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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Pain. 2021 Apr 1;162(4):1082–1094. doi: 10.1097/j.pain.0000000000002119

Ubiquitin-mediated receptor degradation contributes to development of tolerance to MrgC agonist-induced pain inhibition in neuropathic rats

Qian Huang a, Neil C Ford a, Xinyan Gao a, Zhiyong Chen a, Ruijuan Guo a, Srinivasa N Raja a, Yun Guan a,b, Shaoqiu He a,†
PMCID: PMC7969388  NIHMSID: NIHMS1638903  PMID: 33110031

1. Introduction

Neuropathic pain results in considerable social and economic burden for patients [26; 48]. Opioid therapies are commonly used to treat neuropathic pain but are associated with severe central adverse effects and addiction [32; 37; 39; 49]. Therefore, researchers and drug developers are working to identify novel molecular targets in the peripheral nervous system, especially those on nociceptive sensory neurons in the trigeminal ganglia and dorsal root ganglia (DRG).

Recent preclinical studies suggest that subtype C of Mas-related G-protein-coupled receptor (MrgC) is a promising candidate. MrgC and the human homolog MrgX1 have restricted expression in primary sensory neurons, and MrgC may exert a dual role in pain control [20]. First, activation of MrgC or MrgX1 inhibits high-voltage-gated calcium currents [28–30], reduces neurotransmitter release from central terminals of DRG neurons [19], and attenuates persistent pain in various animal models [16; 19; 30]. Additionally, MrgC may function as a positive modulator of μ-opioid receptor (MOR) and enhance morphine analgesia mediated by MORs on DRG neurons. Thus, it may limit dose-dependent side effects of morphine [20; 51; 56].

The repeated dosing of an agonist targeting G-protein–coupled receptors (GPCRs) often induces tachyphylaxis and tolerance to its cellular actions, reducing drug efficacy. Tolerance development has been shown for many GPCRs after chronic drug treatment [6; 23; 33; 34]. In particular, morphine tolerance after prolonged or repeated exposure has been studied extensively. Yet, the mechanism remains incompletely understood. Phosphorylation, endocytosis, recycling, heterodimerization, ubiquitination, and degradation of opioid receptors in sensory neurons may all affect the overall development of tolerance to opiate drugs [18; 52; 54].

Intrathecal administration of MrgC agonists such as JHU58 (a dipeptide agonist) and BAM8-22 (a large peptide agonist) can acutely inhibit persistent pain-related behavior in animal models [19; 30]. For example, JHU58 attenuates neuropathic mechanical and heat hypersensitivity through activation of MrgC on the central terminals of DRG neurons [19; 29; 30]. Yet, it is not known whether repeated administration of MrgC agonist induces tolerance to its pain-inhibitory effects. Using JHU58 as a pharmacologic tool, we examined whether repeated intrathecal drug treatment induces acute tolerance in rats with an L5 spinal nerve ligation (SNL). Additionally, we used HEK293T cells to investigate the molecular mechanisms that might underlie tolerance to the analgesic effects of JHU58 and examined whether inhibition of receptor ubiquitination attenuates the development of analgesic tolerance to JHU58 in SNL rats.

2. Materials and methods

2.1. Animals

Adult male Sprague-Dawley rats (250-300 g; Harlan Bioproducts for Science) were used in behavior tests. Animals were housed under optimal laboratory conditions with a 12-hour light/dark cycle and free access to food and water. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee (Baltimore, MD, USA) as consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals to ensure minimal animal use and discomfort.

2.2. Spinal nerve ligation

We ligated the L5 spinal nerve using a modification of the procedure described previously [15; 21]. The animals were anesthetized with isoflurane (2%, Abbott Laboratories, North Chicago, IL) delivered through a nose cone. Under aseptic conditions, the skin was incised at the midline over the lumbar spine, and the L5, L6, and upper sacral vertebrae were exposed. The left transverse process of the L6 vertebra was removed, and the left L5 spinal nerve was exposed and dissected from the underlying tissue with fine forceps. The left L5 spinal nerve was then tightly ligated with a 6-0 silk suture and cut distally. The muscle layer was approximated with 4-0 chromic gut suture and the skin closed with metal clips. After the surgery, the rats were returned to their cages, kept warm under a heat lamp, and monitored during recovery. Skin staples were removed approximately 1 week after surgery.

2.3. Animal behavioral tests

Animals were habituated to the test environment for 30 minutes before testing was begun on a given day. To minimize experimenter bias, the investigator who performed the behavioral tests was blinded to the drug treatment conditions. Before the behavioral testing, animals were acclimatized to the facilities for 1 week.

2.3.1. Mechanical hypersensitivity

Animals were placed under plastic domes on a mesh floor that allowed full access to the plantar surface of the paws. The up-down method was used to determine mechanical paw withdrawal thresholds (PWTs). We applied a series of von Frey filaments that deliver approximately logarithmic incremental forces (0.38, 0.57, 1.23, 1.83, 3.66, 5.93, 9.13, 13.1 g) to the region between the foot pads in the plantar aspect of the hind paw to test mechanical hypersensitivity [15; 19]. The von Frey filaments were applied for 4 to 6 seconds, starting with the 1.83 g stimulus. If a positive response occurred, the next smaller von Frey filament was used; if a negative response was observed, the next higher force was used. The test was continued until (1) the responses to 5 stimuli were assessed after the first crossing of the withdrawal threshold, or (2) the upper or lower end of the von Frey hair set was reached before a positive or negative response had been obtained. Abrupt paw withdrawal, licking, and shaking were regarded as positive responses. All experimental conditions (animal age, room temperature, time of day for behavioral testing, drug preparation, drug injection, animal handling, etc.) were carefully controlled to maintain consistency across groups.

2.3.2. Heat hypersensitivity

We used the Hargreaves test to measure paw withdrawal latency (PWL) to radiant heat stimuli. Animals were trained (e.g., handling) for 2 to 3 days before data were obtained. In addition, animals were habituated to the test environment for 30 minutes before testing was begun on a given day. Animals were placed under a transparent plastic box on a glass floor. Radiant heat was delivered by a plantar stimulator analgesia meter (IITC model 390, Woodland Hills, CA) through the glass to the hind paw. After acclimatization sessions, the latency for the animal to withdraw its hind paw in response to the heat was measured. Radiant heat was applied to the mid-plantar area of each hind paw three times with 5-minute intervals. A cutoff time of 20 seconds was used to prevent tissue damage. Data from three trials were averaged for analysis.

2.3.3. Rota-rod test

We used the rota-rod test to assess whether drug treatment caused any motor impairment. Rats were acclimatized and trained on a rotating rod (Ugo Basile, Italy) that accelerated from 0 to 30 rpm in 180 seconds. On the day of testing, rat performance on the rod was measured before (pre-drug baseline) and 45 minutes after drug administration. The time (in seconds) that each animal remained on the accelerating rod without falling was recorded [19; 47].

2.4. Intrathecal catheter Implantation

After rats were anesthetized with 2% isoflurane, a small slit was cut in the atlanto-occipital membrane, and a 6- to 7-cm piece of saline-filled PE-10 tubing was inserted. We confirmed intrathecal drug delivery by injecting lidocaine (400 μg/20 μL, Hospira, USA), which resulted in a temporary motor paralysis of the lower limbs [16; 19; 21].

2.5. Tolerance-inducing paradigm

Based on previous studies that used acute tolerance-inducing paradigms [19; 21], we administered drug intrathecally to rats at 4-5 weeks post-SNL (Fig. 1A), a time when mechanical and heat hypersensitivity have reached a maximum and stable level. As in our previous study [19], we used JHU58 at a dose of 1x ED50 (dose estimated to produce 50% maximum possible effect) to compare pre-tolerance and post-tolerance drug effects, whereas we used a 5x ED50 dose to induce tolerance. The pre-tolerance testing (0.1 mM JHU58, 10 μL, i.t.) was conducted at 4 weeks post-SNL. Then, 10 μL of 0.5 mM JHU58 was injected twice/day (i.t.) for 3 consecutive days. The post-tolerance test (0.1 mM JHU58, 10 μL, i.t.) was conducted 1 day after tolerance induction. In experiments that included ubiquitin activation, TAK-243 (0.2 mM, 5 μL, i.t.), a small molecule inhibitor of ubiquitin activating enzyme (UAE), was administered 2 hours before each tolerance-inducing dose of JHU58. TAK-243 is a new UAE inhibitor used in animal studies [25; 57], but to our knowledge, it has not been used in studies of the nervous system. We calculated the dose of TAK-243 (0.2 mM, 5 μL, i.t.) for intrathecal injection in rats based on a previous study of this drug in cancer treatment [25]. We did not observe notable side effects (e.g., motor deficit) of TAK-243 at this dose in our pilot experiment.

Figure 1. Repeated intrathecal drug administration produced tolerance to JHU58-induced pain inhibition in nerve-injured rats.

Figure 1.

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(A) The tolerance-inducing protocol. (B) At 4-5 weeks after rats underwent an L5 spinal nerve ligation (SNL), intrathecal (i.t.) administration of JHU58 (0.1 mM, 10 μL) inhibited mechanical hypersensitivity of the hind paw ipsilateral to the injured (left) side, as indicted by the reversal of a decrease in paw withdrawal threshold (PWT) to mechanical stimuli. JHU58 inhibition of mechanical hypersensitivity largely diminished after repeated i.t. injections with tolerance-inducing doses of JHU58 (0.5 mM, 10 μL, twice/day, 3 days, n = 5-10/group). (C) Before induction of tolerance, JHU58 (0.1 mM, 10 μL, i.t.) reversed the decrease in paw withdrawal latency (PWL) to heat stimuli of the ipsilateral hind paw. The inhibition of heat hypersensitivity by the same dose of JHU58 was decreased after tolerance induction (n = 5/group). PWT and PWL were measured at 45 minutes post-injection. Data are presented as mean + SEM with overlaid individual data points. **P < 0.01, ***P < 0.001 versus pre-JHU58; #P < 0.05 versus pre-tolerance; two-way mixed model ANOVA followed by Bonferroni post hoc test.

2.6. Plasmid construction

All primers and oligonucleotides used for the construction of plasmids are listed in Supplementary Table 1. The cDNA of MrgC was cloned into a pCMV-HA/Myc plasmid (Clontech), and the epitope was positioned at the N-terminus of receptors. MrgC and β-arrestin-2 were inserted into a NaboBiT™ PPI plasmid (Clontech). The small subunit (SmBiT) was positioned at the C-terminus of receptors and the large subunit (LgBiT) was positioned at the N-terminus of β-arrestin-2.

2.7. Cell culture and transfection

HEK293T cells were cultured in growth medium that consisted of 90% DMEM, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) at 37°C in the presence of 95% O2 and 5% CO2. Using Lipofectamine 2000 (Thermo-Fisher Scientific), we transfected the cells with 2–4 μg plasmid/35 mm dish or 0.1–0.2 μg plasmid/96-well microplate well and cultured them for 1–2 days before experiments. We routinely assessed the cells with DAPI staining to confirm the absence of mycoplasma.

2.8. Immunohistochemistry

HEK293T cells cotransfected with Myc-MrgC11 expression plasmids were preincubated with mouse anti-Myc antibody (1:100; M4439, Sigma-Aldrich) and/or LysoTracker Red DND-99 (1:500; Thermo Fisher Scientific) for 30 minutes at 37°C. Cells were then treated with 5 μM BAM 8-22 or HU58 for 30 minutes. Cells were fixed with 4% paraformaldehyde and 0.2% picric acid. The fixed cells were then immunostained with anti-Rab11a antibody (1: 200, Cell Signaling Technology). For secondary antibodies, we used Alexa 568–conjugated goat antibody to rabbit (A11011, Molecular Probes), and Alexa 488–conjugated goat antibody to mouse (A10667, Molecular Probes). All secondary antibodies were diluted 1:100 in blocking solution. The slides were examined, and immunostaining images were obtained with a Zeiss 700 scanning confocal microscope.

2.9. Cell surface biotinylation and immunoblotting

The transfected HEK293T cells were incubated in extracellular solution (in mM: 150 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 10 HEPES, 10 sucrose) alone or extracellular solution containing 5 μM BAM 8-22 or JHU58 with or without sulfo-NHS-LC-biotin (0.5 mg/mL, Pierce) for 45 minutes at 37°C, and then in phosphate-buffered saline (with Ca2+/Mg2+) containing biotin for 45 minutes at 4°C. After the reaction was stopped, cells were lysed in ice-cold RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.5 mg/mL bovine serum albumin). The lysates were then precipitated with streptavidin. Samples were subjected to SDS-PAGE, transferred to membranes, and probed with antibodies to hemagglutinin (HA; Cell Signaling Technology, 1:2000), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, 1:50,000), or Myc (Cell Signaling Technology, 1:4000). Protein bands were detected with enhanced chemiluminescence (ECL, Bio-Rad). Western blots were imaged with the ImageQuant LAS 4000 (GE Healthcare Life Sciences) and analyzed with ImageJ 1.46a software.

2.10. Biotinylation assay of receptor endocytosis and recycling

To assay receptor endocytosis [20; 22], we biotinylated the transfected HEK293T cells with 1.5 mg/mL sulfo-NHS-SS-biotin (Pierce). JHU58 (5 μM) were present throughout all steps and incubations except for the 4°C biotinylation reaction. Cells were then incubated at either 4°C to block membrane trafficking or 37°C for various times to allow endocytosis to occur. The remaining surface biotin was then cleaved by reducing its disulfide linkage with glutathione cleavage buffer (50 mM glutathione in 75 mM NaCl and 10 mM EDTA containing 1% bovine serum albumin and 0.075 N NaOH) twice for 15 minutes each at 4°C. Cell membranes were prepared, and biotinylated proteins were precipitated. The recycling of MrgC was measured biochemically by the loss of internalized receptors specifically labeled with cleavable (disulfide-linked) biotin. The transfected HEK293T cells were treated with glutathione cleavage buffer and surface biotinylated as above and then transferred to 37°C for 30 minutes to allow endocytosis to occur. Cells were cooled to 4°C to stop membrane trafficking, and the remaining surface biotin was quantitatively cleaved with glutathione. Cultures were then returned to serum-free growth medium containing 50 mM glutathione at 37°C (or 4°C) for various times to allow internalized receptors to recycle before the cells were cooled to 4°C and incubated with glutathione cleavage buffer. Residual biotinylated (internalized) receptors were isolated and detected by immunoblot analysis.

2.11. Immunoprecipitation

Cells and tissues were lysed in ice-cold RIPA buffer. The suspended lysate was immunoprecipitated at room temperature for 10 minutes with protein G Dynabeads (Thermo-Fisher Scientific) that had been preincubated for 10 minutes with 0.5–2 μg of mouse anti-Myc (Sigma-Aldrich) or anti-HA antibody (Sigma-Aldrich) at room temperature. Immunoprecipitates were collected and aspirated. The Dynabeads were then resuspended in RIPA buffer, washed, and incubated in SDS sample buffer for 10 minutes at 70°C. The precipitate was immunoblotted as described above. Antibody against Myc (Cell Signaling Technology, 1:2000) or ubiquitin (Cell Signaling Technology, 1:2000) was used.

2.12. β-arrestin recruitment assay

β-arrestin recruitment was measured by the NanoBiT complementation assay (Promega Corporation). The assay was carried out according to the manufacturer’s instructions [20]. Briefly, HEK293T cells were seeded into 96-well plates (Corning) at 2 × 104 cells/well and then co-transfected the following day with the different NanoBiT plasmids (1:1 ratio of interacting pairs). For MrgC/β-arrestin, we used rat MrgC containing C-terminal SmBiT- (MrgC-SmBiT) and LgBiT-tagged N-terminal β-arrestin-2 (LgBiT-β-arrestin-2). At 24 hours after transfection, growth medium was exchanged with Opti-MEM I, and Nano-Glo Live Cell Substrate was added. Cells were then incubated for 15 minutes at 37°C, and luminescence was measured with a FlexStation reader (Molecular Devices) at 37°C. After thermal equilibration, JHU58 in Opti-MEM I was injected, and measurements continued. At least 24 hours after transfection, luminescence was measured on a FlexStation reader (Molecular Devices) as described above. Data were normalized to that of morphine and analyzed with the sigmoidal dose-response function of GraphPad Prism 6.0.

2.13. Data and statistical analysis

Statistical analyses were carried out with GraphPad Prism software or IBM SPSS Statistics 21. We performed normality testing of the data with the D’Agostino & Pearson test (n > 20) in Graphpad Prism. When the sample size was small (n < 10), we used the Kolmogorov-Smirnov test instead. Data that followed a normal distribution are expressed as mean ± SEM. A Student’s t-test was used to compare data consisting of two groups. One-way ANOVA followed by the Bonferroni post hoc test was used for comparisons of data consisting of three or more groups. Comparisons of two or more factors across multiple groups were analyzed by two-way ANOVA followed by the Bonferroni post hoc test. Data that did not follow a normal distribution were analyzed with nonparametric ANOVA (Friedman and Kruskal-Wallis) and expressed as medians. Post-hoc tests (Wilcoxon Matched Pairs Test and Mann-Whitney U Test) were used to analyze specific data points. The methods for statistical comparisons and results in each study are given in the figure legends, and additional details are provided in Supplementary Table 2. Statistical significance was set as P < 0.05. Sample sizes were consistent with those of a previous publication [21]. Representative data are from biological experiments that were replicated at least three times with similar results.

