The orphan receptor GPR139 signals via Gq/11 to oppose opioid effects
2020; Elsevier BV; Volume: 295; Issue: 31 Linguagem: Inglês
10.1074/jbc.ac120.014770
ISSN1083-351X
AutoresHannah M. Stoveken, Stefano Zucca, Ikuo Masuho, Brock Grill, Kirill A. Martemyanov,
Tópico(s)Diabetes Treatment and Management
ResumoThe interplay between G protein–coupled receptors (GPCRs) is critical for controlling neuronal activity that shapes neuromodulatory outcomes. Recent evidence indicates that the orphan receptor GPR139 influences opioid modulation of key brain circuits by opposing the actions of the µ-opioid receptor (MOR). However, the function of GPR139 and its signaling mechanisms are poorly understood. In this study, we report that GPR139 activates multiple heterotrimeric G proteins, including members of the Gq/11 and Gi/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the Gq/11 pathway and thereby distinctly regulates cellular effector systems, including stimulation of cAMP production and inhibition of G protein inward rectifying potassium (GIRK) channels. Electrophysiological recordings from medial habenular neurons revealed that GPR139 signaling via Gq/11 is necessary and sufficient for counteracting MOR-mediated inhibition of neuronal firing. These results uncover a mechanistic interplay between GPCRs involved in controlling opioidergic neuromodulation in the brain. The interplay between G protein–coupled receptors (GPCRs) is critical for controlling neuronal activity that shapes neuromodulatory outcomes. Recent evidence indicates that the orphan receptor GPR139 influences opioid modulation of key brain circuits by opposing the actions of the µ-opioid receptor (MOR). However, the function of GPR139 and its signaling mechanisms are poorly understood. In this study, we report that GPR139 activates multiple heterotrimeric G proteins, including members of the Gq/11 and Gi/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the Gq/11 pathway and thereby distinctly regulates cellular effector systems, including stimulation of cAMP production and inhibition of G protein inward rectifying potassium (GIRK) channels. Electrophysiological recordings from medial habenular neurons revealed that GPR139 signaling via Gq/11 is necessary and sufficient for counteracting MOR-mediated inhibition of neuronal firing. These results uncover a mechanistic interplay between GPCRs involved in controlling opioidergic neuromodulation in the brain. The mammalian brain features a staggering number of neuromodulatory systems that often converge on the same neurons and must be integrated and balanced through a system of opposing signaling and feedback mechanisms (1Huang Y. Thathiah A. Regulation of neuronal communication by G protein-coupled receptors.FEBS Lett. 2015; 589 (25980603): 1607-161910.1016/j.febslet.2015.05.007Crossref PubMed Scopus (52) Google Scholar). Most neuromodulators act on G protein–coupled receptors (GPCRs), an extensive protein family that triggers responses by engaging second messenger systems and ion channels. GPCR signals are relayed through an array of heterotrimeric G proteins comprised of 16 Gα subunits (2Milligan G. Kostenis E. Heterotrimeric G-proteins: a short history.Br. J. Pharmacol. 2006; 147 (16402120): S46-S5510.1038/sj.bjp.0706405Crossref PubMed Scopus (259) Google Scholar). The G protein(s) activated by a GPCR dictates the nature of the signal by engaging specific downstream targets. Ultimately, neuronal responses are built from cross-talk between GPCRs engaged by neuromodulatory inputs (1Huang Y. Thathiah A. Regulation of neuronal communication by G protein-coupled receptors.FEBS Lett. 2015; 589 (25980603): 1607-161910.1016/j.febslet.2015.05.007Crossref PubMed Scopus (52) Google Scholar, 3Greengard P. The neurobiology of slow synaptic transmission.Science. 