Functional diversification of cell signaling by GPCR localization
2024; Elsevier BV; Volume: 300; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2024.105668
ISSN1083-351X
AutoresMatthew J. Klauer, Blair K. A. Willette, Nikoleta G. Tsvetanova,
Tópico(s)Pancreatic function and diabetes
ResumoG protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a critical class of regulators of mammalian physiology. Also known as seven transmembrane receptors (7TMs), GPCRs are ubiquitously expressed and versatile, detecting a diverse set of endogenous stimuli, including odorants, neurotransmitters, hormones, peptides, and lipids. Accordingly, GPCRs have emerged as the largest class of drug targets, accounting for upward of 30% of all prescription drugs. The view that ligand-induced GPCR responses originate exclusively from the cell surface has evolved to reflect accumulating evidence that receptors can elicit additional waves of signaling from intracellular compartments. These events in turn shape unique cellular and physiological outcomes. Here, we discuss our current understanding of the roles and regulation of compartmentalized GPCR signaling. G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a critical class of regulators of mammalian physiology. Also known as seven transmembrane receptors (7TMs), GPCRs are ubiquitously expressed and versatile, detecting a diverse set of endogenous stimuli, including odorants, neurotransmitters, hormones, peptides, and lipids. Accordingly, GPCRs have emerged as the largest class of drug targets, accounting for upward of 30% of all prescription drugs. The view that ligand-induced GPCR responses originate exclusively from the cell surface has evolved to reflect accumulating evidence that receptors can elicit additional waves of signaling from intracellular compartments. These events in turn shape unique cellular and physiological outcomes. Here, we discuss our current understanding of the roles and regulation of compartmentalized GPCR signaling. The classical view had been that GPCR signaling takes place at the cell surface (1Sriram K. Insel P.A. G protein-coupled receptors as targets for approved drugs: how many targets and how many drugs?.Mol. Pharmacol. 2018; 93: 251-258Google Scholar). The agonist-bound receptor undergoes a conformational change that promotes the activation of the heterotrimeric protein complex, Gαβγ. G proteins are classified into four main families according to their Gα subunit. Gαs and Gαi/o interact with adenylyl cyclase enzymes to stimulate or inhibit the production of cyclic AMP (cAMP), respectively. Gαq activates the mobilization of calcium via phospholipase C (PLC), while Gα12/13 signals vias Rho GTPase stimulation. These second messengers initiate signaling cascades that lead to the regulation of key kinases and transcription factors that coordinate a cellular response. For instance, cAMP activates protein kinase A (PKA). Following ligand binding, the GPCR undergoes post-translational modifications, including phosphorylation of its intracellular loops and/or the C-terminal tail by GPCR kinases (GRKs) (2Gurevich V.V. Gurevich E.V. GPCR signaling regulation: the role of GRKs and arrestins.Front. Pharmacol. 2019; 10: 125Google Scholar). This in turn facilitates the recruitment of β-arrestins one and 2. Classically, the association of arrestins with the receptor was presumed to terminate its signaling in two ways: by sterically hindering interactions with the G protein and by promoting GPCR internalization through the scaffolding of the AP2 adaptor complex and clathrin (2Gurevich V.V. Gurevich E.V. GPCR signaling regulation: the role of GRKs and arrestins.Front. Pharmacol. 2019; 10: 125Google Scholar, 3Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. beta-Arrestin: a protein that regulates beta-adrenergic receptor function.Science. 1990; 248: 1547-1550Google Scholar, 4Cahill 3rd, T.J. Thomsen A.R. Tarrasch J.T. Plouffe B. Nguyen A.H. Yang F. et al.Distinct conformations of GPCR-β-arrestin complexes mediate desensitization, signaling, and endocytosis.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 2562-2567Google Scholar). Subsequently, the internalized receptor is sorted and degraded in lysosome or recycled back to the plasma membrane for additional cycles of activation (Fig. 1A). This traditional model has undergone significant revision in the past 2 decades that highlights the immense complexity of the cascades and their resulting signaling outputs. For example, it is now well-appreciated that the functions of arrestins are multifaceted and extend beyond receptor desensitization. Arrestin-coordinated signaling scaffolds downstream of GPCR activation give rise to responses that are distinct from the ones driven by the G protein. While not the subject of the current review, the phenomenon of "arrestin-biased signaling" is of high physiological and pharmacological significance (5Lefkowitz R.J. Shenoy S.K. Transduction of receptor signals by beta-arrestins.Science. 2005; 308: 512-517Google Scholar, 6Shenoy S.K. Lefkowitz R.J. β-Arrestin-mediated receptor trafficking and signal transduction.Trends Pharmacol. Sci. 2011; 32: 521-533Google Scholar). A more recent shift in the classical model involves the discovery that receptors can activate G proteins from intracellular compartments. Early evidence supporting the activation of mammalian G proteins inside the cell emerged from studies of the metabotropic glutamate receptor, mGluR5, and the hormone receptors, TSHR (thyroid stimulating hormone receptor) and PTHR (parathyroid hormone receptor). There, biochemical and pharmacological manipulations were utilized to demonstrate that G protein-dependent production of the second messengers Ca2+ and cAMP is blunted in the absence of activation of the intracellular receptor fractions (7Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Google Scholar, 8Jong Y.J. Kumar V. Kingston A.E. Romano C. O'Malley K.L. Functional metabotropic glutamate receptors on nuclei from brain and primary cultured striatal neurons. Role of transporters in delivering ligand.J. Biol. Chem. 2005; 280: 30469-30480Google Scholar, 9Calebiro D. Nikolaev V.O. Gagliani M.C. de Filippis T. Dees C. Tacchetti C. et al.Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.PLoS Biol. 2009; 7e1000172Google Scholar). However, these studies relied on indirect and on in vitro methods to examine intracellular GPCR signaling (global endocytic blockade, cell fractionation, temporal correlation between receptor endocytosis and second messenger kinetics). The first direct demonstration of endosomal GPCR and G protein activity emerged from experiments utilizing nanobody-based conformational biosensors in live cells. A study employed two biosensors: Nanobody 80 (Nb80) raised against purified active beta2-adrenergic receptor (β2AR), and Nb37 recognizing a catalytic intermediate of the G protein activation cycle (10Irannejad R. Tomshine J.C. Tomshine J.R. Chevalier M. Mahoney J.P. Steyaert J. et al.Conformational biosensors reveal GPCR signalling from endosomes.Nature. 2013; 495: 534-538Google Scholar). The nanobodies co-localized with over-expressed β2AR at the cell surface and on early endosomes following ligand-induced receptor activation and internalization (10Irannejad R. Tomshine J.C. Tomshine J.R. Chevalier M. Mahoney J.P. Steyaert J. et al.Conformational biosensors reveal GPCR signalling from endosomes.Nature. 2013; 495: 534-538Google Scholar). The same strategy has since been applied to directly demonstrate the intracellular GPCR/G protein activity for several other receptors, most notably also for GPCRs expressed at native levels (11Godbole A. Lyga S. Lohse M.J. Calebiro D. Internalized TSH receptors en route to the TGN induce local G(s)-protein signaling and gene transcription.Nat. Commun. 2017; 8: 443Google Scholar, 12Stoeber M. Jullié D. Lobingier B.T. Laeremans T. Steyaert J. Schiller P.W. et al.A genetically encoded biosensor reveals location bias of opioid drug action.Neuron. 2018; 98: 963-976.e965Google Scholar, 13Puri N.M. Romano G.R. Lin T.Y. Mai Q.N. Irannejad R. The organic cation transporter 2 regulates dopamine D1 receptor signaling at the Golgi apparatus.Elife. 2022; 11https://doi.org/10.7554/eLife.75468Google Scholar, 14Irannejad R. Pessino V. Mika D. Huang B. Wedegaertner P.B. Conti M. et al.Functional selectivity of GPCR-directed drug action through location bias.Nat. Chem. Biol. 2017; 13: 799-806https://doi.org/10.1038/nchembio.2389Google Scholar, 15Nash C.A. Wei W. Irannejad R. Smrcka A.V. Golgi localized β1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy.Elife. 2019; 8https://doi.org/10.7554/eLife.48167Google Scholar). What are the roles of intracellular G protein signaling? This question is only beginning to be addressed, but emerging evidence supports that intracellular receptor activity drives discrete cellular functions. In parallel genome- and proteome-wide investigations carried out on native adrenergic receptors in HEK293 cells it was demonstrated that endosomal β2AR/cAMP signaling gives rise to unique transcriptional and phospho-signaling (16Tsvetanova N.G. Trester-Zedlitz M. Newton B.W. Peng G.E. Johnson J.R. Jimenez-Morales D. et al.Endosomal cAMP production broadly impacts the cellular phosphoproteome.J. Biol. Chem. 2021; 297100907Google Scholar, 17Tsvetanova N.G. Von Zastrow M. Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.Nat. Chem. Biol. 2014; 10: 1061-1065Google Scholar). Two orthogonal approaches were utilized to pinpoint the functional contributions of endosomal signaling: acute pharmacological inhibition of endocytosis and local cAMP production via a photoactivatable adenylyl cyclase ("opto-cyclase") tethered to different subcellular sites. With these, it was found that the entire gamut of transcriptional changes driven by adrenergic receptor activation is selectively induced from the endosome (17Tsvetanova N.G. Von Zastrow M. Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.Nat. Chem. Biol. 2014; 10: 1061-1065Google Scholar). Likewise, endosomal GPCR/cAMP was reported as the primary driver of protein phosphorylation via PKA (16Tsvetanova N.G. Trester-Zedlitz M. Newton B.W. Peng G.E. Johnson J.R. Jimenez-Morales D. et al.Endosomal cAMP production broadly impacts the cellular phosphoproteome.J. Biol. Chem. 2021; 297100907Google Scholar). Notably, by carefully matching the light stimulation conditions to yield comparable cAMP production in their opto-cyclase experiments, the authors demonstrated that the location of signaling alone, rather than the total amount of second messenger, dictates these unique outcomes (16Tsvetanova N.G. Trester-Zedlitz M. Newton B.W. Peng G.E. Johnson J.R. Jimenez-Morales D. et al.Endosomal cAMP production broadly impacts the cellular phosphoproteome.J. Biol. Chem. 2021; 297100907Google Scholar, 17Tsvetanova N.G. Von Zastrow M. Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.Nat. Chem. Biol. 2014; 10: 1061-1065Google Scholar). Similar to the β2AR, intracellular activity from endogenous TSHR/Gαs is required for the regulation of discrete PKA substrates and for the induction of several known downstream transcriptional target genes (9Calebiro D. Nikolaev V.O. Gagliani M.C. de Filippis T. Dees C. Tacchetti C. et al.Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.PLoS Biol. 2009; 7e1000172Google Scholar, 11Godbole A. Lyga S. Lohse M.J. Calebiro D. Internalized TSH receptors en route to the TGN induce local G(s)-protein signaling and gene transcription.Nat. Commun. 2017; 8: 443Google Scholar). Hence, this paradigm extends beyond a single GPCR, and it remains to be determined if the same principle applies to all receptors that signal via cAMP production. Therefore, rather than being strictly plasma membrane-delimited, receptor signaling is compartmentalized, as the cell can functionally differentiate the subcellular location of GPCR/G protein activation when initiating various responses (Fig. 1B). Although also referred to as "location bias" or "spatial bias," this review uses the term "spatial encoding" of GPCR signaling to refer to this phenomenon. To date, reports have linked virtually all sub-cellular compartments to GPCR activity transduced via different Gα subunits (18Jong Y.I. Harmon S.K. O'Malley K.L. GPCR signalling from within the cell.Br. J. Pharmacol. 2018; 175: 4026-4035Google Scholar). Examples of organelles that harbor signaling receptors include the early endosome (7Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Google Scholar, 10Irannejad R. Tomshine J.C. Tomshine J.R. Chevalier M. Mahoney J.P. Steyaert J. et al.Conformational biosensors reveal GPCR signalling from endosomes.Nature. 2013; 495: 534-538Google Scholar), very early endosome (19Sposini S. Jean-Alphonse F.G. Ayoub M.A. Oqua A. West C. Lavery S. et al.Integration of GPCR signaling and sorting from very early endosomes via opposing APPL1 mechanisms.Cell Rep. 2017; 21: 2855-2867Google Scholar), Golgi (12Stoeber M. Jullié D. Lobingier B.T. Laeremans T. Steyaert J. Schiller P.W. et al.A genetically encoded biosensor reveals location bias of opioid drug action.Neuron. 2018; 98: 963-976.e965Google Scholar, 13Puri N.M. Romano G.R. Lin T.Y. Mai Q.N. Irannejad R. The organic cation transporter 2 regulates dopamine D1 receptor signaling at the Golgi apparatus.Elife. 2022; 11https://doi.org/10.7554/eLife.75468Google Scholar, 14Irannejad R. Pessino V. Mika D. Huang B. Wedegaertner P.B. Conti M. et al.Functional selectivity of GPCR-directed drug action through location bias.Nat. Chem. Biol. 2017; 13: 799-806https://doi.org/10.1038/nchembio.2389Google Scholar), endoplasmic reticulum (20Jong Y.J. Kumar V. O'Malley K.L. Intracellular metabotropic glutamate receptor 5 (mGluR5) activates signaling cascades distinct from cell surface counterparts.J. Biol. Chem. 2009; 284: 35827-35838Google Scholar), mitochondrion (21Suofu Y. Li W. Jean-Alphonse F.G. Jia J. Khattar N.K. Li J. et al.Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E7997-E8006Google Scholar), and nucleus (22Boivin B. Lavoie C. Vaniotis G. Baragli A. Villeneuve L.R. Ethier N. et al.Functional beta-adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes.Cardiovasc. Res. 2006; 71: 69-78Google Scholar, 23Kumar V. Jong Y.J. O'Malley K.L. Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release.J. Biol. Chem. 2008; 283: 14072-14083Google Scholar). The ever-growing list of active intracellular GPCRs is comprised of receptors that couple to Gαs, Gαq, and Gαi. As we discuss below, there are ongoing efforts to shed light on the consequences and regulation of intracellular GPCRs, but based on the information at hand, the functions and mechanisms of spatial encoding are likely to be diverse. Examples of mechanisms that may promote diverse outcomes from intracellular GPCRs include the following.•Intracellular compartments can harbor receptors that couple to different G proteins: A straightforward scenario for how intracellular receptor activation can lead to discrete outcomes involves GPCR signaling via distinct G proteins. The early endosome, one of the best characterized signaling compartments to date, has been linked to the acute activation of both Gαs- (e.g., β2AR (10Irannejad R. Tomshine J.C. Tomshine J.R. Chevalier M. Mahoney J.P. Steyaert J. et al.Conformational biosensors reveal GPCR signalling from endosomes.Nature. 2013; 495: 534-538Google Scholar, 16Tsvetanova N.G. Trester-Zedlitz M. Newton B.W. Peng G.E. Johnson J.R. Jimenez-Morales D. et al.Endosomal cAMP production broadly impacts the cellular phosphoproteome.J. Biol. Chem. 2021; 297100907Google Scholar)) and Gαi-coupled receptors (e.g., delta- and mu-opioid receptors (12Stoeber M. Jullié D. Lobingier B.T. Laeremans T. Steyaert J. Schiller P.W. et al.A genetically encoded biosensor reveals location bias of opioid drug action.Neuron. 2018; 98: 963-976.e965Google Scholar)).•Receptors that signal from the same compartment and couple to the same G protein exhibit different signaling kinetics: In the case of certain receptors (e.g., Vasopressin receptor 2 (V2R) and PTHR (24Thomsen A.R.B. Plouffe B. Cahill 3rd, T.J. Shukla A.K. Tarrasch J.T. Dosey A.M. et al.GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling.Cell. 2016; 166: 907-919Google Scholar, 25Wehbi V.L. Stevenson H.P. Feinstein T.N. Calero G. Romero G. Vilardaga J.P. Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gβγ complex.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 1530-1535Google Scholar)), the endosomal population gives rise to persistent cAMP production. In contrast, other GPCRs (10Irannejad R. Tomshine J.C. Tomshine J.R. Chevalier M. Mahoney J.P. Steyaert J. et al.Conformational biosensors reveal GPCR signalling from endosomes.Nature. 2013; 495: 534-538Google Scholar, 16Tsvetanova N.G. Trester-Zedlitz M. Newton B.W. Peng G.E. Johnson J.R. Jimenez-Morales D. et al.Endosomal cAMP production broadly impacts the cellular phosphoproteome.J. Biol. Chem. 2021; 297100907Google Scholar) undergo acute and reversible G protein/cAMP activation on endosomes. Whether the distinct temporal dynamics of the endosomal cAMP signal encode additional functional information remains to be determined.•Receptors that signal from the same compartment via the same G protein take different trafficking routes to get to their destination: Although the TSHR and beta1-adrenergic receptor (β1AR) both activate Gαs at the Golgi (11Godbole A. Lyga S. Lohse M.J. Calebiro D. Internalized TSH receptors en route to the TGN induce local G(s)-protein signaling and gene transcription.Nat. Commun. 2017; 8: 443Google Scholar, 14Irannejad R. Pessino V. Mika D. Huang B. Wedegaertner P.B. Conti M. et al.Functional selectivity of GPCR-directed drug action through location bias.Nat. Chem. Biol. 2017; 13: 799-806https://doi.org/10.1038/nchembio.2389Google Scholar), these receptors traffic via distinct mechanisms. The TSHR undergoes ligand-dependent internalization into early endosomes followed by retrograde trafficking to the Golgi. On the other hand, the β1AR is delivered to the Golgi through the biosynthetic pathway. Therefore, it is likely that the Golgi activity and residence of each receptor is subject to unique regulation.•The same receptor signals from multiple intracellular compartments: Lastly, some receptors stimulate G protein signaling from multiple organelles. The Gαq-coupled GPCR mGluR5 elicits calcium responses from the ER and nuclear membranes (20Jong Y.J. Kumar V. O'Malley K.L. Intracellular metabotropic glutamate receptor 5 (mGluR5) activates signaling cascades distinct from cell surface counterparts.J. Biol. Chem. 2009; 284: 35827-35838Google Scholar, 26Sergin I. Jong Y.I. Harmon S.K. Kumar V. O'Malley K.L. Sequences within the C terminus of the metabotropic glutamate receptor 5 (mGluR5) are responsible for inner nuclear membrane localization.J. Biol. Chem. 2017; 292: 3637-3655Google Scholar). Two opioid receptors, delta (DOR) and mu (MOR), activate Gαi signaling from early endosomes and the Golgi (12Stoeber M. Jullié D. Lobingier B.T. Laeremans T. Steyaert J. Schiller P.W. et al.A genetically encoded biosensor reveals location bias of opioid drug action.Neuron. 2018; 98: 963-976.e965Google Scholar). The gamut of unique downstream responses that can be generated by the same signal originating from each of these organelles is unexplored. Recent studies have highlighted the role of endomembrane signaling in numerous physiological and pathophysiological processes and have suggested that spatial encoding of GPCR signaling can be exploited to improve therapeutic strategies that target these pathways. We highlight several of these reports below and reference additional studies that further support this model (27Jimenez-Vargas N.N. Pattison L.A. Zhao P. Lieu T. Latorre R. Jensen D.D. et al.Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7438-e7447Google Scholar, 28Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9https://doi.org/10.1126/scitranslmed.aal3447Google Scholar, 29Don-Salu-Hewage A.S. Chan S.Y. McAndrews K.M. Chetram M.A. Dawson M.R. Bethea D.A. et al.Cysteine (C)-x-C receptor 4 undergoes transportin 1-dependent nuclear localization and remains functional at the nucleus of metastatic prostate cancer cells.PLoS One. 2013; 8e57194Google Scholar, 30Gorvin C.M. Rogers A. Hastoy B. Tarasov A.I. Frost M. Sposini S. et al.AP2σ mutations impair calcium-sensing receptor trafficking and signaling, and show an endosomal pathway to spatially direct G-protein selectivity.Cell Rep. 2018; 22: 1054-1066Google Scholar, 31Bénard G. Massa F. Puente N. Lourenço J. Bellocchio L. Soria-Gómez E. et al.Mitochondrial CB₁ receptors regulate neuronal energy metabolism.Nat. Neurosci. 2012; 15: 558-564Google Scholar, 32Yarwood R.E. Imlach W.L. Lieu T. Veldhuis N.A. Jensen D.D. Klein Herenbrink C. et al.Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 12309-12314Google Scholar, 33Wang Q. Zhang H. Xu H. Guo D. Shi H. Li Y. et al.5-HTR3 and 5-HTR4 located on the mitochondrial membrane and functionally regulated mitochondrial functions.Sci. Rep. 2016; 637336Google Scholar, 34Revankar C.M. Cimino D.F. Sklar L.A. Arterburn J.B. Prossnitz E.R. A transmembrane intracellular estrogen receptor mediates rapid cell signaling.Science. 2005; 307: 1625-1630Google Scholar, 35Jimenez-Vargas N.N. Gong J. Wisdom M.J. Jensen D.D. Latorre R. Hegron A. et al.Endosomal signaling of delta opioid receptors is an endogenous mechanism and therapeutic target for relief from inflammatory pain.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 15281-15292Google Scholar, 36Tadevosyan A. Vaniotis G. Allen B.G. Hébert T.E. Nattel S. G protein-coupled receptor signalling in the cardiac nuclear membrane: evidence and possible roles in physiological and pathophysiological function.J. Physiol. 2012; 590: 1313-1330Google Scholar, 37Joyal J.S. Nim S. Zhu T. Sitaras N. Rivera J.C. Shao Z. et al.Subcellular localization of coagulation factor II receptor-like 1 in neurons governs angiogenesis.Nat. Med. 2014; 20: 1165-1173Google Scholar, 38Latorre R. Hegron A. Peach C.J. Teng S. Tonello R. Retamal J.S. et al.Mice expressing fluorescent PAR(2) reveal that endocytosis mediates colonic inflammation and pain.Proc. Natl. Acad. Sci. U. S. A. 2022; 119https://doi.org/10.1073/pnas.2112059119Google Scholar). Work on GPCRs coupled to each of the main Gα proteins supports that compartmentalized receptor activity critically shapes physiology across tissues. In the brain, key neuronal processes are mediated by mGluR5/Gαq signaling. It was found that the metabotropic glutamate receptor is enriched in the perinuclear region of hippocampal neurons, and this intracellular population is required to elicit sustained Gαq/Ca2+ signaling and long-term depression (LTD) after chemical and low-frequency stimulation (39Purgert C.A. Izumi Y. Jong Y.J. Kumar V. Zorumski C.F. O'Malley K.L. Intracellular mGluR5 can mediate synaptic plasticity in the hippocampus.J. Neurosci. 2014; 34: 4589-4598Google Scholar). Similarly, intracellular signaling is the driver of essential physiology for another neuronal GPCR, the melatonin type 1 receptor (MT1R), which couples to Gαi. Stimulation of MT1R on the mitochondrial membrane of cerebrocortical neurons has neuroprotective effects via blockade of cytochrome c release and associated caspase activation (21Suofu Y. Li W. Jean-Alphonse F.G. Jia J. Khattar N.K. Li J. et al.Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E7997-E8006Google Scholar). Intracellular GPCR/G protein activation is also important outside of the central nervous system. In kidney cells, this persistent activity of endosomal V2R leads to the translocation of aquaporin water channels and epithelial ion channels to the apical membrane, resulting in increased water and sodium permeability (40Feinstein T.N. Yui N. Webber M.J. Wehbi V.L. Stevenson H.P. King Jr., J.D. et al.Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin.J. Biol. Chem. 2013; 288: 27849-27860Google Scholar). While physiologically advantageous in the contexts outlined above, intracellular GPCRs have also been implicated as drivers of disease. For example, selective stimulation of Golgi-localized β1AR in cardiac myocytes results in cardiac hypertrophy through Gαs-dependent induction of phosphoinositide hydrolysis (15Nash C.A. Wei W. Irannejad R. Smrcka A.V. Golgi localized β1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy.Elife. 