2.14. Materials

Stock solutions were freshly prepared as instructed by the manufacturer. BAM8-22 and JHU58 were diluted in saline or extracellular solution. JHU58 was synthesized at the Johns Hopkins University. BAM8-22 was purchased from Tocris Bioscience (Bristol, UK). TAK-243 was obtained from Chemietek (Indianapolis, IN, USA). Other drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used: anti-Myc (Cell Signaling Technology, Cat# 2276, RRID: AB_331783, Cat# 2278, RRID: AB_490778), anti-HA (Cell Signaling Technology, Cat# 3724, RRID: AB_1549585), anti-ubiquitin (Cell Signaling Technology, Cat# 43124, RRID: AB_2799235), anti-GLUT1 (Cell Signaling Technology, Cat# 12939, RRID: AB_2687899), anti-Rab11a (Cell Signaling Technology, Cat# 5589, RRID: AB_10693925), anti-actin (Millipore, Cat# MAB1501, RRID: AB_2223041), and anti-GAPDH (Millipore, Cat# ABS16, RRID: AB_10806772).

3. Results

3.1. Tolerance to JHU58-induced pain inhibition

We first examined whether tolerance to JHU58-induced pain inhibition develops after repeated intrathecal drug injections in SNL rats. Before tolerance induction, SNL rats exhibited a significant increase in ipsilateral PWT 45 minutes after intrathecal injection of JHU58 (0.1 mM, 10 μL). This effect was significantly decreased after rats were administered 3 days of twice daily JHU58 injections (0.5 mM, 10 μL, i.t., n = 4–10/group, Fig. 1A, B). Similarly, repeated injections decreased the ability of JHU58 (i.t., 0.1 mM, 10 μL, n = 5) to inhibit heat hypersensitivity, as measured by PWL (Fig. 1C). Together, these findings suggest that tolerance developed to JHU58-induced inhibition of both mechanical and heat hypersensitivity in SNL rats.

3.2. Acute treatment with MrgC agonist induces endocytosis of MrgC

Our recent study suggested that acute treatment with MrgC agonist can induce endocytosis of mouse MrgC11 [20]. To test whether rat MrgC is also internalized after agonist exposure in vitro, we labeled Myc-MrgC on the membrane of HEK293T cells with monoclonal anti-Myc antibody. The Myc peptide tag was placed at the N-terminus of the receptor and therefore was exposed to the extracellular space when receptors were on the cell surface. Under control conditions, the prelabeled MrgC was present mostly on the cell surface. After a 45-minute stimulation with BAM8-22 (5 μM, n = 25) or JHU58 (5 μM, n = 32), the prelabeled surface Myc-MrgC was internalized and redistributed into intracellular vesicle-like structures (control, n = 34, Fig. 2A, B).

Figure 2. Acute agonist treatment led to internalization of MrgC.

Figure 2.

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(A) In control cells, Myc-MrgC (green) was present at the cell surface. Representative images show internalization of Myc-MrgC in HEK293T cells after 45 minutes of exposure to JHU58 (5 μM) or BAM8-22 (5 μM). Scale bar: 10 μm. (B) Quantitative data show the ratio of internalized Myc-MrgC staining to total surface immunostaining (n = 34 for control, n = 32 for JHU58, n = 25 for BAM8-22). (C, D) In HEK293T cells that were transfected with Myc-MrgC, immunoblotting (IB) and quantitative data showed that the levels of MrgC on the cell surface declined after 45-minute treatment with JHU58 (5 μM, n = 4 per group) and BAM8-22 (5 μM, n = 4 per group). GLUT1 at the biotinylated cell surface fraction was used to normalize MrgC expression. Data are presented as mean + SEM with overlaid individual data points. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; one-way ANOVA followed by Bonferroni post hoc test.

We further confirmed internalization of MrgC by using immunoblotting and quantified the receptors present on the cell surface. In HEK293T cells transfected with Myc-MrgC, the receptors that remained on the cell surface after BAM8–22 (5 μM, n = 4 per group) or JHU58 (5 μM, n = 4 per group) treatment were biotinylated and precipitated with immobilized streptavidin [13; 22]. Glucose transporter 1 (GLUT1) is highly expressed in plasma membrane of HEK293T cells and was used as an internal control for surface protein expression [9]. Accordingly, we used GLUT1 to normalize Myc-MrgC expression in the biotinylated cell surface fraction. Both BAM8-22 and JHU58 significantly reduced Myc-MrgC expression on the cell surface at 45 minute post-treatment, as compared to that in the control group (Fig. 2C, D).

3.3. Acute treatment with JHU58 targets the receptor into the recycling pathway

In Myc-MrgC-expressing HEK293T cells, few MrgC receptors co-localized with lysosome-like compartments labeled by LysoTracker probe after 90-minute treatment with JHU58 (5 μM, n = 36, Fig. 3A, B). Furthermore, most internalized MrgC was present in the recycling endosomes (n = 38, Fig. 3A, B), which were stained with antibody to Rab11a, a recycling endosomal antigen [55]. Thus, after acute agonist treatment, most of the internalized MrgC receptors were not sorted into the degradation pathway.

Figure 3. Internalized MrgCs were sorted into the recycling pathway.

Figure 3.

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(A) Upper: Myc-MrgCs (arrows) that were internalized after acute JHU58 exposure were not sorted into lysosome-like compartments labeled by LysoTracker (arrowhead). Lower: Internalized Myc-MrgCs colocalized with Rab11a-labeled recycling-endosomes (arrowheads). Scale bar: 20 μm. (B) The mean percent of receptor-containing vesicles co-labeled by LysoTracker (n = 36) and by Rab11a (n = 38), normalized to the total number of receptor-containing vesicles. (C) Immunoblotting showed a loss of biotinylated receptors after a second biotin cleavage, providing a measure of receptor recycling. (D) Quantitative data analysis of receptor recycling showed that the percentage of internalized MrgC was significantly decreased after 60 minutes of recycling time (n = 4 for all groups). Data are presented as mean + SEM with overlaid individual data points. ***P < 0.001 versus control; ###P < 0.001 versus indicated group; one-way ANOVA followed by Bonferroni post hoc test.

To directly test whether the internalized receptors are reinserted back into the plasma membrane, we performed biotinylation assays of receptor recycling in HEK293T cells (n = 4, Fig. 3C). Indeed, internalized MrgCs were mostly reinserted into the plasma membrane after 60 minutes (n = 4, Fig. 3D). These results suggest quick recycling of MrgC to the plasma membrane after endocytosis.

3.4. Chronic exposure to JHU58 enhances MrgC and β-arrestin-2 coupling and increases receptor ubiquitination

The β-arrestin-2 pathway may play an important role in the development of side effects and analgesic tolerance to morphine [2; 3; 8]. We utilized a NanoBiT complementation assay (Promega) to examine whether MrgC activation recruits β-arrestin-2 to the receptor [10]. In NanoBiT titration experiments, stimulation of MrgC with JHU58 (5 μM, n = 3–4 experiments) for 20 minutes induced little increase in the NanoLuc luminescence. Modest β-arrestin coupling was observed only at the highest JHU58 dose. This evidence indicates that acute treatment with JHU58 induces little MrgC and β-arrestin-2 coupling (Fig. 4A, B). In contrast, chronic exposure to JHU58 (24 hours) significantly and dose dependently increased the recruitment of β-arrestin-2 to MrgC (Fig. 4B).

Figure 4. Chronic exposure of transfected HEK293T cells to JHU58 increased the coupling between MrgC and β-arrestin-2.

Figure 4.

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(A) HEK293T cells were transfected with both Myc-MrgC and β-arrestin-2 plasmids. Schematic representation of the experimental design to monitor β-arrestin recruitment with the NanoBiT complementation assay. (B) Concentration response curves of JHU58 for stimulation of MrgC and β-arrestin-2 coupling before and after acute JHU58 treatment in cells that co-express MrgC-SmBiT and LgBiT-β-arrestin-2 (n = 3–4 experiments). ***P < 0.001 versus control, two-way mixed model ANOVA. (C) Immunoprecipitation and Western blot analysis were used to detect ubiquitinated MrgC. HEK293T cells were stimulated with JHU58 (5 μM) for 1.5 or 24 hours. MrgC was immunoprecipitated with an anti-Myc-tag antibody. Ubiquitin and MrgC were blotted with specific antibodies (n = 6 for all groups). (D) Quantitative data analysis of MrgC ubiquitination in cells exposed to JHU58 for 1.5 or 24 hours. The values were normalized to the maximum level at 24 hours after JHU58 exposure. Box-and-whisker plots show median (horizontal line), interquartile range (box), maximum and minimum values (whiskers). ***P < 0.001 versus control; #P < 0.05 versus indicated group, Mann-Whitney U test. (E, F) In HEK293T cells that were transfected with HA-MrgC, immunoblotting (IB) and quantitative data showed that the levels of MrgC on the cell surface declined after 1.5 and 24 hours of JHU58 treatment (5 μM, n = 4 for all groups). Data are presented as mean + SEM with overlaid individual data points. ***P < 0.001 versus control; ##P < 0.001 versus indicated group; one-way ANOVA followed by Bonferroni post hoc test.

Prolonged interaction of β-arrestin with GPCRs may promote receptor ubiquitination [45], a crucial mechanism that sorts GPCRs into lysosomes for degradation [43]. Therefore, we investigated the effects of β-arrestin and MrgC coupling on the fate of MrgC by examining receptor ubiquitination in HEK293T cells. Immunoprecipitation followed by Western blot analysis showed that the ubiquitination of MrgC was significantly increased when cells were treated with JHU58 (5 μM) for 24 hours (Fig. 4C, D). Compared to the 24-hour treatment, 90 minutes of JHU58 exposure induced much less MrgC ubiquitination (n = 6 for all groups, Fig. 4C, D). The ubiquitinated membrane protein is mostly targeted to the lysosomes for degradation [43]. Correlating with the increased level of receptor ubiquitination by JHU58, 24-hour JHU58 treatment induced more significant downregulation of MrgC on the cell membrane than did 90-minute drug treatment (n = 4 for all groups, Fig. 4E, F).

3.5. TAK-243 inhibits JHU58-induced ubiquitination of MrgC

TAK-243 is a potent small-molecule inhibitor of the UAE [1; 25; 57]. Thus, we examined the effects of TAK-243 on ubiquitin conjugation of MrgC by conducting immunoprecipitation followed by Western blot analysis. Ubiquitination of MrgC was significantly increased when cells were treated with 5 μM JHU58 for 24 hours (Fig. 5A, B). Co-treatment with 1 μM TAK-243 significantly decreased the MrgC ubiquitination induced by chronic JHU58 exposure (n = 4 for all groups, Fig. 5A, B). Ubiquitin is known to target proteins for degradation. By conducting biotinylation assays of cell-surface MrgC, we found that co-treatment with TAK-243 prevented the JHU58-induced downregulation of MrgC on the cell surface (n = 4 for all groups, Fig. 5C, D).

Figure 5. TAK-243 inhibited the chronic JHU58-induced ubiquitination and downregulation of surface MrgC in HEK293T cells.

Figure 5.

