Functional Analysis of ret Oncogene with Multiple Endocrine Neoplasia Type 2

多发性内分泌瘤（MEN）2A和2B突变激活的Ret细胞内信号通路的分析由于其组成性激活，具有多发性内分泌瘤（MEN）2A或2B突变的ret原癌基因可以高效转化NIH 3 T3细胞。我们通过MEN 2A、2B突变激活的Ret和胶质细胞源性神经营养因子（GDNF）分析了细胞内信号通路。结果表明，它们都诱导了由Ret、Shc和Grb 2蛋白组成的信号转导复合物。此外，GDNF明显激活了人神经母细胞瘤细胞中的Ras-MAPK通路。Ret主要表达为两种在羧基末端序列上不同的同种型：长同种型（1114个氨基酸）和短同种型（1072个氨基酸）。长同种型含有用于结合Shc PTB结构域但不结合其SH 2结构域的共有序列，而短同种型具有用于结合两个结构域的共有序列。体外结合试验显示MEN 2A-Ret蛋白的长同种型和MEN 2B-Ret蛋白的两种同种型优先结合Shc PTB结构域。另一方面，MEN 2A-Ret的短同种型与PTB和SH 2结构域结合。在神经母细胞瘤细胞表达的两种异构体的Ret，其激活GDNF也导致在这两个域的结合。GDNF和MEN 2A突变通过诱导其二聚化来激活Ret，而MEN 2B突变增加Ret催化活性而不二聚化。因此，我们的研究结果表明，Ret二聚化可能需要结合的Shc SH 2结构域的短亚型。

Analysis of the intracellular signaling pathway through Ret activated by multiple endocrine neoplasia (MEN) 2A and 2B mutations.The ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A or 2B mutation can transform NIH3T3 cells with high efficiencies as a consequence of its constitutive activation. We analyzed the intracellular signaling pathway through Ret activated by MEN 2A,2B mutations, and glial-cell-line-derived neurotrophic factor (GDNF). The results showed that all of them induce a signal transducing complex consisting of Ret, Shc, and Grb2 proteins. In addition, GDNF clearly activated a Ras-MAPK pathway in human neuroblastoma cells. Ret is expressed mainly as two isoforms that differ in the carboxy-terminal sequence : a long isoform (1114 amino acids) and a short isoform (1072 amino acids). The long isoform contains the consensus sequence for binding of the Shc PTB domain but not of its SH2 domain, whereas the short isoform has the consensus sequences for binding of both domains. In vitro binding assay revealed that the long isoform of the MEN2A-Ret protein and both isoforms of the MEN2B-Ret protein bound preferentially to the Shc PTB domain. On the other hand, the short isoform of MEN2A-Ret bound to the PTB and SH2 domains. In neuroblastoma cells expressing both isoforms of Ret, its activation by GDNF also resulted in the binding of both domains. GDNF and MEN2A mutations activate Ret by inducing its dimerization, whereas the MEN2B mutation increases Ret catalytic activity without dimerization. Our results thus suggest that Ret dimerization might be required for binding of the Shc SH2 domain to the short isoform.