Development of a novel cell-based, In-Cell Western/ERK assay system for the high-throughput screening of agonists acting on the delta-opioid receptor (original) (raw)
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Cell. Mol. Life Sci., 2014
Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E max values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.
BMC pharmacology, 2002
In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned delta-opioid receptor (delta-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive. Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed delta-ORs are stimulated by the delta-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the delta...
Behavioural brain research, 2015
Opioids produce antinociception by activation of G protein signaling linked to the mu-opioid receptor (MOPr). However, opioid binding to the MOPr also activates β-arrestin signaling. Opioids such as DAMGO and fentanyl differ in their relative efficacy for activation of these signaling cascades, but the behavioral consequences of this differential signaling are not known. The purpose of this study was to evaluate the behavioral significance of G protein and internalization dependent signaling within ventrolateral periaqueductal gray (vlPAG). Antinociception induced by microinjecting DAMGO into the vlPAG was attenuated by blocking Gαi/o protein signaling with administration of pertussis toxin (PTX), preventing internalization with administration of dynamin dominant-negative inhibitory peptide (dyn-DN) or direct inhibition of ERK1/2 with administration of the MEK inhibitor, U0126. In contrast, the antinociceptive effect of microinjecting fentanyl into the vlPAG was not altered by admin...
Life Sciences, 2013
Aims-Chronic administration of cocaine attenuates delta opioid receptor (DOPR) signaling in the striatum and the desensitization is mediated by the indirect actions of cocaine on dopamine D 1 receptors (D 1 R). In addition, DOPR and D 1 R co-exist in some rat striatal neurons. In the present study, we examined the underlying mechanism of DOPR desensitization by D 1 R activation. Main methods-NG 108-15 cells stably expressing HA-rat D 1 receptor (HA-D 1 R) and Chinese hamster ovary (CHO) cells stably expressing both FLAG-mouse DOPR (FLAG-DOPR) and HAD 1 R were used as the cell models. Receptor binding, [ 35 S]GTP S binding, receptor phosphorylation and western blot were conducted to examine DOPR affinity, expression, internalization, downregulation, desensitization, phosphorylation and phosphorylated ERK1/2. Key findings-Pretreatment with either the DOPR agonist DPDPE or the D 1 R agonist SKF-82958 for 30 min attenuated DPDPE-stimulated [ 35 S]GTP S binding to G proteins, demonstrating homologous and heterologous desensitization of the DOPR, respectively. SKF-82958 pretreatment did not affect the level of DOPR or affinity of DOPR antagonist or agonists, nor did it induce phosphorylation, internalization or down-regulation of the DOPR in the CHO-FLAG-DOPR/HA-D 1 R cells. Pretreatment of cells with inhibitors of PKA, MEK1 and PI3K, but not PKC, attenuated SKF-82958-induced desensitization of the DOPR. The D 1 R agonist SKF-82958 enhanced phosphorylation of ERK1/2, and pretreatment with inhibitors of MEK1 and PI3K, but not PKA and PKC reduced the effect. These results indicate that activation of ERK1/2 and/or PKA, but not PKC, is involved in D 1 receptor-induced heterologous desensitization of the DOPR. Significance-This study provides possible mechanisms underlying D 1 R activation-induced DOPR desensitization.
Journal of Neurochemistry, 2002
Although it is well-established that G proteincoupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellularsignal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with~6-, or K-opioid receptors and ERK1-or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corre-sponding~-([D-A1a 2,Me-Phe4 ,Gly-ol 5] enkephalin) -, 6-([D-Pen2,o-Pen5] enkephalin) -, or K-(U69593) -selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a~3yscavenger, CD8-8adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h)opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-i3-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and ,ey subunits of G~, 0proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity. Key Words: Opioid receptor-Mitogen-activated protein kinase-Extracellular signal-regulated protein kinase-GTP binding regulatory protein-Ras-Epidermal growth factor. J. Neurochem. 70, 635-645 (1998).
Opioid modulation of ERK activity is ras dependent and involves Gβγ subunits
Journal of Neurochemistry
Although it is well-established that G proteincoupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellularsignal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with~6-, or K-opioid receptors and ERK1-or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corre-sponding~-([D-A1a 2,Me-Phe4 ,Gly-ol 5] enkephalin) -, 6-([D-Pen2,o-Pen5] enkephalin) -, or K-(U69593) -selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a~3yscavenger, CD8-8adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h)opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-i3-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and ,ey subunits of G~, 0proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity. Key Words: Opioid receptor-Mitogen-activated protein kinase-Extracellular signal-regulated protein kinase-GTP binding regulatory protein-Ras-Epidermal growth factor. J. Neurochem. 70, 635-645 (1998).
Activation profiles of opioid ligands in HEK cells expressing delta opioid receptors
BMC neuroscience, 2002
The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only delta opioid receptors. Activation profiles of most of these ligands at delta opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. Naltrexone and nalorphine were classified as antagonists at delta opioid receptor. The other ligands studied were agonists at delta opioid receptors and demonstrated IC50 values of 0.1 nM to 2 microM, maximal inhibition of 39-77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol > or = fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine > or = met-enkephalin > morphine = dezocine. For the first time these data describe and compare the functio...
Activation profiles of opioid ligands in HEK cells expressing d opioid receptors
Bmc Neurosci, 2002
Background: The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. Results: Naltrexone and nalorphine were classified as antagonists at δ opioid receptor. The other ligands studied were agonists at δ opioid receptors and demonstrated IC 50 values of 0.1 nM to 2 µM, maximal inhibition of 39-77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol ≥ fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine ≥ metenkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at δ opioid receptors. Conclusions: The data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at δ opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at δ opioid receptors. Background Opioid receptors belong to the large superfamily of G protein coupled receptors [1]. Three major types of opioid receptors (µ, δ and κ) have been cloned and each one is associated with several physiological and behavioural roles. The focus of the present work is the δ opioid receptor. Delta opioid receptors play a modulatory role in analgesia, hibernation, autonomic nervous system function, neuroendocrine system function, mood driven behaviours and olfaction [2]. Thus, understanding the requirements for activation of δ opioid receptors can lead to identification and design of novel drugs acting through this receptor for treatment of various physiological problems. While designing drugs with selectivity to a single receptor can be a desirable approach in drug design, another approach may be to design drugs that act on more than one receptor, but with differential activity on each receptor. These would be drugs that act as an agonist at one receptor
Activation profiles of opioid ligands in HEK cells expressing δ opioid receptors
BMC Neuroscience - BMC NEUROSCI, 2002
BACKGROUND: The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. RESULTS: Naltrexone and nalorphine were classified as antagonists at δ opioid receptor. The other ligands studied were agonists at δ opioid receptors and demonstrated IC50 values of 0.1 nM to 2 μM, maximal inhibition of 39–77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol ≥ fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine ≥ met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relat...
Pharmacology Biochemistry and Behavior, 2007
The clinical use of opioids is limited by the development of tolerance and physical dependence. Opioid tolerance and dependence are believed to result from complex adaptations in the CNS, representing a form of neural plasticity. Extracellular signal-regulated kinases (ERKs) are involved in many forms of neural plasticity, and therefore could also be involved in the development of opioid tolerance and dependence. In this study, we investigated the effect of a systemically bioavailable MEK (ERK kinase) inhibitor, SL327, upon the development and the expression of tolerance to and dependence on morphine in mice. In tolerance and dependence development studies, two strains of mice were treated daily for 8 or 9 days with 5 mg/kg morphine s.c. Tolerance development was assessed by tail flick latency. Withdrawal was then precipitated by subcutaneous injection of 2 mg/ kg naloxone s.c. and signs recorded. Co-administration of 50 mg/kg SL327 i.p. prior to morphine administration had no effect on the development of tolerance or withdrawal signs. To study possible effects of ERK inhibition on the expression of tolerance and dependence, mice were implanted with 75 mg morphine pellets s.c. Tolerance and dependence were assessed as previously described. An acute i.p. injection of 50 mg/kg SL327 after 4 days of morphine exposure had no effect on the expression of either morphine tolerance or physical dependence. To verify that this dose of SL327 inhibited morphine-induced ERK modulation, mice received an acute i.p. injection of 50 mg/kg SL327 prior to morphine administration, and sacrificed 30 min. later. Western blots demonstrated that SL327 did inhibit morphine-induced ERK modulation. Taken together, these data suggest that unlike many other observed forms of neural plasticity, the ERK signaling cascade is not involved in the development or expression of opioid tolerance and dependence.