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(A) Immunoblotting showed that JHU58 increased ubiquitination of MrgC in HEK293T cells after 24 hours of exposure; this effect was inhibited by co-treatment with TAK-243 (1 μM). (B) Quantitative data analysis of ubiquitinated MrgC after drug treatments (n = 4 for all groups). The values were normalized to the maximum level at 24 hours after JHU58 (5 μM) exposure. Box-and-whisker plots show median (horizontal line), interquartile range (box), maximum and minimum values (whiskers). ***P < 0.001 versus control (no drug treatment); #P < 0.05 versus indicated group. Mann-Whitney U test. (C) Immunoblotting showed that 24 hours of JHU58 (5 μM) exposure reduced MrgC on the cell surface; this effect was prevented by co-treatment with TAK-243 (1 μM). (D) Quantitative data analysis of surface MrgC after drug treatments (n = 4 for all groups). The values were normalized to the control. Data are presented as mean + SEM with overlaid individual data points. *P < 0.05 versus control; one-way ANOVA followed by Bonferroni post hoc test.

3.6. TAK-243 attenuates the development of JHU58 tolerance in SNL rats

After showing that TAK-243 inhibits MrgC ubiquitination in vitro (Fig. 5), we examined whether this UAE inhibitor can also attenuate JHU58 tolerance in vivo. Before induction of tolerance, intrathecal injection of JHU58 (0.1 mM, 10 μL) significantly increased ipsilateral PWT to mechanical stimuli from the pre-injection level in SNL rats (Fig. 6A, B). SNL rats were then pretreated with intrathecal injections of TAK-243 (0.2 mM, 5 μL, i.t.) or vehicle (n = 5-7/group) 1 hour before receiving tolerance-inducing doses of JHU58 (0.5 mM, 10 μL, twice/day, i.t., Fig. 6A) for 3 days. Unlike the vehicle-pretreated rats, rats that were pretreated with TAK-243 did not develop tolerance to JHU58-induced inhibition of mechanical hypersensitivity (Fig. 6C). TAK-243 alone did not change ipsilateral PWT of SNL rats or fall time (at 45 minutes after injection) in the rota-rod test (Supplementary Fig 1A, B, n = 6-7/group), suggesting that it may not induce significant impairment of motor coordination or locomotor function at this dose.

Figure 6. TAK-243 inhibited the development of JHU58 tolerance in nerve-injured rats.

Figure 6.

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(A) Protocol for TAK-243 pretreatment and the JHU58 tolerance-inducing protocol. (B) Before tolerance induction, intrathecal injection of JHU58 (0.1 mM, 10 μL) significantly increased the paw withdrawal threshold (PWT) in both vehicle- and TAK-243-pretreated SNL rats (vehicle, n = 6-7/group; TAK-243, n = 5-7/group). (C) After repeated treatment with tolerance-inducing doses of JHU58 (0.5 mM, 10 μL, i.t., twice/day, 3 days), SNL rats pretreated with vehicle (n = 5) no longer exhibited an increase in PWT after i.t. injection with JHU58 (0.1 mM, 10 μL), but the JHU58-induced increase in PWT persisted in rats that had received TAK-243 pretreatment (0.2 mM, 5 μL, i.t., 2 hours before first JHU58 injection, n = 5-6/group). (D) Intrathecal injection of JHU58 (0.1 mM, 10 μL) increased ipsilateral paw withdrawal latency (PWL) to heat stimuli in both TAK-243– (n = 6) and vehicle-pretreated (n = 7) SNL rats before tolerance induction. (E) After induction of JHU58 tolerance, the inhibition of heat hypersensitivity by intrathecal JHU58 (0.1 mM, 10 μL) was maintained in SNL rats that received TAK-243 pretreatment (0.2 mM, 5 μL, i.t., 2 hours before first JHU58 injection, n = 6), but not in SNL rats pretreated with vehicle (n = 6). Data are presented as mean + SEM with overlaid individual data points. *P < 0.05, ***P < 0.001 versus pre-JHU58; ###P < 0.001 versus indicated group; two-way mixed model ANOVA followed by Bonferroni post hoc test.

In a separate study, JHU58 (0.1 mM, 10 μL, i.t.) significantly increased ipsilateral PWL to heat stimuli from pre-JHU58 level in SNL rats before tolerance induction, but not after tolerance was established (Fig. 6D, E). Pretreatment with TAK-243, but not vehicle, reduced the development of tolerance to JHU58-induced inhibition of heat hypersensitivity (n = 5-7/group, Fig. 6E). Thus, the ability of JHU58 (0.1 mM, 10 μL, i.t.) to inhibit both mechanical and thermal hypersensitivity persisted in the TAK-243-pretreated group after the vehicle-treated group had developed tolerance.

3.7. Acute morphine analgesia is decreased in SNL rats with JHU58 tolerance

Co-treatment with a low dose of MrgC agonist may acutely enhance morphine analgesia [20; 56]. Here, we questioned whether tolerance to MrgC agonist alters morphine analgesia. Morphine (3 μg, 10 μL, i.t.) inhibited mechanical hypersensitivity in SNL rats before the induction of JHU58 tolerance, as indicated by a significant increase of ipsilateral PWT from pre-morphine baseline (n = 5). However, the same dose of morphine did not significantly increase ipsilateral PWT in SNL rats after JHU58 tolerance (n = 7, Fig. 7A, B). The percent of the maximum possible effect (%MPE) achieved by both JHU58 and morphine (n = 5-7/group) was significantly lower in rats with JHU58 tolerance than it was before tolerance induction (Fig. 7C). Importantly, after the JHU58 tolerance-induction protocol, morphine-induced inhibition of paw mechanical hypersensitivity was largely intact in SNL rats pretreated with TAK-243 (0.2 mM, 5 μL, i.t.), but not in those pretreated with vehicle (n = 5-8/group, Fig. 7D, E). In a separate study, pretreatment with TAK-243, but not vehicle, during the JHU58 tolerance-induction protocol, also preserved morphine-induced inhibition of paw heat hypersensitivity in SNL rats (n = 6-7/group, Fig. 7F, G).

