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Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines
S Chappuis-Flament1, A Pasini, G De Vita
1Laboratoire de Génétique, CNRS UMR 5641, Domaine Rockefeller, Lyon, France.
Abstract:
The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and affected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disulfide homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.
Insights
RET gene mutations in cysteines cause cancer and Hirschsprung disease. Cysteine substitutions create constitutive RET activity, with varying effects on cell surface levels and dimerization, explaining diverse disease phenotypes.
Area of Science:
- Genetics
- Molecular Biology
- Oncology
Background:
- The RET gene encodes a receptor tyrosine kinase crucial for kidney and nervous system development.
- Germline mutations in RET cysteines (Cys609, 611, 618, 620, 634) cause multiple endocrine neoplasia type 2A (MEN 2A), familial medullary thyroid carcinoma (FMTC), and Hirschsprung disease (HSCR).
- Some MEN 2A kindreds also exhibit HSCR, with affected individuals carrying single RET mutations at codons 609, 618, or 620.
Purpose of the Study:
- To investigate the functional consequences of cysteine substitutions in the RET receptor.
- To determine how specific Cys-to-Arg mutations affect RET kinase activity, dimerization, and cell surface localization.
- To elucidate the molecular mechanisms underlying the diverse clinical phenotypes associated with RET cysteine alterations.
Main Methods:
- Introduced Cys-to-Arg mutations into wild-type RET at codons 609, 618, 620, 630, and 634.
- Assessed constitutive catalytic activity and aberrant disulfide homodimerization of mutant RET proteins.
- Quantified RET activation levels in rodent fibroblasts and pheochromocytoma PC12 cells.
- Performed biochemical analysis to determine RET cell surface levels and covalent dimer formation.
Main Results:
- All tested RET mutations (Cys609, 611, 618, 620, 634) induced constitutive catalytic activity via aberrant disulfide homodimerization.
- Mutations at codons 630 and 634 demonstrated stronger RET activation compared to mutations at codons 609, 618, and 620.
- Mutations at codons 618 and 620, and to a lesser extent 609, significantly reduced cell surface RET levels and decreased covalent dimer formation.
Conclusions:
- RET cysteine substitutions lead to constitutive kinase activity through abnormal homodimerization.
- The varying degrees of RET activation and cell surface reduction by different mutations explain the spectrum of MEN 2A, FMTC, and HSCR phenotypes.
- Mutations at codons 609, 618, and 620 exert dual effects, activating RET while simultaneously reducing its cell surface presence and dimerization.