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. 2015 Jun 16;21(7):591–598. doi: 10.1111/cns.12409

Modulation of mTOR Activity by μ‐Opioid Receptor is Dependent upon the Association of Receptor and FK506‐Binding Protein 12

Yan Wang 1,3, Yan‐Hui Ge 1, Yan‐Xia Wang 1, Ting Liu 1, Ping‐Yee Law 2, Horace H Loh 2, Hong‐Zhuan Chen 1,✉, Yu Qiu 1,✉
PMCID: PMC4477277  NIHMSID: NIHMS686258  PMID: 26096047

Summary

Aims

Mechanistic/mammalian target of rapamycin (mTOR) activation by μ‐opioid receptor (OPRM1) participates in antinociceptive tolerance, hyperalgesia, and physical dependence. Our previous study also showed that mTOR activation by OPRM1 could attenuate β amyloid oligomers‐induced neurotoxicity. OPRM1 is demonstrated to interact with FK506‐binding protein 12 (FKBP12). It is our great interest to investigate whether OPRM1‐mediated mTOR signaling is related to receptor‐FKBP12 association.

Methods

The activities of mTOR and its downstream effector p70 S6K were measured by immunoblotting their phosphorylation status. The interaction of receptor with mTOR was detected by co‐immunoprecipitation and immunofluorescence.

Results

OPRM1 activation by morphine‐induced time‐dependent mTOR activation. PI3K‐specific inhibitor LY294002 only blocked the late phase of mTOR activation. However, morphine‐induced mTOR activation was totally blocked at all time points in cells expressing FKBP12 association‐deficient mutant receptor. FKBP12 knockdown also blocked morphine‐induced mTOR activation. Further analysis demonstrated that morphine treatment enhanced the association of receptor with phosphorylated mTOR, whereas decreased association was observed after FKBP12 knockdown, mTOR inhibition or in cells expressing FKBP12 association‐deficient mutant.

Conclusions

OPRM1‐FKBP12 association played a key role in OPRM1‐mediated mTOR activation, which could underlie the mechanisms of multiple physiological and pathological processes. Thus, our findings provide new avenue to modulating these processes.

Keywords: FK506‐binding protein 12, Mechanistic/mammalian target of rapamycin, Protein interaction, μ‐opioid receptor

Introduction

μ‐opioid receptor (OPRM1) plays a decisive role in antinociception 1. Opioids are still the most effective medication for pain control. However, the clinical use of opioids, especially for long‐term use, is hampered by the development of antinociceptive tolerance, hyperalgesia, physical dependence, and addiction. Although the mechanisms underlying these phenomena are not fully resolved and still under intensive investigation, the importance of mechanistic target of rapamycin (mTOR) in these processes gradually emerged 2, 3, 4.

mTOR, a serine/threonine protein kinase, primarily controls transcription, protein synthesis, and cell growth, cell proliferation, cell motility 5, 6. In central nervous system (CNS), mTOR signaling has emerged as a critical integrator of neuronal activity and synaptic inputs and plays a pivotal role in neuronal plasticity and the process of learning and memory 7, 8. mTOR forms two structurally and functionally distinct complexes: mTOR complex 1 (mTORC1) and mTORC2. mTORC1 contains the defining components raptor and proline‐rich protein kinase B (Akt) substrate 40 kDa. mTOR responds to a phosphatidic acid‐mediated signal to transmit a positive signal to p70 S6 kinases (p70 S6K) 9. Phosphorylation of p70 S6K at Thr389 serves as a characterized downstream effector of mTOR 10, 11. Another well‐characterized downstream effector is the eukaryotic translation initiation factor‐4E‐binding protein 1 (4E‐BP1) 12. Both p70 S6K and 4E‐BP1 interact with mRNAs and regulate mRNA translation initiation and progression, which are the control steps of protein synthesis 13.