2019; 8https://doi.org/10.7554/eLife.48167Google Scholar). Likewise, nerve injury in spinal dorsal horn neurons leads to an increase in the population of perinuclear mGluR5 and neuropathic pain (41Vincent K. Cornea V.M. Jong Y.I. Laferriere A. Kumar N. Mickeviciute A. et al.Intracellular mGluR5 plays a critical role in neuropathic pain.Nat. Commun. 2016; 710604Google Scholar). The demonstration that intracellular GPCR/G protein signaling has physiological and pathophysiological implications has raised the intriguing possibility that this phenomenon can be exploited therapeutically. One strategy would rely on the distinct pharmacodynamic properties of existing compounds in order to selectively target the receptor at a given compartment. Of specific interest are the affinity, hydrophobicity, and endocytic efficacy of the ligand. Indeed, several studies have provided proof-of-principle for the utility of such approaches. Long-acting agonists that remain bound to the PTHR following internalization into endosomes stimulate sustained Gαs/cAMP signaling and elicit elevated serum calcium and serum vitamin D (7Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Google Scholar, 42White A.D. Pena K.A. Clark L.J. Maria C.S. Liu S. Jean-Alphonse F.G. et al.Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.Sci. Signal. 2021; 14eabc5944Google Scholar). This contrasts with an agonist that dissociates rapidly from the receptor and does not induce endosomal signaling (7Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Google Scholar, 43Horwitz M.J. Tedesco M.B. Sereika S.M. Syed M.A. Garcia-Ocaña A. Bisello A. et al.Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)2 vitamin D.J. Bone Miner Res. 2005; 20: 1792-1803Google Scholar, 44Horwitz M.J. Tedesco M.B. Sereika S.M. Hollis B.W. Garcia-Ocaña A. Stewart A.F. Direct comparison of sustained infusion of human parathyroid hormone-related protein-(1-36) [hPTHrP-(1-36)] versus hPTH-(1-34) on serum calcium, plasma 1,25-dihydroxyvitamin D concentrations, and fractional calcium excretion in healthy human volunteers.J. Clin. Endocrinol. Metab. 2003; 88: 1603-1609Google Scholar). Further, a pharmacologic approach relying on agonists and antagonists with distinct hydrophobicity has been applied to selectively inactivate the pathological functions of several intracellular GPCRs. As one example, the increase in neuropathic pain following perinuclear mGluR5 activation in the model of nerve injury that we discuss above can be attenuated by the application of a membrane-permeant mGluR5 antagonist (41Vincent K. Cornea V.M. Jong Y.I. Laferriere A. Kumar N. Mickeviciute A. et al.Intracellular mGluR5 plays a critical role in neuropathic pain.Nat. Commun. 2016; 710604Google Scholar). Similarly, beta-adrenergic receptor antagonists that can access the Golgi-localized population of β1ARs are effective at preventing cardiac hypertrophy compared to antagonists that cannot permeate the plasma membrane (15Nash C.A. Wei W. Irannejad R. Smrcka A.V. Golgi localized β1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy.Elife. 2019; 8https://doi.org/10.7554/eLife.48167Google Scholar). The range of endocytic efficacies of GPCR agonists presents yet another important tool to manipulate compartmentalized receptor activity. For the β2AR, it was reported that dopamine, a ligand that does not induce robust internalization, leads to weaker transcriptional signaling compared to epinephrine, a ligand that drives receptor endocytosis (17Tsvetanova N.G. Von Zastrow M. Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.Nat. Chem. Biol. 2014; 10: 1061-1065Google Scholar). In addition to targeting the receptor itself, the transporters responsible for ligand entry into the cells and into the lumen of organelles could also be manipulated for therapeutic purposes. Catecholamines are transported into the cell via organic cation transporters (OCT proteins). Inhibition of OCT3 with corticosterone in cultured cardiac myocytes abolishes norepinephrine-induced β1AR activation at the Golgi and subsequent cardiac hypertrophy (15Nash C.
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