies, Gaithersburg, MD). Transfections were performed by calcium phosphate precipitation, as described elsewhere (27). To obtain coexpression of GDNFR-{alpha} (G418-R) together with ret constructs (mycophenolic acid-R), cells were cotransfected with 1 µg GDNFR-{alpha} and 10 µg ret constructs and were selected for plasmid expression by growth in 400 µg/ml G418 (Gibco BRL). Soft agar colony assay was performed as reported (27); colonies were scored at 10 days. The expression of Ret and GDNFR-{alpha} in each cell line was verified by Western blotting and RT-PCR, respectively. Briefly, total RNA was isolated from each cell line by the acid guanidinium thiocyanate phenol method (28) and then subjected to ribonuclease-free deoxyribonuclease digestion (Promega Corp., Madison, WI). RNAs (1 µg) were reverse transcribed with the Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer Cetus, Branchburg, NJ), and the complementary DNA (cDNA) products were PCR-amplified using standard conditions (Perkin-Elmer Cetus). The primers used for GDNFR-{alpha} were the following: forward, 5'-CGGTTAACAGCAGGTTGTCAGA-3'; and reverse, 5'-GTGTGGGGATCTCATTCTCAGAC-3'.

The PCR conditions included an initial denaturation step at 94 C for 4 min, followed by 20 cycles at 94 C for 1 min, 58 C for 2 min, 72 C for 2 min, and a final extension step at 72 C for 5 min. Amplified products (1/10 of the reaction mixture) were analyzed by electrophoresis on 1% agarose gel and hybridized with a GDNFR-{alpha} probe excised from the pSJA45-GDNFR-{alpha} plasmid. The expected size of the reaction product was 801 bp. RT-PCR reactions, performed without previous reverse transcription, gave negative results, demonstrating that the amplification was not caused by contaminating DNA. The same RNAs were subjected to RT-PCR amplification using primers specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for messenger RNA (mRNA) control. The amplification product for GAPDH cDNA is 174 bp. The primer set flanks intron 3 of the gene, so that cDNA and the eventual genomic DNA amplification products can be distinguished by size (29). [3H]thymidine incorporation assays were performed as described previously (30). NIH 3T3 transfectants, grown to confluence in 24-well plates (Corning Costar Corp., Acton, MA), were serum-starved for 24 h and then treated with GDNF (Alomone Laboratories, Jerusalem, Israel) for 24 h in the presence of 4 µCi [3H]thymidine/ml. Data are expressed as mitogenic index, calculated as the fraction of stimulation obtained in the presence of GDNF, with respect to the stimulation obtained with 1% calf serum. The t test was used for the statistical ANOVA.

Protein studies
GDNF was purchased from Alomone Laboratories. Immunoprecipitation and immunoblotting experiments were performed as described elsewhere (30). Briefly, cells were lysed in a buffer containing 50 mM HEPES (pH 7.5), 1% (vol/vol) Triton X-100, 50 mM NaCl, 5 mM EGTA, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride, and 0.2 µg/ml each of aprotinin and leupeptin. Lysates were clarified by centrifugation at 10,000 x g for 15 min, and the supernatant was processed for immunoblotting or for immunoprecipitation. Protein concentration was estimated with the Bio-Rad kit (Bio Rad Laboratories, Hercules, CA). To analyze tyrosine-phosphorylation of Ret products, equal amounts of proteins were immunoprecipitated and assayed for phosphotyrosine (pTyr) content. Anti-Ret is a polyclonal antibody directed against the tyrosine-kinase domain of Ret (30), and the anti-pTyr is a monoclonal antibody (4G10) purchased from Upstate Biotechnology (Lake Placid, NY). To detect the pTyr content of the Shc proteins, cells were serum-starved overnight, stimulated or not with GDNF, and lysed as described above. Equal amounts of total proteins were immunoprecipitated with a polyclonal anti-Shc antibody (Upstate Biotechnology). The immunocomplexes were divided in two aliquots and probed with either the anti-pTyr antibody or with the anti-Shc antibody. Immunoblots were stained with appropriate secondary antibodies and revealed with the Amersham ECL system (Amersham Life Sciences, Buckinghamshire, UK). Treatment of the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) (Boheringer, Mannheim, Germany) was performed as previously reported (19). Briefly, cells were incubated with 1 U/ml PI-PLC (Boheringer, Mannheim) at 37 C for 45 min, washed three times with serum-free medium, and processed for Ret phosphorylation experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We evaluated the response to GDNF of wild-type Ret and of various Ret mutants associated with HSCR (ret/HSCR972 and ret/HSCR32), or with MEN2 syndromes [ret/Cys634 (MEN2A), ret/Met918 (MEN2B), and ret/Cys620 (FMTC)]. Figure 1Go is a schematic representation of the mutants assayed. To obtain a full response to GDNF, we engineered NIH 3T3 cells coexpressing the different ret constructs and GDNFR-{alpha}. Marker (G418)-selected mass populations were obtained. All the generated cell lines expressed comparable levels of Ret proteins (see below). Furthermore, all the cell lines expressed similar levels of GDNFR-{alpha}, as demonstrated by a semiquantitative RT-PCR assay (Fig. 2Go). First, we evaluated the tyrosine phosphorylation of the different Ret mutants after 5 min triggering with 50 ng/ml GDNF (Fig. 3Go). As reported previously (9, 11), Ret/Cys634, Ret/Cys620, and Ret/Met918 (but not wild type Ret) proteins showed a basal level of tyrosine phosphorylation. As expected, GDNF induced prompt phosphorylation of wild-type Ret products. Among the active Ret mutants, only Ret/Met918 was further stimulated by GDNF triggering. GDNFR-{alpha} is associated with the plasma membrane through a GPI tail (19). It has been reported that treatment of cells expressing GDNFR-{alpha} with PI-PLC, which is able to digest the GPI anchor, dramatically affects Ret response to GDNF (19). We have used this assay to demonstrate that the observed effects were specific. Figure 3Go shows that pretreatment of the cells with PI-PLC greatly diminished GDNF-induced tyrosine phosphorylation of both-wild type Ret and Ret/Met918. As expected, HSCR-associated Ret mutants, Ret/HSCR972 and Ret/HSCR32, were devoid of basal kinase activity. In addition, the responsiveness to GDNF of HSCR-associated Ret mutants was much lower, with respect to wild-type Ret proteins (Fig. 3Go).



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  		Figure 1. Schematic representation of the Ret mutants analyzed in this study. This figure depicts the Ret receptor kinase structure with the signal peptide (SP), the cadherin-like (CAD), cysteine-rich (CYS), transmembrane (TM), and the tyrosine kinase domains (TK). The Ret mutations that are associated with various human diseases and are analyzed in this study are indicated.

 


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  		Figure 2. RT-PCR identification of GDNFR-{alpha} expression RNAs derived from NIH 3T3 cells transfected with GDNFR-{alpha} and the various Ret mutants have been subjected to RT-PCR for the identification of GDNFR-{alpha} expression. Amplified products (1/10 of the reaction mixture) were analyzed by electrophoresis on 1% agarose gel and hybridized with a GDNFR-{alpha} probe excised from the pSJA45-GDNFR-{alpha} plasmid. The expected size of the reaction product was 801 bp. All the samples were negative when the PCR amplification was performed without previous reverse transcription, to verify that the results were caused by amplification of RNA and not of contaminating DNA (not shown). The same RNAs were subjected to RT-PCR amplification using primers specific for the GAPDH gene for mRNA control. The amplification products were run on a 2% agarose gel and were stained with ethidium bromide.

 


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  		Figure 3. Tyrosine phosphorylation of Ret induced by GDNF NIH 3T3 cells transfected with a plasmid encoding GDNFR-{alpha} and ret, ret/Cys634, ret/Met918, ret/Cys620, ret/HSCR972, or ret/HSCR32, were treated for 5 min at 37 C with 50 ng/ml GDNF. Two milligrams of protein lysates were immunoprecipitated with a polyclonal antibody to Ret; half of the immunoprecipitate was immunoblotted with anti-Ret and half with anti-pTyr monoclonal antibodies. Where indicated, cells were pretreated with PI-PLC before incubating with GDNF. Ret proteins were detected as 145-kDa and 160-kDa bands. The 160-kDa species represents the mature glycosylated protein present on the cell surface, whereas the 145-kDa form is an immature precursor. As previously reported, reduced accumulation of the mature 160-kDa species was observed in the Cys620 mutant and, even more dramatically, with the HSCR32 mutation (11 18 ). The results are typical and are representative of at least four independent experiments.

 
To verify these results, we evaluated Ret phosphorylation in cell lines expressing only the Ret mutants when stimulated with GDNF plus a soluble form of GDNFR-{alpha} (Jing et al., unpublished). Also in this experiment, Ret/Met918, differently from the other Ret mutants, responded to the stimulation (data not shown). In these conditions, the addition of soluble GDNFR-{alpha} was essential for efficient Ret stimulation; indeed, a very weak response was obtained when cells were stimulated with GDNF in the absence of soluble GDNFR-{alpha} (data not shown).

Ret activation results in proliferation of NIH 3T3 cells (2, 30). Thus, we used a thymidine incorporation assay to determine whether GDNF-induced Ret triggering leads to mitogenesis. GDNF dose-dependently induced DNA synthesis in NIH 3T3 cells coexpressing Ret and GDNFR-{alpha} (Fig. 4Go) but not in untransfected cells (data not shown). GDNF had a potent mitogenic effect on Ret/Met918-expressing cells but not on Ret/Cys620- and Ret/Cys634-expressing cells (Fig. 4Go). Consistent with the protein phosphorylation data, cells expressing Ret/HSCR32 (Fig. 4Go) and Ret/HSCR972 (data not shown) did not respond to GDNF. To evaluate further these mitogenic effects, we compared the colony-forming efficiency in soft agar of Ret/Met918-, Ret/Cys634-, and Ret/HSCR32-expressing cells in the presence and absence of GDNF. As reported previously (9), cells expressing Ret/Met918 and Ret/Cys634 showed a high clonogenic efficiency in soft agar. Ten days after plating, the GDNF-stimulated Ret/Met918 colonies had increased in number (80% vs. 45% of the plated cells) and in average size (Fig. 5Go). In contrast, GDNF failed to affect the colony-forming efficiency of Ret/Cys634-cells, in terms of number (80% of plated cells) and size (Fig. 5Go). Ret/HSCR32-expressing cells did not form colonies in soft agar, either in the presence or in the absence of GDNF (data not shown).



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  		Figure 4. Mitogenic effect of GDNF on NIH 3T3 cells expressing wild-type and mutant Ret receptors. [3H]thymidine incorporation assay was performed on NIH 3T3 cells grown in DMEM supplemented with 10% calf serum. Briefly, NIH 3T3 transfectants, grown to confluence in 24-well plates (Costar), were serum-starved for 24 h and then treated with increased amounts of GDNF for 24 h in the presence of 4 µCi/ml [3H]thymidine. Each experimental point is the result of triplicate assays, and the results represent the average of two independent experiments. Data are expressed as mitogenic index, calculated as the average fraction of stimulation obtained in the presence of GDNF, with respect to the stimulation obtained with 1% calf serum [(GDNF cpm - background cpm)/(1% calf serum cpm - background cpm)] x 100. Error bars are indicated: variations between each experimental point were less than 20% of the average results. ANOVA indicated that both growth rate differences between Ret and Ret/Met918 cells (P < 0.01) and between Ret/Met918 and Ret/Cys634 (P < 0.005) were significant.

 


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  		Figure 5. Soft agar growth assay cells (2 x 104) were plated in soft agar in 60-mm culture dishes in the presence (+) or the absence (-) of 50 ng/ml GDNF, and the colony formation was scored at 10 days. A, Microphotographs of the colonies grown in soft-agar are reported (magnification x150); B, bar-charts, reporting the colony-forming efficiency (10 days after plating), calculated with the formula: [number of colonies (larger than 64 cells) formed/number of plated cells] x100. These results are typical and representative of at least three independent experiments.

 
Little is known about the signal transduction pathways responsible for the mitogenic activity of Ret. It has been demonstrated in constitutively active ret mutants that the Shc adaptor is a substrate for the Ret kinase (31, 32, 33, 34, 35). Moreover, coupling to Shc is an important step in Ret mitogenic signaling, because mutation of tyrosine 1062 (the Shc binding site) abrogates the transforming activity of the Ret/Cys634 and Ret/Met918 mutants (36). Thus, we stimulated wild-type and mutant Ret-expressing cells to see whether GDNF was able to induce Shc phosphorylation. Ret-expressing cells were serum-starved and stimulated with 50 ng/ml GDNF. Cells were lysed, and tyrosine phosphorylation of Shc was evaluated by immunoprecipitation with anti-Shc antibodies, followed by staining of the immunoprecipitates with anti-pTyr antibodies. As shown in Fig. 6Go, upon addition of GDNF to the cultures, Shc phosphorylation promptly increased in the NIH 3T3-Ret cells. As expected, basal Shc phosphorylation was higher in Ret/Met918-expressing cells than in the wild-type Ret cells; however, this basal activation was further increased by GDNF triggering. Also, Ret/Cys634 cells had a basal level of Shc phosphorylation, but GDNF treatment did not increase Shc phosphorylation (Fig. 6Go). Ret/Cys620- and Ret/HSCR-expressing cells were totally unresponsive to GDNF (data not shown).



