Loss of Morphine Reward and Dependence in Mice Lacking G Protein–Coupled Receptor Kinase 5

doi:10.1016/j.biopsych.2014.01.021

Biological Psychiatry

Volume 76, Issue 10, 15 November 2014, Pages 767-774

https://doi.org/10.1016/j.biopsych.2014.01.021 Get rights and content

Background

The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide in vivo evidence for the involvement of G protein–coupled receptor kinases (GRKs) in opioid dependence in addition to their roles in agonist-selective mu-opioid receptor (MOR) phosphorylation.

Methods

In vivo MOR phosphorylation was examined by immunoprecipitation and nanoflow liquid chromatography–tandem mass spectrometry analysis. Using the hot-plate and conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5.

Results

Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast, morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administration and developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine was ineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the MOR.

Conclusions

These findings show for the first time that MOR phosphorylation is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining the analgesic properties of opioid drugs at an effective level.

Section snippets

Antibodies

The phosphorylation-independent rabbit monoclonal anti-MOR antibody (UMB-3) was obtained from Epitomics (Burlingame, California) (18). The guinea pig polyclonal phosphosite-specific antibody anti-pS375 (GM375-2) and the phosphorylation-independent guinea pig polyclonal anti-MOR antibody (GP6) were generated and extensively characterized in a previous study 18, 19. The phosphosite-specific antibody for the T370-phosphorylated form of MOR (GM370-1) was generated against the IRQN(20)REHP sequence

Hierarchical Multisite Phosphorylation of MORs In Vivo

To facilitate detection of multisite phosphorylated MORs in vivo, we first generated guinea pig polyclonal anti-pT370, anti-pS375, and anti-pT379 antibodies as well as the phosphorylation-independent rabbit monoclonal anti-MOR antibody (UMB-3). In UMB-3 immunoprecipitates from brain homogenates prepared from MOR^+/+ mice treated with the high-efficacy agonist etonitazene, we clearly detected phosphorylation of multiple sites, including T370, S375, and T379 (Figure 1A). By contrast, morphine

Discussion

For many G protein–coupled receptors, the agonist-driven phosphorylation of intracellular serine and threonine clusters directly regulates their affinity for beta-arrestin. Accumulating evidence suggests that distinct GRKs are preferentially recruited to and phosphorylate receptors as a function of their ligand-induced conformation 24, 28, 29. In this manner, different GRKs act as sensors that detect active receptor conformations stabilized by different ligands. This preferential recruitment of

