ICANCER RESEARCH 57, 391-395, Februaiy 1, 1997J Advances in Brief

The Different RET-activating Capability of Mutations of Cysteine 620 or Cysteine 634 Correlates with the Multiple Endocrine Neoplasia Type 2 DiseasePhenotype1

Francesca Carlomagno, Giuliana Salvatore, Anna Maria Cirafici, Gabriella De Vita, Rosa Marina Melillo, Vittorio de Franciscis, Marc Billaud, Aifredo Fusco, and Massimo Santoro2

Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, do Dipartimento di Biologia e Patologia Cellulare e Molecolare. Facoltâ di Medicina e Chirurgia, Università di Napoli Federico II, via S. Pansini 5. 80131 Naples, Italy (F. C.. G. S.. A. M. C.. G. D. V.. R. M. M., V. d. F.. M. S.): Laboratoire de Genétique,CentreNational de Ia Recherche Scientifique UMR564I, Domaine Rockefeller, UniversitéClaudeBernard Lyon I, 8 avenue Rockefeller. 69372 Lyon Cedex 08, France fM. B.J; and Dipartimento di Medicina Sperimentale e Clinica. Facoltà di Medicina e Chirurgia di Catanzaro, Università di Reggio Calabria, via T. Campanella 5. 88100 Casanzaro, Italy (A. Fl

Abstract (MEN2A), MEN2B, and FMTC syndromes. Although there is a certain degree of overlap, each disease has a distinct phenotype. Distinct point mutations of RET, a tyrosine-kinase receptor encoding MEN2B is characterized by MTCs, pheochromocytomas, skeletal gene, are responsible for the inheritance of multiple endocrine neoplasia abnormalities, and ganglioneuromas of the intestinal tract. MEN2A is type 2 syndromes (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC). In particular, MEN2A is a more complex and ag characterized by MTCs, pheochromocytomas, and parathyroid alter gressive disease than FMTC, being characterized by pheochromocytomas ations. Finally, FMTC is a closely related but less severe disorder that and parathyroid alterations, in addition to medullary thyroid carcinomas. shows as its only feature an inherited predisposition to MTC (9). The The mutations associated with MEN2A and FMTC affect one of five diversity of patterns of involved tissues corresponds to differences in cysteineresidues mapping in the extracellular domain of the Ret protein. the nature and position of the underlying RET mutation. The mutation However, recent studies have indicated that MEN2A and FMTC disease responsible for MEN2B is a Met-918--Thr substitution in RET tyro phenotypes correlate with the position of mutations in RET. Mutations of sine kinase domain (10, 11). On the other side, MEN2A and FMTC Cys-634 are more frequent in families with MEN2A, whereas Cys-620 mutations consist of the substitution of any of several amino acids for mutations are very rarely found in MEN2Apatients and, in contrast, are one of five cysteines (residues 609, 61 1, 618, 620, and 634) mapping frequently found in FMTC patients. We have reported previously that in the cysteine-rich region of the RET extracellular domain (12, 13). mutations of Cys-634 constitutively activate the RET transforming poten A strong correlation between disease phenotypes and the position of tial by causing a disulfide bridge-mediated homodimerization. Here, we report that the mutation Cys-620---+Tyr is able to cause a constitutive the mutated RET cysteine codon has been demonstrated. Specifically, dimerization of Ret, with consequent activation of its kinase and tram Cys-634 is the most frequently affected residue in families with forming activities, to a lower extent than mutation of Cys-634. We suggest MEN2A (about 80% of reported cases). Mutations of Cys-620 and that the difference In ability to activate RET shown by mutations associ Cys-618, in contrast, are rarely associated with the MEN2A pheno ated with FMTC and MEN2A represents the molecular basis of the type, whereas they account for about 60% of FMTC cases (14, 15). A phenotypic diversity between the two syndromes. different behavior of RET mutants having Cys-634 or Cys-620/Cys 618 substitutions is also indicated by the observation that codon 620 Introduction or 618 but not codon 634 mutations have been described in those rare cases in which congenital megacolon or Hirschsprung's disease co The RET proto-oncogene encodes a tyrosine kinase transmembrane segregates with a MEN2 phenotype (16). Another observation sup receptor named Ret (1). The presence of its transcripts in migrating porting the concept that FMTC is a genetically distinct disease, neural crest cells and in central and peripheral nervous systems (2, 3), although related to MEN2A, is the recent finding of two novel RET and the effects of targeted disruption of RET, which causes a devel mutations, Glu-768---*Asp and Val-804—Leu, both mapping in the opmental defect of the enteric nervous system (4), indicate that RET intracellular domain, in some FMTC cases but not in MEN2A cases is involved in differentiation and/or proliferation of nervous cells. (17, 18). Consistently, mutations causing a reduced RET activity are associated In contrast to other inherited cancer syndromes, characterized by with the developmental defect of enteric neurons involved in congen “lossof function― of tumor suppressor genes, MEN2 syndromes are ital megacolon or Hirschsprung's disease (5, 6); moreover, it has been the first example of familial tumoral diseases associated with “gainof reported that a neurotrophic molecule, the GDNF,3 is a functional function―mutations of a dominant oncogene, i.e. , the RET gene. We ligand for Ret: GDNF is able to bind to a cell surface protein, named and others (19, 20) have demonstrated that RET alleles carrying GDNFRa, and to stimulate Ret tyrosine kinase activity (7, 8). MEN2A mutations of Cys-634 are endowed with transforming poten Point mutations of RET cause the inheritance of the MEN type 2A tial. A constitutive activation of the tyrosine kinase function accounts for the oncogenic potential of RET-MEN2A. Indeed, the loss of one Received 9/26/96; accepted 12/17/96. The costs of publication of this article were defrayed in part by the payment of page cysteine residue causes the generation of disulfide bridge-stabilized charges. This article must therefore be hereby marked advertisement in accordance with Ret dimers; the covalent association between two Ret molecules, by 18 U.S.C. Section 1734 solely to indicate this fact. mimicking ligand-induced dimerization, activates their tyrosine ki @ This study was supported by the Associazione Italiana per Ia Ricerca sul Cancro; by the Progetto Finalizzato. Consiglio Nazionale delle Ricerche, Applicazioni Cliniche della nase and transforming activities (19, 20). Ricerca Oncologica, Sottoprogetto 2, Biologia Molecolare; by the Association de Recher An unresolved question is how similar mutations of the same gene che sur Ic Cancer; and by the Ligue Nationale Contre le Cancer. are able to cause different disease phenotypes, i.e., FMTC and 2 To whom requests for reprints should be addressed. Phone: 39-81-7463056; Fax: 39-81-7463037. MEN2A. To address this point, we have analyzed the functional 3 The abbreviations used are: GDNF, glial cell line-derived neurotrophic factor; MEN, consequences of one of the RET mutations more typically associated multiple endocrine neoplasia; FMTC, familial medullary thyroid carcinoma; MTC, mcd ullary thyroid carcinoma; LTR, long terminal repeat; wt, wild type; CAT, chloramphen with FMTC, i.e, that affecting Cys-620. Here, we demonstrate that icol acetyltransferase; FFU, focus-forming unit; NGFI-A. NGF-induced-cDNA-A. substitution of Cys-620 activates RET, but to a lower extent than 391

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substitution of Cys-634. These results offer a plausible explanation of introduced during the cloning steps. The primers used were the following (the the correlation between the position of RET mutation and the associ introduced mutation is shown in parentheses): forward, 5'-AAGTGCTTC ated disease phenotype. (TAC)GAGCCCGAA-3', and reverse, 5'-C1TFCAGCATCTfCACGGC-3', for the right PCR; forward, 5'-CAGCTGCTFGTAACAGTG-3', and reverse, Materials and Methods 5'-11@CGGGCTC(GTA)GAAGCA-CTT-3', for the left PCR. The pNGFI-A CAT plasmid contains sequences from position —1150to +200 of the Cells and Transfection Experiments. NIH 3T3 cells were grown in NGFI-A promoter, fused to the CATgene (22). DMEM (Life Technologies, Inc.) supplemented with 10% fetal calf serum Protein Studies. Immunoprecipitationandimmunoblottingexperiments (Life Technologies, Inc.) and were transfected using the calcium phosphate were performed as reported previously (25). Briefly, cells were lysed in a precipitation method as described elsewhere (21). Transformed foci were buffer containing 50 nmiHEPES, pH 7.5, 1%(vlv) Triton X-100, 50 mMNaCl, scored at 3 weeks. Transforming efficiency was calculated in FFUs per pmol 5 mMEGTA, 50 mMNaF,20 mMsodium PP1.1 mMsodium vanadate,2 mM of added DNA after normalization for the efficiency of colony formation in phenylmethylsulfonyl fluoride, 0.2 @.tgeachof aprotinin and leupeptin per ml. parallel dishes subjected to marker selection in mycophenolic acid. Marker Lysates were clarified by centrifugation at 10,000 X g for 15 mm, and the selected mass populations of transfected NIH 313 cells were obtained by supernatant was processed for immunoblotting or for immunoprecipitation. growing the transfectants in the presence of mycophenolic acid. Soft agar Protein concentration was estimated by a modified Bradford assay (Bio-Rad). colony assay was performed as reported (21);colonies were scored at 2 weeks. When required, 6% gels were run under nonreducing conditions by omitting To assess theirtumorigenicpotential,I X l0@transfectedcells were injected f3-mercaptoethanolfrom the gel loading buffer. Polyclonal antibodies to the s.c. into athimic nude mice. For each sample, five mice received injections in Ret tyrosine kinase domain have been described previously (25). Immunoblots each flank. Tumor appearance was followed every 4 days and was considered were subsequently stained with appropriate secondary antibodies and revealed when tumors reached, across the longest dimension, a size greater than 10mm. with the Amersham ECL system. The immunocomplex kinase assay was For transienttransfectionassays, 3 X 10@PC12cells were plated in tissue performed as published previously (19). Briefly, comparable amounts of Ret culture dishes 60 mm in diameter. Transfection was performed using the were immunoprecipitated and incubated at 4°Cfor 2 mm in 50 @.dofa buffer lipofectin reagent following the manufacturer's instructions (Life Technolo containing 0.1% Triton, 20 mMHEPES, 150 mrviNaCI, 10% glycerol, 15 msi gies, Inc.). The pNGFI-A-CAT plasmid (22) was chosen as a target because of MgCl,, 15 mr@iMnCI2,20 pCi of [y-32P]ATP(3,000 Ci/mmol; Amersham its very low basal level of activity in PCI2 cells (lower than 0.5% of chlor Corp.) in the presence or absence of 5 jiM of unlabeled ATP. Samples were amphenicol conversion; Ref. 23). Transfections were carried out with 2 @gof subjected to 7.5 and 6% SDS-PAGE under reducing and nonreducing condi reporter plasmid together with 0.3 or 2 @gof the RET constructs. The same tions, respectively (19). DNA concentration was reached by adding various amounts of the LTR control vector. Cell extracts were prepared 60 h after transfection, and CAT Results and Discussion activity was analyzed by TLC with 95% chloroform-5% methanol, as de scribed previously (23). Each experimental point was cut from the TLC plate Substitutions of Cys-620 with Arg, Tyr, or Phe have been described and counted. For each experiment, the percentage of conversion to the acety in MEN2 patients (14). We introduced the Cys-620---@Tyr mutation lated form of [‘4Cichloramphenicol was then calculated. The results were into an expression vector carrying the RET wt cDNA to obtain the plotted as promoter induction relative to the induction exerted by the LTR vector alone. LTR-RET@620 plasmid. The structure of the normal Ret protein and Expression Plasmids. The position of the mutated cysteine is indicated by the position of the two cysteine (634 and 620) mutants analyzed in this the exponent of the name of the different constructs used in this study. The study are represented in Fig. 1. The activity of these constructs was expression vectors LTR@RETwtandLTR@RETcY@@(RET-MEN2A),carry evaluated in two different cell systems, NIH 3T3 and PC 12. ing a Cys-634--@Tyr mutation, are described elsewhere (19). To obtain the A focus-forming assay on NIH 3T3 cells was performed by trans Cys-620—@Tyr(TGC—÷TAC)mutation,PCR fragments containing the re fecting LTR-RET wt, LTR@RETC62O, and LTR@RETs634 plas quired mutation were generated by recombinant PCR using LTR-RET ‘N@asa mids. The Cys-620--@Tyr mutation was able to activate RET trans template. PCR reactions were performed according to Higuchi (24). Briefly, forming potential with an efficiency (about 2 X iO@FFUs/pmol of two primary PCR reactions (a “left―anda “right―reaction)were performed, transfected DNA) similar to that of LTR-RET@―@634,whereas, as using standard PCR conditions (AmpliTaq, Perkin-Elmer Corporation). This reported (19, 20), RETW( was unable to induce the formation of yielded two products overlapping in the sequence corresponding to the reverse primer of the left PCR and the forward primer of the right PCR, and the transformed foci (Table 1). mutation was introduced as part of these overlapping primers. Ten ng of the Marker-selected mass populations of NIH 3T3 cells transfected purified PCR products of the left and the right primary PCR reactions, purified with the different RET constructs were obtained. Although able to by agarose gel electrophoresis, were annealed and elongated with 5 PCR cycles efficiently induce transformed foci formation in NIH 3T3 cells, (95°Cfor1 mm, 37°Cfor2 mm, and 72°CforI mm), followed by 15 cycles RETCYS62O induced a morphology characterized by an higher fraction ofa secondary PCR using as primers the 5'- and 3'-most oligonucleotides. The of flat cells and a lower percentage of round refractile and spindle recombinant PCR products were cloned in the pT7Blue T vector (Novagen) shaped cells. RET Cys62O.transfected cells also displayed a reduced and completely sequenced using the Sequenase Kit (United States Biochemical growth rate when compared to RET CYs634expressing cells (data not Corporation). Finally, the fragment containing the mutation was excised by shown). To better evaluate their transformed phenotype, a soft-agar digestion with appropriate restriction enzymes (NdeI and EcoRI) and cloned in the LTR-RET“toobtain the L@@[email protected] resulting expres colony assay was performed by comparing the colony-forming effi sion vector was sequenced in both strands of the regions that underwent ciency of RET Cys-620 and RET CYs634..expressing cells to that of NIH genetic manipulations to verify that the predicted structures were achieved 3T3@RETwCandvector-transfected NIH 3T3 cells. As reported (19), after the recombination procedures and that no additional mutations were cells expressing RETCYS.6@showed an high clonogenic efficiency in

@ I- 1@1@

@ 1072

@ RE@l@ —I I IIIlIlIllhIlIIIIIIIIIIIIIIIIllhIllh1I sp CAD CYSTM Is Fig. 1. Schematic representation of the 1072-amino acid-long Ret protein. The signal peptide (SP), cadherin domain (CAD), cysteine-rich region (CYS), transmembrane (TM), and tyrosine kinase (TK) domains are indicated. The two mutations analyzed in this study are indicated. 392

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ACFIVATIONOFRETBY MUTATIONOFCYSTEINE620

TablefibroblastsTransfected 1 Transforming efficiency of different RET constructs inNiH 3T3 constructs in the PC12 cell line by evaluating their ability to activate aga?'LTR DNAFFUs/pmol of DNAaSoft the expression of the pNGFI-A-CAT plasmid. NGFI-A is an imme vector diate-early response gene, the expression of which is rapidly induced RET― growth factor; in these cells, active forms of RET are able to induce It has been demonstrated that the mechanism by which Cys-634 transcription of genes involved in neuronal differentiation (23). Thus, mutations activate the kinase function of Ret is the formation of we investigated the relative effects of RET Cys620 and RET Cys-634 disulfide-linked Ret homodimers; these dimers are the active kinase

Cys 634 Cys 620 C @c,

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I' I * 0@

._ B . - @‘4@ l-.o@ @o @ __j a:505°wc@Ic') W('4

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M9 2 0.3 2

Fig. 2. Biological activity of RE7@YS@2OandREI'@'@ mutants. A and B, anchorage-independent growth of RetCy@2OandRet'―634transfectants. The two RET constructs were transfected in NIH 3T3 cells, and marker-selected mass populations were obtained. Cells (2 X l0@)were plated in soft agar in 60-mm culture dishes, and the colony formation was scored at 15 days: high- (X 150; A) and low (X50; B)-magnification microphotographs of the colonies grown in soft agar are shown. These results are typical and representative of at least tlsree independent experiments. C, induction of the NGFI-A promoter in PC12 cells by wt and mutant REF versions. PCI2 cells were transfected with 2 @&gofpNGF1-A-CAT and 0.3 or 2.0 @.tgofthe RET constructs. Sixty h after transfection, total proteins were isolated and promoter induction determined by CAT assay. Bar graph of the relative induction, foldincreasesabovethebasalactivityofpNGFI-ACATreportergenetransfectedalone.Theresultsrepresenttheaverageoftwoseparateexperimentsperformedinduplicate.Variation between experiments was less than 15% of the mean. 393

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rsduclng non-reducing carrying these mutations. Moreover, recently, similar results have 1 I I been obtained by analyzing the activity of a Ret@@618 and a A B RetCY@@@mutant,both associated more frequently with an FMTC (5 (5 phenotype.4 The relationship between tissue involvement and RET mutation may, indeed, reflect a different sensitivity of the affected cell types to mutations activating RET to a different extent. For instance, thyroid C-cells may have a low transformation threshold, being sen sitive also to the low activity of Cys-620 mutation. In contrast, alteration of only certain amino acids (634 in the case of MEN2A and anti-RET 918 in the case of MEN2B) has a sufficiently high activating effect on . 4 Ret to cause a neoplastic transformation of adrenal chromaffin cells. Indeed, a lower susceptibility of adrenal tissue to the transforming potential of RET, as compared to thyroid C cells, is also indicated by the fact that even in the MEN2A phenotype, pheochromocytomas are less frequently observed than MTCs (9) and by the lower frequency of RET mutations in sporadic pheochromocytomas than in sporadic C 0 D C.) N medullary thyroid carcinomas (27).

0 0 0 (5 Acknowledgments

Kinass Assay We are gratefulto Prof. G. Vecchio for his continuousand enthusiastic support during the course of this work. We are indebted to Dr. Moses Chao for

-AlP +ATP the pNGFI-A plasmid.

Fig. 3. Expression and immunocomplex kinase assay of the different Ret mutants. References Equal amounts of Ret proteins (3 mg of total cellular lysate) were immunoprecipitated from RetC@2Oand RetC@6@@@transfectants.The immunoprecipitates were divided in five I. Takahashi, M., Buma, Y., Iwamoto, 1., Inaguma, Y., Ikeda, H., and Hiai, H. Cloning equal aliquots. One aliquot was immunoblotted with anti-Ret antibody; the molecular and expression of the ret proto-oncogene encoding a tyrosine kinase with two weights of the M, 145.000and 160,000 Ret species are indicated (A). Another aliquot was potential transmembrane domains. Oncogene, 3: 571—578,1988. subjected to electrophoretic separation under nonreducing conditions and immunobloued 2. Pachnis, V., Mankoo, B., and Costantini, F. Expression of the c-RET proto-oncogene with anti-Ret antibody to check for dimer formation; the positions of Ret monomer and during mouse embryogenesis. Development (Camb.), I 19: 1005—1017,1993. homodimer are indicated (B). Two aliquots were subjected to an in vitro kinase assay in 3. Avantaggiato, V., Dathan, N. A., Grieco, M., Fabien, N., Lazzaro, D., Simeone, A., the presenceor absenceof 5 psi unlabeledAlP (C). Finally,anotheraliquotof the Fusco, A., and Santoro, M. Developmental expression of the RET proto-oncogene. immunoprecipitates was subjected to a kinase assay and run under nonreducing conditions Cell Growth & Differ., 5: 305-3 11, 1994. 4. Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F., and Pachnis, V. (D). These results are typical and representative of at least three independent experiments. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature (Lond.), 367: 380—383,1994. 5. Pasini, B., Borrello, M. G., Greco, A., Bongarzone, I., Luo, Y., Mondellini, P., (19, 20). We postulated that a similar mechanism could be responsible Alberti, L., Miranda, C., Arighi, E., Bocciardi, R., Sen. M., Barone, V., Radice, M. 1., for the activation of Ret@@62°mutant;therefore, Ret products were Romeo, G., and Pierotti, M. Loss of function effect of RET mutations causing Hirschsprung disease. Nat. Genet., 10: 35—40,1995. analyzed by a SDS-PAGE under nonreducing conditions to detect the 6. Carlomagno, F., Dc Vita, G., Berlingieri, M. T., de Franciscis, V., Melillo, R. M., presence of Ret dimers. Under these conditions, both Ret@@@ and Colantuoni, V., Kraus. M. H., Di Fiore, P. P., Fusco, A., and Santoro, M. Molecular Ret@'@620displayed an additional species of Mr @300,000,corre heterogeneity of RET loss of function in Hirschsprung's disease. EMBO J., 15: 2717—2725,1996. sponding to Ret homodimers (19, 20). However, a significantly lower 7. Treanor, J. J. S., Goodman, L., de Sauvage, F., Stone, D. M., Poulsen, K. T., Beck, accumulation of Ret dimers in RET Cys620with respect to RET Cys-634 C. D.,Gray,C.,Armanini,M.P.,Pollock,R.A.,Hefti,F., Phillips,H.S.,GOddard, A., Moore, M. W., Buj-Bello, A., Davies, A. M., Asai, N., Takahashi, M., Vandlen, transfectants was observed (Fig. 3B). Similarly, when the products of R., Henderson, C. E., and Rosenthal, A. Characterization of a multicomponent an immunocomplex kinase assay were run under nonreducing condi receptor for GDNF. Nature (Lond.), 382: 80—83, 1996. tions, a lower amount of the autophosphorylated Mr @300,000Ret 8. Jing, S., Wen, D., Yu, Y., Hoist, P. L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, z.. Cupples,R.,Louis,J.C., Hu,S.,Altrock,B.W.,andFox,G.M.GDNF-induced dimeric form was detected in RETCYS62Othan in RETCYS634 cells activation of the Ret protein tyrosine kinase is mediated by GDNFR-a, a novel (Fig. 3D). Because it has been reported that these homodimers are receptor for GDNF. Cell, 85: 1113—1124,1996. composed by the mature Mr 160,000 protein (20), these results are 9. Smith, D. P., Eng, C., and Ponder, B. A. J. Mutations of the RET proto-oncogene in the multiple endocrine neoplasia type 2 syndromes and Hirschsprung's disease. J. consistent with the reduced amount ofthe Mr 160,000 species detected Cell. Sci, 18: 43—49,1994. in NIH 3T3-RET@@620. 10. Hofstra, R. M. W., Landsvater, R. M., Ceccherini, I., Stulp, R. P., Stelwagen, T., Luo, Y., Pasini, B., Hoppener. J. w. M., van Amstel, H. C. P., Romeo, G., Lips, C. J. M., Data reported here demonstrate that, as previously reported for and Buys,C. H. C. M. A mutationin the RET proto-oncogeneassociatedwith Cys-634, Cys-620 mutation caused an activation of RET; thus, a RET multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. gain of function mechanism is involved also in tumoral diseases Nature (Lond.), 367: 375—376,1994. I I. Carlson. K. M., Dou, S., Chi, D., Scavarda, N.. Toshima, K., Jackson, C. E., associated with this mutation. As in the case of Cys-634 mutation, a wells, S. A., Goodfellow, P., and Donis-Keller, H. Single missense mutation in constitutive dimerization, mediated by disulfide bridges, was caused the tyrosine-kinase catalytic domain of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2B. Proc. NatI. Acad. Sci. USA, 91: 1579— by Cys-620 mutation. However, both the amount of Ret homodimers 1583,1994. and Ret kinase activity were definitely lower in Ret@'@62°thanin 12. Mulligan, L. M., Kwok, J. B. J., Healey, C. S., Elsdon, M. J., Eng, C., Gardner, E., Ret@'@634transfectants. A likely explanation for these differences is Love, D. R., Mole, S. E., Moore, J. K., Papi, L., Ponder, M. A., Telenius, H., Tunnacliffe, A., and Ponder, B. A. J. Germ-line mutations of the RET proto-oncogene the lower abundance, in the case of Ret@62° mutant, of the Mr in multiple endocrine neoplasia type 2A. Nature (Lond.), 363: 458—460, 1993. 160,000 Ret protein form, the one involved in the formation of Ret 13. Donis-Keller, H., Dou, S., Chi, D., Carlson, K. M., Toshima, K., Lairmore, 1. C., dimers (20). Alternatively, it is possible that the mutation of Cys-620 Howe, J. R., Moley, J. F., Goodfellow, P., and Wells, S. A. Mutations in the RE! proto-oncogene are associated with MEN2A and FMTC. Hum. Mol. Gen., 2: influences at the same time the ability of Ret to form homodimers and 851—856,1993. the maturation or the half-life of the Mr 160,000 protein form. 14. Mulligan, L. M., Eng, C., Henley, C. S., Clayton, D., Kwok, J. B. J., Gardner, E., The lower activity of RetC>@@62Ocomparedto RetCY@6@providesan explanation for the different disease phenotypes observed in patients 4 M. Billaud, personal communication. 394

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1997 American Association for Cancer Research. The Different RET-activating Capability of Mutations of Cysteine 620 or Cysteine 634 Correlates with the Multiple Endocrine Neoplasia Type 2 Disease Phenotype

Francesca Carlomagno, Giuliana Salvatore, Anna Maria Cirafici, et al.

Cancer Res 1997;57:391-395.

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