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  		Figure 6. Tyrosine phosphorylation of Shc induced by GDNF stimulation of different Ret mutants. NIH 3T3 cells, coexpressing GDNFR-{alpha} and Ret, Ret/Cys634, or Ret/Met918, were stimulated for 5 min with 50 ng/ml GDNF. One milligram of total lysate was immunoprecipitated with anti-Shc antibodies. Half of the immunoprecipitate was immunoblotted with anti-pTyr (upper panel) and the other half with anti-Shc (lower panel). The results are typical and representative of at least three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we used NIH 3T3 cells expressing different Ret mutants and GDNFR-{alpha} to evaluate the responsiveness of these mutants to GDNF. Our data demonstrate that the kinase and the biological activities of Ret and Ret/MEN2B, although partly constitutive, are triggered by GDNF. Indeed, GDNF treatment induced an increase of Shc phosphorylation and DNA duplication in both Ret and Ret/MEN2B cells. GDNF even induced the growth of Ret/MEN2B cells in soft agar. In contrast, Ret/Cys634 and Ret/Cys620 failed to respond to GDNF.

It is well established that mutations detected in MEN2A, MEN2B, and FMTC convert ret into a dominant oncogene by activating its intrinsic kinase activity and transforming potential. MEN2A is characterized by the occurrence of medullary thyroid carcinoma, phaeochromocytoma, and hyperparathyroidism; MEN2B is an early onset and very aggressive disease associated with medullary thyroid carcinoma, phaeochromocytoma, ganglioneuromas of the intestinal tract, mucosal neuromas, and skeletal abnormalities. Finally, medullary thyroid carcinoma is the only FMTC disease phenotype (for reviews, see Refs. 37, 38). The different clinical manifestations of these syndromes have been attributed to the different molecular mechanisms underlying ret activation in the three conditions. In MEN2A, Ret activation is triggered by a disulfide-bond-mediated dimerization of the receptor that results in constitutive activation of the kinase (9, 10). A similar mechanism (i.e. dimerization) has been proposed for cases of FMTC caused by cysteine mutations. However, there is a strong correlation between specific Ret mutations and MEN2A and FMTC phenotypes. In fact, mutation of codon 634 is responsible for more than 80% of cases of MEN2A, and mutations of cysteines other than codon 634 are more frequently associated with FMTC (8). This correlation may be related to the observation that the FMTC-associated mutations are less efficient than the MEN2A-associated mutation in inducing Ret activation; a scarse supply of Ret to the plasma membrane is associated with a lower level of dimerization and low Ret activation (11, 12, 13). Also the MEN2B mutation, which affects the substrate recognition pocket of the receptor, activates the Ret kinase, not by inducing the formation of Ret dimers (9), but by altering the catalytic specificity of the receptor (9, 14). It is likely that the different responsiveness to ligand triggering observed here, between MEN2B and FMTC/MEN2A Ret products, largely depends on the different mechanisms by which these mutations activate Ret. At the present state of knowledge, we may only speculate about why GDNF is not able to trigger activation of MEN2A and FMTC mutants. Trupp and co-workers (21) demonstrated that the Cys634 (MEN2A) mutation does not alter GDNF-GDNFR-{alpha} binding to Ret; and thus, the possibility of impaired binding can be excluded, at least for this mutant. Because GDNF-GDNFR-{alpha} binding has been reported to activate Ret by inducing its dimerization (19), a more plausible explanation is that the MEN2A and FMTC mutants, which are constitutively dimerized, are already maximally activated and cannot be further stimulated by GDNF. Alternatively, mutations of Cys634 and Cys620 could alter the folding of the extracellular domain of Ret, thus impairing the structural changes required to transmit the GDNF-GDNFR-{alpha} signal to the intracytoplasmic domain of Ret.

