The other side of opioid receptor signalling: regulation by protein-protein interaction

Opiate drugs mediate their analgesic, euphoriant, and rewarding effects by activating opioid receptors. Pharmacological and molecular studies have demonstrated the existence of three opioid receptor subtypes, mu, delta, and kappa-that couple predominantly to Gi/Go types of G proteins to regulate the activity of a diverse array of effector systems. Ample experimental evidence has demonstrated that these receptors can physically interact with a variety of accessory proteins, confirming that signal transduction of the opioid receptors is not restricted to heterotrimeric G protein activation. Such interactions can alter the effectiveness of agonist-driven cell signalling, determine the signals generated and alter the trafficking, targeting, fine tuning and cellular localization of these receptors by providing a scaffold that links the receptors to the cytoskeletal network. The current review will summarize opioid receptor interacting partners and their role as currently understood. Increasing knowledge of the mechanisms by which these interactions are regulated is expected to address problems related to phenomena such as pain perception, tolerance and dependence that occur upon chronic opiate administration and define whether disruption of such interactions may contribute to the development of novel therapeutic strategie

Functional expression of mammalian opioid receptors in insect cells and high-throughput screening platforms for receptor ligand mimetics

Lepidopteran cell lines have been engineered to constitutively express high levels of mouse δ opioid receptors either alone or in combination with human Gα16 protein. Biochemical and pharmacological studies demonstrate that these lines contain all the mediator G proteins and downstream effectors required for opioid receptor function, including phospholipase C, and that expression of exogenous Gα16 does not contribute significantly to increased receptor responses upon activation. The activation of the phospholipase C pathway in the transformed cells upon stimulation with known receptor ligands results in easily and quantitatively measurable increases in free intracellular calcium, which can be monitored by automated fluorescent methods, while the addition of specific antagonists blocks the agonist-induced responses. Therefore, the transformed lepidopteran cell lines can be used as sensitive high-throughput screening platforms for fast detection of δ opioid receptor ligand mimetics (agonists and antagonists) in collections of natural products and synthetic compounds. © Birkhäuser Verlag, Basel, 2005

The κ-opioid receptor-induced autophagy is implicated in stress-driven synaptic alterations.

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

Novel Anosmia-Inducing Compounds for Environmentally Friendly Mosquito Vector Control: Structural Determinants of ORco Ligands Antagonizing Odorant Receptor Function

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. © FASEB

Phosphorylation of RGS13 by the Cyclic AMP-dependent Protein Kinase Inhibits RGS13 Degradation

Regulators of G-protein signaling (RGS) proteins are scaffolds that control diverse signaling pathways by modulating signalosome formation and by accelerating the GTPase activity of heterotrimeric G proteins. Although expression of many RGS proteins is relatively low in quiescent cells, transcriptional and post-translational responses to environmental cues regulate both their abundance and activity. We found previously that RGS13, one of the smallest RGS proteins in the family, inhibited cyclic AMP-dependent protein kinase (PKA)-induced gene expression through interactions with the transcription factor cAMP-response element-binding (CREB) protein. Here, we show that PKA activation also leads to increased steady-state RGS13 expression through RGS13 phosphorylation, which inhibits RGS13 protein degradation. RGS13 turnover was significantly reduced in cells stimulated with cAMP, which was reversed by expression of the PKA-specific inhibitory peptide PKI. RGS13 phosphorylation was diminished by mutation of an N-terminal Thr residue (T41) identified as a phosphorylation site by mass spectrometry. Mutation of Thr41 in RGS13 to Ala (T41A) reduced steady-state RGS13 levels and its ability to inhibit M2 muscarinic receptor-mediated Erk phosphorylation compared with wild-type RGS13 by attenuating the protective effect of cAMP on RGS13 degradation. RGS13 underwent ubiquitylation, indicating that it is a likely target of the proteasome. These studies are the first to demonstrate post-translational mechanisms controlling the expression of RGS13. Stabilization of RGS13 through PKA-mediated phosphorylation could enhance RGS13 functions, providing negative feedback regulation that promotes cellular desensitization

Prognostic significance of signal transducer and activator of transcription 5 and 5b expression in Epstein–Barr virus-positive patients with chronic lymphocytic leukemia

Signal transducer and activator of transcription (STAT) proteins have been intensively studied in hematologic malignancies, and the efficacy of agents against STATs in lymphomas is already under research. We investigated the expression of total STAT5 and STAT5b in peripheral blood samples of patients with chronic lymphocytic leukemia (CLL) in correlation with the presence of Epstein–Barr Virus (EBV) and its major oncoprotein (latent membrane protein 1, LMP1). The EBV load was measured in the peripheral blood by real-time PCR for the BXLF1 gene and the levels of LMP1 by PCR and ELISA. Western blotting was performed for total STAT5 and STAT5b in protein extracts. STAT5b was only expressed in patients (not in healthy subjects) and STAT5 but particularly STAT5b expression was correlated with the presence of the virus (77.3% vs. 51.2%, P = 0.006 for STAT5b) and to the expression of LMP1 (58.3% vs. 21.6%, P = 0.011 for STAT5b). Moreover, the expression of STAT5b and the presence of EBV and LMP1 were strongly negatively correlated with the overall survival of the patients (log-rank test P = 0.011, 0.015, 0.006, respectively). Double positive (for EBV and STAT5b) patients had the lowest overall survival (log-rank test P = 0.013). This is the first report of a survival disadvantage of EBV+ patients with CLL, and the first time that STAT5b expression is correlated with survival. The correlation of STAT5 expression with the presence of the virus, along with our survival correlations defines a subgroup of patients with CLL that may benefit from anti-STAT agents. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd