Internalized TSH receptors en route to the TGN induce local Gs-protein signaling and gene transcription

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  • Institute of Pharmacology and Toxicology and Bioimaging Center, University of Wuerzburg, Wuerzburg, Germany.


G-protein-coupled receptors (GPCRs) constitute the largest family of receptors for hormones and neurotransmitters1, 2. They are implicated in several human diseases and are major pharmacological targets3. For all these reasons, their signaling mechanisms have been intensively investigated. Signaling by GPCRs is initiated by binding of an agonist to a receptor1, 2, 7. The ensuing conformational change in the receptor is then relayed via activation of heterotrimeric G-proteins to effectors located at the cell membrane1, 2, 7. Among these, adenylyl cyclases play a major role in GPCR signaling by generating the soluble second messenger cyclic AMP (cAMP), which is implicated in the regulation of a plethora of cellular functions, such as gene transcription and cell proliferation1, 2, 7. These effects of cAMP are mediated by activation of cAMP effectors, which include guanine nucleotide exchange proteins activated by cAMP (Epac), cyclic nucleotide gated (CNG) ion channels and protein kinase A (PKA)8. In particular, the translocation of PKA catalytic subunit to the nucleus in response to GPCR activation regulates gene transcription via phosphorylation of transcription factors of the cAMP response element binding protein(CREB)/activating transcription factor (ATF) family9. Although cAMP is a small diffusible molecule, it has been hypothesized that cAMP/PKA signaling is compartmentalized such that cAMP and/or PKA would exert their effects only close to their site of production/activation8, 10, 11, 12, 13, 14. While this hypothesis is consistent with a number of experimental observations, a direct proof that cAMP/PKA microcompartments are involved in GPCR signaling is still missing8, 12. Although several GPCRs rapidly internalize upon agonist stimulation and traffic through various intracellular compartments, signaling by GPCRs has long been believed to occur only at the cell surface. The only exception was represented by the G-protein-independent and -arrestin-dependent activation of the mitogen-activated protein kinase (MAPK) pathway, which was shown to happen also on endosomal membranes15. However, recent findings have challenged this classical model of GPCR signaling by suggesting that internalized GPCRs can activate G-protein-dependent pathways at intracellular sites4, 5, 6, 16, 17, 18, 19, 20, 21.
The first direct evidence that internalized ligand/receptor complexes can stimulate cAMP production after their internalization came from studies of two typical protein hormone receptors: one by our group on the 4
thyroid stimulating hormone receptor (TSHR)4 and one by another group on the parathyroid hormone (PTH) receptor5. These studies independently suggested that GPCRs are able of inducing a second, persistent phase of cAMP production once internalized. Importantly, recent data suggest that signaling by internalized GPCRs is biologically relevant21, 22, as it has been implicated in the regulation of gene transcription22 or even in the ultimate biological effects of a hormone21. However, the mechanisms linking GPCR signaling at intracellular sites to these biological effects are insufficiently understood23, 24, 25, 26.
Most of the available information regarding the site of intracellular GPCR signaling comes from experiments in which receptors have been overexpressed in heterologous cell lines. In these experiments, PTH5 and 2-adrenergic6 receptors have been found to colocalize with Gs-proteins on early endosomes. Moreover, a retrograde trafficking of PTH and vasopressin V2 receptors to the trans-Golgi network (TGN) has been shown to terminate cAMP signaling20, 27. These data, together with direct evidence for the β2-adrenergic receptor using conformational biosensors6, suggest early endosomes as the site of intracellular cAMP signaling. However, since trafficking mechanisms may differ substantially among cell types and might be affected by the level of expression of the receptor28, it remains to be demonstrated whether the same mechanisms also operate on endogenous receptors in their native context. Furthermore, important differences might exist among individual receptors. Indeed, in our initial study on the TSHR, we found, based on immunofluorescence, colocalization between internalized TSH/TSHR complexes and Gs-protein in the Golgi/TGN4. This raises the intriguing possibility that, besides on early endosomes, GPCR signaling might also occur at other intracellular compartments.
In this study, we address these important questions by following the internalization/trafficking of an endogenous receptor in real time, using the TSHR in primary thyroid cells as a model, while directly visualizing the intracellular sites of Gs-protein activation and monitoring local cAMP/PKA signaling. We find that internalized TSHRs induce Gs-protein activation in a retromer-coated compartment (RCC) that is an extension of the TGN and mediates retrograde trafficking to this organelle, which is accompanied by a late phase of cAMP/PKA signaling at the Golgi/TGN. Interfering with receptor internalization, retrograde trafficking or the organization of the Golgi/TGN impair nuclear signaling in response to TSH. These data 5 indicate a new potential role for the TGN as a central hub for GPCR trafficking and signaling and provide a new mechanism to explain the cellular effects of GPCR signaling at intracellular sites.


Original languageEnglish
Article number443
JournalNature Communications
Publication statusPublished - 5 Sep 2017


  • Fluorescence imaging, G protein-coupled receptors, Hormone receptors