Lee-Yuan Liu-Chen - Academia.edu (original) (raw)
Papers by Lee-Yuan Liu-Chen
Frontiers in Neuroscience, Nov 3, 2022
Frontiers in Bioscience, 2009
The number of cell surface opioid receptors reflects a delicate balance between biosynthesis path... more The number of cell surface opioid receptors reflects a delicate balance between biosynthesis pathway and endocytosis pathway. The post-activation endocytic events such as internalization, recycling and degradation have been well-documented; however, only a few studies have been conducted on the regulatory events occurring along the protein biosynthesis pathway, including protein folding, endoplasmic reticulum (ER) export, ER-associated degradation, vesicular trafficking and membrane targeting and insertion. Accumulated in vitro evidence has demonstrated that expression of the opioid receptors, either wild-type or mutated, is subject to regulation by prolonged treatment with cell-permeant ligands that exert their regulatory effects post-transcriptionally. These hydrophobic ligands, both agonists and antagonists, were found to act in the ER like ER-resided molecular chaperones to positively affect stability, folding efficiency and/or ER export rate of newly-synthesized receptor proteins. Moreover, a number of observations demonstrated that long-term opioid antagonists up-regulated the receptors in vivo, in accord with the in vitro findings. Potential therapeutic applications of the chaperone-like function of opioid ligands are discussed.
Biochemical and Biophysical Research Communications, 2008
The mu opioid receptor (MOR) in the rat and mouse caudate putamen (CPu) and thalamus was demonstr... more The mu opioid receptor (MOR) in the rat and mouse caudate putamen (CPu) and thalamus was demonstrated as diffuse and broad bands by western blot with polyclonal antibodies against a Cterminal peptide of MOR, which were absent in the cerebellum and brains of MOR-knockout mice. The electrophoretic mobility of MOR differed in the two brain regions with median relative molecular masses (Mr's) of 75 kDa (CPu) vs. 66 kDa (thalamus) for the rat, and 74 kDa (CPu) vs. 63 kDa (thalamus) for the mouse, which was due to its differential N-glycosylation. Rat MOR in CPu was found mainly associated with low-density cholesterol-and ganglioside M1 (GM1)-enriched fractions (lipid rafts), while the MOR in the thalamus was present in rafts and non-rafts without preference. Cholesterol reduction by methyl-β-cyclodextrin decreased DAGMO-induced [ 35 S]GTPγS binding in rat CPu membranes to a greater extent than in the thalamus membranes.
Life Sciences, 2011
In recent years it has become apparent that sex is a major factor involved in modulating the phar... more In recent years it has become apparent that sex is a major factor involved in modulating the pharmacological effects of exogenous opioids. The kappa opioid receptor (KOPR) system is a potential therapeutic target for pain, mood disorders and addiction. In humans mixed KOPR/ MOPR ligands have been found to produce greater analgesia in women than men. In contrast, in animals, selective KOPR agonists have been found to produce greater antinociceptive effects in males than females. Collectively, the studies indicate that the direction and magnitude of sex differences of KOPR-mediated antinociception/analgesia are dependent on species, strain, ligand and pain model examined. Of interest, and less studied, is whether sex differences in other KOPRmediated effects exist. In the studies conducted thus far, greater effects of KOPR agonists in males have been found in neuroprotection against stroke and suppression of food intake behavior. On the other hand, greater effects of KOPR agonists were found in females in mediation of prolactin release. In modulation of drugs of abuse, sex differences in KOPR effects were observed but appear to be dependent on the drug examined. The mechanism(s) underlying sex differences in KOPR-mediated effects may be mediated by sex chromosomes, gonadal hormonal influence on organization (circuitry) and/or acute hormonal influence on KOPR expression, distribution and localization. In light of the diverse pharmacology of KOPR we discuss the need for future studies characterizing the sexual dimorphism of KOPR neural circuitry and in examining other behaviors and processes that are modulated by the KOPR.
Bioorganic & Medicinal Chemistry Letters, 2012
The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alt... more The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alter the course of mood, anxiety and addictive disorders or reduce response to stress. In a search for highly potent and selective KOPR partial agonists as pharmacological tools, we have modified 12epi-salvinorin A, a compound which we have previously observed to be a KOPR partial agonist. Five analogues of 12-epi-salvinorin A were synthesized and their effects on G protein activation as well as β-arrestin2 recruitment were evaluated. Only 12-epi-salvinorin A (1) partially activated signaling through G proteins, yet acted as a full agonist in the β-arrestin 2 DiscoveRx assay. Other salvinorin analogues tested in these functional assays were full agonists in both assays of KOPR activation. By comparison, the non-selective opioid ligand nalbuphine, known to be a partial agonist for G-protein activation, was also a partial agonist for the β-arrestin mediated signaling pathway activated through KOPR.
Science (New York, N.Y.), Jun 22, 2018
A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is centra... more A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.
