Mechanisms of Development of Multiple Endocrine Neoplasia Type 2 ...

Mechanisms of Development of Multiple Endocrine Neoplasia Type 2 and Hirschsprung's Disease by ret Mutations M. Takahashi, N. Asai, T. Iwashita, H. Murakami, and S. Ito Department of Pathology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan

Abstract The ret proto-oncogene encodes a receptor tyrosine kinase whose ligands belong to the glial cell line-derived neurotrophic factor (GDNF) protein family. Its germline mutations are responsible for the development of multiple endocrine neoplasia (MEN) types 2A and 2B and Hirschsprung's disease (HSCR). MEN2A and MEN2B mutations result in the constitutive activation of Ret by different molecular mechanisms. MEN2A mutations involve cysteine residues present in the Ret extracellular domain and induce disulfide-linked Ret dimerization on the cell surface. MEN2B mutations were identified in methionine 918 in the tyrosine kinase domain and activate Ret without dimerization, probably due to a conformational change of its catalytic core region. In contrast to MEN2 mutations, HSCR mutations represent loss of function mutations. We found that most of HSCR mutations detected in the extracellular domain impair the Ret cell surface expression. More interestingly, ret mutations in cysteines 618 and 620 were reported in several families who developed both MEN2A and HSCR. It was suggested that these mutations might have two biological effects on Ret function, leading to the development of different clinical phenotypes in the same patients.

Introduction The ret proto-oncogene encodes a receptor tyrosine kinase that contains a cadherin-related sequence in the extracellular domain (Takahashi 1995). It was recently demonstrated that glial cell line-derived neurotrophic factor (GDNF) and neurturin, which define a new protein family, can induce Ret tyrosine phosphorylation, indicating that these two neurotrophic factors represent Ret ligands. It is interesting to note that these factors mediate their actions through a multicomponent receptor system composed of ligand-binding glycosyl-phosphatidylinositol (GPI)-linked proteins and Ret (Buj-Bello et al. 1997; Jing et al. 1996; Klein et al. 1997; Treanor et al. 1996). This unique Recent Results in Cancer Research, Vol. 154 © Springer-Verlag Berlin· Heidelberg 1998

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multicomponent receptor system plays an important role in the survival and/ or differentiation of a variety of central and peripheral neurons. Germline mutations of the ret proto-oncogene are associated with the development of three hereditary neoplastic disorders: multiple endocrine neoplasia (MEN)2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) (Carlson et al. 1994; Donis-Keller et al. 1993; Hofstra et al. 1994; Mulligan et al. 1993). MEN2A and MEN2B share the clinical features of medullary thyroid carcinoma and pheochromocytoma, and FMTC is characterized by the development of medullary thyroid carcinoma alone. MEN2B is distinguished from MEN 2A and FMTC by a more complex phenotype including mucosal neuroma, hyperganglionosis of the gastrointestinal tract, and marfanoid habitus. The MEN2A and FMTC mutations resulted mainly in nonconservative substitutions for six cysteines (cysteines 609, 611, 618, 620, 630, and 634) in the Ret extracellular domain, whereas the MEN2B mutation was detected in methionine 918 in the tyrosine kinase domain. Germline mutations of ret are also responsible for Hirschsprung's disease (HSCR), which is characterized by the absence of intrinsic ganglion cells in the distal gastrointestinal tract (Pasini et al. 1996). HSCR mutations, including missense, nonsense, and frame shift mutations, are scattered along the whole coding sequence of ret and account for 50% of familial and 10%-20% of sporadic cases of HSCR. Based on the length of the aganglionic segment, HSCR is classified into two subtypes, short-segment HSCR and long-segment HSCR; ret mutations appear to be associated with long segment HSCR. In addition, three mutations in cysteine 618 or 620 were reported in several families who developed both MEN2A and HSCR. To assess the correlation between genotype and phenotype, we introduced most mutations reported in these diseases into ret cDNA which was transfected into NIH 3T3 cells. Characterization of the nature of the mutant Ret proteins provided interesting insights into the mechanisms of development of MEN2A, MEN2B, FMTC, and HSCR.