Figure 7. Morphine analgesia was decreased in JHU58-tolerant rats.

Figure 7.

Open in a new tab

(A) Protocol for TAK-243 pretreatment and JHU58 tolerance induction. (B) Intrathecal morphine (3 μg, 10 μL) significantly increased ipsilateral paw withdrawal threshold (PWT) in SNL rats before JHU58 tolerance induction. The effect of morphine was decreased in SNL rats that received tolerance-inducing doses of JHU58 (0.5 mM, 10 μL, i.t., twice/day, 3 days). Pre-tolerance, n = 5; post-tolerance, n = 7. (C) The percent maximum possible effect (%MPE) of JHU58 and morphine to inhibit mechanical hypersensitivity were each significantly decreased after JHU58 tolerance was established (JHU58, n = 7; morphine, n = 5). %MPE = [(post-drug PWT) – (pre-drug PWT)] / [(cut-off) – (pre-drug PWT)] x 100. (D) In the absence of JHU58 tolerance, morphine (3 μg, 10 μL, i.t.) significantly increased PWT in both vehicle- and TAK-243-treated SNL rats (vehicle, n = 7; TAK-243, n = 7). (E) After the JHU58 tolerance-inducing protocol, morphine (3 μg, 10 μL, i.t.) significantly increased PWT in SNL rats that were pretreated with TAK-243 (0.2 mM, 5 μL, i.t., 2 hours before first JHU58 injection, n = 5-6/group), but not in those pretreated with vehicle (n = 5-8/group). (F) In the absence of JHU58 tolerance, morphine (3 μg, 10 μL, i.t.) reversed the decrease in ipsilateral paw withdrawal latency (PWL) to heat stimuli equally in SNL rats pretreated with vehicle ( n = 7) and those pretreated with TAK-243 (n = 6). (G) After the JHU58 tolerance-inducing protocol, the inhibition of heat hypersensitivity by morphine remained intact in SNL rats pretreated with TAK-243 (0.2 mM, 5 μL, i.t., n = 6), but not in those pretreated with vehicle (n = 6). Data are presented as mean + SEM with overlaid individual data points. *P < 0.05, **P < 0.01, ***P < 0.001 versus pre-tolerance; ##P < 0.01, ###P < 0.001 versus indicated group; two-way mixed model ANOVA followed by Bonferroni post hoc test.

4. Discussion

In previous studies we have shown that activation of rat MrgC and mouse MrgC11 at central terminals of primary sensory neurons inhibits neuropathic pain-related behavior in animal models [16; 19; 20]. Here, we demonstrated that tolerance develops to the pain inhibitory effects of MrgC agonist JHU58 after repeated intrathecal administration to SNL rats. Our findings further unravel potential cellular mechanisms underlying JHU58 tolerance.

Although activation of rat MrgC by acute agonist treatment promoted receptor endocytosis followed by targeting to the recycling pathway, chronic exposure to JHU58 increased coupling of MrgC to β-arrestin-2, which led to the addition of ubiquitin molecules to lysine residues of MrgC (receptor ubiquitination) and fewer receptors on the cell surface. Importantly, TAK-243, a UAE inhibitor, attenuated cellular ubiquitin conjugation of MrgC, as well as the reduction of surface MrgC after prolonged JHU58 treatment in vitro. Furthermore, the inhibitory effects of JHU58 on mechanical and heat hypersensitivities in behavior studies both persisted in the TAK-243-pretreated SNL rats after tolerance had developed in vehicle-pretreated rats, illustrating the important role of ubiquitin modification in JHU58 tolerance in vivo.

MrgC is expressed mostly in small-diameter primary sensory neurons whose central processes terminate in the spinal cord dorsal horn, but it is not expressed in any intrinsic neurons or glial cells of the central nervous system [20; 29]. Identifying cellular mechanisms of tolerance to the pain inhibitory effects of MrgC agonists is important for future development of therapeutic drugs. GPCRs activated by selective agonists can be sorted into distinct trafficking pathways [14; 17; 31; 53]. Recycling of receptors to the plasma membrane after endocytosis promotes rapid receptor resensitization for retaining receptor-mediated signal transduction. In contrast, targeting of receptors to lysosomes causes proteolytic downregulation, which would decrease signal transduction and agonist action [7; 14]. We found that MrgC internalized after acute agonist exposure was not redistributed to the lysosome-like compartments. Furthermore, we confirmed that co-localization of internalized MrgC and Rab11a, a specific marker of recycling endosomes [5], was high in HEK293T cells after JHU58 treatment. Together, these findings suggest that a large proportion of internalized MrgC may be sorted for rapid recycling, in accordance with results in a previous study of mouse MrgC11 [20]. Thus, the endocytosis of rat MrgC should facilitate the sorting of receptors into the recycling pathway. However, SNL rats developed tolerance after repeated JHU58 treatments in our current study. Thus, MrgC may not fit the traditional model whereby internalization leads to enhanced receptor resensitization and reduced tolerance [7; 14; 38].

To directly test whether MrgC receptors are indeed being trafficked in the recycling pathway, in a future study, we would need to transfect HEK293T cells with a dominant negative (S25N) form of Rab11 to block the endosomal recycling pathway. Then we could determine whether trafficking of internalized MrgC to recycling endosomes is prevented [5; 25; 57]. Because no available antibodies are specific for labeling cell membrane MrgC, the endocytosis and trafficking of MrgC in native DRG neurons remains to be determined. Importantly, due to differences in JHU58 dose, exposure time and experimental setting, it is difficult to correlate findings from in vitro cellular study with drug actions observed in vivo. Accordingly, internalization of MrgC at the central terminals of DRG neurons and the ubiquitination of MrgC in the spinal cord after long-term intrathecal agonist treatment (e,g., continuous intrathecal infusion of JHU58 at low-dose) warrants future in vivo examination, so that findings from our in vitro cell biology studies can be correlated with the drug effects observed in animal behavior tests.

