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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. [ABSTRACT FROM AUTHOR]

R2502 --> 691.3 --> 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 Differ22: 398‐407 2. Wauson EM, Dbouk HA, Ghosh AB, Cobb MH (2014) Trends Endocrinol Metab25: 274‐2823. 3. Georganta EM, Tsoutsi L, Gaitanou M, Georgoussi Z (2013) J Neurochem127: 329‐341 4. Kibaly C, Xu C, Cahill CM, Evans CJ, Law PY (2019) Nat Rev Neurosci20: 5‐18 5. Georgoussi Z and Karoussiotis C (2019) FASEB J. Supplement Issue Vol.33 [ABSTRACT FROM AUTHOR]

Previous studies have shown that RGS4 associates with the C-termini of μ- and δ-opioid receptors in living cells and plays a key role in Gi/Go protein coupling selectivity and signalling of these receptors [12,20]. To deduce whether similar effects also occur for the κ-opioid receptor (κ-ΟR) and define the ability of members of the Regulators of G protein Signaling (RGS) of the B/R4 subfamily to interact with κ-ΟR subdomains we generated glutathione S-transferase fusion peptides encompassing the carboxyl-termini of κ-OR (κ-CT). Results from pull down experiments indicated that RGS2 and RGS4 directly interact within different domains of the κ-CT. Co-precipitation studies in living cells indicated that RGS2 and RGS4 associate with κ-ΟR constitutively and upon receptor activation and confer selectivity for coupling with a specific subset of G proteins. Expression of both members, RGS2 and/or RGS4, in 293 F cells attenuated κ-agonist mediated-adenylyl cyclase inhibition and extracellular signal regulated kinase (ERK1,2) phosphorylation with a different amplitude in their modulatory effect in κ-ΟR signaling. Our findings demonstrate that RGS2 and RGS4 are new interacting partners that play key roles in G protein coupling to negatively regulate κ-ΟR signaling. [ABSTRACT FROM AUTHOR]

G protein‐coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. This overview emphasises the GPCRs of the nervous system, which are the research focus of the members of ERNEST COST action (CA18133) working group ‘Biological roles of signal transduction’. First, the (patho)physiological role of the nervous system GPCRs in the modulation of synapse function is discussed. We then debate the (patho)physiology and pharmacology of opioid, acetylcholine, chemokine, melatonin and adhesion GPCRs in the nervous system. Finally, we address the orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed to deorphanize them. [ABSTRACT FROM AUTHOR]

R4433 --> 537.15 --> Insect repellents are important means of personal protection against bites from mosquitoes carrying various pathogens. Odorant‐dependent behaviours are triggered by odor ligands bound to odorant receptors. Insect odorant receptors are heterotetrameric ligand‐gated cation channels composed of an obligatory subunit, ORco, and one of many variable subunits, ORx, to which odorant ligands bind. Upon expression ex vivo, ORco forms homomeric channels gated by specific ligands acting as channel agonists. In past studies, we identified ligands of natural origin that bind to the African malaria mosquito vector Anopheles gambiae ORco (AgamORco). Some of these ligands function as specific ORco channel antagonists, orthosteric or allosteric relative to a predicted ORco agonist binding site, and cause severe inhibition of the olfactory function in a variety of mosquito species. Given that ORco is evolutionarily conserved, we hypothesized that it should be feasible to develop an AgamORco‐based screening platform allowing fast identification of new ORco ligands acting as mosquito odorant receptor antagonists causing anosmia‐like deficits to mosquitoes. We are now reporting on the compilation of common structural features of previously identified orthosteric AgamORco antagonists and generation of a ligand‐based pharmacophore. In silico screening of a collection of volatile compounds of natural origin resulted in identification of several AgamORco ligand hits. Parallel cell‐based screening of the same compounds against AgamORco was also employed in order to identify ligands acting as AgamORco antagonists. The screening resulted in identification of several ligands inhibiting AgamORco channel function ex vivo by at least 40% and inducing anosmic behaviors to Aedes albopictus mosquitoes in vivo. To test the pharmacophore model for binding site specificity, binding competition assays were undertaken, which distinguished the identified antagonists into orthosteric and allosteric ones. Direct comparisons of the pharmacophore predictions with the results of the cell‐based screen revealed that the pharmacophore predicted correctly 100% of the orthosteric antagonists and none of the allosteric ones. These results confirm the pharmacophore's value for in silico prediction of AgamORco orthosteric ligands. Upon combination with the AgamORco‐specific cell‐based screening platform, the pharmacophore provides a valuable tool for fast identification of multiple, ecologically friendly vector control agents. Finally, to confirm the predicted AgamORco agonist binding site and determine the binding orientation of orthosteric antagonists in it, we are assessing the interactions of these ligands with specific amino acids in the AgamORco binding pocket by site‐specific mutagenesis. [ABSTRACT FROM AUTHOR]