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

Abstract

A new paradigm of G-protein-coupled receptor (GPCR) signaling at intracellular sites has recently emerged, but the underlying mechanisms and functional consequences are insufficiently understood. Here, we show that upon internalization in thyroid cells, endogenous TSH receptors traffic retrogradely to the trans-Golgi network (TGN) and activate endogenous Gs-proteins in the retromer-coated compartment that brings them to the TGN. Receptor internalization is associated with a late cAMP/protein kinase A (PKA) response at the Golgi/TGN. Blocking receptor internalization, inhibiting PKA II/interfering with its Golgi/TGN localization, silencing retromer or disrupting Golgi/TGN organization all impair efficient TSH-dependent cAMP response element binding protein (CREB) phosphorylation. These results suggest that retrograde trafficking to the TGN induces local Gs-protein activation and cAMP/PKA signaling at a critical position near the nucleus, which appears required for efficient CREB phosphorylation and gene transcription. This provides a new mechanism to explain the functional consequences of GPCR signaling at intracellular sites and reveals a critical role for the TGN in GPCR signaling.

When trafficking and signaling mix: How subcellular location shapes G protein‐coupled receptor activation of heterotrimeric G proteins

“…We propose that these layers function together to determine the integrated cellular response these studies reported a second phase of GPCR and G protein activation in endosomes, with a brief (seconds to about a minute) refractory period separating the arrival of receptors in endosomes from the second activation phase. 46,53,54,56,57 Limitations of the present evidence. While there is now reasonable evidence that some GPCRs initiate G protein signaling after endocytosis, endomembrane signaling by G proteins is not proven and the present evidence supporting it has limitations and caveats.…”

“…Ligand trapping in the endosome lumen is thought to prolong or enhance GPCR responses by favoring ligand rebinding after dissociation from the receptor, and G protein activation at endosomes may differ inherently from that at the plasma membrane because of location‐specific differences in activation or regulatory machineries engaged (see below). GPCR‐G protein activation in endosomes (or the trans‐Golgi network after endocytic delivery) can also preferentially promote a subset of downstream effects such as GPCR‐dependent transcriptional responses . Studies of GPCR‐dependent transcriptional induction identified an additional function of endosomal G protein activation by GPCRs in enhancing cellular discrimination between similar ligands and increasing reliability of the cellular response …”

“…Activation is thought to occur in two basic ways: In the first, the Golgi (or trans-Golgi network (TGN))associated receptor pool arrives by cotrafficking with ligand from the plasma membrane. 56 In the second, the relevant pool of GPCRs is derived by retention or retrieval during biosynthetic trafficking; in this case, ligand access is thought to be achieved through transmembrane transporters 69,98 or, for sufficiently hydrophobic ligands, direct permeation through the membrane bilayer. 54,69 A glutamate-activated GPCR, mGluR5, localizes at steady state primarily to the endoplasmic reticulum and outer nuclear membrane of neurons.…”

Fluorescent ligands: Bringing light to emerging GPCR paradigms

“…Stimulation of the TSH receptor results in a sustained increase in intracellular cAMP levels, and by blocking internalisation through the use of dynamin inhibitors, this sustained response was lost (Calebiro et al, ). Further work demonstrated that this sustained response mediated by internalised receptors was occurring in the trans‐Golgi network, and this localised perinuclear cAMP signalling was required for efficient CREB‐mediated gene transcription (Godbole et al, ). Taken together, these data revealed the ability for the TSH receptor to signal from the endosomes and Golgi apparatus in primary cells, providing real insight into the mechanisms of TSH receptor action in an endogenous system.…”

“…Another exciting recent development in the GPCR field has been the discovery that receptors can signal from intracellular compartments, including endosomes (Calebiro, Godbole, Lyga, & Lohse, 2015;Irannejad et al, 2013) or the trans-Golgi network (Godbole, Lyga, Lohse, & Calebiro, 2017). Fluorescent ligands have been used to monitor GPCR internalisation and have found a niche in labelling untagged or endogenously expressed GPCRs (Brand, Klutz, Jacobson, Fredholm, & Schulte, 2008;Lam et al, 2018;Margathe et al, 2016;Stoddart, Vernall, Briddon, Kellam, & Hill, 2015).…”