Recent studies have suggested that interactions between GPCRs and β-arrestins could profoundly affect tolerance. Ligands to GPCRs, including β2-adrenoreceptor, CXCR4 chemokine receptors, and MORs, can activate arrestin functions [27; 44; 50]. Although acute JHU58 treatment induced minimal MrgC and β-arrestin-2 interaction in the NanoBiT complementation assay, prolonged drug treatment led to significant MrgC and β-arrestin-2 coupling. At this stage, it is unclear how JHU58 switched from a β-arrestin nonbiased ligand to a biased ligand after prolonged treatment, leading to increased coupling between MrgC and β-arrestin. Future studies may use β-arrestin-2 siRNAs and knockout mice to determine roles of the β-arrestin-2 pathway in MrgC agonist tolerance development.

Ubiquitin regulates the endocytic trafficking and signal transduction of many GPCRs. Yet, no study has assessed the effect of blocking GPCR ubiquitination on the development of analgesic tolerance. Recently, TAK-243 (MLN7243) was identified as a specific first-in-class inhibitor of UAE [25; 57]. In our study, ubiquitination of MrgC was increased when the cells were treated with JHU58 for 24 hours. However, inhibiting the ubiquitin-proteasome system with TAK-243 prevented ubiquitination and downregulation of surface MrgC induced by prolonged JHU58 exposure in vitro. Importantly, pretreatment with TAK-243 (0.2 mM, 5 μL, i.t.) in vivo significantly inhibited tolerance to JHU58-induced inhibition of mechanical and heat hypersensitivity in SNL rats. Collectively, these findings provide converging biologic and pharmacologic evidence for an important role of ubiquitin-related modulation in the development of JHU58 tolerance. It remains to be determined whether tolerance also develops to other MrgC and human MrgX1 agonists. Additionally, the minimal effective dose for TAK-243 to attenuate tolerance in vivo and to inhibit chronic agonist-induced MrgC ubiquitination in vitro must also be ascertained. Besides UAE, or E1s, ubiquitination of a protein also involves two other classes of enzymes, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s [24; 35; 41; 42]. The roles of E2s and E3s in JHU58 and morphine cross-tolerance warrant further investigation.

Cross-tolerance may occur when long-term exposure to one drug results in the development of tolerance to another drug in the same pharmacologic class [12]. It often happens when two drugs target the same receptor [36]. Nevertheless, cross-tolerance can also occur between two compounds that target distinct GPCRs, such as opioid and cannabinoid receptors [40; 46]. The degree of cross-tolerance may imply the level of physical interaction between the two receptors [12; 40; 46]. Previous studies have indicated that MORs interact and oligomerize with MrgC or C11, which may allow MrgC agonists to positively modulate MOR function [4; 20]. However, pharmacologic evidence for cross-tolerance between MOR and MrgC agonists remains unclear. In this study, morphine analgesia was decreased in SNL rats that had become tolerant to JHU58. Furthermore, intrathecal pretreatment with TAK-243, which reduced JHU58 tolerance, also attenuated the cross-tolerance to morphine analgesia in SNL rats. Thus, the cross-tolerance to morphine can also be blocked by pretreatment with the UAE inhibitor. Based on previous studies [19; 21], we used a high-dose paradigm (5x ED50) to induce acute JHU58 tolerance. Future study is needed to determine if tolerance also develops after continuous intrathecal infusion of JHU58 at lower doses (e.g., 1x ED50 by osmotic pump) that mimic regimens used in clinic.

In summary, our study suggests that repeated intrathecal injections of JHU58 may lead to development of tolerance to its pain inhibitory effect in neuropathic rats. Importantly, the UAE inhibitor TAK-243 inhibited JHU58-induced ubiquitination and downregulation of surface MrgC, and attenuated JHU58 tolerance in SNL rats in vivo. Ubiquitin is thought to influence the trafficking and function of more than 40 mammalian GPCRs, including opioid receptors [11]. Whether TAK-243 can block tolerance to agonists of these receptors, especially MOR agonists such as morphine, still must be determined. Studies are also needed to ascertain the intracellular mechanisms of β-arrestin and ubiquitin-proteasome pathways and how they affect MrgC and human MrgX1 trafficking and signaling transduction in DRG neurons. The knowledge gained can be used for developing strategies to attenuate tolerance by blocking ubiquitin-mediated receptor degradation.

Supplementary Material

Supplementary Materials: figures
Supplementary Materials: tables

Acknowledgments:

This study was conducted at the Johns Hopkins University School of Medicine. The authors thank Claire F. Levine, MS, ELS (scientific editor, Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University), for editing the manuscript. This work was facilitated by the Pain Research Core funded by the Blaustein Fund and the Neurosurgery Pain Research Institute at the Johns Hopkins University. The authors declare no competing interests. There are no other relationships that might lead to a conflict of interest in the current study.

Funding: This work was supported by a seed grant for Stimulating and Advancing ACCM Research (StAAR) from the Department of Anesthesiology and Critical Care Medicine at Johns Hopkins University (S.H.), and grants from the National Institutes of Health [NS070814 (Y.G.), NS110598 (Y.G.), NS117761 (Y.G), NS026363 (S.N.R.)]. Funders had no role in study design, data collection, or data interpretation, or in the decision to submit the work for publication.

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Supplementary Materials

Supplementary Materials: figures
NIHMS1638903-supplement-Supplementary_Materials__figures.docx (144KB, docx)
Supplementary Materials: tables
NIHMS1638903-supplement-Supplementary_Materials__tables.docx (37.4KB, docx)

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