Mechanism of Ret Activation by MEN2A and FMTC Mutations We investigated the transforming activity of Ret with a MEN2A or FMTC mutation found in cysteine 609, 611, 618, 620, 630, or 634. Of these cysteine mutations, cysteine 634 mutations are known to be strongly associated with the MEN2A phenotype, whereas cysteine 609, 618, and 620 mutations were found in about 70% cases of FMTC (Pander and Smith 1996). We introduced a total of 18 mutations into six cysteine residues (Fig. 1) and found that all mutant ret cDNAs had the ability to transform NIH 3T3 cells at variable levels (Ito et al. 1997). The transforming activity of cysteine 634 mutant proteins was approximately three- to five-fold higher than that of cysteine 609, 611, 618, or 620 mutant proteins. The activity of cysteine 630 mutant proteins was slightly lower than that of cysteine 634 mutant proteins. In addition, different amino acid substitution for the same cysteine displayed com-

Mechanisms of Development of Multiple Endocrine Neoplasia Type 2

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c-Ret

Fig. 1. Ret protein. 5, signal sequence; CAD, cad herin-like domain; CYS, cysteine-rich region; TM, transmembrane domain; TK, tyrosine kinase domain. Eighteen cysteine mutations and a MEN-2B mutation (Met918-> Thr) are shown

parable transforming activity, suggesting that the degree of activity depends on the position of cysteine rather than the substituted amino acids. Our results showing that cysteine 634 mutant proteins had the highest transforming activity are consistent with the observation that there is a strong association between cysteine 634 mutations and the development of pheochromocytoma in MEN2A. On the other hand, the low transforming activity of cysteine 609, 618, and 620 mutant pr6teins appears to correlate with the FMTC phenotype. Western blot analysis of the mutant Ret proteins revealed that the level of expression of the 175-kDa Ret form present on the cell surface was directly proportional to the transforming activity of each mutant protein [Ito et al. 1997] . The level of expression the 175-kDa Ret form with a cysteine 609, 611, 618, or 620 mutation was approximately five- to ten-fold lower than that of the 175-kDa form with a cysteine 634 mutation. Since the expression of the 155-kDa Ret form present in the cytoplasmic membrane fraction was not affected by each cysteine mutation, this result suggests that cysteine 609, 611, 618, or 620 mutations severely impair the transport of Ret to the plasma membrane or its correct maturation. As we and others have already reported (Asai et al. 1995; Borrello et al. 1995; Iwashita et al. 1996a; Santoro et al. 1995), cysteine 634 mutations are able to activate Ret by inducing homodimers of the 175-kDa Ret form on the cell surface (Fig. 2). Although cysteine 609, 611, 618, and 620 mutations were also able to form homodimers of the 175-kDa form, the amount of homodimers with these mutations was very low. Thus the levels of expression of the 175-kDa Ret form with each cysteine mutation and of its homodimers appear to correlate with both the transforming activity and the clinical phenotype.

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Difference in Activation Mechanisms by the MEN2A Versus MEN2B Mutation Unlike the MEN2A and FMTC mutations, the MEN2B mutation does not induce Ret dimerization. Since the MEN2B mutation is present in the catalytic core region of the Ret kinase domain, it could induce a conformational change of its region, resulting in Ret activation (Fig. 2; Iwashita et al. 1996a). Ret is translated as two isoforms of 1114 amino acids (long isoform) and 1072 amino acids (short isoform). The 51 carboxy-terminal amino acids of the long isoform are replaced by the nine unrelated amino acids of the short isoform by alternative splicing in the 3' region. Interestingly, the transforming activity of the long isoform with the MEN2B mutation was approximately ten fold higher than that of the short isoform, although the transforming activity of both isoforms with the MEN2A mutation showed similar transforming activity (Asai et al. 1996). Thus the carboxy-terminal tail may regulate the transforming activity of Ret with the MEN2B mutation. To investigate further the different activation mechanisms by MEN2A versus MEN2B mutations, we mutated tyrosine residues present in the tyrosine

c-Ret

c V-®864 V-®9S2

®-V 1062®V

V~

~~ Signal

v-® 8

~~Signal

Fig. 2. Mechanisms of Ret activation by the MEN2A (center) or MEN2B mutation (right). When one cysteine residue (C) in the extracellular domain is replaced by other amino acids (X) due to a MEN2A mutation, it is anticipated that another cysteine which should form an intramolecular disulfide bond with the mutated cysteine becomes free and forms an aberrant intermolecular disulfide bond, leading to ligand-independent Ret dimerization. However, since the MEN2B mutation (Met918 -> Thr) is present in the catalytic core region in the kinase domain, it could induce a conformational change of its region, reSUlting in Ret activation without dimerization. In addition, tyrosine 905 and tyrosines 864 and 952 are required for the transforming activity of MEN2A-Ret and MEN2B-Ret, respectively. Tyrosine 1062 represents a binding site of Shc adaptor proteins that are crucial for the transforming activity of both MEN2A-Ret and MEN2B-Ret


