title: Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist.
abstract: Several lines of evidence indicate that constraining intramolecular interactions between transmembrane domains are required to maintain G protein-coupled receptors in an inactive conformation in the absence of agonist. For the glycoprotein hormone receptors, which harbor a long amino-terminal ectodomain responsible for hormone binding, it has been suggested that the ectodomain could contribute to these negative constraints. To test this hypothesis, we expressed at the surface of COS-7 cells mutants of the TSH receptor in which variable portions of the amino-terminal ectodomain are replaced by a 19-residue tag from bovine rhodopsin. Whereas none of the rhodopsin-tagged truncated mutants could be activated by saturating concentrations of TSH, the constructs with the shortest amino-terminal extension displayed increased constitutive activity toward the cAMP pathway, when compared with the wild-type holoreceptor. The shortest truncated construct was strongly activated by the introduction of mutations in transmembrane segment VI (D633A), or in the third intracellular loop (A623I) of the receptor. The magnitude of the stimulation was similar to that observed when the same mutations were introduced in the intact wild-type receptor. On the contrary, the shortest truncated construct was unaffected by activating mutations affecting residues of the extracellular loop region (I486F, I568T) or the top of transmembrane segment VII (del658-661). Together, our results are compatible with a model in which activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to a true agonist.
authors.affiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, Campus Erasme, B-1070 Bruxelles, Belgium. gvassert@ulb.ac.be
authors.forename: Virginie
authors.initials: V
authors.lastname: Vlaeminck-Guillem
authors.forename: Su-Chin
authors.initials: SC
authors.lastname: Ho
authors.forename: Patrice
authors.initials: P
authors.lastname: Rodien
authors.forename: Gilbert
authors.initials: G
authors.lastname: Vassart
authors.forename: Sabine
authors.initials: S
authors.lastname: Costagliola
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D000242:Cyclic AMP / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D011989:Receptors, Thyrotropin / Q000502:physiology*; D012243:Rhodopsin / Q000737:chemistry; D013329:Structure-Activity Relationship; D013972:Thyrotropin / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D012243:Rhodopsin; D000242:Cyclic AMP