2001; 294 (11691979): 1024-103010.1126/science.294.5544.1024Crossref PubMed Scopus (680) Google Scholar). Understanding the logic and mechanisms of this GPCR signaling coordination is critical yet poorly explored. Recently, we uncovered a novel regulatory GPCR system that revolves around GPR139 antagonizing the μ-opioid receptor (MOR) (4Wang D. Stoveken H.M. Zucca S. Dao M. Orlandi C. Song C. Masuho I. Johnston C. Opperman K.J. Giles A.C. Gill M.S. Lundquist E.A. Grill B. Martemyanov K.A. Genetic behavioral screen identifies an orphan anti-opioid system.Science. 2019; 365 (31416932): 1267-127310.1126/science.aau2078Crossref PubMed Scopus (15) Google Scholar). MOR couples to the Gi/o class of heterotrimeric G proteins to inhibit production of second messenger cAMP and to activate G protein inwardly rectifying potassium (GIRK) channels (5Al-Hasani R. Bruchas M.R. Molecular mechanisms of opioid receptor-dependent signaling and behavior.Anesthesiology. 2011; 115 (22020140): 1363-138110.1097/ALN.0b013e318238bba6Crossref PubMed Scopus (490) Google Scholar). It has been heavily targeted therapeutically for its desirable analgesic effects (6Chen Y. Mestek A. Liu J. Hurley J.A. Yu L. Molecular cloning and functional expression of a μ-opioid receptor from rat brain.Mol. Pharmacol. 1993; 44 (8393525): 8-12PubMed Google Scholar, 7Matthes H.W. Maldonado R. Simonin F. Valverde O. Slowe S. Kitchen I. Befort K. Dierich A. Le Meur M. Dollé P. Tzavara E. Hanoune J. Roques B.P. Kieffer B.L. 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Our initial examination of the intersection between MOR and GPR139 revealed that multiple aspects of MOR signaling were impinged by GPR139, which could be aided by a physical interaction between the receptors (4Wang D. Stoveken H.M. Zucca S. Dao M. Orlandi C. Song C. Masuho I. Johnston C. Opperman K.J. Giles A.C. Gill M.S. Lundquist E.A. Grill B. Martemyanov K.A. Genetic behavioral screen identifies an orphan anti-opioid system.Science. 2019; 365 (31416932): 1267-127310.1126/science.aau2078Crossref PubMed Scopus (15) Google Scholar). For instance, GPR139 enhanced β-arrestin recruitment to activated MOR and moderated its coupling to GIRK channels (4Wang D. Stoveken H.M. Zucca S. Dao M. Orlandi C. Song C. Masuho I. Johnston C. Opperman K.J. Giles A.C. Gill M.S. Lundquist E.A. Grill B. Martemyanov K.A. Genetic behavioral screen identifies an orphan anti-opioid system.Science. 2019; 365 (31416932): 1267-127310.1126/science.aau2078Crossref PubMed Scopus (15) Google Scholar). However, the exact mechanism(s) of how GPR139 exerts an inhibitory influence on MOR and its relevance to the regulation of neuronal responses remains unknown. The biology and signaling mechanisms of GPR139 are also poorly understood. It is an orphan GPCR expressed in select circuits in the brain, most notably in the areas implicated in motivation and reward (13Liu C. Bonaventure P. Lee G. Nepomuceno D. Kuei C. Wu J. Li Q. Joseph V. Sutton S.W. Eckert W. Yao X. Yieh L. Dvorak C. Carruthers N. Coate H. et al.GPR139, an orphan receptor highly enriched in the habenula and septum, is activated by the essential amino acids l-tryptophan and l-phenylalanine.Mol. Pharmacol. 2015; 88 (26349500): 911-92510.1124/mol.115.100412Crossref PubMed Scopus (29) Google Scholar, 14Matsuo A. Matsumoto S. Nagano M. Masumoto K.H. Takasaki J. Matsumoto M. Kobori M. Katoh M. Shigeyoshi Y. Molecular cloning and characterization of a novel Gq-coupled orphan receptor GPRg1 exclusively expressed in the central nervous system.Biochem. Biophys. Res. Commun. 