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 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1997 American Association for Cancer Research. ACTIVATION OF RET BY MUTATION OF CYSTEINE 620 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