By controlling the protein synthesis in the CNS, mTOR has been found to regulate multiple biological processes which are mediated by opioid receptor. It is shown to play a key role in addiction‐related behaviors such as reward seeking and excessive drug intake 14. The activation of mTOR and p70 S6K in hippocampus by morphine is shown to be positively correlated with the acquisition of morphine conditioned place preference (CPP), a learning paradigm for assessing drug reward 4. Rapamycin, a selective inhibitor of mTOR, prevents the acquisition of CPP 4, 15. Activation of OPRM1 in rat spinal dorsal horn neurons results in an increase in mTOR,p70 S6K and a decrease in 4E‐BP1 activities, and the inhibition of mTOR activity by rapamycin or siRNA blocks both induction and maintenance of morphine tolerance and hyperalgesia 3. Our recent study has demonstrated that the attenuation of β amyloid oligomers‐induced neurotoxicity by OPRM1 activation is mediated through mTOR signaling 16.

mTOR belongs to the phosphatidylinositol 3‐kinase (PI3K)‐related kinase family. The best‐characterized activation input to mTORC1 is through PI3K/Akt signaling pathway. On the other hand, rapamycin can specifically and effectively block the activity of mTORC1 by binding to FK506‐binding protein 12 (FKBP12) to form a complex with mTOR 17. FKBP12 belongs to the family of the immunophilins which are protein chaperones to guide proper folding and assembly that provide functional stability to multiprotein macromolecules 18. Moreover, FKBP12 is expressed 10‐ to 50‐fold more highly in CNS and peripheral nervous system than in immune system 19, 20.

In our previous study, we have demonstrated that OPRM1 specifically interacts with FKBP12 and the receptor–FKBP12 complex increases after morphine treatment 21. Moreover, we have showed that PI3K signaling pathway is involved in mTOR signaling induced by OPRM1 activation 16. In this study, we further demonstrated that the association of FKBP12 with OPRM1 played a key role in mTOR activation.

Materials and Methods

Materials

Morphine and naloxone were supplied by the National Institute on Drug Abuse. Rapamycin, H‐D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2 (CTAP), and mouse monoclonal anti‐β‐actin antibody were purchased from Sigma Chemical Co (St. Louis, MO, USA). Mouse HA.11 monoclonal antibody (clone 16B2) was purchased from Covance (Berkeley, CA, USA). All the other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All the cell culture reagents were purchased from GIBCO (Grand Island, NY, USA).

Cell Culture

HEK293 cells stably transfected with hemagglutinin (HA) epitope tagged rat OPRM1 (HA‐OPRM1) or its mutant at Pro353 to Ala (HA‐OPRM1P353A) were maintained in Eagle's minimum essential medium (MEM) with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin plus 200 μg/mL G418 at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Western Blot

After treatment, cells were extracted with cell lysis buffer (20 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X‐100, 2.5 mM sodium pyrophosphate, 1 mM β‐glycerophosphate, 1 mM sodium vanadate, 1 μg/mL leupeptin, and 1 ×  protease inhibitor cocktail [Sigma]). After centrifuging at 12,000 × g for 5 min, sample loading buffer was added to the supernatants and boiled for 5 min. Approximately 30–40 μg of protein was subjected to SDS‐PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% milk and then incubated with primary antibodies against mTOR phosphorylated at Ser2448 (1:1000), mTOR (1:1000), p70 S6K phosphorylated at Thr389 (1:1000), p70 S6K (1:1000), and β‐actin (1:5000) overnight at 4°C. Membranes were then incubated with goat anti‐rabbit/mouse HRP‐conjugated secondary antibody for 1 h at room temperature, visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical, Rockford, IL, USA), and analyzed by Image J (National Institutes of Health, Bethesda, MD, USA) software.

Knockdown of FKBP12

HEK293 cells stably expressing OPRM1 were transfected with short interfering RNA (siRNA) corresponding to the target sequence GCTTGAAGATGGAAAGAAA of FKBP12 gene (Qiagen, Valencia, CA, USA) or a scrambled siRNA as control at the final concentration of 50 nm using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions 22; 24 h later, cells were analyzed as indicated. The effect of siRNA on protein expression was determined by Western blot.

Co‐Immunoprecipitation

After treatment, cells in 100 mm dish were extracted with 300 μL lysis buffer (0.1% Triton X‐100, 50 mM Tris·HCl pH 8.0, 100 mM NaCl, 10% glycerol, 10 mM EDTA, 10 mM NaF, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyrophosphate, 1 mM sodium vanadate, and 1 ×  protease inhibitor). After centrifuging at 12,000 × g for 5 min, 1 μL mouse anti‐HA antibodies and 30 μL protein‐G‐agarose (Pierce, Rockford, IL, USA) were added to the supernatants and rotated overnight. Antibodies specific for phospho‐mTOR Ser2448, mTOR and HA were used for immunoblotting.

Confocal Imaging

HEK293 cells stably expressing OPRM1, OPRM1P353A, and OPRM1 with FKBP12 knockdown were treated with 1 μM morphine for 5 min, fixed with prewarmed 3%‐4% formaldehyde for 20 min at room temperature. Then, the cells were washed with PBS three times and blocked with 10% normal goat serum in PBS. Then, the cells were permeabilized by 0.3% Triton X‐100 and stained with rabbit antiphospho‐mTOR Ser2448 antibody (Clone D9C2, 1:200) and mouse anti‐HA antibody (1:1000), washed with PBS for five times and stained with Alexa 488‐conjugated goat anti‐mouse and Alexa 594‐conjugated goat anti‐rabbit antibodies (1:500, Molecular Probes, Eugene, OR, USA). Cells were viewed and captured with laser confocal microscope (Carl Zeiss LSM 510, Oberkochen, Germany). Colocalization was analyzed using MetaMorph Microscopy Automation and Image Analysis Software (Sunnyvale, CA, USA) to determine the percentage of receptor overlapping phosphorylated mTOR.

Statistical Analysis

Data were presented as mean ± standard error of the mean (SEM). The results were analyzed by one‐way analysis of variances (ANOVA) followed by Dunnett's test to compare with control or Bonferroni's test to compare all pairs or two‐way anova followed by Bonferroni's test using GraphPad Prism 5.0 statistical analysis software (La Jolla, CA, USA). P < 0.05 was considered statistically significant.

Results

Morphine‐Induced mTOR Activation is not fully Mediated by PI3K Signaling Pathway

In a previous study, we have demonstrated that morphine induces mTOR activation 16. Here, we continue to characterize this morphine action by investigating the time‐dependent effect of morphine on mTOR activation. When OPRM1‐expressing HEK293 cells were treated with 1 μM morphine, marked increase of mTOR phosphorylation was demonstrated (Figure 1A upper panel and B). Robust increase of mTOR phosphorylation started within 5 min of morphine exposure and lasted for at least 2 h. 5 min, 15 min, 30 min, 1 h, and 2 h of morphine treatment resulted in 1.43 ± 0.11‐, 1.50 ± 0.05‐, 1.57 ± 0.09‐, 1.61 ± 0.09‐, 1.50 ± 0.09‐fold increase of mTOR phosphorylation, respectively. To confirm the effect of PI3K signaling pathway, PI3K‐specific inhibitor LY294002 was employed. Unexpectedly, in the presence of the inhibitor, 5 min of morphine treatment still resulted in 1.42 ± 0.06‐fold increase of mTOR phosphorylation. But LY294002 did block the phosphorylation of mTOR when the cells were treated with morphine for more than 15 min (Figure 1A middle panel and B). Further analyses demonstrated that Akt was activated after 15 and 30 min of morphine treatment (Figure S1A,B). These data demonstrated that the morphine‐induced mTOR activation was not fully mediated by PI3K signaling pathway. The morphine‐induced mTOR activation could be divided into two phases, and only the late phase was related to PI3K signaling pathway.

Figure 1.

