1. RET Multiple Endocrine Neoplasia Type 2 (MEN2) Brooke Martin 3/20/08

2. History of RET Discovered in 1985 by the transfection of NIH3T3 cells with DNA from T cell lymphoma cells RET stands for –rearranged during transfection Can have either loss of function or gain of function mutations

3. RET Properties Transmembrane protein and RTK Mainly found in precursors of the urogenital system and neural crest Has 3 isoforms –short (RET9), middle(RET43), and long (RET51) Homodimer –Also pairs with GFR (growth factor receptor)α 1, 2, 3, and 4 Ligands –GDNF (glial-derived neurotrophic factor), neurturin (NTN), persephin (PSP), and artemin

4. GDNF Family with Receptors SIGMA-ALDRICH

5. RET Properties Continued Gene found on long arm of chromosome 10 at 11.2 Gene has 21 exons Cadherin part must bind with Ca 2+ in order for RET to work 5 phosphotyrosine residues –2 more in long isoform

6. Protein Structures of RET UnphosphorylatedPhosphorylated J. Biol. Chem. v281, p.33577-33587

7. What Does RET Do Normally? Helps with kidney development and enteric nervous system Also implicated in cell differentiation and apoptosis

8. Knockout Mice Knockouts had no neurons in the gut, superior cervical ganglia, no kidneys at all or malformed and malfunctioning RET null mutation die shortly after birth No endocrine organs affected in MEN 2 Heterozygotes have no apparent defects

9. MEN 2-multiple endocrine neoplasia type 2 Inherited form of cancer and very rare First to be discovered in 1993 that MEN 2 was caused by germline mutations Three subtypes –MEN 2A and MEN 2B –Familial medullary thyroid carcinoma (FMTC) Autosomal dominant RET constitutively active Endocrine glands affected –Adrenal, parathyroid, and thyroid Gain of function mutations

10. MEN 2A Codons effected 630, 634,(exon 11) or 609, 611, 618, 620 (exon10) Mutation to 634 worst 90% of MEN 2A have this. 50% have change from a cysteine to argenine How RET is active Ligand-independent dimerization, loss of disulfide bond between dimers Tumors developed Pheochromocytoma, MTC Incidence 100% get MTC, 50% pheochromocytoma, 15-30% parathyroid hyperplasia

11. MEN 2B Codons affected 918, change from methionine to threonine (exon 16) 883 in TK domain but rare (5%) Most aggressive of the three 95% have the M918T mutation What keeps RET active Made to look unlike an RTK from mutation, changes autophosphorylation Tumors developed and incidence 100% MTC, 50% pheochromocytoma, very rare parathyroid hyperplasia Pattern of phosphorylation The proteins phosphorylated differs form MEN 2A

12. FMTC Have the same mutations as 2A in extracellular domain but also can be in TK domain at 768, 790, 791,(exon 13); 804, 844,(exon 14); or 891 (exon15) Have mild C cell disease Low transforming activity can predispose to FMTC rather than MEN 2A

13. Treatment and Testing Thyroidectomy –Before age 6 or 6-12 depending on mutation –If MEN 2B, needed before a year old –Have to take thyroid for the rest of life Chemotherapy not effective Microarray (best) Direct sequencing or single-strand conformational polymorphism Drugs being tested to disrupt RET kinase activity –Needs a higher concentration though

14. Questions?