Molecular Mechanisms of RET Receptor–Mediated Oncogenesis in Multiple Endocrine Neoplasia 2B

Abstract

Multiple endocrine neoplasia 2B (MEN 2B) is an inherited syndrome of early onset endocrine tumors and developmental anomalies. The disease is caused primarily by a methionine to threonine substitution of residue 918 in the kinase domain of the RET receptor (2B-RET); however, the molecular mechanisms that lead to the disease phenotype are unclear. In this study, we show that the M918T mutation causes a 10-fold increase in ATP binding affinity and leads to a more stable receptor-ATP complex, relative to the wild-type receptor. Further, the M918T mutation alters local protein conformation, correlating with a partial loss of RET kinase autoinhibition. Finally, we show that 2B-RET can dimerize and become autophosphorylated in the absence of ligand stimulation. Our data suggest that multiple distinct but complementary molecular mechanisms underlie the MEN 2B phenotype and provide potential targets for effective therapeutics for this disease.

Transcript Level Modulates the Inherent Oncogenicity of RET/PTC Oncoproteins

“…We have recently suggested that a quantitative increase in RET kinase activity may be responsible for the greater phenotypic severity of MEN 2B (RET M918T) relative to other MEN 2 mutants (13). Further, we have recently shown that MEN 2A and 2B up-regulate a similar compliment of genes; however, the more aggressive MEN 2B form induces a higher level of expression of these genes due to its increased kinase activity (36).…”

“…Immunoprecipitation and immunoblotting were done according to previously published methods (13). Briefly, lysed cells were separated into cytosolic and membrane-associated fractions by centrifugation at 12,000 Â g. Cytosolic fractions were immunoprecipitated with 0.8 Ag antibody and 20 AL protein A/G slurry (Santa Cruz Biotechnology).…”

“…Colony formation assays were done as described previously (13). Approximately 2 Â 10 4 cells were resuspended in 0.2% top agar in DMEM and plated on 0.4% bottom agar in medium.…”

GENETICS IN ENDOCRINOLOGY: The genetics of phaeochromocytoma: using clinical features to guide genetic testing

“…Interestingly, a case of familial phaeochromocytoma described in 1886 was shown to result from a germline RET mutation 121 years later (9). MEN2 is an autosomal dominantly inherited disorder and is divided into three clinical subtypes, two of which (MEN2A and MEN2B) are characterised by the development of medullary thyroid cancer (MTC) and (10)(11)(12). Individuals with MEN2A have about a 50% risk of developing aPCA but the mean age at diagnosis of MTC is earlier than that of aPCA (w40 years) and so, compared with individuals with germline SDHB, SDHD or VHL mutations, individuals with MEN2 are less likely to present with sporadic non-syndromic phaeochromocytoma.…”

Interplay between Ret and Fap-1 regulates CD95-mediated apoptosis in medullary thyroid cancer cells

“…RET M918T, which is also the most frequent somatic mutation in sporadic MTCs is prognostic of aggressive disease thus indicating a strong carcinogenic potential [3,7]. Substitution of methionine 918 with threonine results in increased Ret kinase activity, release of auto-inhibition, increased ligandindependent formation of activated monomers and dimers, and change in substrate specificity contributing to the receptor oncogenic activity [8,9]. Preclinical studies have provided evidence that Ret oncoproteins may represent a therapeutic target exploitable in subsets of thyroid tumors [5,10,11].…”