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Opioid receptors are G protein-coupled receptors (GPCRs) that regulate activity within peripheral, subcortical and cortical circuits involved in pain, reward, and aversion processing. Opioid receptors are expressed in both presynaptic terminals where they inhibit neurotransmitter release and postsynaptic locations where they act to hyperpolarize neurons and reduce activity. Agonist activation of postsynaptic receptors at the plasma membrane signal via ion channels or cytoplasmic second messengers. Agonist binding initiates regulatory processes that include phosphorylation by G protein receptor kinases (GRKs) and recruitment of beta-arrestins that desensitize and internalize the receptors. Opioid receptors also couple to effectors from endosomes activating intracellular enzymes and kinases. In contrast to postsynaptic opioid receptors, receptors localized to presynaptic terminals are resistant to desensitization such that there is no loss of signaling in the continuous presence of opioids over the same time scale. Thus, the balance of opioid signaling in circuits expressing pre- and postsynaptic opioid receptors is shifted toward inhibition of presynaptic neurotransmitter release during continuous opioid exposure. The functional implication of this shift is not often acknowledged in behavioral studies. This review covers what is currently understood about regulation of opioid/nociceptin receptors, with an emphasis on opioid receptor signaling in pain and reward circuits. Importantly, the review covers regulation of presynaptic receptors and the critical gaps in understanding this area, as well as the opportunities to further understand opioid signaling in brain circuits.
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The I₁ imidazoline receptor and its candidate protein imidazoline receptor antisera-selected (IRAS)/Nischarin are linked to μ opioid receptor (MOR) functions associated with MOR trafficking. We previously demonstrated that IRAS may play an important role in the development of morphine tolerance and physical dependence in vivo. However, the eﬀects of IRAS on morphine psychological dependence are not fully understood. To extend these studies, we investigated the impact of IRAS on morphine dependence in conditioned place preference (CPP) experiments and explored the underlying mechanisms. Knockout of IRAS enhanced the acquisition and reinstatement of morphine-induced CPP. Conditional-knockout of IRAS in the nucleus accumbens (NAc) reproduced higher CPP, and overexpression of IRAS in the NAc rescued the increased morphine-induced CPP in IRAS^-/- mice. IRAS^-/- mice showed dramatic cAMP-dependent protein kinase (PKA) activation, upregulation of the phosphorylation of the AMPA receptor GluR1-S845 and NMDA receptor NR1-S897 in the NAc after CPP experiment. Moreover, knockout of IRAS induced an increase in spontaneous excitatory postsynaptic current (sEPSC) frequency and a decrease in the AMPA/NMDA ratio in the NAc after chronic morphine treatment. The selective AMPA receptor antagonist NBQX could inhibit morphine CPP in WT mice, while its effect was significantly reduced in IRAS^-/- mice. Together, our results demonstrate that IRAS contributes to the regulation of morphine dependence and that the alteration of glutamatergic transmission in the NAc may participate in the effect of IRAS.
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Post-translational modifications (PTMs) are key events in signal transduction since they affect protein function by regulating their abundance and/or activity. PTMs involve the covalent attachment of functional groups to specific amino acids. Since they tend to be generally reversible, PTMs serve as regulators of signal transduction pathways. G-protein-coupled receptors (GPCRs) are major signaling proteins that undergo multiple types of PTMs. In this Review, we focus on the opioid receptors, members of GPCR family A, and highlight recent advances in the field that have underscored the importance of PTMs in the functional regulation of these receptors. Since opioid receptor activity plays a central role in the development of tolerance and addiction to morphine and other drugs of abuse, understanding the molecular mechanisms regulating receptor activity is of fundamental importance.
Biased agonism of clinically approved μ-opioid receptor agonists and TRV130 is not controlled by binding and signaling kinetics
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Taking the example of the μ-opioid receptor (μ-OR), which has been extensively studied in relation to bias, it has been demonstrated that β-arrestin2 knock out in mice led to decreased side-effects such as respiratory depression and constipation, as well as reducing morphine tolerance but not dependence (Bohn et al., 2000; Raehal et al., 2005). Further, morphine reward and dependence have been shown to be driven by GRK5 but not GRK3 whereas morphine-induced locomotor activity is dependent on GRK6 (Glück et al., 2014; Raehal et al., 2009). Recently, G protein biased agonists for the μ-OR have been discovered that have analgesic properties comparable to morphine, yet with reduced side-effects such as nausea and constipation in rodents and humans (Chen et al., 2013; DeWire et al., 2013; Manglik et al., 2016; Soergel et al., 2014), and reduced respiratory depression in rodents (DeWire et al., 2013; Manglik et al., 2016).
Binding and signaling kinetics have previously proven important in validation of biased agonism at GPCRs. Here we provide a comprehensive kinetic pharmacological comparison of clinically relevant μ-opioid receptor agonists, including the novel biased agonist oliceridine (TRV130) which is in clinical trial for pain management. We demonstrate that the bias profile observed for the selected agonists is not time-dependent and that agonists with dramatic differences in their binding kinetic properties can display the same degree of bias. Binding kinetics analyses demonstrate that buprenorphine has 18-fold higher receptor residence time than oliceridine. This is thus the largest pharmacodynamic difference between the clinically approved drug buprenorphine and the clinical candidate oliceridine, since their bias profiles are similar. Further, we provide the first pharmacological characterization of (S)-TRV130 demonstrating that it has a similar pharmacological profile as the (R)-form, oliceridine, but displays 90-fold lower potency than the (R)-form. This difference is driven by a significantly slower association rate. Finally, we show that the selected agonists are differentially affected by G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) expression. GRK2 and GRK5 overexpression greatly increased μ-opioid receptor internalization induced by morphine, but only had modest effects on buprenorphine and oliceridine-induced internalization. Overall, our data reveal that the clinically available drug buprenorphine displays a similar pharmacological bias profile in vitro compared to the clinical candidate drug oliceridine and that this bias is independent of binding kinetics suggesting a mechanism driven by receptor-conformations.
This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.
Toward Directing Opioid Receptor Signaling to Refine Opioid Therapeutics
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Knockdown of siRNA for βarr2 but not for βarr1 prevented morphine tolerance in mice in the hot plate assay (14) . In the hot plate test, GRK6-KO mice displayed equivalent tolerance compared with WT mice when tested for tolerance development to morphine (20), as did GRK5-KO mice (19). GRK3-KO mice showed no improvement of tolerance upon long-term daily morphine administration, although fentanyl-treated mice exhibited significantly less tolerance relative to WT control animals in the hot plate test (24).
The mu opioid receptor (MOR) is a diversely regulated target for the alleviation of pain in the clinical setting. However, untoward side effects such as tolerance, dependence, respiratory suppression, constipation, and abuse liability detract from the general activation of these receptors. Studies in genetically modified rodent models suggest that activating G protein signaling pathways while avoiding phosphorylation of the receptor or recruitment of β-arrestin scaffolding proteins could preserve the analgesic properties of MOR agonists while avoiding certain side effects. With the development of novel MOR “biased” agonists, which lead to preferential activation of G protein pathways over receptor phosphorylation, internalization, or interaction with other effectors, this hypothesis can be tested in a native, physiological setting. Overall, it is clear that the MOR is not a simple on-off switch and that the diverse means by which the receptor can be regulated may present an opportunity to refine therapeutics for the treatment of pain.
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Paradoxically, morphine-induced tolerance appears to involve a mechanism independent of S375 phosphorylation [42]. In addition, the importance of GRK3 and GRK5 for MOR phosphorylation and its relation to morphine reward and dependence was demonstrated in vivo [40]. Dephosphorylation of MOR at S375 occurs shortly after agonist clearance and MOR is fully dephosphorylated after 8 h, whereas analgesic tolerance to morphine persists even after 12 h [42].
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Archival ReportLoss of Morphine Reward and Dependence in Mice Lacking G Protein–Coupled Receptor Kinase 5

