The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein

Abstract

Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations a ecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disul®de-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This ®nding speci®cally correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion a ecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is a ected by mutation type.

A novel type of mutation in the cysteine rich domain of the RET receptor causes ligand independent activation

“…In contrast to the missense mutation in codon 634 the two duplication mutations leave the ®rst extracellular cysteine at the same distance from the cell surface as in the wt-RET (Figure 4). Also the recently published deletion of six base pairs (Bongarzone et al, 1999), which represents an activating mutation too, still leaves the cysteine 634 in place (Figure 4). Thus the distance of the cysteine relative to the plasma membrane seems not to be essential.…”

“…The disul®de linkage between cysteine 630 and the new cysteine would be the same as in the wild type and may be preferred, one cysteine remaining free and this may become involved in interchain disul®de linkage. For the D6 bp-RET the distance of the two cysteines may be too small to facilitate intrachain disul®de linkage (Bongarzone et al, 1999).…”

An in-frame complex germline mutation in the juxtamembrane intracellular domain causing RET activation in familial medullary thyroid carcinoma

“…www.endocrinology-journals.org precipitation, as previously described (Bongarzone et al 1999). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum for 72 h. NIH3T3 cells (2!10 5 ) were transfected with calcium-phosphate coprecipitation using 1 mg plasmid DNA and 25 mg mouse carrier DNA.…”

“…Immunoprecipitates from both wild-type and mutant cell lines were subjected to SDS-PAGE under reducing and non-reducing conditions, as previously described (Bongarzone et al 1999).…”

2813 Comprehensive genomic profiling of clinically advanced medullary thyroid carcinoma

“…RET M918T was the most common RET alteration, observed in 62% (21/34) of patients [17][18][19]. RET C618G/R/S was detected in three cases (9%), RET C634R and E632_L633del each in two cases (6%); RET V804M or C620R was each present in one tumor (3%) [9,[20][21][22][23][24][25]. One case harbored both a RET amplification and RET M918T [26], and no other cases had more than one RET alteration.…”