Functional Analysis of ret Oncogene with Multiple Endocrine Neoplasia Type 2

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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.