Figure 1

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Activation of mTOR by morphine was not fully blocked by PI3K inhibitor but was absent in expressing FKBP12 association‐deficient mutant OPRM1P353A. HEK293 cells stably expressing OPRM1 and OPRM1P353A were treated with 1 μM morphine for various time intervals. OPRM1‐expressing cells were also pretreated with 10 μM LY294002 for 30 min before morphine treatment. Cell extracts were analyzed by immunoblotting with specific phospho‐mTOR antibody at Ser2448. (A) Representative blots. (B) Quantification of the relative densities. **P < 0.01, ***P < 0.001 versus 0 min, also versus the corresponding time points of OPRM1P353A. #P < 0.05, versus 0 min for LY294002‐pretreated OPRM1. $P < 0.05, between LY294002‐pretreated OPRM1 and OPRM1P353A at 5 min. &&P < 0.01, &&&P < 0.001 between OPRM1 and LY294002‐pretreated OPRM1 at the corresponding time points. Experiments were repeated 3–5 times.

The Initial mTOR Activation by Morphine is dependent on OPRM1 association with FKBP12

Our previous data identified that FKBP12 is an OPRM1 association protein in mammalian cells and Pro353 residue in the carboxyl tail of OPRM1 is involved in the interaction 21. Site mutation of Pro353 to Ala (OPRM1P353A) abolished the association of FKBP12 with OPRM1 21. FKBP12 binding to rapamycin regulates the activities of mTOR and its downstream effectors, which is highly conserved from yeast to humans 5, 6. Thus, we investigated whether the interaction of FKBP12 with OPRM1 could affect the morphine‐induced mTOR activation. As shown in Figure 1A lower panel and 1B, morphine did not activate mTOR in HEK293 cells expressing OPRM1P353A at all time points, demonstrating that receptor association with FKBP12 played an important role in morphine‐induced mTOR activation and could be the prerequisite for PI3K‐mediated mTOR activation. The activation of p70 S6K, one of mTOR downstream effectors, was measured by detecting its phosphorylation at Thr389. In cells expressing wild‐type OPRM1, phosphorylation of p70 S6K was increased to 2.09 ± 0.51‐fold over basal level after 5 min treatment of morphine (Figure 2A,B), whereas the increase of p70 S6K phosphorylation was not observed in cells expressing OPRM1P353A mutant (Figure 2A,C). To further confirm the effects of FKBP12 association with OPRM1 on morphine‐induced mTOR activation, the endogenous FKBP12 of OPRM1‐expressing cells was knocked down by specific siRNA. As shown in Figure 2D,E, knockdown of endogenous FKBP12 abolished morphine‐induced activation of mTOR and p70 S6K, whereas treatment with scrambled siRNA did not affect the activities of mTOR and p70 S6K activated by morphine.

Figure 2.

Figure 2

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Activation of mTOR and its downstream effector p70 S6K by morphine was dependent on receptor association with FKBP12 and receptor activation. (A–C) HEK293 cells stably expressing OPRM1 or OPRM1P353A were preincubated with 10 μM CTAP for 10 min or not and further treated with 1 μM morphine for 5 min. Then, cell extracts were analyzed by immunoblotting with antibodies against mTOR phosphorylated at Ser2448 (p‐mTOR), total mTOR, p70 S6K phosphorylated at Thr389 (p‐p70 S6K), total p70 S6K (A), and the relative densities were quantified (B, C). *P < 0.05 versus control (ctrl); #P < 0.05 versus cells treated with morphine. (D–E) OPRM1‐expressing cells were transfected with siRNA of FKBP12 for 24 h, and then, the cells were treated with 1 μM morphine for 5 min. Cell extracts were analyzed by immunoblotting with specific antibody against mTOR phosphorylated at Ser2448 (p‐mTOR) (D), and the relative densities were quantified (E). *P < 0.05, **P < 0.01, ***P < 0.001. Experiments were repeated 3–5 times.

Next, we investigated whether morphine‐induced mTOR activation was a direct consequence of OPRM1 activation. As shown in Figure 2A,B, morphine‐induced phosphorylation of mTOR and p70 S6K was almost totally inhibited by preincubation with selective OPRM1 antagonist CTAP. Moreover, treatment with nonselective antagonist naloxone alone did not affect the activity of mTOR (Figure S2A,B). These results indicated that the effect of morphine was dependent on OPRM1 activation.

OPRM1 Associates with Activated mTOR through Receptor Interaction with FKBP12

To further explore whether morphine‐induced activation of mTOR is mediated by direct interplay between OPRM1 and FKBP12, the receptor signaling complex after activation was immunoprecipitated and analyzed. As shown in Figure 3A,B, mTOR existed in the receptor complex in the absence of morphine. After morphine treatment, the amount of total mTOR in receptor complex did not change, but phosphorylated mTOR increased in activated receptor complex in cells expressing wild‐type OPRM1 (3.05 ± 0.40‐fold over basal level). Whereas in OPRM1P353A‐expressing cells in which the interaction of FKBP12 with receptor is absent or in wild‐type OPRM1‐expressing cells with FKBP12 knockdown, mTOR was still present in the receptor complex without morphine treatment, however, morphine treatment did not change the amount of phosphorylated mTOR in receptor complex. Pretreatment with rapamycin which binds to FKBP12 and inhibits mTOR activity also abolished the morphine‐induced increase of phosphorylated mTOR in receptor complex.

Figure 3.

Figure 3

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The activation of mTOR in receptor complex was dependent on the association of OPRM1 with FKBP12. HEK293 cells stably expressing OPRM1 or mutant OPRM1P353A were treated with 1 μM morphine for 5 min. OPRM1‐expressing cells were also pretreated with 1 μM rapamycin for 10 min or were transfected with siRNA for FKBP12 for 24 h before morphine treatment. OPRM1 signaling complexes were immunoprecipitated with HA antibody and immunoblotted with phospho‐mTOR, mTOR antibodies. (A) Representative blots. (B) Relative densities of morphine‐treated samples were quantified by comparing with that of corresponding controls (B). **P < 0.01 versus OPRM1‐expressing cells. Experiments were repeated 3 times.

The increased activation of mTOR in receptor signaling complex was also confirmed by immunofluorescence. As expected, after treatment of OPRM1‐expressing cells with 1 μM morphine for 5 min, OPRM1 and phosphorylated mTOR were observed to be colocalized in cluster mainly on cell membrane and in cytoplasm (Figure 4A). Quantification analysis demonstrated that the colocalization of receptor and phosphorylated mTOR was increased 1.27 ± 0.05‐fold over basal level (Figure 4B). These results were consistent with the association of phosphorylated mTOR and OPRM1 in response to receptor activation demonstrated by immunoprecipitation assay. Such increased colocalization of OPRM1 and phosphorylated mTOR was absent or significantly reduced in cells expressing mutant OPRM1P353A, as well as in OPRM1‐expressing cells with FKBP12 level knocked down by si‐RNA treatment or with rapamycin pretreatment (Figure 4A,B). These data indicated that receptor‐associated FKBP12 promoted the phosphorylation of mTOR within receptor complex.

Figure 4.

Figure 4

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Colocalization of activated mTOR and OPRM1. HEK‐OPRM1 cells and HEK‐OPRM1P353A cells were treated with 1 μM morphine for 5 min. OPRM1‐expressing cells were also pretreated with 1 μM rapamycin for 10 min or were transfected with siRNA of FKBP12 for 24 h before morphine treatment. Then, the cells were stained with phospho‐mTOR Ser2448 antibody for activated mTOR and HA antibody for receptor (A) and the colocalization of activated mTOR and receptor was quantified (B). **P < 0.01. 3 individual slides were performed in each group, and 3–6 cells per slide were analyzed.

