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Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior.

ABSTRACT: Kappa opioid receptor (KOR) agonists show promise in ameliorating disorders, such as addiction and chronic pain, but are limited by dysphoric and aversive side effects. Clinically beneficial effects of KOR agonists (e.g., analgesia) are predominantly mediated by heterotrimeric G protein signaling, whereas ?-arrestin signaling is considered central to their detrimental side effects (e.g., dysphoria/aversion). Here we show that Regulator of G protein Signaling-12 (RGS12), via independent signaling mechanisms, simultaneously attenuates G protein signaling and augments ?-arrestin signaling downstream of KOR, exhibiting considerable selectivity in its actions for KOR over other opioid receptors. We previously reported that RGS12-null mice exhibit increased dopamine transporter-mediated dopamine (DA) uptake in the ventral (vSTR), but not dorsal striatum (dSTR), as well as reduced psychostimulant-induced hyperlocomotion; in the current study, we found that these phenotypes are reversed following KOR antagonism. Fast-scan cyclic voltammetry studies of dopamine (DA) release and reuptake suggest that striatal disruptions to KOR-dependent DAergic neurotransmission in RGS12-null mice are restricted to the nucleus accumbens. In both ventral striatal tissue and transfected cells, RGS12 and KOR are seen to interact within a protein complex. Ventral striatal-specific increases in KOR levels and KOR-induced G protein activation are seen in RGS12-null mice, as well as enhanced sensitivity to KOR agonist-induced hypolocomotion and analgesia-G protein signaling-dependent behaviors; a ventral striatal-specific increase in KOR levels was also observed in ?-arrestin-2-deficient mice, highlighting the importance of ?-arrestin signaling to establishing steady-state KOR levels in this particular brain region. Conversely, RGS12-null mice exhibited attenuated KOR-induced conditioned place aversion (considered a ?-arrestin signaling-dependent behavior), consistent with the augmented KOR-mediated ?-arrestin signaling seen upon RGS12 over-expression. Collectively, our findings highlight a role for RGS12 as a novel, differential regulator of both G protein-dependent and -independent signaling downstream of KOR activation.

SUBMITTER: Gross JD

PROVIDER: S-EPMC6785087 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior.

Gross Joshua D JD Kaski Shane W SW Schmidt Karl T KT Cogan Elizabeth S ES Boyt Kristen M KM Wix Kim K Schroer Adam B AB McElligott Zoe A ZA Siderovski David P DP Setola Vincent V

Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 20190529 10

Kappa opioid receptor (KOR) agonists show promise in ameliorating disorders, such as addiction and chronic pain, but are limited by dysphoric and aversive side effects. Clinically beneficial effects of KOR agonists (e.g., analgesia) are predominantly mediated by heterotrimeric G protein signaling, whereas β-arrestin signaling is considered central to their detrimental side effects (e.g., dysphoria/aversion). Here we show that Regulator of G protein Signaling-12 (RGS12), via independent signaling ...[more]

PMID: 31141817

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Project description:Kappa Opioid receptors（KORs） belong to family A of G-protein–coupled receptors (GPCRs), and the involvement of their ligands in analgesia and sedation has been extensively studied. KORs, which are expressed in the central and peripheral nervous systems are well known to exert potent anti-nociceptive effects by negatively modulating excitatory neurotransmission. The present study reveals that acute activation of KOR weaken SERT function via protein kinase B/Akt (Akt) and calcium calmodulin dependent kinase II (CaMKII) dependent signaling pathways. Therefore, studying phosphorylation will be helpful to elucidate the cellular mechanisms and downstream effector(s). Studies correlating the dysphoric effects of KOR agonists to the G-protein–independent activation of the p38 MAPK pathway suggest that identification of KOR agonists biased to G-protein–mediated signaling could lead to the development of therapeutics with reduced side effects. Therefore, to better understand the cellular mechanisms and downstream effector(s) by which KOR ligands produce these effects, we employed dimethyl labeling quantitative proteomic approach in this study. In this study, three monoclonal HEK293T cell lines with KOR over-expression were successfully constructed as our experimental models. Then, mass spectrometry-based quantitative approach, based on dimethyl labeling and TiO2 beads-coupled phosphopeptides enrichment, was utilized to analyze the KOR-mediated dynamic phosphorylation network in these three KOR-over-expressed monoclonal cell lines disturbed by LPK-26 or U50,488 treatments. Further Substrate-kinase prediction analysis of 212 up-regulated phosphosites indicated that 18 kinases, such as AKT1 and p70S6K, were involved in the up-regulation of KOR phosphorylation. The up-regulated phosphorylation of AKT1 and p70S6K were further confirmed by western blot, which was consistent with our mass spectrometry results. In summary, quantitative phosphoproteomic study of KOR-over-expressed cells upon agonist stimulation paves the way to systematical investigation of KOR-mediated phosphorylation network dynamics, which will be helpful to research about targeted therapy of KOR related diseases and mechanism study of analgesia addiction.

Project description:Kappa opioid receptor (KOR) agonists are potentially useful as analgesic and anti-pruritic agents, for prevention and treatment of substance use disorders, and for treatment of demyelinating diseases. However, side effects of KOR agonists, including psychotomimesis, dysphoria, and sedation, have caused early termination of clinical trials. Understanding the signaling mechanisms underlying the beneficial therapeutic effects and the adverse side effects may help in the development of KOR agonist compounds. In this review, we summarize the current knowledge in this regard in five sections. First, studies conducted on mutant mouse lines (GRK3-/-, p38alpha MAPK-/-, β-arrestin2-/-, phosphorylation-deficient KOR) are summarized. In addition, the abilities of four distinct KOR agonists, which have analgesic and anti-pruritic effects with different side effect profiles, to cause KOR phosphorylation are discussed. Second, investigations on the KOR agonist nalfurafine, both in vitro and in vivo are reviewed. Nalfurafine was the first KOR full agonist approved for clinical use and in the therapeutic dose range it did not produce significant side effects associated with typical KOR agonists. Third, large-scale high-throughput phosphoproteomic studies without a priori hypotheses are described. These studies have revealed that KOR-mediated side effects are associated with many signaling pathways. Fourth, several novel G protein-biased KOR agonists that have been characterized for in vitro biochemical properties and agonist biases and in vivo behavior effects are described. Lastly, possible mechanisms underlying KOR-mediated CPA, hypolocomotion and motor incoordination are discussed. Overall, it is agreed upon that the analgesic and anti-pruritic effects of KOR agonists are mediated via G protein signaling. However, there is no consensus on the mechanisms underlying their side effects. GRK3, p38 MAPK, β-arrestin2, mTOR pathway, CB1 cannabinoid receptor and protein kinase C have been implicated in one side effect or another. For drug discovery, after initial in vitro characterization, in vivo pharmacological characterizations in various behavior tests are still the most crucial steps and dose separation between beneficial therapeutic effects and adverse side effects are the critical determinant for the compounds to be moved forward for clinical development.

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Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior.

Publications

Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior.

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