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kinase domain. When tyrosine 905 that is conserved at the same position in all tyrosine kinases was changed to phenylalanine, the transforming activity of Ret with a MEN2A mutation (Cys634 ---'> Arg; designated MEN2A-Ret) was completely abolished, suggesting that phosphorylation of this tyrosine is essential for its activity (Iwashita et al. 1996a). On the other hand, this tyrosine mutation did not affect the transforming activity of Ret with the MEN2B mutation (MEN2E-Ret) at all. To look for tyrosine residues required for the activity of MEN2B-Ret, we further changed several tyro sines in the kinase domain to phenylalanine. When tyrosine 864 or 952 was replaced with phenylalanine, the transforming activity of MEN2E-Ret significantly decreased. In addition, the double mutation of these two tyrosines completely abolished the activity of MEN2B-Ret, but not of MEN2A-Ret, indicating that tyrosine residues essential for the transforming activity differ between MEN2A-Ret and MEN2B-Ret (Iwashita et al. 1996a). In addition to these tyrosine residues, we identified tyrosine 1062 as a binding site of Shc adaptor proteins that could play an important role in the activity of both MEN2A-Ret and MEN2B-Ret proteins. Possible activation mechanisms of Ret by the MEN2A or MEN2B mutation are summarized in Fig. 2.

Mechanism of Ret Dysfunction by HSCR Mutations It is known that ret is an important gene for the development of enteric neu-

rons because ret knockout mice completely lack ganglion cells in the intestine (Schuchardt et al. 1997). To date, a variety of mutations, including missense, nonsense, and frame shift mutations, have been identified in both the extracellular and intracellular domains of Ret in HSCR (Chakravarti 1996). Pasini et al. (1995) reported that some mutations detected in the kinase domain impair the Ret kinase activity as expected. We and others investigated the biological effects of several HSCR mutations reported in the Ret extracellular domain and found that these mutations severely impair Ret cell surface expression (Carlomagno et al. 1996; Iwashita et al. 1996b). Based on its length, the aganglionic segment is classified into two groups: short -segment HSCR and long-segment HSCR. Our results also showed that long-segment HSCR mutations more severely impair transport of Ret to the plasma membrane than a short-segment HSCR mutation, suggesting that the level of its cell surface expression may correlate with the HSCR phenotype (Iwashita et al. 1996b). In addition, the fact that ret heterozygous deletions were detected in both familial and sporadic HSCR patients suggests that haploinsufficiency for ret is crucial for development of HSCR. Since the cell surface expression of Ret with long-segment HSCR mutations was very low, a haploinsufficiency effect might be also postulated in the cases of missense mutations found in the Ret extracellular domain. Thus it seems likely that sufficient GDNF binding to Ret on the cell surface of enteric neuroblasts is required for their full differentiation during embryogenesis.

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Cys 609 Cys 618 Mutations Cys 620

Dimerization of Ret

Impairment of Ret Cell Surface Expression

Development of MTC

Hirschsprung's Disease

Fig. 3. Biological effects of cysteine 609, 618, and 620 mutations on Ret function. These mutations might have different biological effects on Ret function, depending on the cell type expressing Ret. MTC medullary thyroid carcinoma

Another interesting finding is that there are several families who develop both MEN2A and FMTC (Ponder and Smith 1996). In these families, substitution of arginine or serine for cysteine 618 and of arginine for cysteine 620 in ret was identified. In addition, mutations of cysteine 609 reported in MEN2A were found in sporadic cases of HSCR. These findings suggest that the same cysteine mutations are able to induce both MEN2A and HSCR. As mentioned already, the transforming activity of Ret with cysteine 609, 618, or 620 mutations was very low compared with the activity of Ret with cysteine 634 mutations (Ito et al. 1997). The low activity of Ret with cysteine 609, 618 or 620 mutations correlated with the low expression of the 175-kDa Ret form present on the cell surface, indicating that these mutations impair the Ret cell surface expression in the same way as HSCR mutations affecting the Ret extracellular domain. These results thus suggest that cysteine 609, 618, and 620 mutations have two biological effects on Ret function (Fig. 3; Ito et al. 1997). First, these mutations induce ligand-independent Ret homodimers at low levels that are sufficient for tumorigenesis of thyroid C cells, leading to the development of MEN2A or FMTC. Second, they also severely impair Ret cell surface expression in enteric neurons. This could result in the premature arrest of differentiation of enteric neuroblasts during embryogenesis, leading to the development of HSCR.


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Conclusion We have studied and elucidated molecular mechanisms of development of MEN2A, MEN2B, FMTC, HSCR, and papillary thyroid carcinoma by ret mutations. However, further studies of the intracellular signaling pathway via the mutant Ret proteins could be essential to better understand the correlation between genotype and phenotype correlation observed in these diseases. In addition, ret is known to playa crucial role in the development of the enteric nervous system and kidney and in survival of various neurons. In this regard, the recent identification of GDNF and neurturin as Ret ligands may be an important step in promoting the study of Ret signaling in neuronal cells and embryonic kidney.

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