U50,488H-induced Internalization of the Human κ Opioid Receptor Involves a β-Arrestin- and Dynamin-dependent Mechanism
1999; Elsevier BV; Volume: 274; Issue: 17 Linguagem: Inglês
10.1074/jbc.274.17.12087
ISSN1083-351X
AutoresJianguo Li, Lai-Yi Luo, Jason G. Krupnick, Jeffrey Benovic, Lee‐Yuan Liu‐Chen,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAgonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat κ opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human κ receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat κ opioid receptor. U50,488H-induced human κ receptor internalization was time- and concentration-dependent, with 30–40% of the receptors internalized following a 30-min exposure to 1 μmU50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50,488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50,488H-induced internalization. In contrast, incubation with sucrose (0.4–0.8 m) significantly reduced U50,488H-induced internalization of the κ receptor. While co-expression of the wild type GRK2, β-arrestin, or dynamin I had no effect on κ receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, β-arrestin (319–418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of β-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human κ receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat κ receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human κ opioid receptor in Chinese hamster ovary cells occurs via a GRK-, β-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the κ receptor is not required for activation of MAP kinase. Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat κ opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human κ receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat κ opioid receptor. U50,488H-induced human κ receptor internalization was time- and concentration-dependent, with 30–40% of the receptors internalized following a 30-min exposure to 1 μmU50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50,488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50,488H-induced internalization. In contrast, incubation with sucrose (0.4–0.8 m) significantly reduced U50,488H-induced internalization of the κ receptor. While co-expression of the wild type GRK2, β-arrestin, or dynamin I had no effect on κ receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, β-arrestin (319–418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of β-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human κ receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat κ receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human κ opioid receptor in Chinese hamster ovary cells occurs via a GRK-, β-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the κ receptor is not required for activation of MAP kinase. G protein-coupled receptor human κ opioid receptor G protein-coupled receptor kinase Chinese hamster ovary cells Chinese hamster ovary cells stably transfected with the cloned human κ opioid receptor CHO cell line stably transfected with the rat κ opioid receptor the rat κ opioid receptor β2-adrenergic receptor muscarinic cholinergic receptor mitogen-activated protein kinase 488H, (−)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide guanosine 5′-3-(thio)-triphosphate Opioid receptors are members of the G protein-coupled receptor (GPCR)1 family and can be classified into at least three types, μ, δ, and κ, based on pharmacological (for review, see Ref. 1Pasternak G.W. The Opiate Receptors. Humana Press, Clifton, NJ1988Crossref Google Scholar), anatomical (2Mansour A. Khachaturian H. Lewis M.E. Akil H. Watson S.J. 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Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9914-9919Crossref PubMed Scopus (315) Google Scholar). While κ opioid receptor internalization has not been extensively investigated, the potent nonselective opioid agonist etorphine did not induce internalization of the mouse κ opioid receptor expressed in HEK293 cells (38Chu P. Murray S. Lissin D. von Zastrow M. J. Biol. Chem. 1997; 272: 27124-27130Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). We have previously established a CHO cell line stably transfected with the human κ opioid receptor (CHO-hkor) (45Zhu J. Luo L.-Y. Chen C. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 1997; 282: 676-684PubMed Google Scholar, 46Zhu J. Luo L.Y. Mao G.F. Ashby B. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 1998; 285: 28-36PubMed Google Scholar). These cells exhibit the expected binding affinity and specificity for opioid ligands, and activation by κ opioid agonists enhances [35S]GTPγS binding to pertussis toxin-sensitive G proteins and inhibits forskolin-stimulated adenylate cyclase (45Zhu J. Luo L.-Y. Chen C. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 1997; 282: 676-684PubMed Google Scholar, 46Zhu J. Luo L.Y. Mao G.F. Ashby B. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 1998; 285: 28-36PubMed Google Scholar). In addition, we observed that after exposure of CHO-hkor cells to U50,488H, a selective κ opioid agonist, the human κ opioid receptor underwent desensitization and down-regulation (46Zhu J. Luo L.Y. Mao G.F. Ashby B. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 1998; 285: 28-36PubMed Google Scholar). A CHO cell line stably expressing the rat κ opioid receptor (47Li S. Zhu J. Chen C. Chen Y.-W. de Riel J.K. Ashby B. Liu-Chen L.-Y. Biochem. J. 1993; 295: 629-633Crossref PubMed Scopus (177) Google Scholar) was also established. In the present study, we investigated whether agonists promoted internalization of the human and rat κ opioid receptors in CHO cells and, if so, whether GRK, β-arrestin, and dynamin I were involved in this process. In addition, since Daaka et al. (25Daaka Y. Luttrell L.M. Ahn S. Dellarocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar) demonstrated that β-arrestin- and dynamin-mediated internalization of the β2-AR and lysophosphatidic acid receptor was essential for activation of the MAP kinase pathway, we also examined whether agonist-promoted κ receptor internalization played a critical role in activation of MAP kinase. [3H]Diprenorphine (58 Ci/mmol) was purchased from NEN Life Science Products Inc. (Boston, MA). (−)-U50,488H and U69,593 were generous gifts from Upjohn Co. (Kalamazoo, MI). Diprenorphine, etorphine, ethylketocyclazocine, and tifluadom were provided by the National Institute on Drug Abuse. Reagents were purchased from the indicated companies: naloxone, RBI (Natick, MA); dynorphin A (1Pasternak G.W. The Opiate Receptors. Humana Press, Clifton, NJ1988Crossref Google Scholar, 2Mansour A. Khachaturian H. Lewis M.E. Akil H. Watson S.J. Trends Neurosci. 1988; 11: 308-314Abstract Full Text PDF PubMed Scopus (1080) Google Scholar, 3Uhl G.R. Childers S. Pasternak G. Trends Neurosci. 1994; 17: 89-93Abstract Full Text PDF PubMed Scopus (221) Google Scholar, 4Knapp R.J. Malatynska E. Collins N. Fang L. Wang J.Y. Hruby V.J. Roeske W.R. Yamamura H.I. FASEB J. 1995; 9: 516-525Crossref PubMed Scopus (216) Google Scholar, 5von Voigtlander P.F. Lahti R.A. Ludens J.H. J. Pharmacol. Exp. Ther. 1983; 224: 7-12PubMed Google Scholar, 6Dykstra L.A. Gmerek D.E. Winger G. Woods J.H. J. Pharmacol. Exp. Ther. 1987; 242: 413-420PubMed Google Scholar, 7Pfeiffer A. Brantl V. Herz A. 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CHO-hkor cells and CHO-rkor cells express approximately 1.2 pmol of the human κ receptor and 1.0 pmol of the rat κ receptor per mg of membrane protein, respectively, as determined by [3H]diprenorphine binding (46Zhu J. Luo L.Y. Mao G.F. Ashby B. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 1998; 285: 28-36PubMed Google Scholar). CHO-hkor or CHO-rkor cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 10% fetal calf serum, 0.1 mg/ml geneticin, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere consisting of 5% CO2 and 95% air at 37 °C. Opioid receptor binding on intact cells was conducted according to Ref. 51Toll L. J. Pharmacol. Exp. Ther. 1992; 260: 9-15PubMed Google Scholar. Briefly, binding was performed on CHO-hkor cells cultured in 24-well plates with [3H]diprenorphine in Krebs-Ringer HEPES buffer solution (110 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, 25 mm glucose, 55 mm sucrose, 10 mm HEPES, pH 7.4). Binding of 2 nm [3H]diprenorphine to untreated CHO-hkor cells in the presence or absence of 1 μm diprenorphine was compared at three different temperatures: 0 °C, 12 °C, and room temperature (∼22 °C). Binding reached equilibrium in 4 h at 0 °C, 3 h at 12 °C, and 1 h at room temperature. At equilibrium, similar binding levels were attained at the three temperatures. Saturation [3H]diprenorphine binding to intact cells was performed at room temperature for 1 h and theK d of [3H]diprenorphine was determined to be ∼2 nm, which is much higher than theK d of [3H]diprenorphine binding to κ receptors in membranes conducted in 50 mm Tris/HCl buffer (∼0.2 nm) (45Zhu J. Luo L.-Y. Chen C. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 1997; 282: 676-684PubMed Google Scholar). CHO-hkor cells cultured in 24-well plates were incubated with U50,488H for the indicated intervals (up to 60 min) at 37 °C. Culture medium was removed and the cells were washed three times on ice with ice-cold phosphate-buffered saline (pH 7.0). Total receptor levels were assessed by binding with 2 nm [3H]diprenorphine in the presence or absence of 1 μm diprenorphine, while surface receptors were measured by binding with 2 nm[3H]diprenorphine in the presence or absence of 1 μm dynorphin A(1–17). Typically, binding was performed at room temperature for 60 min. Diprenorphine, a hydrophobic ligand, can bind to both cell surface and intracellular receptors, whereas dynorphin A(1–17), a hydrophilic ligand, binds only to the cell surface receptors. Thus, the difference between total receptor binding and cell surface receptor binding represents binding to the intracellular receptor pool. An increase in intracellular [3H]diprenorphine binding over the basal level following agonist exposure provides a quantitative measure of internalized receptors. CHO-hkor cells grown in 100-mm dishes were transiently transfected with 8 μg of bovine GRK2 (52Benovic J.L. DeBlasi A. Stone W.C. Caron M.G. Lefkowitz R.J. Science. 1989; 246: 235-240Crossref PubMed Scopus (328) Google Scholar) in pcDNA3.1 Zeo(+), GRK2-K220R (53Kong G. Penn R. Benovic J. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar) in pcDNA3.1 Zeo(+), bovine β-arrestin (54Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (900) Google Scholar) in pcDNA 3.1 Zeo(+), β-arrestin (319–418) (55Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) in pcDNA3, or dynamin I or dynamin I-K44A (48van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (587) Google Scholar, 49Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar) in pcDNA3 using LipofectAMINE (50 μl) following the manufacturer's instructions. Control cells were transfected with pcDNA 3.1 Zeo(+) or pcDNA3 and exhibited no opioid receptor binding (data not shown). Following transfection (∼18 h) the cells were incubated with fresh medium and allowed to recover 24–28 h before being reseeded in 24-well dishes and allowed to grow an additional 24 h. The cells were then analyzed for agonist-induced receptor internalization. CHO-hkor or CHO-rkor cells were transferred to 24-well plates and changed to serum-free medium for 2 h to overnight to reduce basal MAP kinase phosphorylation. Cells were treated with or without an agonist at 37 °C for 10 min and then lysed by addition of Laemmli sample buffer. Aliquots of the lysates were separated on SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Phosphorylated MAP kinase was detected by Western blot using phosphospecific MAP kinase antibodies and enzyme-linked chemiluminescence with the PhosphoPlus p44/42 MAP Kinase (Thr202/Tyr204) Antibody Kit according to the manufacturer's instructions. For comparison of multiple groups, data were analyzed by analysis of variance to determine if there were significant differences among groups. If so, Scheffe F test was performed to determine whether there we
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