Whatever the mechanism of action, our data suggest that the tissue availability of the ligand may influence the expression of the biological effects of the MEN2B mutation, as well as the aggressiveness of the associated disease phenotype. When stimulated by GDNF, Ret/Met918 was a very potent oncoprotein. Thus, it is conceivable that the presence of the ligand may affect some of the disease phenotypes that characterize the MEN2B syndrome, such as the presence of gastro-intestinal neuromas, the skeletal abnormalities, and the lack of parathyroid involvement. Studies are in progress in our laboratory to ascertain whether the observations reported here are applicable to the NTN, the other functional ligand of Ret (25, 26). Other FMTC-associated mutations affect the Ret kinase domain, and not cysteine residues, thus leading to an activation of its transforming activity (39). Moreover, a novel intracytoplasmic mutation (codon 883) has been detected in some MEN2B families (40). We are currently investigating whether the ligand affects the activity of these mutants.

Two mechanisms of oncogenic activation of receptor tyrosine kinases have been proposed. In some cases, e.g. ret/PTC oncogenes, the extracellular encoding domain of the receptor is replaced by 5'-encoding sequences of heterologous genes (2). Similar to mutants of trk or met protooncogenes (for a review, see Ref. 41), these chimeric receptors cannot be modulated by their ligand. In other instances, single amino acid substitutions can activate receptor tyrosine kinases. Examples are ret mutations in MEN2 syndromes, and also met oncogene point mutations that have recently been described in both familial and sporadic tumors. In particular, the substitution of the methionine residue, corresponding to the ret/MEN2B residue (Met918), has been found in the met gene in papillary renal carcinomas (42). All these mutant receptor tyrosine kinases retain their extracellular encoding domain and, therefore, could still be able to interact with their ligand. Thus, ret/MEN2B could be a paradigmatic example of how the biological activity of these mutant receptors (and the associated disease phenotypes) might be influenced by the ligand.

It is well known that mutations of the Ret receptor responsible for HSCR are dispersed throughout the gene (15, 16). Cases of HSCR can be caused by large deletions of the gene, small intragenic deletions or insertions, splicing alterations, or nonsense mutations, which obviously cause a loss of function of Ret. Other cases are caused by more subtle missense mutations (for a review, see Ref. 43). In this study, we have analyzed two HSCR mutants belonging to the last category. Ret/HSCR32 and Ret/HSCR972 mutants showed a very low level of tyrosine phosphorylation and a scarse biological response to GDNF triggering. These results confirm, in a physiological setting (i.e., conditions under which the receptor is stimulated by its specific ligand) that HSCR mutations impair ret function by affecting its responsiveness to the ligand. Whereas the quiescence of the Ret/HSCR972 mutant is probably caused by impaired kinase function (17, 18), the lack of response to GDNF of the HSCR32 mutant is probably caused by its incorrect exposure on the outer cell surface (18). The HSCR972 mutant retained a low response to GDNF, rather than a complete knock-out, suggesting that even a partial loss of Ret function may lead to HSCR.

In conclusion, these findings indicate that the GDNF-Ret interaction plays a role in the establishment of a MEN2B or HSCR phenotype and suggest that GDNF and GDNFR-{alpha} are possible modifier genes in the expression of these syndromes. Moreover, the responsiveness of some of the Ret mutants to GDNF raises the possibility of devising therapeutic approaches that could intervene in this pathway.


   Acknowledgments
 
We thank Marc Billaud for critical reading of the manuscript, Anna Maria Cirafici for her contribution to this work, and Fabrizio Santoro for his help in the statistical analysis. We are indebted to Jean Gilder for editing the text and to Francesco Ciliberti for the artwork. The authors dedicate this manuscript to the memory of the late Prof. Gaetano Salvatore, who continuously and enthusiastically supported this work.


   Footnotes
 
1 This study was supported by the Associazione Italiana per la Ricerca sul Cancro and by European Community Grant BMH4-CT96–0814. Back

2 These authors contributed equally to the work. Back

3 Recipient of a Fogarty Scholar fellowship of the National Institutes of Health, Bethesda, Maryland. Back

Received December 12, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Takahashi M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H, Hiai H 1988 Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene 3:571–578[Medline]
  2. Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi R, Bongarzone I, Pierotti MA, Della Porta G, Fusco A, Vecchio G 1990 PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell 60:557–563[CrossRef][Medline]
  3. Mulligan LM, Kwok JBJ, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA, Telenius H, Tunnacliffe A, Ponder BAJ 1993 Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 363:458–460[CrossRef][Medline]
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