BMC Research Notes, 2020
Objective Selective kappa opioid receptor (KOR) agonists were shown to produce a dose-dependent d... more Objective Selective kappa opioid receptor (KOR) agonists were shown to produce a dose-dependent depression of brain-stimulation reward (BSR) in the rat intracranial self-stimulation (ICSS) tests. However, limited studies using mice produced less conclusive results. Here the effects of U50,488H were re-examined on BSR in mice with a larger cohort of animals. Results Forty C57BL/6J male mice were implanted with the electrodes in medial forebrain bundle. About a week after surgery, mice were subject to ICSS training. Only eighteen passed the two-phase procedures, at which point they readily spun the wheels to obtain reinforcing effect of BSR, and were used for the ICSS tests. Compared with saline (s.c.), U50,488H (2 mg/kg, s.c.) did not have effects on the BSR thresholds within 1 h post-treatment, while it decreased the maximum wheel-spinning rates in a time-dependent manner. In contrast, cocaine (5 mg/kg, s.c.) decreased the BSR thresholds time-dependently without affecting the maximu...
peptides differentially regulate the human κ opioid receptor
Neuropsychopharmacology, 2018
Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clini... more Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clinical application was limited by dysphoria and hallucinations. Nalfurafine, a clinically used KOR agonist, does not cause dysphoria or hallucinations at therapeutic doses in humans. We found that in CD-1 mice nalfurafine produced analgesic and anti-scratch effects dose-dependently, like the prototypic KOR agonist U50,488H. In contrast, unlike U50,488H, nalfurafine caused no aversion, anhedonia, or sedation or and a low level of motor incoordination at the effective analgesia and anti-scratch doses. Thus, we established a mouse model that recapitulated important aspects of the clinical observations. We then employed a phosphoproteomics approach to investigate mechanisms underlying differential KOR-mediated effects. A large-scale mass spectrometry (MS)-based analysis on brains revealed that nalfurafine perturbed phosphoproteomes differently from U50,488H in a brain-region specific manner after 30-min treatment. In particular, U50,488H and nalfurafine imparted phosphorylation changes to proteins found in different cellular components or signaling pathways in different brain regions. Notably, we observed that U50,488H, but not nalfurafine, activated the mammalian target of rapamycin (mTOR) pathway in the striatum and cortex. Inhibition of the mTOR pathway by rapamycin abolished U50,488H-induced aversion, without affecting analgesic, anti-scratch, and sedative effects and motor incoordination. The results indicate that the mTOR pathway is involved in KOR agonist-induced aversion. This is the first demonstration that phosphoproteomics can be applied to agonist-specific signaling of G protein-coupled receptors (GPCRs) in mouse brains to unravel pharmacologically important pathways. Furthermore, this is one of the first two reports that the mTOR pathway mediates aversion caused by KOR activation.
We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse ... more We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (KOPR) at residues S356, T357, T363 and S369. Here, we found that agonist (U50,488H)-dependent KOPR phosphorylation at all the residues were mediated by Gi/oα proteins and multiple protein kinases [GRKs2, 3, 5 and 6 and protein kinase C (PKC)]. In addition, PKC activation by phorbol ester induced agonist-independent KOPR phosphorylation. Compared with U50,488H, PKC activation promoted much higher S356/T357 phosphorylation, much lower T363 phosphorylation and similar levels of S369 phosphorylation. Following U50,488H, GRKs, but not PKC, were involved in agonist-induced KOPR internalization. In contrast, PKC activation caused a lower level of agonist-independent KOPR internalization, compared to U50,488H. U50,488H-induced activation of extracellular signal regulated kinase 1/2 (ERK1/2) was G protein-, but not β-arrestin-, dependent. After U50,488H, GRK-mediated, but n...
Neuropharmacology, 2016
Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of f... more Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of fear learning behavior. Moreover, recent studies from our laboratory have shown that acute nicotine impaired fear extinction and safety learning in mice. However, the effects of chronic nicotine and withdrawal on fear extinction are unknown. Therefore, the current experiments were conducted to investigate the effects of chronic nicotine as well as withdrawal from chronic nicotine on contextual fear extinction in mice. C57BL6/J mice were given contextual fear conditioning training and retention testing during chronic nicotine administration. Mice then received contextual fear extinction either during chronic nicotine or during withdrawal from chronic nicotine. Our results showed that contextual fear extinction was impaired both during chronic nicotine administration and subsequent withdrawal. However, it was also observed that the effects of prior chronic nicotine disappeared after 72 h in...
The Biochemical journal, Jan 3, 2015
Phosphorylation sites of KOPR following treatment with the selective agonist U50,488H were identi... more Phosphorylation sites of KOPR following treatment with the selective agonist U50,488H were identified after affinity purification, SDS-PAGE, in-gel digestion with Glu-C and LC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated S356, T357, T363 and S369 in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pS356/pT357, pT363, and pS369, respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pT363, pS369 and pS356/pT357 antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. S369 phosphorylation affected T363 phosphorylation and vice versa and T363 or S369 phosphorylation was important for S356/T357 phosphorylation, revealing phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced h...
