title: Persistent cAMP signaling by internalized TSH receptors occurs in thyroid but not in HEK293 cells.
abstract: G-protein-coupled receptors (GPCRs) have long been believed to activate G proteins only on the cell surface. However, we have recently shown that, in thyroid cells, the GPCR for the thyroid-stimulating hormone (TSH) can continue stimulating cAMP production after cointernalization with TSH. cAMP signaling by internalized TSH receptors (TSHRs) was persistent, whereas that by cell-surface TSHRs was apparently transient, but the reasons for the transient signaling by cell-surface TSHRs were not investigated. Here, we developed and used fluorescence resonance energy transfer (FRET)-based methods to precisely compare the kinetics of TSH binding and dissociation from cell-surface TSHRs with those of the subsequent termination of cAMP signaling directly in living cells. Our results indicate that both TSH binding to human TSHRs expressed in a human embryonic kidney cell line (HEK 293) and the ensuing cAMP signals are rapidly and fully reversible (t(1/2,off)=2.96±1.04 and 2.70±0.73 min, respectively). The FRET measurement of TSH binding was specific, as shown by the lack of a detectable interaction between TSH and the β(2)-adrenergic receptor expressed in control cells. Enhancing TSHR internalization by β-arrestin 2 overexpression did not modify the reversibility of TSHR-cAMP signaling. These findings strengthen the view that the cointernalization of TSH-TSHR complexes to a signaling compartment present in thyroid, but not in HEK 293 cells, is responsible for persistent cAMP signaling.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany.
authors.forename: Ruth C
authors.initials: RC
authors.lastname: Werthmann
authors.forename: Silvia
authors.initials: S
authors.lastname: Volpe
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: Davide
authors.initials: D
authors.lastname: Calebiro
mesh_terms: D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D011989:Receptors, Thyrotropin / Q000378:metabolism*; D015398:Signal Transduction; D013961:Thyroid Gland / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism
chemical_list: D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP