μ-Opioid Receptors: Correlation of Agonist Efficacy for Signalling with Ability to Activate Internalization

McPherson, Jamie; Rivero, Guadalupe; Baptist, Myma; Llorente, Javier; Al-Sabah, Suleiman; Krasel, Cornelius; Dewey, William L.; Bailey, Christopher; Rosethorne, Elizabeth M.; Charlton, Steven J.; Henderson, Graeme; Kelly, Eamonn

doi:10.1124/mol.110.066613

Cited by 248 publications

(360 citation statements)

References 35 publications

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“…When compared with other mu agonists, morphine induces less internalization of the mu opioid receptor (Borgland et al, 2003;McPherson et al, 2010;Zheng et al, 2011). This suggests that the prototypical role of b-arrestin 2 in initiating receptor internalization does not explain how the effects of morphine, but not of other mu agonists, are altered in mice lacking b-arrestin 2.…”

Section: Discussionmentioning

confidence: 98%

Evidence that Behavioral Phenotypes of Morphine in β-arr2−/− Mice Are Due to the Unmasking of JNK Signaling

Mittal

Tan

Egbuta

et al. 2012

Neuropsychopharmacol

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The altered behavioral effects of morphine, but not most other mu agonists, in mice lacking b-arrestin 2, suggest that this scaffolding protein regulates the signaling cascade of this commonly used analgesic. One of the cascades that could be regulated by b-arrestin 2 is cJun-N-terminal kinase (JNK), which binds with b-arrestin 2 and modulates the analgesic effects of morphine. Using neurons lacking b-arrestin 2 (b-arr2À/À) to examine this interaction, we found that b-arr2À/À neurons show altered intracellular distribution of JNK and cJun, and that morphine, but not fentanyl, increased the nuclear localization of the phosphorylated, therefore activated, form of cJun, a JNK target in dorsal root ganglia neurons. This suggests that deleting b-arrestin 2 affects the JNK cascade. We therefore examined whether some of the behavioral phenotypes of mice lacking b-arrestin 2 could be a result of altered JNK signaling. Indeed, two different JNK inhibitors reversed the enhanced analgesic effect of morphine, a known phenotype of b-arr2À/À mice, to + / + levels. Both the reduced locomotor effect of morphine and the psychomotor sensitization to repeated morphine administration in b-arr2À/À mice were also returned to + / + levels by inhibiting JNK. In contrast, the behavioral effects of fentanyl were neither genotype-dependent nor affected by JNK inhibition. Furthermore, a PKC inhibitor had a similar effect as inhibiting JNK in reducing the enhanced analgesic effect of morphine in b-arr2À/À mice to + / + levels. In summary, removing b-arrestin 2 reveals mu receptor activation of the JNK cascade in a ligand-specific manner explaining several behavioral phenotypes of b-arr2À/À mice.

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Section: Discussionmentioning

confidence: 98%

Evidence that Behavioral Phenotypes of Morphine in β-arr2−/− Mice Are Due to the Unmasking of JNK Signaling

Mittal

Tan

Egbuta

et al. 2012

Neuropsychopharmacol

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“…11 It is also possible that the efficacy discrepancies reflect subtle bias in ligand signaling to one pathway over another in different tissues, and it is worth noting that endomorphins have recently been reported to show such bias in assays of β-arrestin recruitment. 25,26 Continuous application of opioid agonists usually results in desensitization of receptor signaling, a complex process that may involve receptor phosphorylation and sequestration. The apparent stability of the membrane potential assay led us to explore whether it could be used to investigate µ-opioid receptor desensitization.…”

Section: Discussionmentioning

confidence: 99%

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Knapman

Santiago

et al. 2013

SLAS Discovery

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Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.

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“…Very different EC 50 values for partial agonists also have been reported for m-opioid receptors (McPherson et al, 2010;Nickolls et al, 2011), histamine H 4 receptors (Nijmeijer et al, 2012), and b 1 -adrenoceptors (Casella et al, 2011). This suggests that effective bias may be obtained through combinations of efficacies and affinities for the receptor as it interacts with different pathways.…”

Section: Quantifying Biasmentioning

confidence: 98%

The Effective Application of Biased Signaling to New Drug Discovery

Kenakin

2015

Mol Pharmacol

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The ability of agonists to selectively activate some but not all signaling pathways linked to pleiotropically signaling receptors has opened the possibility of obtaining molecules that emphasize beneficial signals, de-emphasize harmful signals, and concomitantly deemphasize harmful signals while blocking the harmful signals produced by endogenous agonists. The detection and quantification of biased effects is straightforward, but two important factors should be considered in the evaluation of biased effects in drug discovery. The first is that efficacy, and not bias, determines whether a given agonist signal will be observed; bias only dictates the relative concentrations at which agonist signals will appear when they do appear. Therefore, a Cartesian coordinate system plotting relative efficacy (on a scale of Log relative Intrinsic Activities) as the ordinates and Log(bias) as the abscissae is proposed as a useful tool in evaluating possible biased molecules for progression in discovery programs. Second, it should be considered that the current scales quantifying bias limit this property to the allosteric vector (ligand/receptor/coupling protein complex) and that whole-cell processing of this signal can completely change measured bias from in vitro predictions.

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μ-Opioid Receptors: Correlation of Agonist Efficacy for Signalling with Ability to Activate Internalization

Cited by 248 publications

References 35 publications

Evidence that Behavioral Phenotypes of Morphine in β-arr2−/− Mice Are Due to the Unmasking of JNK Signaling

Evidence that Behavioral Phenotypes of Morphine in β-arr2−/− Mice Are Due to the Unmasking of JNK Signaling

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

The Effective Application of Biased Signaling to New Drug Discovery

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