European journal of pharmacology, Jan 6, 2015
Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. Howev... more Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [(35)S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi-G) and receptor internalization (RAi-I) and the degree of functional selectivity between the two [Log RAi-G - logRAi-I, RAi-G/RAi-I and bias factor]. The parameter, RAi, represents a relative...
Life Sciences, 2007
Dynorphins, endogenous peptides for the κ opioid receptor, play important roles in many physiolog... more Dynorphins, endogenous peptides for the κ opioid receptor, play important roles in many physiological and pathological functions. Here, we examined how prolonged treatment with three major prodynorphin peptides, dynorphin A (1-17) (Dyn A), dynorphin B (1-13) (Dyn B) and αneoendorphin (α-Neo), regulated the human kappa opioid receptor (hKOR) stably expressed in Chinese hamster ovary (CHO) cells. Results from receptor binding and [ 35 S]GTPγS binding assays showed that these peptides were potent full agonists of the hKOR with comparable receptor reserve and intrinsic efficacy to stimulate G proteins. A 4-h incubation with α-Neo at a concentration of ∼600× EC 50 value (from [ 35 S]GTPγS binding) resulted in receptor down-regulation to a much lower extent than the incubation with Dyn A and Dyn B at comparable concentrations (∼10% vs. ∼65%). Extending incubation period and increasing concentrations did not significantly affect the difference. The plateau level of α-Neo-mediated receptor internalization (30 min) was significantly less than those of Dyn A and Dyn B. Omission of serum from the incubation medium or addition of peptidase inhibitors into the serum-containing medium enhanced α-Neo-, but not Dyn A-or Dyn B-, mediated receptor down-regulation and internalization; however, the degrees of α-Neo-induced adaptations were still significantly less than those of Dyn A and Dyn B. Thus, these endogenous peptides differentially regulate KOR after activating the receptor with similar receptor occupancy and intrinsic efficacy. Both stability in the presence of serum and intrinsic capacity to promote receptor adaptation play roles in the observed discrepancy among the dynorphin peptides.
Neuroscience Letters, 2014
It has been shown previously that norbinaltorphimine (norBNI) and 5΄-guanidinonaltrindole (5΄-GNT... more It has been shown previously that norbinaltorphimine (norBNI) and 5΄-guanidinonaltrindole (5΄-GNTI), long-acting kappa opioid receptor (KOPR) antagonists, cause frenzied scratching in mice [1;2]. In the current study, we examined if zyklophin, a short-acting cyclic peptide KOPR antagonist, also elicited scratching behavior. When injected s.c. in the nape of the neck of male Swiss-Webster mice, zyklophin at doses of 0.1, 0.3 and 1 mg/kg induced dose-related hindleg scratching of the neck between 3 and 15 min after injection. Pretreating mice with norBNI (20 mg/kg, i.p.) at 18-20 hr before challenge with zyklophin (0.3 mg/kg) did not markedly affect scratching. Additionally, KOPR −/− mice given 0.3 mg/kg of zyklophin displayed similar levels of scratching as wild-type animals. The absence of KOPR in KOPR −/− mice was confirmed with ex vivo radioligand binding using [ 3 H]U69,593. Taken together, our data suggest that the presence of kappa receptors is not required for the excessive scratching caused by zyklophin. Thus, zyklophin, similar to the structurally different KOPR antagonist 5΄-GNTI, appears to act at other targets to elicit scratching and potentially the sensation of itch.
Neuropsychopharmacology, 2013
Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activatio... more Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR À / À) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KOR lox/lox). Autoradiography demonstrated complete ablation of KOR binding in the KOR À / À mutants, and reduced binding in the DAT-KOR lox/lox mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR À / À mice appeared normal in the open field and light/dark box tests, DAT-KOR lox/lox mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR À / À mutants, but was exaggerated in DAT-KOR lox/lox mutants. Increased sensitivity to cocaine in the DAT-KOR lox/lox mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR À / À mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.
