The orphan receptor GPR139 signals via Gq/11 to oppose opioid effects

Abstract

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 G_q/11 and G_i/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the G_q/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 G_q/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.

Keywords: G protein-coupled receptor (GPCR); G protein-coupled receptor 139 (GPR139); GIRK channel; GPCR signaling; adenylate cyclase (adenylyl cyclase); brain; cell signaling; cellular regulation; heterotrimeric G protein; medial habenula; neuron; opiate opioid; opioids; orphan receptor.

Figure 1.

GPR139 G protein–coupling profile. A, schematic representation of the G protein BRET fingerprinting assay in HEK293 cells. B–F, GPR139 activity on the G_i/o class (B and C), G_q/11 class (D), G_s class (E), or G_12/13 class (F) of G proteins. Amplitude and rate of activation were measured upon application of 10 μm JNJ-63533054, indicated by an arrow. Shown are the maximum amplitude (G) and the initial activation rate (H) for each G protein by GPR139. A–F, data are mean ± S.E. (error bars) of three independent experiments. G and H, mean of representative data ± S.D.

Figure 2.

GPR139 signaling to downstream effectors. A, schematic of downstream signaling pathways examined. B, calcium mobilization was measured using the CalFluxVTN calcium biosensor in response to treating HEK293 cells with 10 μm JNJ-63533054. C, the maximal amplitude of the Ca²⁺ signal across concentrations of JNJ-63533054. D, thallium flux through GIRK1/2 channels in HEK293 cells upon treatment with 10 μm JNJ-63533054. E, the initial activation rate of thallium flux calculated across doses of JNJ-63533054. F, time course of GPR139 influence on cAMP. G, dose dependence of GPR139 activation on cAMP modulation. Data are mean ± S.E. (error bars) of 3–4 independent experiments.

Figure 3.

GPR139 signals selectively through G_q/11 to secondary effectors in cultured cells. Shown are changes in forskolin-stimulated cAMP (A–D) or ML-297-mediated GIRK1/2 activation (E–H) in response to GPR139 activation. A, effect of JNJ-63533054 on FSK-stimulated cAMP response. B, effect of pretreatment with G_q inhibitor YM-254890 on GPR139-mediated cAMP modulation. C and D, quantification of the maximum amplitude (C) and the initial activation rates (D) from A and B. E, effect of GPR139 on thallium flux through GIRK1/2 channels activated by ML-297. F, quantification of the initial activation rates from E. Student's t test was used: ***, p < 0.001. G, thallium flux in GPR139-expressing cells either additionally expressing G₁₁ or pretreated with 10 μm YM-254890. H, quantification of the initial activation rates from G. Data are mean ± S.E. (error bars) of 3–5 independent experiments. C, D, and H, one-way analysis of variance with Tukey's post hoc test: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Shaded areas in A, B, E, and G are S.E.

Figure 4.

GPR139 signals via G_q/11 to oppose opioid-mediated inhibition of neuronal firing in medial habenula. A, representative firing traces recorded from medial habenular neurons in brain slices. B and C, change in firing frequency upon treatment with MOR agonist DAMGO. Slices were pretreated with vehicle (B) or 10 μm JNJ-63533054 (blue trace) (C) or with 10 μm JNJ-63533054 and 10 μm YM-254890 (green trace). D, maximum change in firing was quantified for (B and C). Data are mean ± S.E. of 8–10 neurons/trace; one-way analysis of variance with Tukey's post hoc test: ns, not significant; *, p < 0.05; **, p < 0.01.

Figure 5.

GPR139 signaling mechanisms. A, GPR139 activates both G_q/11 and G_i/o classes of G proteins, but signaling to secondary effectors is mediated by G_q/11. B, GPR139 counteracts MOR signaling by opposing regulation of the downstream effectors AC and GIRK channels.