title: Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation.
abstract: The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor.
authors.affiliation: Department of Internal Medicine, University of Leipzig, 04103 Leipzig, Germany.
authors.forename: Joerg
authors.initials: J
authors.lastname: Schaarschmidt
authors.affiliation: Department of Internal Medicine, University of Leipzig, 04103 Leipzig, Germany,; Department of Proteomics, Helmholtz-Centre for Environmental Research, 04318 Leipzig, Germany.
authors.forename: Marcus B M
authors.initials: MBM
authors.lastname: Nagel
authors.affiliation: Department of Internal Medicine, University of Leipzig, 04103 Leipzig, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Huth
authors.affiliation: Department of Internal Medicine, University of Leipzig, 04103 Leipzig, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Jaeschke
authors.affiliation: Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37235.
authors.forename: Rocco
authors.initials: R
authors.lastname: Moretti
authors.affiliation: Institute of Materials Science, Max Bergmann Center of Biomaterials, TU Dresden, 01069 Dresden, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Hintze
authors.affiliation: Department of Proteomics, Helmholtz-Centre for Environmental Research, 04318 Leipzig, Germany,; Faculty of Biosciences, Pharmacy and Psychology, Institute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany,; Aalborg University, Department of Chemistry and Bioscience, Fredrik Bajers Vej 7, 9220 Aalborg, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: von Bergen
authors.affiliation: Department of Proteomics, Helmholtz-Centre for Environmental Research, 04318 Leipzig, Germany,; Department of Bioanalytics, University of Applied Sciences and Arts of Coburg, 96450 Coburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kalkhof
authors.affiliation: Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37235.
authors.forename: Jens
authors.initials: J
authors.lastname: Meiler
authors.affiliation: Division of Endocrinology and Metabolism and Arnie Charbonneau Cancer Institute University of Calgary, Calgary, Alberta T2N 1N4, Canada. Electronic address: ralf.paschke@ucalgary.ca.
authors.forename: Ralf
authors.initials: R
authors.lastname: Paschke
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D059748:Proteolysis; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D011989:Receptors, Thyrotropin
keywords: G protein-coupled receptor (GPCR); cell signaling; computer modeling; mass spectrometry (MS); protein cross-linking; receptor structure-function; site-directed mutagenesis; structural biology; surface plasmon resonance (SPR)