![Research paper thumbnail of Molecular Basis of Differences in (−)(trans)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide-Induced Desensitization and Phosphorylation between Human and Rat κ-Opioid Receptors Expressed in Chinese Hamster Ovary Cells](https://attachments.academia-assets.com/88665977/thumbnails/1.jpg)
](Molecular Pharmacology, 2002
The agonist (Ϫ)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide [(Ϫ... more The agonist (Ϫ)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide [(Ϫ)U50,488H] caused desensitization of the human-opioid receptor (hkor) and Flagtagged hkor (Flag-hkor) but not the rat-opioid receptor (rkor) and Flag-tagged rkor (Flag-rkor) stably expressed in CHO cells as assessed by guanosine 5Ј-O-(3-[ 35 S]thiotriphosphate) binding. In addition, (Ϫ)U50,488H stimulation enhanced phosphorylation of the Flag-hkor, but not Flag-rkor. (Ϫ)U50,488H-induced phosphorylation of the Flag-hkor was reduced by expression of the dominant negative mutant GRK2-K220R, demonstrating the involvement of G protein-coupled receptor kinases (GRKs). However, expression of GRK2 and arrestin-2 or GRK3 and arrestin-3 did not result in desensitization or phosphorylation of the Flag-rkor after (Ϫ)U50,488H pretreatment. To understand the molecular basis of the species differences, we constructed two Flag-tagged chimeric receptors, Flag-h/rkor and Flag-r/hkor, in which the C-terminal domains of Flag-hkor and Flag-rkor were switched. When stably expressed in CHO cells, Flag-r/hkor, but not Flag-h/rkor, was desensitized and phosphorylated after exposure to (Ϫ)U50,488H, indicating that the C-terminal domain plays a critical role in the differences. We then generated a Flag-hkor mutant, in which S358 was mutated to N (Flag-hkorS358N) and a Flag-rkor mutant, in which N358 was substituted with S (Flag-rkorN358S). Although Flag-hkorS358N was not phosphorylated or desensitized by (Ϫ)U50,488H stimulation, Flag-rkorN358S underwent (Ϫ)U50,488H-induced desensitization with slightly increased phosphorylation. These results indicate that there are differences in (Ϫ)U50,488H-induced desensitization and phosphorylation between the hkor and the rkor. In addition, the Cterminal domain plays a crucial role in these differences and the 358 locus contributes to the differences. Our findings suggest caution in extrapolating studies on-opioid receptor regulation from rats to humans.
Molecular Pharmacology, 2008
Abbreviations: 7TMR, seven transmembrane domain receptor; β 2-AR, β 2-adrenergic receptor; CHO ce... more Abbreviations: 7TMR, seven transmembrane domain receptor; β 2-AR, β 2-adrenergic receptor; CHO cells, Chinese hamster ovary cells; CHO-FLAG-hKOR, CHO cell lines stably expressing FLAG-hKOR; CHO-FLAG-hKOR-10KR, CHO cell lines stably expressing FLAG-hKOR-10KR; CYLD, cylindromatosis tumor suppressor gene; DN, dominant negative mutant; Dyn A, dynorphin A (1-17); ER, Endoplasmic reticulum; FLAG-hKOR, Flag-tagged human κ opioid receptor; hKOR, human κ opioid receptor; hKOR-10KR, a FLAG-hKOR mutant in which all ten
Life Sciences, 2008
Several approaches have been taken for these in vivo studies. In many studies, the use of semiqua... more Several approaches have been taken for these in vivo studies. In many studies, the use of semiquantitative immuno-electron microscopy is the approach of choice. Endogenous opioid receptors display differential subcellular distributions with µ opioid receptor (MOPR) being mostly present on the plasma membrane and δand κ-opioid receptors (DOPR and KOPR, respectively) having a significant intracellular pool. Etorphine and DAMGO cause endocytosis of the MOPR, but morphine does not, except in some dendrites. Interestingly, chronic inflammatory pain and morphine treatment promote trafficking of intracellular DOPR to the cell surface which may account for the enhanced antinociceptive effects of DOPR agonists. KOPR has been reported to be associated with secretory vesicles in the posterior pituitary and translocated to the cell surface upon salt loading along with the release of vasopressin. The study of endogenous opioid receptors using in vivo models has produced some interesting results that could not have been anticipated in vitro. In vivo studies, therefore, are essential to provide insight into the mechanisms underlying opioid receptor regulation.
![Research paper thumbnail of Sex Difference in -Opioid Receptor (KOPR)-Mediated Behaviors, Brain Region KOPR Level and KOPR-Mediated Guanosine 5'-O-(3-[35S]Thiotriphosphate) Binding in the Guinea Pig](https://attachments.academia-assets.com/88665974/thumbnails/1.jpg)
](Journal of Pharmacology and Experimental Therapeutics, 2011
We examined if sex differences in KOPR pharmacology exists in guinea pigs, which are more similar... more We examined if sex differences in KOPR pharmacology exists in guinea pigs, which are more similar to humans in the expression level and distribution of KOPR in the brain than rats and mice. The KOPR agonist U50,488H produced a dose-dependent increase in abnormal postures and immobility with greater effects in males than females. Males also showed greater U50,488H-induced antinociception in the paw pressure test than females. Pretreatment with KOPR antagonist norBNI blocked U50,488H-induced abnormal body postures and antinociception. In contrast, inhibition of cocaine-induced hyperactivity by U50,488H was more effective in females than males. Thus, sex differences in the effects of U50,488H are endpoint-dependent. We then examined if sex differences in KOPR level and KOPR-mediated G protein activation in brain regions may contribute to the observed differences using quantitative in vitro autoradiography of [ 3 H]U69,593 binding to the KOPR and U50,488H-stimulated [ 35 S]GTPS binding. Compared to females, males exhibited greater [ 3 H]U69,593 binding in the deep layers of somatosensory and insular cortices, claustrum, endopiriform nucleus, periaqueductal gray, and substantial nigra. Concomitantly, U50,488H-stimulated [ 35 S]GTPS binding was greater in males than females in the superficial and deep layers of somatosensory and insular cortices, caudate putamen, claustrum, medial geniculate nucleus and cerebellum. In contrast, compared to males, females showed greater U50,488Hstimulated [ 35 S]GTPS binding in the dentate gyrus, and a trend of higher [ 35 S]GTPS binding in the hypothalamus. These data demonstrate that males and females differ in KOPR expression and KOPR-mediated G-protein activation in distinct brain regions, which may contribute to the observed sex differences in KOPR-mediated pharmacology.
