title: Lack of in vitro constitutive activity for four previously reported TSH receptor mutations identified in patients with nonautoimmune hyperthyroidism and hot thyroid carcinomas.
abstract: OBJECTIVE
  Constitutively activating mutations (CAMs) of the TSHR are the major cause for nonautoimmune hyperthyroidism. Re-examination of constitutive activity previously determined in CHO cell lines recently demonstrated the caveats for the in vitro determination of constitutive TSHR activity, which leads to false positive conclusions regarding the molecular origin of hyperthyroidism or hot thyroid carcinomas.
  
  
  METHODS
  Mutations L677V and T620I identified in hot thyroid carcinomas were previously characterized in CHO and in 3T3-Vill cell lines, respectively, stably expressing the mutant without determination of TSHR expression. F666L identified in a patient with hot thyroid nodules, I691F in a family with nonautoimmune hyperthyroidism and F631I identified in a hot thyroid carcinoma were not characterized for their in vitro function. Therefore, we decided to (re)evaluate the in vitro function of these five TSHR variants by determination of cell surface expression, and intracellular cAMP and inositol phosphate levels and performed additionally linear regression analyses to determine basal activity independently from the mutant's cell surface expression in COS-7 and HEK(GT) cells.
  
  
  CONCLUSIONS
  Only one (F631I) of the five investigated TSHR variants displayed constitutive activity for G(α) s signalling and showed correlation with the clinical phenotype. The previous false classification of T620I and L677V as CAMs is most likely related to the fact that both mutations were characterized in cell lines stably expressing the mutated receptor construct without assessing the respective receptor number per cell. Other molecular aetiologies for the nonautoimmune hyperthyroidism and/or hot thyroid carcinomas in these three patients and one family should be elucidated.
authors.affiliation: Department for Internal Medicine, Endocrinology and Nephrology, University of Leipzig, Leipzig, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Jaeschke
authors.forename: Sandra
authors.initials: S
authors.lastname: Mueller
authors.forename: Markus
authors.initials: M
authors.lastname: Eszlinger
authors.forename: Ralf
authors.initials: R
authors.lastname: Paschke
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006980:Hyperthyroidism / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000378:metabolism*; D013964:Thyroid Neoplasms / Q000235:genetics*
chemical_list: D011989:Receptors, Thyrotropin; D000242:Cyclic AMP