Different kinases desensitize the human delta-opioid receptor (hDOP-R) in the neuroblastoma cell line SK-N-BE upon peptidic and alkaloid agonists.

In a previous work, we described a differential desensitization of the human delta-opioid receptor (hDOP-R) by etorphine (a non-selective and alkaloid agonist) and delta-selective and peptidic agonists (DPDPE ([D-Pen(2,5)]enkephalin) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH(2))) in the neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different kinases in this differential regulation. First, selective chemical inhibitors of PKA, PKC and tyrosine kinases were used and we showed a significant reduction of etorphine-induced opioid receptor desensitization by the bisindolylmaleimide I (PKC inhibitor) while genistein (tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by H89 pretreatment, no modification of opioid receptor desensitization was observed whatever the agonist used. Second, we further studied the role of G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2 isoform was expressed in the SK-N-BE cells. Next, the neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by etorphine suggesting that this kinase was involved in the regulation of hDOP-R activated only by etorphine. Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser(363) antibody. While all agonists were potent to increase phosphorylation of opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser(363) phosphorylation only after peptidic agonists pretreatment. In conclusion, hDOP-R desensitization upon etorphine exposure relies on the GRK2, PKC and tyrosine kinases while DPDPE and deltorphin I mediate desensitization at least via tyrosine kinases. Although the Ser(363) was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this amino acid.

Tracking the opioid receptors on the way of desensitization.

Opioid receptors belong to the super family of G-protein coupled receptors (GPCRs) and are the targets of numerous opioid analgesic drugs. Prolonged use of these drugs results in a reduction of their effectiveness in pain relief also called tolerance, a phenomenon well known by physicians. Opioid receptor desensitization is thought to play a major role in tolerance and a lot of work has been dedicated to elucidate the molecular basis of desensitization. As described for most of GPCRs, opioid receptor desensitization involves their phosphorylation by kinases and their uncoupling from G-proteins realized by arrestins. More recently, opioid receptor trafficking was shown to contribute to desensitization. In this review, our knowledge on the molecular mechanisms of desensitization and recent progress on the role of opioid receptor internalization, recycling or degradation in desensitization will be reported. A better understanding of these regulatory mechanisms would be helpful to develop new analgesic drugs or new strategies for pain treatment by limiting opioid receptor desensitization and tolerance.

High-purity selection and maintenance of gene expression in human neuroblastoma cells stably over-expressing GFP fusion protein. Application for opioid receptors desensitization studies.

Chronic use of opiates such as morphine is associated with drug tolerance, which is correlated with the desensitization of opioid receptors. This latter process involves phosphorylation of opioid receptors by G protein-coupled receptors kinases (GRKs) and subsequent uncoupling by beta-arrestins. To explore these molecular mechanisms, neuronal cell lines, endogenously expressing the opioid receptors, provide an ideal cellular model. Unfortunately, there are two major drawbacks: (1) these cells are refractory to cDNA introduction, resulting in low transfection efficiency; (2) continuous culturing of transfected cells invariably leads to phenotypic drift of the cultures even after an antibiotic selection. So, these cells were dropped in favor of heterologous expression systems, which are easier to transfect but whose relevance as adequate cellular model for studying opioid receptor regulation should be questioned, as recently demonstrated by [Haberstock-Debic, H., Kim, K.A.,Yu, Y.J., von Zastrow, M., 2005. Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J. Neurosci. 25, 7847-7857]. In this work, we describe a method, based on fluorescence-activated cell sorting (FACS), to select and maintain a high proportion of transfected SK-N-BE cells (a neuronal cell line endogenously expressing human Delta-Opioid Receptor (hDOR)), expressing the beta-arrestin1 fused to green fluorescent protein (GFP). While in functional experiments, we were not able to observe a major effect in non-sorted SK-N-BE cells expressing beta-arrestin1-GFP, the enrichment by 18-fold with FACS resulted in a robust increase of beta-arrestin1-GFP expression associated with strong hDOR desensitization. Moreover, this method also allows to counteract the phenotypic drift and to maintain a high-purity selection of SK-N-BE cells expressing beta-arrestin1-GFP. Thus, this approach provides a valuable tool for exploring opioid receptors desensitization in neuronal cells.

Novel genes as primary triggers for polygenic hypertension.

OBJECTIVES: The discovery of causative genes leading to hypertension in animal models can reveal new mechanistic insights into blood pressure (BP) regulations. Previously, we isolated segments that harbor BP quantitative trait loci (QTLs) on rat chromosome 10 as defined by congenic strains made from crosses of inbred hypertensive Dahl salt-sensitive (DSS) and normotensive Lewis rats. The aim of the current study was to identify hypertension-causing genes for each QTL. METHODS: Molecular analysis was performed. RESULTS: A systematic and comprehensive molecular analysis divulged particular genes that carry nonconserved mutations. Specifically, the proline rich 11 gene is likely responsible for C10QTL5. C10QTL1 is one of five genes, namely Benzodiazepine receptor associated protein 1, Loc689764, myotubularin related protein 4, protein phosphatase 1E, PP2C domain containing and ring finger protein 43. Loc100363423 with no known function is a candidate for C10QTL3. The ATP-binding cassette, subfamily A (ABC1), member 8a gene is probably responsible for C10QTL2. CONCLUSIONS: Primary genes initiating polygenic hypertension are those not known to be involved in BP modulation. Novel pathways towards BP homeostasis appear to underlie the functionality of C10QTL5, C10QTL1 and C10QTL3 and C10QTL2. Moreover, these genes may become innovative targets for the diagnosis and therapeutics of essential hypertension.

Differential ß-arrestin-dependent conformational signaling and cellular responses revealed by angiotensin analogs.

The angiotensin type 1 receptor (AT1R) and its octapeptide ligand, angiotensin II (AngII), engage multiple downstream signaling pathways, including those mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins) and those mediated by ß-arrestin. Here, we examined AT1R-mediated Gα(q) and ß-arrestin signaling with multiple AngII analogs bearing substitutions at position 8, which is critical for binding to the AT1R and its activation of G proteins. Using assays that discriminated between ligand-promoted recruitment of ß-arrestin to the AT1R and its resulting conformational rearrangement, we extend the concept of biased signaling to include the analog's propensity to differentially promote conformational changes in ß-arrestin, two responses that were differentially affected by distinct G protein-coupled receptor kinases. The efficacy of AngII analogs in activating extracellular signal-regulated kinases 1 and 2 correlated with the stability of the complexes between ß-arrestin and AT1R in endosomes, rather than with the extent of ß-arrestin recruitment to the receptor. In vascular smooth muscle cells, the ligand-induced conformational changes in ß-arrestin correlated with whether the ligand promoted ß-arrestin-dependent migration or proliferation. Our data indicate that biased signaling not only occurs between G protein- and ß-arrestin-mediated pathways but also occurred at the level of the AT1R and ß-arrestin, such that different AngII analogs selectively engaged distinct ß-arrestin conformations, which led to specific signaling events and cell responses.

ßarrestin1-biased agonism at human δ-opioid receptor by peptidic and alkaloid ligands.

We have previously reported on the differential regulation of the human δ-opioid receptor (hDOR) by alkaloid (etorphine) and peptidic (DPDPE and deltorphin I) ligands, in terms of both receptor desensitization and post-endocytic sorting. Since ßarrestins are well known to regulate G protein-coupled receptors (GPCRs) signaling and trafficking, we therefore investigated the role of ßarrestin1 (the only isoform expressed in our cellular model) in the context of the hDOR. We established clonal cell lines of SK-N-BE cells over-expressing ßarrestin1, its dominant negative mutant (ßarrestin1(319-418)), and shRNA directed against endogenous ßarrestin1. Interestingly, both binding and confocal microscopy approaches demonstrated that ßarrestin1 is required for hDOR endocytosis only when activated by etorphine. Conversely, functional experiments revealed that ßarrestin1 is exclusively involved in hDOR desensitization promoted by the peptides. Taken together, these results provide substantial evidence for a ßarrestin1-biased agonism at hDOR, where ßarrestin1 is differentially involved during receptor desensitization and endocytosis depending on the ligand.

Study of G protein-coupled receptor/ß-arrestin interactions within endosomes using FRAP.

ß-arrestins, through their scaffolding functions, are key regulators of G protein-coupled receptor (GPCR) signaling and intracellular trafficking. However, little is known about the dynamics of ß-arrestin/receptor interactions and how these complexes, and complexes with other regulatory proteins, are controlled in cells. Here, we use yellow fluorescent protein (YFP)-tagged ß-arrestin 2 and a fluorescence recovery after photobleaching (FRAP) imaging approach to probe the real-time interaction of ß-arrestin with a GPCR, the bradykinin type 2 receptor (B2R). We provide a detailed protocol to assess the avidity of ß-arrestin2-YFP for B2R within endosomes in HEK293 cells. ß-arrestin2-YFP associated with internalized receptors is photobleached with intense light, and fluorescence recovery due to the entry of nonbleached ß-arrestin2-YFP is monitored over time as a measure of the rate exchange of ß-arrestin2-YFP within the endosome. This approach can be extended to other GPCR/ß-arrestin complexes and their putative regulators to provide information about the kinetics of similar protein-protein interactions in cells. Moreover, these techniques should provide insight into the role of ß-arrestins in the intracellular trafficking and signaling of GPCRs.