Frontiers in Neuroscience, Nov 3, 2022
Frontiers in Bioscience, 2009
The number of cell surface opioid receptors reflects a delicate balance between biosynthesis path... more The number of cell surface opioid receptors reflects a delicate balance between biosynthesis pathway and endocytosis pathway. The post-activation endocytic events such as internalization, recycling and degradation have been well-documented; however, only a few studies have been conducted on the regulatory events occurring along the protein biosynthesis pathway, including protein folding, endoplasmic reticulum (ER) export, ER-associated degradation, vesicular trafficking and membrane targeting and insertion. Accumulated in vitro evidence has demonstrated that expression of the opioid receptors, either wild-type or mutated, is subject to regulation by prolonged treatment with cell-permeant ligands that exert their regulatory effects post-transcriptionally. These hydrophobic ligands, both agonists and antagonists, were found to act in the ER like ER-resided molecular chaperones to positively affect stability, folding efficiency and/or ER export rate of newly-synthesized receptor proteins. Moreover, a number of observations demonstrated that long-term opioid antagonists up-regulated the receptors in vivo, in accord with the in vitro findings. Potential therapeutic applications of the chaperone-like function of opioid ligands are discussed.
Biochemical and Biophysical Research Communications, 2008
The mu opioid receptor (MOR) in the rat and mouse caudate putamen (CPu) and thalamus was demonstr... more The mu opioid receptor (MOR) in the rat and mouse caudate putamen (CPu) and thalamus was demonstrated as diffuse and broad bands by western blot with polyclonal antibodies against a Cterminal peptide of MOR, which were absent in the cerebellum and brains of MOR-knockout mice. The electrophoretic mobility of MOR differed in the two brain regions with median relative molecular masses (Mr's) of 75 kDa (CPu) vs. 66 kDa (thalamus) for the rat, and 74 kDa (CPu) vs. 63 kDa (thalamus) for the mouse, which was due to its differential N-glycosylation. Rat MOR in CPu was found mainly associated with low-density cholesterol-and ganglioside M1 (GM1)-enriched fractions (lipid rafts), while the MOR in the thalamus was present in rafts and non-rafts without preference. Cholesterol reduction by methyl-β-cyclodextrin decreased DAGMO-induced [ 35 S]GTPγS binding in rat CPu membranes to a greater extent than in the thalamus membranes.
Life Sciences, 2011
In recent years it has become apparent that sex is a major factor involved in modulating the phar... more In recent years it has become apparent that sex is a major factor involved in modulating the pharmacological effects of exogenous opioids. The kappa opioid receptor (KOPR) system is a potential therapeutic target for pain, mood disorders and addiction. In humans mixed KOPR/ MOPR ligands have been found to produce greater analgesia in women than men. In contrast, in animals, selective KOPR agonists have been found to produce greater antinociceptive effects in males than females. Collectively, the studies indicate that the direction and magnitude of sex differences of KOPR-mediated antinociception/analgesia are dependent on species, strain, ligand and pain model examined. Of interest, and less studied, is whether sex differences in other KOPRmediated effects exist. In the studies conducted thus far, greater effects of KOPR agonists in males have been found in neuroprotection against stroke and suppression of food intake behavior. On the other hand, greater effects of KOPR agonists were found in females in mediation of prolactin release. In modulation of drugs of abuse, sex differences in KOPR effects were observed but appear to be dependent on the drug examined. The mechanism(s) underlying sex differences in KOPR-mediated effects may be mediated by sex chromosomes, gonadal hormonal influence on organization (circuitry) and/or acute hormonal influence on KOPR expression, distribution and localization. In light of the diverse pharmacology of KOPR we discuss the need for future studies characterizing the sexual dimorphism of KOPR neural circuitry and in examining other behaviors and processes that are modulated by the KOPR.
Bioorganic & Medicinal Chemistry Letters, 2012
The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alt... more The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alter the course of mood, anxiety and addictive disorders or reduce response to stress. In a search for highly potent and selective KOPR partial agonists as pharmacological tools, we have modified 12epi-salvinorin A, a compound which we have previously observed to be a KOPR partial agonist. Five analogues of 12-epi-salvinorin A were synthesized and their effects on G protein activation as well as β-arrestin2 recruitment were evaluated. Only 12-epi-salvinorin A (1) partially activated signaling through G proteins, yet acted as a full agonist in the β-arrestin 2 DiscoveRx assay. Other salvinorin analogues tested in these functional assays were full agonists in both assays of KOPR activation. By comparison, the non-selective opioid ligand nalbuphine, known to be a partial agonist for G-protein activation, was also a partial agonist for the β-arrestin mediated signaling pathway activated through KOPR.