Discussion

In the present study, we demonstrated that morphine‐induced time‐dependent mTOR activation in HEK293 cells stably expressing OPRM1. Even though PI3K signaling played an important role in mTOR activation, the effect could not be fully blocked by PI3K inhibitor which only inhibited mTOR activation at the late phase, whereas mTOR activation by OPRM1 was blocked in cells expressing the mutant receptor incapable of binding to FKBP12 at all time points.

As a GPCR, OPRM1 could activate PI3K/Akt signaling pathway through Gβγ subunits 23. The OPRM1‐induced activation of mTOR has been attributed to the activation of PI3K/Akt by our and others’ studies 3, 4, 16. The activation of Akt by morphine has been reported to occur after 15 to 30 min treatment of morphine 24, 25. In our study, we also demonstrated that Akt was not activated at 5 min of morphine treatment, which is consistent with others’ results. Our current study further demonstrated that PI3K inhibitor did not affect the initial mTOR action induced by morphine. These data suggest that the mTOR activation at 5 min of morphine treatment was not mediated through PI3K/Akt signaling.

Our previous data identified FKBP12 as a specific OPRM1 association protein 21. Conditional knockout of FKBP12 enhances mTOR‐raptor interaction, long‐term potentiation, memory, and perseverative and repetitive behavior 26. Our results showed that basal mTOR activity was increased in cells transfected with FKBP12 siRNA (Figure 2D,E), which is consistent with previous study 26. However, morphine‐induced mTOR activation was absent in these cells, implicating that morphine‐induced mTOR activation is not dependent on mTOR‐raptor interaction. Morphine‐induced activation of mTORC1 was blocked in cells expressing mutant receptor deficient in interacting with FKBP12, indicating that morphine‐induced mTOR activation is dependent on the association of FKBP12 with the receptor.

Further analysis of receptor complex manifested that the association of receptor and mTOR existed without receptor activation, but the phosphorylated mTOR in the receptor complex was specifically increased after receptor activation by morphine. However, such increased association of phosphorylated mTOR was diminished in mutant receptor or in wild‐type receptor with FKBP12 knockdown without affecting the association of mTOR and receptor. Rapamycin also blocked the increase of phosphorylated mTOR associated with the receptor. Such effects were further confirmed by colocalization of OPRM1 with phosphorylated mTOR. These results demonstrate that receptor's association with FKBP12 played a critical role of FKBP12 in mTOR activation by morphine and was unnecessary for the association between mTOR and receptor. The association between receptor and mTOR has not been reported and merits further investigation.

Blockade of receptor with antagonist naloxone did not affect mTOR activation. Neither the activated mTOR in receptor complex nor increase in the colocalization of activated mTOR and OPRM1 was observed in cells treated with naloxone (Figure S3A,B). Moreover, mTOR association with the receptor could not initiate mTOR activation upon morphine treatment when the interaction between FKBP12 and OPRM1 was impaired. Thus, mTOR activation is dependent on receptor activation and FKBP12 association with the receptor. In that the phosphorylation of mTOR by PI3K could also be blocked in the absence of receptor‐FKBP12 interaction, we hypothesize that FKBP12 may act as an adaptor to toggle OPRM1 and mTOR and play a role in mediating the effect of PI3K.

Conclusions

Our current study demonstrated the critical role of receptor‐FKBP12 association in OPRM1‐induced mTOR signaling. PI3K acts in concert with receptor‐associated FKBP12 to modulate mTOR activity. Our study uncovered a new intracellular pathway of mTOR activation through OPRM1. As modulation of mTOR signaling by OPRM1 has been shown to be involved in multiple physiological and pathological processes, our findings may further clarify the underlying mechanisms. Therefore, our study could provide a possible target for modulating these processes.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Figure S1 The activation of Akt by morphine. HEK293 cells stably expressing OPRM1 were treated with 1 μM morphine for various time intervals. Cell extracts were analyzed by immunoblotting with specific phospho‐Akt antibody at Ser473 (A) and the relative densities were quantified (B). *, P < 0.05, **, P < 0.01 versus 0 min.

