Drug discovery to counteract antinociceptive tolerance with mu-opioid receptor endocytosis

Morphine antinociceptive tolerance is highly correlated with its poor ability to promote mu-opioid– receptor (MOR) endocytosis. Our objective was to discover a novel positive allosteric modulator of MOR to enhance morphine-induced MOR endocytosis. We used high-throughput screening to identify several cardiotonic steroids as positive allosteric modulators of morphine-induced MOR endocytosis having high potency and efficacy, independently of Na+/K+-ATPase inhibition. Convallatoxin was found to enhance morphine-induced MOR endocytosis through an adaptor protein 2/clathrin-dependent mechanism without regulating G protein- or β-arrestin-mediated pathways. Both F243 and I292 residues of MOR were essential to the effect of convallatoxin on MOR endocytosis. Co-treatment with chronic morphine and convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance in morphine-tolerant mice. These findings suggest that cardiotonic steroids are potentially therapeutic for morphine side effects and open a new avenue for the study of MOR trafficking.


Introduction
Opioids have long been used to treat severe pain (Waldhoer et al., 2004). However, long-term use leads to tolerance, dependence, and addiction . Opioids primarily activate three GPCRs of the G i subtype: the mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively). Although the mechanisms of opioid-induced analgesia are not well defined, it is now clear that activated opioid receptors are able to utilize both G-protein-dependent and G-proteinindependent signaling pathways (Williams et al., 2013). Furthermore, it is generally believed that opioid analgesics exert their pharmacological effects mainly by acting at MOR (Loh et al., 1998).
The general mechanism of opioid tolerance remains controversial (Al-Hasani and Bruchas, 2011); however, unlike other high-efficacy opioids such as  , N-MePhe 4 , Gly-ol]-enkephalin (DAMGO), etonitazene, etorphine, and fentanyl (Duttaroy and Yoburn, 1995), morphine acted as a poor MOR internalizing agonist (Keith et al., 1996) and developed tolerance through a unique molecular mechanism (Grecksch et al., 2011). Furthermore, morphine tolerance could be limited by enhancing receptor endocytosis (Martini and Whistler, 2007). Previous studies indicated that a mutant MOR with altered recycling (RMOR), which underwent endocytosis after morphine treatment, was associated in vitro with reduced tolerance and with reduced cAMP superactivation, a cellular hallmark of withdrawal (Finn and Whistler, 2001). Compared with WT mice, RMOR knock-in mice showed potentiated morphine antinociception but less tolerance and withdrawal, indicating a beneficial effect of MOR internalization in morphine analgesia (Kim et al., 2008). Furthermore, opioid combinations of morphine with low-dose DAMGO (He et al., 2002) or methadone (He and Whistler, 2005), which are MOR agonists with substantial MOR internalization ability, diminished both morphine tolerance and dependence in rats. However, when using a mixture of agonists, it is difficult to strongly conclude that MOR endocytosis contributes to morphine tolerance/dependence because numerous sets of MOR signaling pathways are modified by each agonist (Alvarez et al., 2002;Blanchet et al., 2003;Enquist et al., 2012;He et al., 2009;Milan-Lobo and Whistler, 2011) according to the so-called agonistselective theory (Zheng et al., 2010). A previous study indicated that morphine induces MOR endocytosis in mutant L83I (mouse orthologue of human L85I) without altering binding affinity or cAMP signaling. However, cellular tolerance to morphine is reduced (Cooke et al., 2015;Ravindranathan et al., 2009), suggesting an alternative strategy to reduce morphine tolerance by specifically enhancing morphine-induced MOR endocytosis. In this study, we established a highthroughput screening assay to identify signaling-specific positive allosteric modulators (PAMs) to augment morphine-induced MOR endocytosis and validated the ability of the identified molecules to regulate morphine effects in both cell and animal models.

Cardiotonic steroids (CTSs) augment morphine-induced MOR endocytosis. To identify PAMs
promoting morphine-induced MOR endocytosis, we screened a 480-natural-compound library in the presence of morphine, using a sensitive enzyme complementation assay for MOR endocytosis in human osteosarcoma U2OS cells expressing human MOR (U2OS-MOR). Three CTSs, gitoxigenin, bufalin, and convallatoxin, all capable of inhibiting Na + /K + -ATPase (Prassas and Diamandis, 2008), were retrieved from the primary screens ( Figure 1A, 1B), and their potency and efficacy tested.
Concentration-response curves for morphine-induced MOR endocytosis were obtained in the absence or presence at 1 μM of each CTS ( Figure 1C). Compared with morphine alone, the maximal response (E max ) of morphine-induced MOR endocytosis was significantly increased by these three CTSs, but not the half-maximum effective concentration (EC 50 ) ( Figure 1C). None of the CTSs affected morphine potency (α-factor), but they increased morphine efficacy (β-factor) by 2.8-3.7-fold . We further examined the enhancement of morphine-induced MOR endocytosis by convallatoxin, which is a blood-brain barrier-penetrating CTS (Gozalpour et al., 2014), using live-cell imaging of CHO-K1 cells expressing MOR-CopGFP. Convallatoxin significantly augmented morphine-induced MOR internalization ( Figure 1G), whereas morphine or convallatoxin alone had no effect. Additionally, we observed similar results in vivo using immunofluorescent staining for MOR and the plasma-membrane marker wheat germ agglutinin (WGA) in dorsal root ganglion (DRG) neurons obtained from mice co-treated with morphine and convallatoxin ( Figure 1H). Moreover, the potentiating effect of convallatoxin was only observed in MOR-expressing cells, indicating an opioid receptor subtype selectivity of CTSs ( Figure 1I). In contrast, convallatoxin failed to potentiate the MOR-endocytotic efficacy of methadone, an opioid effective in promoting MOR endocytosis but with a chemical structure distinct from that of morphine (Alvarez et al., 2002), indicating a potential probe dependence of convallatoxin ( Figure 1-Supplementary Figure 3). Here, we present the first validation of CTSs as unique, small-molecule enhancers of opioid-induced MOR endocytosis.
We evaluated the ability of CTSs to alter three signaling pathways: MOR-mediated G proteinindependent signaling (β-arrestin recruitment), G protein-dependent signaling (adenylyl cyclase inhibition), and G protein-coupled inwardly rectifying potassium (GIRK) channel activation (another G protein-dependent signaling pathway known to contribute to MOR-mediated analgesia) (Marker et al., 2004;Nockemann et al., 2013). The CTSs failed to modulate morphine-induced β-arrestin-2 recruitment to MOR as determined by the PathHunter enzyme complementation assay in CHO-K1  Figure 2H). Expression of MOR protein was not affected either by convallatoxin, morphine, or their combination ( Figure 2I). Thus, CTSs did not alter morphineinduced β-arrestin-2 recruitment, adenylyl cyclase inhibition, or GIRK-channel activation. Furthermore, the effect of CTSs on MOR endocytosis was retained even after these two pathways were inhibited.
However, CTSs did enhance morphine-induced MOR endocytosis in an AP2/clathrin-dependent manner without altering protein expression.
Identification of the convallatoxin binding site on MOR. To analyze the potential binding site of CTSs on MOR, the co-crystal structure of opioid agonist BU72 and mouse MOR was applied to computational docking studies (Huang et al., 2015b;Sounier et al., 2015). We first predicted the morphine binding conformation. After 20 ns of equilibrium molecular dynamics, morphine revealed a binding interaction with MOR different from that existing before equilibration (Figure 3- Supplementary   Figure 1). This resulted in a conformational change at MOR that exposed further binding sites (binding sites I, II, and III) surrounding the MOR transmembrane (TM) helices 1, 2, 5, 6, and 7 (TM1, TM2, TM5, TM6, and TM7; Figure 3A). Convallatoxin was then docked to these binding sites to Convallatoxin treatment diminishes morphine tolerance in an AP2/clathrin-dependent manner in mice. To assess the effect of convallatoxin on morphine-produced antinociception, tail-flick tests after acute and chronic treatments were performed as shown in Figure 4A-4D. Acute morphine and morphine + convallatoxin displayed a similar potency (ED 50 : morphine, 2.6 ± 0.3 mg/kg; morphine + convallatoxin, 2.8 ± 0.7 mg/kg) and magnitude of antinociception ( Figure 4A, 4C). However, chronic morphine + convallatoxin resulted in greater potency (ED 50 : morphine, 10.6 ± 1.2 mg/kg; morphine + convallatoxin, 6.4 ± 1.2 mg/kg) and magnitude ( Figure 4D) of antinociception, and greater MOR endocytosis ( Figure 4G) relative to chronic morphine alone. These results indicate that chronic treatment with convallatoxin reduces morphine antinociceptive tolerance. The experiments described in Figure  Tolerance may be unavoidable after long-term use of morphine (Labianca et al., 2012), and a strategy to reverse these characteristics should be of benefit for chronic pain management (Becker, 2010). We therefore investigated the effects of acute CTS administration on morphine-tolerant mice receiving chronic morphine injection twice daily for 8 d. On the test day, mice were challenged with morphine, either alone or in combination with convallatoxin, and tested for antinociception with the tail-flick model ( Figure 4E). Morphine-tolerant mice that received acute morphine with convallatoxin showed significantly greater antinociception ( Figure 4F) than mice that received morphine alone.
Furthermore, the dosage of convallatoxin we applied did not change the basal locomotor activity in the open field test (Figure 4-Supplementary Figure 2). Thus, these results indicated that acute CTS treatment was able to reverse morphine antinociceptive tolerance.
Because we found that convallatoxin enhances MOR endocytosis through an AP2/clathrin pathway, we then examined the role of AP2/clathrin on the effect of convallatoxin in morphine antinociception.
Intrathecal electroporation of shRNA silenced either clathrin or AP2 in DRG neurons ( suggesting that the AP2/clathrin adaptor complex mediates the chronic effect of convallatoxin in vivo.

Convallatoxin treatment reduces morphine tolerance in mice with chronic inflammatory pain.
Opioids are used to manage chronic osteoarticular pain, but repeated administration results in the development of antinociceptive tolerance (Fernández-Dueñas et al., 2007). We thus investigated whether a CTS has beneficial effects on morphine antinociception in the complete Freund's adjuvant (CFA)-induced mouse model of rheumatoid arthritis (Nagakura et al., 2003), and mechanical allodynia was measured by the Von Frey test to examine the antinociceptive effects of each treatment on postinoculation days (PIDs) 14, 16, and 18 ( Figure 6A). The threshold of mechanical allodynia in CFAtreated mice decreased significantly during PIDs 14 to 18 compared with saline-treated mice and reached a plateau. In CFA-treated mice, acute treatment with morphine + convallatoxin, but not with morphine alone, on PID 14 diminished mechanical allodynia, and the paw withdrawal threshold was similar to that in the non-CFA-treated groups ( Figure 6A). Furthermore, the morphine + convallatoxin group showed reduced antinociceptive tolerance relative to the morphine group. After daily treatment with morphine or morphine + convallatoxin for 5 d continuously (PID 18), the threshold of mechanical allodynia of these two experimental groups was reduced by approximately 92% ( Figure 6B) and 67% ( Figure 6C), respectively, in CFA-treated mice, whereas convallatoxin itself did not produce any antinociceptive effect. Moreover, the silencing of clathrin and AP2 in DRG neurons of B6 mice significantly decreased the effect of convallatoxin on both acute and chronic morphine antinociception ( Figure 6D), as well as on MOR endocytosis ( Figure 6E). This further supports the conclusion that CTSs enhance morphine efficacy and attenuate the development of antinociceptive tolerance through an AP2/clathrin-dependent pathway in chronic inflammatory pain.

Discussion
CTSs are used to treat cardiac failure and atrial fibrillation through inhibition of Na + /K + -ATPase (Wehrens and Marks, 2004). In addition to acting as Na + /K + -ATPase inhibitors, CTSs may regulate other proteins, potentially enabling them to serve as therapeutic agents. Bufalin selectively reduces the protein levels and intrinsic transcriptional activity of steroid receptor coactivators (SRC)-1 and SRC-3, and has potential as a broad-spectrum cancer inhibitor (Wang et al., 2014). Bufalin has also been shown to inhibit interferon-β expression and tumor necrosis factor signaling, and has thus been proposed as a treatment for inflammatory and autoimmune diseases, respectively (Ye et al., 2011).
In this study, we first identified CTSs as novel PAMs of morphine-induced MOR endocytosis Searching for allosteric modulators of GPCRs is a prominent topic for next-generation drug discovery. MOR-selective PAMs for the cAMP and β-arrestin pathways have been identified, and previous studies have indicated that morphine can selectively induce endocytosis in the mutant L83I (rat orthologue of human L85I) MOR without changing receptor binding affinity or the cAMP pathway, suggesting the existence of PAMs for MOR endocytosis. In the present study, CTSs bound to the F243 and I292 residues located in human MOR TM5 and TM6, which are far from L83, to potentiate morphine-mediated MOR endocytosis, potentially indicating a new allosteric site for MOR endocytosis.
According to docking and molecular-dynamical analysis using mouse MOR, the I290 residue is suggested to form a hydrogen bond with the carbonyl group of the furanone of convallatoxin, and one could imagine that PAMs of MOR might favor signaling pathways related to antinociception and disfavor signaling pathways that mediate unwanted side effects. Such abilities have been observed in allosteric modulators of the muscarinic system (Davis et al., 2009). In the present study, CTSs selectively potentiate morphine-mediated MOR endocytosis without altering the cAMP or β-arrestin pathways, suggesting a new class of PAMs distinguished by signaling bias. Another compelling characteristic of CTSs is their ability to enhance the efficacy (β factor) rather than the potency (α factor) of morphine. In the future, drug development programs will benefit from this behavior because it eliminates the need to change the dosage of morphine when combining it with CTSs.
Upon agonist-induced activation of MOR, G-protein coupling (Zaki et al., 2000) and β-arrestin-2 recruitment (Whistler and von Zastrow, 1998) are believed to be essential for promoting receptor internalization through a linkage of GPCRs to proteins of the endocytotic machinery, including clathrin (Goodman et al., 1996) and AP2 (Laporte et al., 1999). However, morphine-induced G-protein coupling and β-arrestin-2 recruitment were not altered by CTSs in the present study ( Figure 2E, 2F), and blockade of these pathways failed to attenuate the effects of CTSs on morphine-mediated MOR endocytosis ( Figure 2G, 2H). In fact, recent research has shown that β-arrestin-2 recruitment is not necessary for internalization of GPCRs. Several neuronal GPCRs, including the metabotropic glutamate receptor 1 (Dhami et al., 2004), the serotonin 5-HT2A receptor (Gray et al., 2003), and the M2 muscarinic cholinergic receptor (van Koppen and Kaiser, 2003), show β-arrestin-independent receptor internalization. Since clathrin and AP2 are involved with the internalization of numerous proteins, and since CTSs potentiated MOR endocytosis rather than that of DOR or KOR ( Figure 1I), a MOR-specific mechanism for CTSs seems possible (Ritter and Hall, 2009;Soohoo and Puthenveedu, 2013). Furthermore, CTSs enhanced MOR endocytosis through a β-arrestin-2-independent and AP2/clathrin-dependent mechanism, which warrants further investigation.
We investigated the effects of CTSs on acute and chronic morphine in animal models of acute thermal and chronic inflammatory pain. Convallatoxin significantly prevented the development of chronic, morphine-induced tolerance. Additionally, acute convallatoxin administration reversed morphine tolerance in morphine-tolerant mice ( Figure 4). In CFA-induced chronic inflammatory pain, morphine only partially relieved the mechanical allodynia ( Figure 6A). These results agree with previous studies showing that MOR agonists are highly effective in acute pain, but less so in chronic inflammatory and neuropathic pain (Fernández-Dueñas et al., 2007;Mankovsky et al., 2012;Rowbotham et al., 2003). Interestingly, the morphine-induced decrease in mechanical allodynia was enhanced by convallatoxin through AP2/clathrin-dependent pathways (Figure 6), which introduces the possibility that morphine's poor ability to induce MOR endocytosis underlies its lack of efficacy in treating chronic inflammatory pain (Kim et al., 2008). It is worth noting that convallatoxin itself exerted no antinociception ( Figures 4A, 4C, 4D, 4F, and 6A), and that the effects of convallatoxin on morphine-produced antinociception were attenuated in mice with spinal AP2 or clathrin knockdown ( Figures 5, 6D, 6E). In contrast, two other CTSs, ouabain and digitoxin, which have greater potency as Na + /K + -ATPase inhibitors than does convallatoxin (Lamers et al., 1985), acted as weak enhancers of morphine-induced MOR endocytosis relative to other glycosides ( Figure 1-Supplementary Figure 2). However, they have been shown to produce antinociception themselves or to antagonize morphineinduced antinociception (Gonzalez et al., 2012;Masocha et al., 2003;Zeng et al., 1999). It is unlikely that the Na + /K + -ATPase inhibition ability of CTSs is associated with ligand-induced MOR endocytosis because knockdown of Na + /K + -ATPase failed to attenuate the effects of CTSs on morphine-induced MOR endocytosis (Figure 2A, 2B). Furthermore, another CTS, rostafuroxin, which does not inhibit Na + /K + -ATPase, also enhanced morphine-induced MOR endocytosis ( Figure 1E, 1F). We thus suggest that other than Na + /K + -ATPase inhibition, CTSs potentially have a structure-activity relationship related to ligand-induced MOR endocytosis that correlates with MOR-mediated analgesia.
Therefore, it should be beneficial to modify the chemical structures of CTSs to increase ligandinduced MOR analgesia by enhancing ligand-induced MOR endocytosis, while diminishing cardiotoxic effects by reducing Na + /K + -ATPase inhibition. A similar strategy has been used for structural modification of digoxin to reduce cytotoxicity while maintaining inhibitory effects on T-cell differentiation (Huh et al., 2011).
In conclusion, our findings demonstrate that cardiotonic steroids act as novel positive allosteric modulators specifically enhancing morphine-induced MOR endocytosis, which enhances opioid antinociception and decrease morphine tolerance (Figure 7). These studies provide proof-of-concept for the development of signaling-specific opioid allosteric modulators, as well as new insights into clinical opioid-managed pain. The newly discovered regulatory mechanism of ligand-induced MOR endocytosis opens a new avenue in the study of the trafficking of MOR and of other GPCRs.

Materials and methods
Animals. Male, eight-to ten-week-old WT C57BL/6 (B6) mice (  Immunoblotting. Cells were lysed in homogenization buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.5, 0.3 M sucrose) containing protease and phosphatase inhibitor cocktails (Roche). Equal amounts of the samples (20-50 μg) were resolved using 8.5% or 10% SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Blots were incubated with rabbit monoclonal anti-MOR antibody Molecular dynamics (MD) simulation. The MD simulation was carried out to determine the natural conformation of the MOR, using the GROMACS v5.1.2 application (Berendsen et al., 1995). The force field for the whole system was GROMOS 43a1 (Chiu et al., 2009). The protein was restrained in a box of cubic shape whose edges were placed 1 nm from the complex, and an extended simple point charge water model was formed. The system was electrically neutralized by adding 20 Cl − ions. Twostep energy minimization was performed using steepest descent and conjugate gradient algorithms to converge the system up to 10 kJ mol −1 nm −1 . After a short energy minimization step, the system was subjected to NVT (300 K) and NPT (1 bar) equilibration with 100 ps running, and a LINCS algorithm was used to constrain the hydrogen bond lengths (Hess et al., 1997). The time step for the simulation was 2 fs. A cut-off distance of 10 Å was used for all short-range non-bonded interactions and a 12 Å Fourier grid spacing was used in the particle mesh Ewald method for long-range electrostatics. Finally, the restraints of the complex structure were removed and a 10-ns MD calculation was performed. The equilibrium structure was used to identify the binding site region using the Discovery Studio 2016/Define protocol and the binding site editing program (BIOVIA, Inc., San Diego, CA, USA).
Spinal cord surgery. Mice were first anesthetized with ketamine/xylazine i.p. (Sigma). To express copGFP-tagged MOR or mutants, or to reduce clathrin and AP2 expression in the spinal cord and DRG neurons (Wang et al., 2005), MOR expression vectors, or a shRNA against clathrin or AP2, or the universal negative control was dissolved in artificial cerebrospinal fluid, and then injected i.t. into adult mouse spinal cord using Micro-Renathane implantation tubing (Braintree Scientific) inserted into the T11-T13 intervertebral disc. After injection, the NEPA21 electroporator gene transfection system (Nepa Gene) was used to deliver plasmid or shRNA into cells through needle electrodes inserted between the L1 and L6 vertebrae. The poring pulse conditions for electroporation were as follows: 150 V, pulse length 5 ms, inter-pulse interval 50 ms, decay rate 10%, and polarity plus. The transfer pulse conditions were as follows: 20 V, pulse length 50 ms, pulse interval 50 ms, decay rate 40%, and polarity plus and minus (Lee et al., 2014). All behavioral tests were conducted at least 7 d after surgery. We confirmed that mice that received the shRNA universal negative control had no significant tissue injury or behavioral dysfunction.
Tail-flick test. Drug-induced antinociception was evaluated using the Tail (Crocker and Russell, 1984). Briefly, a series of test levels was chosen with equal spacing between each log of drug dose. Then, a series of trials (n ≧ 6) was performed following the rule that the dose is reduced on the next trial if inhibition of the tail-flick response is observed and increased if no inhibition is observed.
Each mouse underwent only one trial. The ED 50 value was derived from the equation ED 50 = Xf + k × d, where Xf was the last dose administered, k was the tabular value, and d was the interval between doses.
To determine the chronic effects of compounds on the development of morphine tolerance, mice were chronically injected s.c. with vehicle, morphine (5 or 10 mg/kg), convallatoxin (0.5 or 1 mg/kg), or morphine (5 or 10 mg/kg) + convallatoxin (0.5 mg/kg) twice daily for 8 d. The antinociceptive effect of each treatment was determined as the radiant-heat tail-flick latency on d 1, and again after the final treatment on d 8. To determine the acute effects of compounds on morphine tolerance, mice were first injected s.c. with morphine at 10 mg/kg, twice daily for 8 d to generate morphine-tolerant mice. At 12 h after the last morphine treatment, mice were administered s.c. with acute vehicle, morphine (10 mg/kg), convallatoxin (1 mg/kg), or morphine (10 mg/kg) + convallatoxin (0.5 or 1 mg/kg) and the radiant-heat tail-flick latency was determined at the indicated time points.
Open-field test. Testing was carried out in an open-field chamber 42 x 42 cm, 35 cm in height, under 120-lx lighting as previously described (Wu et al., 2010). Immediately after administration of morphine at 10 mg/kg, convallatoxin at 0.5 mg/kg, or vehicle, the mice were placed in the open-field apparatus along the wall, and behavior recorded with beam-break technology for 30 min. Data were analyzed with Prism v. 5.0 software (GraphPad).

CFA-induced chronic inflammatory pain and Von Frey hair test. Chronic inflammatory pain was
induced by intraplantar injection of CFA. Briefly, each B6 mouse was injected with 20 μL of either saline or CFA in the footpad of the right hind paw under isoflurane anesthesia on PID 0. From PID 14 to 18, saline-and CFA-injected mice were administered vehicle, morphine (10 mg/kg), convallatoxin (0.5 mg/kg), or morphine (10 mg/kg) + convallatoxin (0.5 mg/kg), s.c., twice daily. Mechanical allodynia was subsequently evaluated using von Frey filaments (range 0.1-1 g; IITC Life Science).
Mice were placed on a mesh floor with 5 × 5 mm holes, covered with a cup to prevent visual stimulation, and allowed to adapt for 1 h prior to testing. On test days, withdrawal responses following hind paw stimulation were measured, with filaments applied to the middle of the plantar surface of the hind paw. Mechanical allodynia was defined as change in the pressure required to induce withdrawal.

Statistics.
Generally, in the present study, cell lines were not validated by genomic testing but had previously been tested for mycoplasma contamination. All mouse and in vitro experiments were repeated multiple times as indicated in the figures and figure legends. No statistical method was used to predetermine sample size. There were no exclusion criteria. No samples, mice, or data points were excluded from the reported analyses. No samples or mice were randomly assigned to experimental groups. Investigators were blinded to group allocation during the experiment and when assessing the outcome. Data are presented as the mean ± SEM. All data were tested for normal distribution and equal variance between groups using Prism v. 5.0 software (GraphPad). Multiple groups were compared using 1-way ANOVA with Newman-Keul's post hoc test or 2-way ANOVA with Bonferroni's post hoc test, whereas pairs of groups were compared using the two-sided Student's t-test in Prism v.         Representative immunofluorescence images (D) and quantification (E) of the distribution of CopGFPtagged MOR and two of its mutants (green) and WGA (red) in mouse dorsal root ganglion (DRG) after drug treatment. F 5,24 = 11.29; p < 0.0001 (1-way ANOVA). The localization of MOR and WGA-labeled plasma membrane is monitored by confocal microscopy. DAPI (blue) is a nuclear marker. Scale bar,   Human F243, V247, T251, A289, and I292 were mutated in this study.     However, its poor ability to induce MOR endocytosis results in morphine tolerance after long-term use.
CTSs bind to the F243 and I292 residues, induce a conformational change in MOR, and thus promote better induction of receptor endocytosis by morphine, accompanied by better antinociception and reduced morphine tolerance.