Background

Methods

Results

Conclusions

Section snippets

Antibodies

Hierarchical Multisite Phosphorylation of MORs In Vivo

Discussion

Pharmacol Ther

Trends Pharmacol Sci

Blood

J Biol Chem

J Biol Chem

Drug Alcohol Depend

Regul Pept

J Biol Chem

Trends Endocrinol Metab

J Biol Chem

J Biol Chem

Neuron

Neuropharmacology

Trends Pharmacol Sci

Prog Neuropsychopharmacol Biol Psychiatry

Neurosci Lett

Neuron

J Biol Chem

Brain Res

Eur J Pharmacol

Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene

Nature

Mu-opioid receptor desensitization: Is morphine different?

Br J Pharmacol

Agonist-selective patterns of micro-opioid receptor phosphorylation revealed by phosphosite-specific antibodies

Br J Pharmacol

Differentiation of opioid drug effects by hierarchical multi-site phosphorylation

Mol Pharmacol

Deciphering micro-opioid receptor phosphorylation and dephosphorylation in HEK293 cells

Br J Pharmacol

Morphine induces terminal micro-opioid receptor desensitization by sustained phosphorylation of serine-375

EMBO J

Mu-opioid receptors: Correlation of agonist efficacy for signalling with ability to activate internalization

Mol Pharmacol

Physiological roles of G protein-coupled receptor kinases and arrestins

Annu Rev Physiol

Archival Report
Loss of Morphine Reward and Dependence in Mice Lacking G Protein–Coupled Receptor Kinase 5