Science (New York, N.Y.), Jun 22, 2018
A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is centra... more A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.
BMC Research Notes, 2020
Objective Selective kappa opioid receptor (KOR) agonists were shown to produce a dose-dependent d... more Objective Selective kappa opioid receptor (KOR) agonists were shown to produce a dose-dependent depression of brain-stimulation reward (BSR) in the rat intracranial self-stimulation (ICSS) tests. However, limited studies using mice produced less conclusive results. Here the effects of U50,488H were re-examined on BSR in mice with a larger cohort of animals. Results Forty C57BL/6J male mice were implanted with the electrodes in medial forebrain bundle. About a week after surgery, mice were subject to ICSS training. Only eighteen passed the two-phase procedures, at which point they readily spun the wheels to obtain reinforcing effect of BSR, and were used for the ICSS tests. Compared with saline (s.c.), U50,488H (2 mg/kg, s.c.) did not have effects on the BSR thresholds within 1 h post-treatment, while it decreased the maximum wheel-spinning rates in a time-dependent manner. In contrast, cocaine (5 mg/kg, s.c.) decreased the BSR thresholds time-dependently without affecting the maximu...
peptides differentially regulate the human κ opioid receptor
Neuropsychopharmacology, 2018
Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clini... more Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clinical application was limited by dysphoria and hallucinations. Nalfurafine, a clinically used KOR agonist, does not cause dysphoria or hallucinations at therapeutic doses in humans. We found that in CD-1 mice nalfurafine produced analgesic and anti-scratch effects dose-dependently, like the prototypic KOR agonist U50,488H. In contrast, unlike U50,488H, nalfurafine caused no aversion, anhedonia, or sedation or and a low level of motor incoordination at the effective analgesia and anti-scratch doses. Thus, we established a mouse model that recapitulated important aspects of the clinical observations. We then employed a phosphoproteomics approach to investigate mechanisms underlying differential KOR-mediated effects. A large-scale mass spectrometry (MS)-based analysis on brains revealed that nalfurafine perturbed phosphoproteomes differently from U50,488H in a brain-region specific manner after 30-min treatment. In particular, U50,488H and nalfurafine imparted phosphorylation changes to proteins found in different cellular components or signaling pathways in different brain regions. Notably, we observed that U50,488H, but not nalfurafine, activated the mammalian target of rapamycin (mTOR) pathway in the striatum and cortex. Inhibition of the mTOR pathway by rapamycin abolished U50,488H-induced aversion, without affecting analgesic, anti-scratch, and sedative effects and motor incoordination. The results indicate that the mTOR pathway is involved in KOR agonist-induced aversion. This is the first demonstration that phosphoproteomics can be applied to agonist-specific signaling of G protein-coupled receptors (GPCRs) in mouse brains to unravel pharmacologically important pathways. Furthermore, this is one of the first two reports that the mTOR pathway mediates aversion caused by KOR activation.
We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse ... more We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (KOPR) at residues S356, T357, T363 and S369. Here, we found that agonist (U50,488H)-dependent KOPR phosphorylation at all the residues were mediated by Gi/oα proteins and multiple protein kinases [GRKs2, 3, 5 and 6 and protein kinase C (PKC)]. In addition, PKC activation by phorbol ester induced agonist-independent KOPR phosphorylation. Compared with U50,488H, PKC activation promoted much higher S356/T357 phosphorylation, much lower T363 phosphorylation and similar levels of S369 phosphorylation. Following U50,488H, GRKs, but not PKC, were involved in agonist-induced KOPR internalization. In contrast, PKC activation caused a lower level of agonist-independent KOPR internalization, compared to U50,488H. U50,488H-induced activation of extracellular signal regulated kinase 1/2 (ERK1/2) was G protein-, but not β-arrestin-, dependent. After U50,488H, GRK-mediated, but n...
Neuropharmacology, 2016
Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of f... more Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of fear learning behavior. Moreover, recent studies from our laboratory have shown that acute nicotine impaired fear extinction and safety learning in mice. However, the effects of chronic nicotine and withdrawal on fear extinction are unknown. Therefore, the current experiments were conducted to investigate the effects of chronic nicotine as well as withdrawal from chronic nicotine on contextual fear extinction in mice. C57BL6/J mice were given contextual fear conditioning training and retention testing during chronic nicotine administration. Mice then received contextual fear extinction either during chronic nicotine or during withdrawal from chronic nicotine. Our results showed that contextual fear extinction was impaired both during chronic nicotine administration and subsequent withdrawal. However, it was also observed that the effects of prior chronic nicotine disappeared after 72 h in...
The Biochemical journal, Jan 3, 2015
Phosphorylation sites of KOPR following treatment with the selective agonist U50,488H were identi... more Phosphorylation sites of KOPR following treatment with the selective agonist U50,488H were identified after affinity purification, SDS-PAGE, in-gel digestion with Glu-C and LC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated S356, T357, T363 and S369 in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pS356/pT357, pT363, and pS369, respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pT363, pS369 and pS356/pT357 antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. S369 phosphorylation affected T363 phosphorylation and vice versa and T363 or S369 phosphorylation was important for S356/T357 phosphorylation, revealing phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced h...
