The δ‐opioid receptor (δ‐OR) couples to Gi/Go proteins to modulate a variety of responses in the nervous system. Τhe regulator of G protein signalling 4 (RGS4) was previously shown to directly interact within the C‐terminal region of δ‐OR using its N‐terminal domain to negatively modulate opioid receptor signalling. Herein, using molecular dynamics simulations and in vitro pull‐down experiments we delimit this interaction to 12 helix 8 residues of δ‐ΟR and to the first 17 N‐terminal residues (NT) of RGS4. Monitoring the complex arrangement and stabilization between RGS4 and δ‐OR by molecular dynamics simulations combined with mutagenesis studies, we defined that two critical interactions are formed: one between Phe329 of helix8 of δ‐ΟR and Pro9 of the NT of RGS4 and the other a salt bridge between Glu323 of δ‐ΟR and Lys17 of RGS4. Our observations allow drafting for the first time a structural model of a ternary complex including the δ‐opioid receptor, a G protein and a RGS protein. Furthermore, the high degree of conservation among opioid receptors of the RGS4‐binding region, points to a conserved interaction mode between opioid receptors and this important regulatory protein.
Recent evidence has shown that G protein-coupled receptors (GPCRs) are direct sensors of the autophagic machinery and opioid receptors regulate neuronal plasticity and neurotransmission with an as yet unclarified mechanism. Using in vitro and in vivo experimental approaches, this study aims to clarify the potential role of autophagy and κ-opioid receptor (κ-OR) signaling in synaptic alterations. We hereby demonstrate that the selective κ-OR agonist U50,488H, induces autophagy in a time-and dose-dependent manner in Neuro-2A cells stably expressing the human κ-OR by upregulating microtubule-associated protein Light Chain 3-II (LC3-II), Beclin 1 and Autophagy Related Gene 5 (ATG5). Pretreatment of neuronal cells with pertussis toxin blocked the above κ-OR-mediated cellular responses. Our molecular analysis also revealed a κ-OR-driven upregulation of becn1 gene through ERK1,2-dependent activation of the transcription factor CREB in Neuro-2A cells. Moreover, our studies demonstrated that sub-chronic U50,488H administration in mice causes profound increases of specific autophagic markers in the hippocampus with a concomitant decrease of several pre-and post-synaptic proteins, such as spinophilin, postsynaptic density protein 95 (PSD-95) and synaptosomal associated protein 25 (SNAP25). Finally, using acute stress, a stimulus known to increase the levels of the endogenous κ-OR ligand dynorphin, we are demonstrating that administration of the κ-ΟR selective antagonist, nor-binaltorphimine (norBNI), blocks the induction of autophagy and the stress-evoked reduction of synaptic proteins in the hippocampus. These findings provide novel insights about the essential role of autophagic machinery into the mechanisms through which κ-OR signaling regulates brain plasticity.
Neuronal autophagy controls the quality of cytoplasmic proteins through degradation of important synaptic proteins and modulates synaptic organization and morphogenesis (1). Evidence has shown that G protein coupled receptors are direct sensors regulating the autophagic machinery (2) and opioid receptors regulate neuronal plasticity and neurotransmission with as yet unclarified mechanism (3,4). Using in vitro and in vivo studies, we demonstrated that κ‐opioid receptor (κ‐OR) agonists induce autophagy via a PTX‐sensitive G protein manner and identified the downstream components involved (5). Our molecular analysis also revealed a κ‐OR‐driven upregulation of becn1 gene through ERK1,2‐dependent activation of the transcription factor CREB in neuronal cells. Moreover, our studies demonstrated that sub‐chronic U50,488H administration in mice causes profound increases of specific autophagic markers exclusively in the hippocampus with a concomitant decrease of several pre‐ and post‐synaptic proteins such as spinophilin, PSD‐95 and SNAP25. Finally, using acute stress, a stimulus known to increase the levels of the endogenous κ‐OR ligand dynorphin, we are demonstrating that administration of the κ‐ΟR selective antagonist, nor‐binaltorhimine, blocks the induction of autophagy and the stress‐evoked reduction of synaptic proteins in the hippocampus. These findings provide novel insights about the essential role of autophagic machinery into the mechanisms through which κ‐OR and its signaling regulates brain plasticity. References 1. Nikoletopoulou V, Papandreou ME, Tavernarakis N (2015) Cell Death Differ 22: 398‐407 2. Wauson EM, Dbouk HA, Ghosh AB, Cobb MH (2014) Trends Endocrinol Metab 25: 274‐2823. 3. Georganta EM, Tsoutsi L, Gaitanou M, Georgoussi Z (2013) J Neurochem 127: 329‐341 4. Kibaly C, Xu C, Cahill CM, Evans CJ, Law PY (2019) Nat Rev Neurosci 20: 5‐18 5. Georgoussi Z and Karoussiotis C (2019) FASEB J. Supplement Issue Vol.33
It is already evident that opioid receptors modulate the neuronal function by regulating neurite outgrowth and neurotransmission with unknown yet mechanisms (1, 2). Autophagy is a lysosomal degradation pathway which eliminates misfolded proteins and dysfunctional organelles essential for neuronal function and homeostasis (3). Recent observations have shown that G‐protein coupled receptors could mediate the autophagic machinery and that methamphetamine induces autophagy in endothelial cells through the κ‐OR (4). However, it is unknown whether specific κ‐opioid agonists mediate these effects and whether opiodergic alterations in neuronal cells could be controlled through induction of autophagy. Our data indicate that administration of κ‐selective agonists increase the levels of the autophagosome marker LC3‐II (Fig. 1) and the pre‐autophagosomal proteins Beclin 1 and ATG5 respectively in a dose and time dependent manner. These effects are reversed upon exposure of neuroblastoma cells and cortical neurons with the opioid receptor antagonist naloxone, suggesting that it is a κ‐OR‐mediated effect. Pre‐treatment of cells with pertussis toxin blocked the κ‐OR mediated Beclin 1 upregulation and LC3‐II formation, suggesting that Gi/Go proteins are involved in κ‐OR induced autophagy. Futhermore, κ‐opioid agonist exposure of primary neuronal cortical cultures decreased the levels of Neurabin II, a neuronal protein enriched in dendritic spines, and the levels of Synaptophysin, a synaptic terminal protein, suggesting that these proteins are engulfed in the κ‐OR mediated autophagic cargo. Collectively, the present results demonstrate a novel regulatory mechanism via which specifιc κ‐OR agonists affect dendritic spine morphology via induction of autophagy.Support or Funding InformationThis work was supported mainly by the GSRT Excellence II grant, 3722 entitled “Neurite Outrgowth: ALternative G‐ protein coupled Opioid receptor Signaling”‐ “NO‐ALGOS” to Z.G. We acknowledge support of this work also by the project “Target Identification and Development of Novel Approaches for Health and Environmental Applications” (MIS 5002514) which is implemented under the Action for the Strategic Development on the Research and Technological Sectors, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co‐financed by Greece and the European Union (European Regional Development Fund). The facilities of the Hellenic Research Infrastructure OPENSCREEN‐GR were used.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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