2005; 331 (15845401): 363-36910.1016/j.bbrc.2005.03.174Crossref PubMed Scopus (42) Google Scholar, 15Süsens U. Hermans-Borgmeyer I. Urny J. Schaller H.C. Characterisation and differential expression of two very closely related G-protein-coupled receptors, GPR139 and GPR142, in mouse tissue and during mouse development.Neuropharmacology. 2006; 50 (16378626): 512-52010.1016/j.neuropharm.2005.11.003Crossref PubMed Scopus (38) Google Scholar). Whereas the endogenous ligand for GPR139 is a lingering question, it has been shown to be activated by the amino acids l-Phe and l-Trp and the neuropeptide α-MSH (13Liu C. Bonaventure P. Lee G. Nepomuceno D. Kuei C. Wu J. Li Q. Joseph V. Sutton S.W. Eckert W. Yao X. Yieh L. Dvorak C. Carruthers N. Coate H. et al.GPR139, an orphan receptor highly enriched in the habenula and septum, is activated by the essential amino acids l-tryptophan and l-phenylalanine.Mol. Pharmacol. 2015; 88 (26349500): 911-92510.1124/mol.115.100412Crossref PubMed Scopus (29) Google Scholar, 16Isberg V. Andersen K.B. Bisig C. Dietz G.P. Bräuner-Osborne H. Gloriam D.E. Computer-aided discovery of aromatic l-α-amino acids as agonists of the orphan G protein-coupled receptor GPR139.J. Chem. Inf. Model. 2014; 54 (24826842): 1553-155710.1021/ci500197aCrossref PubMed Scopus (29) Google Scholar, 17Nøhr A.C. Shehata M.A. Hauser A.S. Isberg V. Mokrosinski J. Andersen K.B. Farooqi I.S. Pedersen D.S. Gloriam D.E. Bräuner-Osborne H. The orphan G protein-coupled receptor GPR139 is activated by the peptides: adrenocorticotropic hormone (ACTH), α-, and β-melanocyte stimulating hormone (α-MSH, and β-MSH), and the conserved core motif HFRW.Neurochem. Int. 2017; 102 (27916541): 105-11310.1016/j.neuint.2016.11.012Crossref PubMed Scopus (20) Google Scholar). Additionally, several synthetic ligands can activate GPR139 with high efficiency (18Dvorak C.A. Coate H. Nepomuceno D. Wennerholm M. Kuei C. Lord B. Woody D. Bonaventure P. Liu C. Lovenberg T. Carruthers N.I. Identification and SAR of glycine benzamides as potent agonists for the GPR139 receptor.ACS Med. Chem. Lett. 2015; 6 (26396690): 1015-101810.1021/acsmedchemlett.5b00247Crossref PubMed Scopus (20) Google Scholar, 19Nøhr A.C. Shehata M.A. Palmer D. Pokhrel R. Vallianou M. Foster S.R. Gentry P.R. Gloriam D.E. Bräuner-Osborne H. Identification of a novel scaffold for a small molecule GPR139 receptor agonist.Sci. Rep. 2019; 9 (30846711): 380210.1038/s41598-019-40085-9Crossref PubMed Scopus (3) Google Scholar, 20Shi F. Shen J.K. Chen D. Fog K. Thirstrup K. Hentzer M. Karlsson J.J. Menon V. Jones K.A. Smith K.E. Smith G. Discovery and SAR of a series of agonists at orphan G protein-coupled receptor 139.ACS Med. Chem. Lett. 2011; 2 (24900311): 303-30610.1021/ml100293qCrossref PubMed Scopus (26) Google Scholar). Studies have also shown that GPR139 engages a variety of second messenger pathways when studied in transfected cells (14Matsuo A. Matsumoto S. Nagano M. Masumoto K.H. Takasaki J. Matsumoto M. Kobori M. Katoh M. Shigeyoshi Y. Molecular cloning and characterization of a novel Gq-coupled orphan receptor GPRg1 exclusively expressed in the central nervous system.Biochem. Biophys. Res. Commun. 2005; 331 (15845401): 363-36910.1016/j.bbrc.2005.03.174Crossref PubMed Scopus (42) Google Scholar, 15Süsens U. Hermans-Borgmeyer I. Urny J. Schaller H.C. Characterisation and differential expression of two very closely related G-protein-coupled receptors, GPR139 and GPR142, in mouse tissue and during mouse development.Neuropharmacology. 