European journal of pharmacology, Jan 6, 2015
Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. Howev... more Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [(35)S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi-G) and receptor internalization (RAi-I) and the degree of functional selectivity between the two [Log RAi-G - logRAi-I, RAi-G/RAi-I and bias factor]. The parameter, RAi, represents a relative...
Life Sciences, 2007
Dynorphins, endogenous peptides for the κ opioid receptor, play important roles in many physiolog... more Dynorphins, endogenous peptides for the κ opioid receptor, play important roles in many physiological and pathological functions. Here, we examined how prolonged treatment with three major prodynorphin peptides, dynorphin A (1-17) (Dyn A), dynorphin B (1-13) (Dyn B) and αneoendorphin (α-Neo), regulated the human kappa opioid receptor (hKOR) stably expressed in Chinese hamster ovary (CHO) cells. Results from receptor binding and [ 35 S]GTPγS binding assays showed that these peptides were potent full agonists of the hKOR with comparable receptor reserve and intrinsic efficacy to stimulate G proteins. A 4-h incubation with α-Neo at a concentration of ∼600× EC 50 value (from [ 35 S]GTPγS binding) resulted in receptor down-regulation to a much lower extent than the incubation with Dyn A and Dyn B at comparable concentrations (∼10% vs. ∼65%). Extending incubation period and increasing concentrations did not significantly affect the difference. The plateau level of α-Neo-mediated receptor internalization (30 min) was significantly less than those of Dyn A and Dyn B. Omission of serum from the incubation medium or addition of peptidase inhibitors into the serum-containing medium enhanced α-Neo-, but not Dyn A-or Dyn B-, mediated receptor down-regulation and internalization; however, the degrees of α-Neo-induced adaptations were still significantly less than those of Dyn A and Dyn B. Thus, these endogenous peptides differentially regulate KOR after activating the receptor with similar receptor occupancy and intrinsic efficacy. Both stability in the presence of serum and intrinsic capacity to promote receptor adaptation play roles in the observed discrepancy among the dynorphin peptides.
Neuroscience Letters, 2014
It has been shown previously that norbinaltorphimine (norBNI) and 5΄-guanidinonaltrindole (5΄-GNT... more It has been shown previously that norbinaltorphimine (norBNI) and 5΄-guanidinonaltrindole (5΄-GNTI), long-acting kappa opioid receptor (KOPR) antagonists, cause frenzied scratching in mice [1;2]. In the current study, we examined if zyklophin, a short-acting cyclic peptide KOPR antagonist, also elicited scratching behavior. When injected s.c. in the nape of the neck of male Swiss-Webster mice, zyklophin at doses of 0.1, 0.3 and 1 mg/kg induced dose-related hindleg scratching of the neck between 3 and 15 min after injection. Pretreating mice with norBNI (20 mg/kg, i.p.) at 18-20 hr before challenge with zyklophin (0.3 mg/kg) did not markedly affect scratching. Additionally, KOPR −/− mice given 0.3 mg/kg of zyklophin displayed similar levels of scratching as wild-type animals. The absence of KOPR in KOPR −/− mice was confirmed with ex vivo radioligand binding using [ 3 H]U69,593. Taken together, our data suggest that the presence of kappa receptors is not required for the excessive scratching caused by zyklophin. Thus, zyklophin, similar to the structurally different KOPR antagonist 5΄-GNTI, appears to act at other targets to elicit scratching and potentially the sensation of itch.
Neuropsychopharmacology, 2013
Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activatio... more Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR À / À) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KOR lox/lox). Autoradiography demonstrated complete ablation of KOR binding in the KOR À / À mutants, and reduced binding in the DAT-KOR lox/lox mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR À / À mice appeared normal in the open field and light/dark box tests, DAT-KOR lox/lox mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR À / À mutants, but was exaggerated in DAT-KOR lox/lox mutants. Increased sensitivity to cocaine in the DAT-KOR lox/lox mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR À / À mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.
Molecular Pharmacology, 2002
The agonist (Ϫ)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide [(Ϫ... more The agonist (Ϫ)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide [(Ϫ)U50,488H] caused desensitization of the human-opioid receptor (hkor) and Flagtagged hkor (Flag-hkor) but not the rat-opioid receptor (rkor) and Flag-tagged rkor (Flag-rkor) stably expressed in CHO cells as assessed by guanosine 5Ј-O-(3-[ 35 S]thiotriphosphate) binding. In addition, (Ϫ)U50,488H stimulation enhanced phosphorylation of the Flag-hkor, but not Flag-rkor. (Ϫ)U50,488H-induced phosphorylation of the Flag-hkor was reduced by expression of the dominant negative mutant GRK2-K220R, demonstrating the involvement of G protein-coupled receptor kinases (GRKs). However, expression of GRK2 and arrestin-2 or GRK3 and arrestin-3 did not result in desensitization or phosphorylation of the Flag-rkor after (Ϫ)U50,488H pretreatment. To understand the molecular basis of the species differences, we constructed two Flag-tagged chimeric receptors, Flag-h/rkor and Flag-r/hkor, in which the C-terminal domains of Flag-hkor and Flag-rkor were switched. When stably expressed in CHO cells, Flag-r/hkor, but not Flag-h/rkor, was desensitized and phosphorylated after exposure to (Ϫ)U50,488H, indicating that the C-terminal domain plays a critical role in the differences. We then generated a Flag-hkor mutant, in which S358 was mutated to N (Flag-hkorS358N) and a Flag-rkor mutant, in which N358 was substituted with S (Flag-rkorN358S). Although Flag-hkorS358N was not phosphorylated or desensitized by (Ϫ)U50,488H stimulation, Flag-rkorN358S underwent (Ϫ)U50,488H-induced desensitization with slightly increased phosphorylation. These results indicate that there are differences in (Ϫ)U50,488H-induced desensitization and phosphorylation between the hkor and the rkor. In addition, the Cterminal domain plays a crucial role in these differences and the 358 locus contributes to the differences. Our findings suggest caution in extrapolating studies on-opioid receptor regulation from rats to humans.
Molecular Pharmacology, 2008
Abbreviations: 7TMR, seven transmembrane domain receptor; β 2-AR, β 2-adrenergic receptor; CHO ce... more Abbreviations: 7TMR, seven transmembrane domain receptor; β 2-AR, β 2-adrenergic receptor; CHO cells, Chinese hamster ovary cells; CHO-FLAG-hKOR, CHO cell lines stably expressing FLAG-hKOR; CHO-FLAG-hKOR-10KR, CHO cell lines stably expressing FLAG-hKOR-10KR; CYLD, cylindromatosis tumor suppressor gene; DN, dominant negative mutant; Dyn A, dynorphin A (1-17); ER, Endoplasmic reticulum; FLAG-hKOR, Flag-tagged human κ opioid receptor; hKOR, human κ opioid receptor; hKOR-10KR, a FLAG-hKOR mutant in which all ten
Life Sciences, 2008
Several approaches have been taken for these in vivo studies. In many studies, the use of semiqua... more Several approaches have been taken for these in vivo studies. In many studies, the use of semiquantitative immuno-electron microscopy is the approach of choice. Endogenous opioid receptors display differential subcellular distributions with µ opioid receptor (MOPR) being mostly present on the plasma membrane and δand κ-opioid receptors (DOPR and KOPR, respectively) having a significant intracellular pool. Etorphine and DAMGO cause endocytosis of the MOPR, but morphine does not, except in some dendrites. Interestingly, chronic inflammatory pain and morphine treatment promote trafficking of intracellular DOPR to the cell surface which may account for the enhanced antinociceptive effects of DOPR agonists. KOPR has been reported to be associated with secretory vesicles in the posterior pituitary and translocated to the cell surface upon salt loading along with the release of vasopressin. The study of endogenous opioid receptors using in vivo models has produced some interesting results that could not have been anticipated in vitro. In vivo studies, therefore, are essential to provide insight into the mechanisms underlying opioid receptor regulation.
Journal of Pharmacology and Experimental Therapeutics, 2011
We examined if sex differences in KOPR pharmacology exists in guinea pigs, which are more similar... more We examined if sex differences in KOPR pharmacology exists in guinea pigs, which are more similar to humans in the expression level and distribution of KOPR in the brain than rats and mice. The KOPR agonist U50,488H produced a dose-dependent increase in abnormal postures and immobility with greater effects in males than females. Males also showed greater U50,488H-induced antinociception in the paw pressure test than females. Pretreatment with KOPR antagonist norBNI blocked U50,488H-induced abnormal body postures and antinociception. In contrast, inhibition of cocaine-induced hyperactivity by U50,488H was more effective in females than males. Thus, sex differences in the effects of U50,488H are endpoint-dependent. We then examined if sex differences in KOPR level and KOPR-mediated G protein activation in brain regions may contribute to the observed differences using quantitative in vitro autoradiography of [ 3 H]U69,593 binding to the KOPR and U50,488H-stimulated [ 35 S]GTPS binding. Compared to females, males exhibited greater [ 3 H]U69,593 binding in the deep layers of somatosensory and insular cortices, claustrum, endopiriform nucleus, periaqueductal gray, and substantial nigra. Concomitantly, U50,488H-stimulated [ 35 S]GTPS binding was greater in males than females in the superficial and deep layers of somatosensory and insular cortices, caudate putamen, claustrum, medial geniculate nucleus and cerebellum. In contrast, compared to males, females showed greater U50,488Hstimulated [ 35 S]GTPS binding in the dentate gyrus, and a trend of higher [ 35 S]GTPS binding in the hypothalamus. These data demonstrate that males and females differ in KOPR expression and KOPR-mediated G-protein activation in distinct brain regions, which may contribute to the observed sex differences in KOPR-mediated pharmacology.