25. 1) Mouse model for Hirschsprung disease/ MEN 2A cancer syndrome; 2) Inactivating effect of Met688Thr mutation in TRKA receptor
Record Control Number:
Quality Validation Date:
Abstract: 1) The Cys620Arg mutation of RET tyrosine kinase receptor is associated with MEN 2A cancer syndrome and Hirschsprung disease. Mouse model reproducing these pathological phenotypes will be fruitful to study disease appearance, evolution and to develop effective therapeutic tools, for these almost always fatal diseases.
2) We have investigated the effect on TRKA receptor activity of a mutation equivalent to the Met918Thr mutation affecting Ret in MEN2B patients. We have introduced the Met688Thr mutation into both the TRKA receptor and TRK-T3 oncogene cDNAs.
Biochemical and biological studies indicate that, contrariwise to the effect exerted in Ret, theMet688Thr mutation causes inactivation of the TRKA receptor. Since these mutations all occur in a highly conserved TK domain of the studied receptors, the reported contrasting result could have a great importance in a context of drugs aimed at the development of specific inhibitors.
1) The RET proto-oncogene is one of the receptor tyrosine kinases, which are cell-surface molecules that transduce signals for cell growth and differentiation. Mutations in the RET gene are associated with the disorders multiple endocrine neoplasia, type 2A (MEN 2A), multiple endocrine neoplasia, type 2B, Hirschsprung disease (HSCR; aganglionic megacolon) and medullary thyroid carcinoma. The Cys620Arg mutation is associated with both MEN 2A and HSRC exerting a dual effect on RET. It leads to a decrease of the receptor at the cell surface and converts RET into a constitutive activated kinase due to the formation of disulphide-linked homodimers. We have introduced Cys620Arg mutation in cloned RET mouse sequences inserted in a appropriate targeting vector to be introduced in ES (embrional stem) cells. ES cell clones containing Cys620Arg mutation will be selected and used to produce mice carrying the mutation in the heterozygous state.
2) With the final goal to explore the consequences of the constitutive TRKA activation in mouse models, we decided to introduce in TRKA point mutations activating other RTK genes and associated with human cancer. We choose the following mutations: 1) TRKA Met668Thr: is the equivalent of the Ret Met668Thr mutation associated with MEN2B syndrome; 2) TRKA Asp668Val: is the equivalent of the Kit Asp816Val mutation associated with mast cell neoplasms. TRKA and TRK-T3 cDNA carrying the mutations have been produced. Biochemical analysis of mutated proteins expressed into mammalian cells showed that, upon NGF treatment, the TRKA Asp668Val receptor undergoes autophosphorylation, similarly to the wild type; on the contrary, the Met688Thr mutant remains unphosphorylated. The constitutive autophosphorylation of the TRK-T3 oncogene is unaffected by the 668 mutations, whereas is completely abolished by the 688 mutation.
Biological studies in NIH3T3 cells revealed that the transforming activity of wild type receptor in the presence of NGF is retained by the 668 mutant, whereas is completely lost by the 688 mutant. Similarly, the TRK-T3 transforming activity is unaffected by the 668 mutation, whereas it is abrogated by the Met688Thr mutations. Altogether these results indicate that, although occurring in regions conserved among different RTKs, the activated mutations investigated exert different effect on TRKA activity.
Subject Descriptors: Biotechnology; Surgery; Medicine
Subject Index Codes: Medicine, Health; Biotechnology
Stage of Development: Intermediate design, research phase
Collaboration Sought: Further research or development support,Licence agreement,Financial support,Information exchange/Training
Collaboration Detail: 1) A mouse carrying a single point mutation in the RET gene responsible of MEN 2A and HSRC diseases to be used to screen compounds direct to correct RET dysfunctions.
2) The Met688Thr TRKA mutant could be exploited by structural biochemistry. The definition of the structural features modified by the mutation would give an important contribution to the understanding of tyrosine kinase receptors mechanism of action.