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Ligand stimulation of a Ret chimeric receptor carrying the activating mutation responsible for the multiple endocrine
C Rizzo1, D Califano, G L Colucci-D'Amato
1Centro di Endocrinologia ed Oncologia Sperimentale del CNR, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli "Federico II," via S. Pansini 5, 80131 Napoli, Italy. defrancisci@cds.unina.it
Abstract:
Inherited activating mutations of Ret, a receptor tyrosine kinase, predispose to multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma. To investigate the effects induced by acute stimulation of Ret, we transfected both PC12 and NIH 3T3 cells with a molecular construct in which the ligand-binding domain of the epidermal growth factor receptor was fused to the catalytic domain of Ret. Acute stimulation of the chimeric receptor induced PC12 cells to express a neuronal-like phenotype. Moreover, we introduced the dominant mutation, responsible for the multiple endocrine neoplasia type 2B, in the catalytic domain of the Ret chimera. Expression of the mutant chimera, in the absence of ligand stimulation, induces the PC12 cells to acquire a flat morphology with short neuritic processes and transforms the NIH 3T3 cells. Stimulation of the mutant chimera with epidermal growth factor causes a drastic overgrowth of long neuritic processes, with the induction of the suc1-associated protein tyrosine phosphorylation in PC12 cells and higher transforming efficiency in NIH 3T3 cells. These data indicate that the gain-of-function MEN2B mutation does not abrogate ligand responsiveness of Ret and suggest that the presence of Ret ligand could play a role in the pathogenesis of the MEN2B syndrome.
Insights
Activating mutations in Ret, a receptor tyrosine kinase, cause MEN2B. This study shows the MEN2B mutation in Ret does not prevent ligand response, suggesting Ret ligand involvement in MEN2B syndrome development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Activating mutations in the Ret receptor tyrosine kinase are linked to Multiple Endocrine Neoplasia (MEN) types 2A and 2B, and familial medullary thyroid carcinoma.
- Understanding the effects of acute Ret stimulation is crucial for deciphering its role in these hereditary cancer syndromes.
Purpose of the Study:
- To investigate the functional consequences of acute Ret stimulation using a chimeric receptor construct.
- To examine the impact of the dominant MEN2B mutation on Ret signaling and cellular phenotype.
- To determine if the MEN2B mutation affects Ret's ligand responsiveness.
Main Methods:
- Transfection of PC12 and NIH 3T3 cells with a chimeric receptor construct (Epidermal Growth Factor Receptor ligand-binding domain fused to Ret catalytic domain).
- Introduction of the MEN2B-associated mutation into the Ret chimera.
- Assessment of cellular morphology, neuronal differentiation, NIH 3T3 cell transformation, and protein tyrosine phosphorylation upon stimulation.
Main Results:
- Acute stimulation of the chimeric Ret receptor induced a neuronal-like phenotype in PC12 cells.
- Expression of the MEN2B mutant chimera, even without ligand, induced PC12 cell flattening and NIH 3T3 cell transformation.
- Epidermal growth factor stimulation of the mutant chimera led to exaggerated neuritic outgrowth and increased NIH 3T3 cell transformation.
Conclusions:
- The gain-of-function MEN2B mutation in Ret does not abolish ligand responsiveness.
- These findings suggest that the presence of Ret ligands may contribute to the pathogenesis of the MEN2B syndrome.