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Characterization of Ret-Shc-Grb2 complex induced by GDNF, MEN 2A, and MEN 2B mutations
M Ohiwa1, H Murakami, T Iwashita
1Department of Pathology, Nagoya University School of Medicine, Japan.
Abstract:
We analyzed the intracellular signalling pathway through Ret activated by glial-cell-line-derived neurotrophic factor (GDNF), multiple endocrine neoplasia (MEN) 2A and 2B mutations. 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. Rat 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 MEN 2A mutations activate Ret by inducing its dimerization, whereas the MEN 2B 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.
Insights
Glial-cell-line-derived neurotrophic factor (GDNF) and MEN 2 mutations activate Ret signaling. Ret dimerization is key for Shc SH2 domain binding, influencing neuroblastoma cell pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncology
Background:
- The Ret receptor tyrosine kinase plays a crucial role in cellular signaling.
- Mutations in Ret are associated with Multiple Endocrine Neoplasia (MEN) types 2A and 2B.
- Glial-cell-line-derived neurotrophic factor (GDNF) is a key activator of Ret signaling.
Purpose of the Study:
- To investigate the intracellular signaling pathway of Ret activated by GDNF and MEN 2 mutations.
- To elucidate the role of Ret isoforms and their interaction with Shc proteins.
- To understand the mechanisms of Ret activation by GDNF and MEN 2 mutations.
Main Methods:
- Analysis of intracellular signaling pathways.
- In vitro binding assays.
- Expression analysis in human neuroblastoma cells.
Main Results:
- GDNF and MEN 2 mutations induce a Ret-Shc-Grb2 signal transducing complex.
- GDNF activates the Ras-MAPK pathway in neuroblastoma cells.
- Ret isoforms exhibit differential binding to Shc PTB and SH2 domains, with implications for MEN 2A and MEN 2B mutations.
Conclusions:
- Ret dimerization induced by GDNF and MEN 2A mutations may be required for Shc SH2 domain binding to the short Ret isoform.
- MEN 2B mutations enhance Ret catalytic activity independently of dimerization.
- These findings provide insights into Ret signaling dysregulation in neuroblastoma and MEN syndromes.