title: Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.
abstract: Parathyroid hormone (PTH) and its related receptor (PTHR) are essential regulators of calcium homeostasis and bone physiology. PTH activates PTHR by interacting with a ligand-binding site localized within the N-terminal extracellular domain (the N-domain) and the domain comprising the seven transmembrane helices and the connecting extracellular loops (the J-domain). PTH binding triggers a conformational switch in the receptor, leading to receptor activation and subsequent cellular responses. The process of receptor activation occurs rapidly, within approximately 1 s, but the binding event preceding receptor activation is not understood. By recording FRET between tetramethyl-rhodamine in PTH(1-34) and GFP in the N-domain of the receptor, we measured the binding event in real time in living cells. We show that the association time course between PTH(1-34) and PTHR involves a two-step binding process where the agonist initially binds the receptor with a fast time constant (tau approximately 140 ms) and then with slower kinetics (tau approximately 1 s). The fast and slow phases were assigned to hormone association to the receptor N- and J domains, respectively. Our data indicate that the slow binding step to the J-domain coincides with a conformational switch in the receptor, also monitored by FRET between the enhanced cyan fluorescent protein and the enhanced yellow fluorescent protein in the PTHR sensor, PTHR enhanced cyan fluorescent protein/enhanced yellow fluorescent protein (PTHR(CFP/YFP)). These data suggest that the conformational change that switches the receptor into its active state proceeds in a sequential manner, with the first rapid binding step event preceding receptor activation by PTH(1-34).
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany.
authors.forename: Marián
authors.initials: M
authors.lastname: Castro
authors.forename: Viacheslav O
authors.initials: VO
authors.lastname: Nikolaev
authors.forename: Dieter
authors.initials: D
authors.lastname: Palm
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Vilardaga
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D010281:Parathyroid Hormone / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1