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Full activation of MEN2B mutant RET by an additional MEN2A mutation or by ligand GDNF stimulation
I Bongarzone1, E Vigano, L Alberti
1Division of Experimental Oncology A, Istituto Nazionale Tumori, Milan, Italy.
Abstract:
Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).
Insights
Dimerization enhances the activity of the RET receptor tyrosine kinase in MEN2B cancer syndrome. This suggests that Ret ligand concentration may influence the MEN2B cancer phenotype.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Germline mutations in the RET gene are linked to Multiple Endocrine Neoplasia types 2A (MEN2A) and 2B (MEN2B).
- MEN2A mutations cause receptor dimerization and constitutive activation, while MEN2B has a specific mutation (Met918Thr) in the tyrosine kinase domain.
- The MEN2B mutation leads to ligand-independent activation, but its mechanism and modulation by dimerization were unclear.
Purpose of the Study:
- To investigate whether dimerization can increase the activity of the Ret protein with the MEN2B mutation.
- To explore potential mechanisms for RetMEN2B activation beyond intramolecular cis-autophosphorylation.
- To understand how Ret ligand concentration might influence the MEN2B cancer phenotype.
Main Methods:
- Constructed a double mutant RET receptor combining MEN2A (Cys634Arg) and MEN2B (Met918Thr) mutations to induce dimerization.
- Exposed cells expressing RetMEN2B to the RET ligand GDNF over time to promote stable dimerization.
- Assessed RetMEN2B activity using in vitro transfection assays and biochemical parameter measurements.
Main Results:
- Experimental induction of dimerization, via a MEN2A mutation or GDNF exposure, fully activated RetMEN2B.
- Both methods of dimerization led to enhanced receptor activity, comparable to full activation.
- These findings demonstrate that RetMEN2B activity can be significantly increased by receptor dimerization.
Conclusions:
- Receptor dimerization is a mechanism for fully activating the RetMEN2B oncoprotein.
- The tissue-specific or concentration-dependent effects of Ret ligands may play a role in modulating the MEN2B cancer phenotype.
- This study provides new insights into the molecular mechanisms underlying MEN2B pathogenesis and potential therapeutic strategies.