title: Interactions between the extracellular domain and the extracellular loops as well as the 6th transmembrane domain are necessary for TSH receptor activation.
abstract: OBJECTIVE
  The molecular mechanisms of TSH receptor (TSHR) activation and intramolecular signal transduction are largely unknown. Deletion of the extracellular domain (ECD) of the TSHR results in increased constitutive activity, which suggests a self-inhibitory interaction between the ECD and the extracellular loops (ECLs) or the transmembrane domains (TMDs). To investigate these potential interactions and to pursue the idea that mutations in the ECD affect the constitutive activity of mutants in the ECLs or TMDs we generated double mutants between position 281 in the ECD and mutants in all three ECLs as well as the 6th TMD.
  
  
  METHODS
  We combined mutation S281D, characterized by an impaired TSH-stimulated cAMP response, with the constitutively activating in vivo mutations I486F (1st ECL), I568T (2nd ECL), V656F (3rd ECL) and D633F (6th TMD). Further, we constructed double mutants containing the constitutively activating mutation S281N and one of the inactivating mutations D474E, T477I (1st ECL) and D633K (6th TMD).
  
  
  RESULTS
  The cAMP level of the double mutants with S281N and the inactive mutants in the 1st ECL was decreased below the level of the inactive single mutants, demonstrating that a constitutively activating mutation in the ECD cannot bypass disruption of signal transduction in the serpentine domain. In double mutants with S281D, basal and TSH-induced cAMP and inositol phosphate production of constitutively active mutants was reduced to the level of S281D.
  
  
  CONCLUSIONS
  The dominance of S281D and the dependence of constitutively activating mutations in the ECLs on the functionally intact ECD strongly suggest that interactions between these receptor domains are required for TSHR activation and intramolecular signal transduction.
authors.affiliation: III Medical Department, University of Leipzig, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Neumann
authors.forename: Maren
authors.initials: M
authors.lastname: Claus
authors.forename: Ralf
authors.initials: R
authors.lastname: Paschke
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011869:Radioligand Assay; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013972:Thyrotropin / Q000494:pharmacology; D014162:Transfection
chemical_list: D007295:Inositol Phosphates; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP