Oligomerization of MrgC11 and μ-opioid receptors in sensory neurons enhances morphine analgesia

Abstract

The μ-opioid receptor (MOR) agonist morphine is commonly used for pain management, but it has severe adverse effects and produces analgesic tolerance. Thus, alternative ways of stimulating MOR activity are needed. We found that MrgC11, a sensory neuron-specific G protein-coupled receptor, may form heteromeric complexes with MOR. Peptide-mediated activation of MrgC11 enhanced MOR recycling by inducing coendocytosis and sorting of MOR for membrane reinsertion. MrgC11 activation also inhibited the coupling of MOR to β-arrestin-2 and enhanced the morphine-dependent inhibition of cAMP production. Intrathecal coadministration of a low dose of an MrgC agonist potentiated acute morphine analgesia and reduced chronic morphine tolerance in wild-type mice but not in Mrg-cluster knockout (Mrg KO) mice. BAM22, a bivalent agonist of MrgC and opioid receptors, enhanced the interaction between MrgC11 and MOR and produced stronger analgesia than did the individual monovalent agonists. Morphine-induced neuronal and pain inhibition was reduced in Mrg KO mice compared to that in wild-type mice. Our results uncover MrgC11-MOR interactions that lead to positive functional modulation of MOR. MrgC shares genetic homogeneity and functional similarity with human MrgX1. Thus, harnessing this positive modulation of MOR function by Mrg signaling may enhance morphine analgesia in a sensory neuron-specific fashion to limit central side effects.

Fig. 1.. Interaction between MrgC11 and MOR.

(A) Immunostaining for MrgC and MOR in DRG neurons of wild-type (WT) mice and rats (n = 3 animals per group). Arrowheads, (strongly) double-labeled cells; arrows, single-labeled cells. Right: Venn diagrams portray cells with strong coexpression. Scale bars, 50 mm. (B) Left: Schematic shows the main principles of the PLA. Right: PLA signal (red) for colocalization of MrgC and MOR antibodies in DRG neurons from WT and Mrg KO mice. DAPI (4’,6-diamidino-2-phenylindole) (blue) counterstained the nuclei. Images are representative of four experiments. Scale bars, 20 mm. (C) Left: Amino acid sequences of Met-ENK, Leu-ENK, BAM22, and BAM8–22 from the same precursor, proenkephalin (PENK), in which the consensus sequence (blue) binds the opioid receptor in the N terminus and the three-amino acid sequence (red) binds MrgC in the C terminus. Right: Diagram showing how BAM22 binds to both MOR and MrgCII. (D) Immunoprecipitation (IP) of Myc and immunoblotting (IB) of solubilized protein extracts derived from HEK293T cells transfected with Myc-MrgC11 and FLAG-MOR. (E) As described in (D), cells cotransfected with Myc-MrgC11 and FLAG-MOR were treated with bath application of full-length BAM22 or BAM1–7. (F) As described in (D), from cells cotransfected with FLAG-MOR and Myc-MrgC11, Myc-MrgA3, or Myc-MrgD. (D to F) Data are representative of three to four experiments.

Fig. 2.. MrgC11 interacts with MOR through the CTD.

(A) Co-IP to examine the interaction between FLAG-MOR and WT Myc-MrgC11 or two mutant MrgC11s in HEK293T cells. In two mutant MrgC11s, the second TM2 region was swapped with TM6 of MrgC11 (Myc-MrgC11-TM2^TM6) or TM2 of MrgD (Myc-MrgC11-TM^2D). (B) The diagram shows WT MrgC11; mutant MrgC11s in which the CTD was substituted with that of MrgA3 (MrgC11-CTD^A3) or MrgD (MrgC11-CT^DD); MrgC11 CTD (MrgC11 ^CTD); and MOR CTD (MOR^CTD). (C) Co-IP to examine the interaction between FLAG-MOR and WT Myc-MrgC11, MrgC11-CTD^A3, and MrgC11-CT^DD mutants in transfected cells. (D) Co-IP to examine the interaction between FLAG-MOR and CTD of MrgC11 (MrgC11^CTD-GFP). (E) Co-IP to examine MrgC11-MOR interaction in the presence of MrgC11^CID-GFP. (F) Left: PLA of HEK293T cells cotransfected with FLAG-MOR and WT Myc-MrgC11, mutant MrgC11-CTD^A3, or MrgC11-CTD^D. Scale bar, 20 μm. Right: Quantification of PLA signal (n = 10 experiments per group). (G and H) Co-IP to examine the interaction between MOR^CTD-GFP and WT Myc-MrgC11 (G) and between Myc-MOR^CTD and MrgC11^CTD-GFP (H). (I) Co-IP to examine whether expression of MOR^ctd-GFP reduces MOR and MrgC11 interaction. (F) ***P < 0.001 versus Myc-MrgC11 by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Values are mean ± SEM. Data in (A), (C) to (E), and (G) to (I) are representative of three to four experiments.

Fig. 3.. MrgC11 activation leads to co-internalization and targeting of surface MORs into the recycling pathway.

(A) Diagram depicts FLAG-MOR and Myc-MrgC11 at the cell surface. Representative images show co-internalization of FLAG-MOR and Myc-MrgC11 in HEK293T cells 45 min after administration of BAM8–22 (5 mM), morphine (5 mM), or both. Arrowheads, internalized receptors. Scale bar, 10 mm. Data are representative of three experiments. (B) Experimental design and quantitative data show the ratio of internalized MrgC11 and MOR versus total surface immunostaining (n = 23 to 40 cells). (C) Immunoblotting and quantitative data of MOR and MrgC11 on the cell surface before and after BAM8–22 (5 mM, n = 11 per group). (D) Left: Representative images show that MORs (arrows) were co-internalized with MrgC11 after BAM8–22 exposure and were not sorted into lysosome-like compartments labeled by LysoTracker (arrowheads). Right: Quantification of receptor-containing vesicles labeled by LysoTracker (n = 26 per group). Scale bar, 10 mm. (E) Left: Immunoblotting showed a change of biotinylated receptors after a second biotin cleavage, providing a measure of receptor recycling. Right: Recycling was quantified by comparing the internalized MrgC11 and MOR at 0 min and 60 min of recycling time (n = 11 per group). (B, C, and E) Two-way ANOVA and Bonferroni post hoc test. ***P < 0.001 versus control; ###P < 0.001. Values are mean ± SEM.

Fig. 4.. MrgC agonist potentiates acute inhibition of cAMP production by morphine.

(A) Dose-response curve of morphine to inhibit forskolin-induced cAMP production in HEK293T cells coexpressing MOR, MrgCII, and cAMP biosensor construct (n = 4 experiments). (B) Dose-response curve of BAM8–22 effect on forskolin-induced cAMP production (n = 3 experiments). (C) Time course of forskolin-induced cAMP production in response to different drug treatments in HEK293T cells (n = 6 to 8 experiments). (D) Quantification of forskolin-induced cAMP production after different drug treatments (n = 7 to 12 experiments). (E) Time course of effects of different drug treatments on superactivation of the cAMP pathway in HEK293T cells after withdrawal from 14-hour chronic morphine (10⁻⁶ M) or vehicle treatment (n = 25 to 27 experiments). (F) Quantification of different drug treatments on chronic morphine- induced cAMP superactivation in cells transfected with MOR alone and in cells cotransfected with MOR and MrgC11 (n = 25 to 29 experiments). (D and F) One-way ANOVA with Bonferroni post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control (blue bar); ##P < 0.01 versus the indicated group. All values are mean ± SEM.

Fig. 5.. MrgC11-MOR interaction reduces MOR and β-arrestin-2 coupling.

(A) Schematic representation of β-arrestin recruitment measured by NanoBiT complementation assay. (B) Dose-response curves of morphine and DAMGO for stimulation of MOR/β-arrestin-2 coupling in HEK293T cells coexpressing MOR-SmBiT and LgBiT-β-arrestin-2 (n = 3 to 4 experiments). (C) Dose-response curves of BAM8–22 for inducing MrgC11/β-arrestin-2 coupling (n = 3 to 4). (D) MrgC11/β-arrestin coupling (n = 20) versus MOR/β-arrestin coupling (n = 40). E) Recruitment of β-arrestin to MOR (n = 12) and its CTD (n = 25). (F) Effects of expression of Myc-MOR^CTD on MOR/β-arrestin coupling (n = 14 to 16). (G) Effects of coexpression of WT and mutant MrgC11s on morphine-induced MOR/β-arrestin-2 (n = 21 to 89). (H) Effects of expression of MrgC11 and MrgC11 C-terminal mutants on morphine- and DAMGO-induced β-arrestin-2 recruitment (n = 10 to 23). (I) Dose-response curves and (J) time course of morphine (100 μM) for stimulation of MOR/β-arrestin-2 coupling in cells transfected with MrgC11 or control plasmid (n = 8 to 12 per group). (D to F) ***P < 0.001 by Student’s t test. (G and H) *P < 0.05, **P < 0.01, and ***P < 0.001 versus control; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 by one-way ANOVA with Bonferroni post hoc test. Values are mean ± SEM.

Fig. 6.. Intrathecal BAM8-22 potentiates morphine analgesia.

(A) Time course of pain inhibition after intrathecal drug administration in tail immersion test (52°C) of WT (n = 9 to 12 per group) and Mrg KO mice (n = 7 to 9 per group). BAM22, a dual agonist to MrgC and opioid receptor. (B) Dose-response curves of pain inhibition [% maximum possible effect (MPE) at 30 min postdrug] by intrathecal morphine, BAM8-22, and combined (1:1 ratio, n = 8 to 9 per group). (C) Isobolographical analysis. The experimental ED50 of the combined drug treatment (0.6 nmol; red dot) falls outside the 95% confidence limits of the theoretic additive ED₅₀ (black line). (D) Effects of coapplication of BAM8-22 (0.2 μM) with a subeffective dose of morphine (0.2 μM) on capsaicin (0.3 μM)–induced [Ca²⁺]i increase in WT and Mrg KO DRG neurons (n = 21 to 79). (E) Time course of changes of the analgesic effect of morphine after repeated treatments [15 mg/kg, subcutaneously (s.c.)] in WT (n = 7) and Mrg KO (n = 9) mice. (F) Effects of BAM8-22 (delivered in two 5 nmol intrathecal injections/day on tolerance days 5 and 6; n = 8) and vehicle (n = 7) on the development of morphine tolerance in WT mice. *P < 0.05, **P < 0.01,and ***P < 0.001; ^###P < 0.001 by two-way mixed model ANOVA and Bonferroni post hoc test. Values are mean ± SEM.

Fig. 7.. Intrathecal morphine-induced pain inhibition is reduced in Mrg KO mice.

(A) Tail-flick latencies of naïve WT (n = 19) and Mrg KO mice (n = 11) in the tail immersion test (52°C). (B) Intrathecal morphine-induced (7.8 nmol, 5 ml, i.t.) heat antinociception in naïve WT (n = 19) and Mrg KO (n = 11) mice. Box-and-whisker plots show median (horizontal line), mean (+), interquartile range (box), and maximum and minimum values (“whiskers”). ***P < 0.001 by Mann-Whitney U test. (C and D) Morphine- induced heat antinociception (7.8 nmol, 5 ml) in naïve WT (n = 8) and Mrg KO (n = 9) mice in Hargreaves test (C) and hot plate test (D). (E) At 2 to 3 days after intraplantar injection of complete Freund’s adjuvant (CFA), effects of morphine (7.8 nmol, i.t.) on the increased paw withdrawal frequency to mechanical stimulation in WT mice (n = 8) and Mrg KO mice (n = 9). (F and G) Morphine (7.8 nmol, i.t.) inhibition of inflammatory heat hypersensitivity in CFA-treated WT and Mrg KO mice in the Hargreaves test (F) and hot plate test (G). (H) At 1 to 2 weeks after CCI, effects of morphine (7.8 nmol, i.t.) on the increased paw withdrawal frequency to mechanical stimulation in WT and Mrg KO mice (n = 6 per group). (I) Effects of morphine (7.8 nmol, i.t.) on heat hypersensitivity in CCI-WT and CCI-Mrg KO mice. (C to I) Values are mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 versus WT or indicated group; #P < 0.05 and ^##P < 0.01 versus predrug by two-way mixed model ANOVA with Bonferroni post hoc test.

Fig. 8.. Hypothetical model illustrating MrgC11 regulation of MOR activity.

(A) Representative traces and quantification show morphine (5 μM) inhibition of capsaicin (Cap; 0.3 μM)–induced [Ca²⁺]i increase in WT and Mrg KO DRG neurons (n = 54 to 117). Values are mean ± SEM. *P < 0.05 and ***P < 0.001 versus control; ^###P < 0.001 by two-way mixed model ANOVA with Bonferroni post hoc test. (B) Top: Patch-clamp recording in a lamina II neuron and representative traces of eEPSCs to paired-pulse test stimulation (500 μA, 0.1 ms, 400-ms interval) before and 5 min after morphine (1 μM). Bottom: Quantification of morphine inhibition of eEPSCs and changes in pairedpulse ratio (n = 8 per group). Box-and-whisker plots show median (horizontal line), mean (+), interquartile range (box), and maximum and minimum values (whiskers). *P < 0.05, **P < 0.01, and ***P < 0.001 versus predrug; ^###P < 0.001 by Mann-Whitney U test. (C) Hypothetical model by which MrgC11 regulates MOR activity. Left: Coactivation of MrgC11 and MOR facilitates G_i-coupled regulation of cAMP and shifts the MOR response to agonist away from β-arrestin-2 signaling pathways that mediate morphine tolerance toward the Gi signaling pathways mediating a therapeutic response. Right: Activation of MrgC11 in the MOR-MrgC11 complex leads to receptor coendocytosis. The internalized receptors are sorted into recycling endosomes.