Figure S2 mTOR was not activated by OPRM1 antagonist naloxone. HEK293 cells stably expressing OPRM1 were treated with 1 μM morphine or 10 μM naloxone for indicated time intervals. The cells were also pretreated with 10 μM naloxone and then treated with 1 μM morphine. Cell extracts were analyzed by immunoblotting with specific phospho‐mTOR antibody at Ser2448 (A) and the relative densities were quantified (B). *P < 0.05, **P < 0.01.

Figure S3 The activated mTOR was not co‐immunoprecipitated or colocalized with OPRM1 after treatment with OPRM1 antagonist naloxone. HEK293 cells stably expressing OPRM1 were treated with 10 μM naloxone for 5 min. (A) OPRM1 signaling complexes were immunoprecipitated with HA antibody or normal serum and immunoblotted with phospho‐mTOR, mTOR, HA antibodies. (B) The cells were stained with phospho‐mTOR Ser2448 antibody for activated mTOR and HA antibody for receptor.

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Acknowledgments

This research was supported by National Natural Science Foundation of China (81173044), International Science & Technology Cooperation Program of China (2011DFA33180), and Shanghai Pujiang Program (11PJ1406200) and in parts by National Institutes of Health grants (DA007339, DA011806).

The first two authors contributed equally to this work.