2006; 50 (16378626): 512-52010.1016/j.neuropharm.2005.11.003Crossref PubMed Scopus (38) Google Scholar, 21Hu L.A. Tang P.M. Eslahi N.K. Zhou T. Barbosa J. Liu Q. Identification of surrogate agonists and antagonists for orphan G-protein-coupled receptor GPR139.J. Biomol. Screen. 2009; 14 (19525486): 789-79710.1177/1087057109335744Crossref PubMed Scopus (32) Google Scholar). However, its coupling to individual G proteins and their role in propagating downstream signals has not been holistically examined, which limits our understanding of the MOR-GPR139 interplay. Here, we present the results of a systematic investigation of G protein–coupling selectivity of GPR139 and its role in signaling to downstream effectors. We also explore how GPR139 regulates MOR effects in a native neuronal setting. We establish that the key effects of GPR139 are mediated by Gq/11 signaling, which opposes the transmission of MOR signals at several intersecting points. Based on our findings, we propose that GPR139 "tunes" cellular responses to modulate opioid effects. To determine the signaling properties of GPR139, we first examined its complete G protein–coupling profile. We used a bioluminescence resonance energy transfer (BRET) GPCR "fingerprinting" approach that permits the comprehensive study of unmodified receptors and unmodified Gα proteins to examine their coupling selectivity (22Masuho I. Ostrovskaya O. Kramer G.M. Jones C.D. Xie K. Martemyanov K.A. Distinct profiles of functional discrimination among G proteins determine the actions of G protein-coupled receptors.Sci. Signal. 2015; 8 (26628681): ra12310.1126/scisignal.aab4068Crossref PubMed Scopus (110) Google Scholar). In this assay, Venus-tagged Gβ1γ2 was paired with the nanoluciferase-based sensor mas-GRK3CT. In the presence of a chosen Gα subunit, GPCR activation triggers dissociation of the G protein heterotrimer, generating the BRET signal (Fig. 1A). Using this strategy, we surveyed GPR139 activity on 15 Gα proteins (Fig. 1, B–F) and recorded both the maximum amplitude and initial activation kinetics for each G protein (Fig. 1, G and H). Interestingly, GPR139 could activate multiple G proteins with varying efficacies and kinetics. Within the Gi/o class, there were no strong preferences for individual subtypes (Fig. 1, B and C). Within the Gq/11 class, only Gq and G11 showed appreciable activation (Fig. 1D). Dose-response studies with representative members further confirmed similar potencies of GPR139 coupling to G11 and Go (Fig. S1). Gs and G12/13 classes were not activated by GPR139 (Fig. 1, E and F). Side-by-side comparison of maximum amplitudes (Fig. 1G) and the activation rates (Fig. 1H) across all classes of G proteins revealed a unique fingerprint-like profile of GPR139 coupling preferences. Together, these results indicate that GPR139 is a dual-specificity receptor capable of activating G proteins of the Gi/o and Gq/11 classes. Because GPR139 can activate multiple G protein classes, which can lead to different cellular responses, we examined the effectors engaged by GPR139. We used a panel of sensors to assay the effect of GPR139 activation on Ca2+ mobilization, ion channel regulation, and cAMP generation (Fig. 2A). When calcium levels were assayed with the BRET-based sensor CalFluxVTN (23Yang J. Cumberbatch D. Centanni S. Shi S.Q. Winder D. Webb D. Johnson C.H. Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca2+ sensing.Nat. Commun. 2016; 7 (27786307): 1326810.1038/ncomms13268Crossref PubMed Scopus (43) Google Scholar), we observed that GPR139 activation triggered a rapid and transient increase in BRET signal (Fig. 2B) suggestive of Ca2+ release from intracellular stores and consistent with previous reports (14Matsuo A. Matsumoto S. Nagano M. Masumoto K.H. Takasaki J. Matsumoto M. Kobori M. Katoh M. Shigeyoshi Y. Molecular cloning and characterization of a novel Gq-coupled orphan receptor GPRg1 exclusively expressed in the central nervous system.Biochem. Biophys. Res. Commun. 2005; 331 (15845401): 363-36910.1016/j.bbrc.2005.03.174Crossref PubMed Scopus (42) Google Scholar, 15Süsens U. Hermans-Borgmeyer I. Urny J. Schaller H.C. Characterisation and differential expression of two very closely related G-protein-coupled receptors, GPR139 and GPR142, in mouse tissue and during mouse development.Neuropharmacology. 2006; 50 (16378626): 512-52010.1016/j.neuropharm.2005.11.003Crossref PubMed Scopus (38) Google Scholar). The application of the Gαq family–selective inhibitor YM-254890 completely blocked the response, indicating that the signal was mediated by Gαq-type proteins (Fig. 2B). Dose-response studies confirmed these observations and established an EC50 of 75 ± 29 nm (Fig. 2C), which is consistent with the EC50 for G11 activation in the G protein BRET assay (119 ± 32 nm) (Fig. S1). Next, we tested the influence of GPR139 on GIRK channels, which are activated by Gβγ released preferentially by Gi/o proteins (24Breitwieser G.E. Szabo G. 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A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.J. Biomol. Screen. 2004; 9 (15634793): 671-67710.1177/1087057104268749Crossref PubMed Scopus (121) Google Scholar). The simultaneous addition of thallium and the GPR139 agonist JNJ-63533054 did not increase the rate of thallium influx compared with the slow, passive entry observed in cells without receptor (Fig. 2D). Dose-response studies quantifying changes in the slope of fluorescence showed no JNJ-63533054–induced changes at any concentration tested, confirming that GPR139 does not activate GIRK channels (Fig. 2E). Positive control experiments with MOR-transfected cells treated with morphine significantly increased the rate of GIRK channel activation, indicating adequate assay sensitivity (Fig. S2, A and B). Finally, we measured the effect of GPR139 on cAMP generation with a biosensor (Fig. 2F). Unexpectedly, we found that GPR139 stimulation dynamically increased cAMP levels. The increase occurred in a dose-dependent manner with an EC50 of 41 ± 20 nm (Fig. 2G). GPR139 expression without agonist stimulation also increased the baseline cAMP levels, suggesting a high level of constitutive activity (Fig. S3). In summary, these results indicate that GPR139 coupling to Gq/11 propagates to engage the canonical Ca2+ release pathway. However, despite the efficient coupling of GPR139 to Gi/o and no coupling to Gs/olf, we observed no GIRK channel activation and paradoxical increase in cAMP production. The seemingly contradictory results between G protein coupling and downstream modulation of cAMP warranted further investigation. First, we tested whether GPR139 can also alter cAMP dynamics after treatment with the AC activator forskolin (FSK). Preincubation with JNJ-63533054 had no effect on cAMP dynamics in control cells (Fig. S4), but in GPR139-transfected cells, we detected a substantial enhancement of cAMP generation in both the amplitude and the initial activation rate (Fig. 3, A, C, and D). Intriguingly, several AC isoforms known to be expressed in HEK293T can be regulated by calcium generated downstream of Gq/11-coupled GPCRs (28Atwood B.K. Lopez J. Wager-Miller J. Mackie K. Straiker A. Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis.BMC Genomics. 2011; 12 (21214938): 1410.1186/1471-2164-12-14Crossref PubMed Scopus (232) Google Scholar, 29Beazely M.A. Watts V.J. Gαq-coupled receptor signaling enhances adenylate cyclase type 6 activation.Biochem. Pharmacol. 2005; 70 (15885660): 113-12010.1016/j.bcp.2005.04.007Crossref PubMed Scopus (15) Google Scholar, 30Sadana R. Dessauer C.W. 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Evidence that direct binding of Gβγ to the GIRK1 G protein-gated inwardly rectifying K+ channel is important for channel activation.Neuron. 1995; 15 (7576656): 1133-114310.1016/0896-6273(95)90101-9Abstract Full Text PDF PubMed Scopus (280) Google Scholar, 26Pfaffinger P.J. Martin J.M. Hunter D.D. Nathanson N.M. Hille B. GTP-binding proteins couple cardiac muscarinic receptors to a K channel.Nature. 1985; 317 (2413367): 536-53810.1038/317536a0Crossref PubMed Scopus (579) Google Scholar, 31Huang C.L. Feng S. Hilgemann D.W. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gβγ.Nature. 1998; 391 (9486652): 803-80610.1038/35882Crossref PubMed Scopus (723) Google Scholar). Interestingly, GIRK channels have also been shown to be impacted by Gq signaling acting via PIP2 and protein kinase C to inhibit channel activity (31Huang C.L. Feng S. Hilgemann D.W. 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Pascual C. Malik A. Yang L. Zou B. Du Y. Sliwoski G. Morrison R.D. Denton J. Niswender C.M. Daniels J.S. Sulikowski G.A. Xie X.S. et al.ML297 (VU0456810), the first potent and selective activator of the GIRK potassium channel, displays antiepileptic properties in mice.ACS Chem. Neurosci. 2013; 4 (23730969): 1278-128610.1021/cn400062aCrossref PubMed Scopus (89) Google Scholar), which requires PIP2 as a cofactor, making it sensitive to Gq-mediated modulation (34Kaufmann K. Romaine I. Days E. Pascual C. Malik A. Yang L. Zou B. Du Y. Sliwoski G. Morrison R.D. Denton J. Niswender C.M. Daniels J.S. Sulikowski G.A. Xie X.S. et al.ML297 (VU0456810), the first potent and selective activator of the GIRK potassium channel, displays antiepileptic properties in mice.ACS Chem. Neurosci. 2013; 4 (23730969): 1278-128610.1021/cn400062aCrossref PubMed Scopus (89) Google Scholar, 35Wydeven N. Marron Fernandez de Velasco E. Du Y. Benneyworth M.A. Hearing M.C. Fischer R.A. Thomas M.J. Weaver C.D. Wickman K. Mechanisms underlying the activation of G-protein-gated inwardly rectifying K+ (GIRK) channels by the novel anxiolytic drug, ML297.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25002517): 10755-1076010.1073/pnas.1405190111Crossref PubMed Scopus (61) Google Scholar). Indeed, expression of GPR139 inhibited GIRK channel opening to 52.3 ± 1.6% of control (Fig. 3, E and F). Overexpression of G11 enhanced this inhibition (Fig. 3, G and H). In contrast, blockade of Gq/11 by YM-254890 diminished the inhibitory effects of GPR139 (Fig. 3, G and H). These effects were selective for GPR139 as MOR had no impact on ML-297–mediated GIRK activation either upon overexpression of G11 or blockade of Gq/11 by YM-254890 (Fig. S5, A and B). We thus conclude that GPR139 negatively regulates GIRK channel opening by engaging the Gq/11 pathway, which predominates over stimulatory Gi/o engagement. We next sought to determine the relevance of GPR139-mediated Gq/11 signaling to counteracting MOR in the endogenous setting by examining spontaneous neuron firing in the medial habenula (mHb), where MOR and GPR139 are co-expressed (4Wang D. Stoveken H.M. Zucca S. Dao M. Orlandi C. Song C. Masuho I. Johnston C. Opperman K.J. Giles A.C. Gill M.S. Lundquist E.A. Grill B. Martemyanov K.A. Genetic behavioral screen identifies an orphan anti-opioid system.Science. 2019; 365 (31416932): 1267-127310.1126/science.aau2078Crossref PubMed Scopus (15) Google Scholar, 36Boulos L.J. Darcq E. Kieffer B.L. Translating the habenula: from rodents to humans.Biol. Psychiatry. 2017; 81 (27527822): 296-30510.1016/j.biopsych.2016.06.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Application of the MOR agonist DAMGO significantly dampened neuronal firing and reversed upon DAMGO washout (Fig. 4, A, B, and D). Pretreatment with JNJ-63533054 did not change the baseline firing rate (Fig. 4A) but completely blocked DAMGO's effects on firing (Fig. 4, A, C, and D). This effect of JNJ-63533054 was specific as it did not block DAMGO influence on firing in GPR139−/− mice (Fig. S6, A and B). Importantly, blockade of Gαq/11 with YM-254890 reversed the effects of the JNJ-63533054 and restored inhibition of firing in response to DAMGO (Fig. 4, A, C, and D). Taken together, these data indicate that GPR139-initiated Gq/11 signaling is sufficient to counteract MOR-mediated effects on mHb neuron firing. Orphan GPCRs comprise ∼100 receptors with unknown ligands and often unknown physiological roles and signaling (37Sriram K. Insel P.A. G protein-coupled receptors as targets for approved drugs: how many targets and how many drugs?.Mol. Pharmacol. 2018; 93 (29298813): 251-25810.1124/mol.117.111062Crossref PubMed Scopus (321) Google Scholar) yet critical involvement in many neuronal functions (38Alavi M.S. Shamsizadeh A. Azhdari-Zarmehri H. Roohbakhsh A. Orphan G protein-coupled receptors: the role in CNS disorders.Biomed. Pharmacother. 2018; 98 (29268243): 222-23210.1016/j.biopha.2017.12.056Crossref PubMed Scopus (32) Google Scholar). Thus, elucidating their functional roles presents a compelling research frontier. GPR139 is an orphan GPCR with poorly understood biology and signaling mechanisms. It is an intriguing receptor for its selective expression pattern across brain circuits and involvement in movement (13Liu C. Bonaventure P. Lee G. Nepomuceno D. Kuei C. Wu J. Li Q. Joseph V. Sutton S.W. Eckert W. Yao X. Yieh L. Dvorak C. Carruthers N. Coate H. et al.GPR139, an orphan receptor highly enriched in the habenula and septum, is activated by the essential amino acids l-tryptophan and l-phenylalanine.Mol. Pharmacol. 2015; 88 (26349500): 911-92510.1124/mol.115.100412Crossref PubMed Scopus (29) Google Scholar) and the effects of alcohol and opioids (4Wang D. Stoveken H.M. Zucca S. Dao M. Orlandi C. Song C. Masuho I. Johnston C. Opperman K.J. Giles A.C. Gill M.S. Lundquist E.A. Grill B. Martemyanov K.A. Genetic behavioral screen identifies an orphan anti-opioid system.Science. 2019; 365 (31416932): 1267-127310.1126/science.aau2078Crossref PubMed Scopus (15) Google Scholar, 39Kononoff J. Kallupi M. Kimbrough A. Conlisk D. de Guglielmo G. George O. Systemic and intra-habenular activation of the orphan G protein-coupled receptor GPR139 decreases compulsive-like alcohol drinking and hyperalgesia in alcohol-dependent rats.eNeuro. 2018; 5 (ENEURO.0153-18.2018) (29971251)10.1523/ENEURO.0153-18.2018Crossref PubMed Scopus (19) Google Scholar). We recently found that GPR139 is molecularly intertwined with MOR and opposes its signaling, offering the first glimpse of a possible physiological role for GPR139 in regulating reward (4Wang D. Stoveken H.M. Zucca S. Dao M. Orlandi C. Song C. Masuho I. Johnston C. Opperman K.J. Giles A.C. Gill M.S. Lundquist E.A. Grill B. Martemyanov K.A. Genetic behavioral screen identifies an orphan anti-opioid system.Science. 2019; 365 (31416932): 1267-127310.1126/science.aau2078Crossref PubMed Scopus (15) Google Scholar). In this report, we provide critical insight into unanswered questions regarding GPR139 signaling mechanisms. W
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