References

  • 1. Matthes HW, Maldonado R, Simonin F, et al. Loss of morphine‐induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu‐opioid‐receptor gene. Nature 1996;383:819–823. [DOI] [PubMed] [Google Scholar]
  • 2. Mazei‐Robison MS, Koo JW, Friedman AK, et al. Role for mTOR signaling and neuronal activity in morphine‐induced adaptations in ventral tegmental area dopamine neurons. Neuron 2011;72:977–990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Xu JT, Zhao JY, Zhao X, et al. Opioid receptor‐triggered spinal mTORC1 activation contributes to morphine tolerance and hyperalgesia. J Clin Invest 2014;124:592–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Cui Y, Zhang XQ, Xin WJ, Jing J, Liu XG. Activation of phosphatidylinositol 3‐kinase/Akt‐mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine‐induced place preference in rats. Neuroscience 2010;171:134–143. [DOI] [PubMed] [Google Scholar]
  • 5. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 2004;18:1926–1945. [DOI] [PubMed] [Google Scholar]
  • 6. Jacinto E, Hall MN. Tor signalling in bugs, brain and brawn. Nat Rev Mol Cell Biol 2003;4:117–126. [DOI] [PubMed] [Google Scholar]
  • 7. Costa‐Mattioli M, Monteggia LM. mTOR complexes in neurodevelopmental and neuropsychiatric disorders. Nat Neurosci 2013;16:1537–1543. [DOI] [PubMed] [Google Scholar]
  • 8. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012;149:274–293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Fang Y, Vilella‐Bach M, Bachmann R, Flanigan A, Chen J. Phosphatidic acid‐mediated mitogenic activation of mTOR signaling. Science 2001;294:1942–1945. [DOI] [PubMed] [Google Scholar]
  • 10. Pullen N, Thomas G. The modular phosphorylation and activation of p70s6k. FEBS Lett 1997;410:78–82. [DOI] [PubMed] [Google Scholar]
  • 11. Saitoh M, Pullen N, Brennan P, Cantrell D, Dennis PB, Thomas G. Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site. J Biol Chem 2002;277:20104–20112. [DOI] [PubMed] [Google Scholar]
  • 12. Gingras AC, Gygi SP, Raught B, et al. Regulation of 4E‐BP1 phosphorylation: a novel two‐step mechanism. Genes Dev 1999;13:1422–1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Ma XM, Blenis J. Molecular mechanisms of mTOR‐mediated translational control. Nat Rev Mol Cell Biol 2009;10:307–318. [DOI] [PubMed] [Google Scholar]
  • 14. Neasta J, Barak S, Ben Hamida S, Ron D. mTOR complex 1: a key player in neuroadaptations induced by drugs of abuse J Neurochem 2014;130:172–184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Lin J, Liu L, Wen Q, et al. Rapamycin prevents drug seeking via disrupting reconsolidation of reward memory in rats. Int J Neuropsychopharmacol 2014;17:127–136. [DOI] [PubMed] [Google Scholar]
  • 16. Wang Y, Wang YX, Liu T, et al. mu‐opioid receptor attenuates abeta oligomers‐induced neurotoxicity through mTOR signaling. CNS Neurosci Ther 2015;21:8–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nat Rev Immunol 2009;9:324–337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Barik S. Immunophilins: for the love of proteins. Cell Mol Life Sci 2006;63:2889–2900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Steiner JP, Dawson TM, Fotuhi M, et al. High brain densities of the immunophilin FKBP colocalized with calcineurin. Nature 1992;358:584–587. [DOI] [PubMed] [Google Scholar]
  • 20. Lyons WE, Steiner JP, Snyder SH, Dawson TM. Neuronal regeneration enhances the expression of the immunophilin FKBP‐12. J Neurosci 1995;15:2985–2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Qiu Y, Zhao W, Wang Y, et al. FK506‐binding protein 12 modulates mu‐opioid receptor phosphorylation and protein kinase Cɛ‐dependent signaling by its direct interaction with the receptor. Mol Pharmacol 2014;85:37–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Giordano A, Romano S, Mallardo M, et al. FK506 can activate transforming growth factor‐beta signalling in vascular smooth muscle cells and promote proliferation. Cardiovasc Res 2008;79:519–526. [DOI] [PubMed] [Google Scholar]
  • 23. Maier U, Babich A, Nurnberg B. Roles of non‐catalytic subunits in gbetagamma‐induced activation of class I phosphoinositide 3‐kinase isoforms beta and gamma. J Biol Chem 1999;274:29311–29317. [DOI] [PubMed] [Google Scholar]
  • 24. Horvath RJ, DeLeo JA. Morphine enhances microglial migration through modulation of P2X4 receptor signaling. J Neurosci 2009;29:998–1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Liu H, Li H, Guo L, et al. Mechanisms involved in phosphatidylinositol 3‐kinase pathway mediated up‐regulation of the mu opioid receptor in lymphocytes. Biochem Pharmacol 2010;79:516–523. [DOI] [PubMed] [Google Scholar]
  • 26. Hoeffer CA, Tang W, Wong H, et al. Removal of FKBP12 enhances mTOR‐Raptor interactions, LTP, memory, and perseverative/repetitive behavior. Neuron 2008;60:832–845. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 The activation of Akt by morphine. HEK293 cells stably expressing OPRM1 were treated with 1 μM morphine for various time intervals. Cell extracts were analyzed by immunoblotting with specific phospho‐Akt antibody at Ser473 (A) and the relative densities were quantified (B). *, P < 0.05, **, P < 0.01 versus 0 min.

Figure S2 mTOR was not activated by OPRM1 antagonist naloxone. HEK293 cells stably expressing OPRM1 were treated with 1 μM morphine or 10 μM naloxone for indicated time intervals. The cells were also pretreated with 10 μM naloxone and then treated with 1 μM morphine. Cell extracts were analyzed by immunoblotting with specific phospho‐mTOR antibody at Ser2448 (A) and the relative densities were quantified (B). *P < 0.05, **P < 0.01.

Figure S3 The activated mTOR was not co‐immunoprecipitated or colocalized with OPRM1 after treatment with OPRM1 antagonist naloxone. HEK293 cells stably expressing OPRM1 were treated with 10 μM naloxone for 5 min. (A) OPRM1 signaling complexes were immunoprecipitated with HA antibody or normal serum and immunoblotted with phospho‐mTOR, mTOR, HA antibodies. (B) The cells were stained with phospho‐mTOR Ser2448 antibody for activated mTOR and HA antibody for receptor.

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