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Differential Effect of Membrane Cholesterol Removal on μ- and δ-Opioid Receptors

2009; Elsevier BV; Volume: 284; Issue: 33 Linguagem: Inglês

10.1074/jbc.m109.030411

ISSN

1083-351X

Autores

Erica S. Levitt, Mary J. Clark, Paul M. Jenkins, Jeffrey R. Martens, John R. Traynor,

Tópico(s)

Neuropeptides and Animal Physiology

Resumo

According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-β-cyclodextrin (MβCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged μ-opioid receptors (HEK FLAG-μ) or δ-opioid receptors (HEK FLAG-δ) with MβCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the μ-opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MβCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing μ-opioid receptors. The effects of MβCD may be due to uncoupling of the μ receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-μ cells, MβCD treatment of HEK FLAG-δ cells had no effect on acute inhibition or sensitization of adenylyl cyclase by δ-opioid agonists. The differential responses of μ- and δ-opioid agonists to cholesterol depletion suggest that μ-opioid receptors are more dependent on cholesterol for efficient signaling than δ receptors and can be partly explained by localization of μ- but not δ-opioid receptors in cholesterol- and caveolin-enriched membrane domains. According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-β-cyclodextrin (MβCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged μ-opioid receptors (HEK FLAG-μ) or δ-opioid receptors (HEK FLAG-δ) with MβCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the μ-opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MβCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing μ-opioid receptors. The effects of MβCD may be due to uncoupling of the μ receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-μ cells, MβCD treatment of HEK FLAG-δ cells had no effect on acute inhibition or sensitization of adenylyl cyclase by δ-opioid agonists. The differential responses of μ- and δ-opioid agonists to cholesterol depletion suggest that μ-opioid receptors are more dependent on cholesterol for efficient signaling than δ receptors and can be partly explained by localization of μ- but not δ-opioid receptors in cholesterol- and caveolin-enriched membrane domains. Membrane cholesterol can alter the function of integral proteins, such as G protein-coupled receptors, through cholesterol-protein interactions and by changes in membrane viscosity (1Gimpl G. Burger K. Fahrenholz F. Biochemistry. 1997; 36: 10959-10974Crossref PubMed Scopus (399) Google Scholar). In addition, cholesterol interacts with other lipids found in the bilayer, particularly sphingolipids (2Slotte J.P. Chem. Phys. Lipids. 1999; 102: 13-27Crossref PubMed Scopus (177) Google Scholar), which allows for tight and organized packing that can precipitate the formation of specialized domains within the plasma membrane (3Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8117) Google Scholar). These domains have become an area of intense research interest and have been termed lipid or membrane rafts (4Pike L.J. J. Lipid Res. 2006; 47: 1597-1598Abstract Full Text Full Text PDF PubMed Scopus (1135) Google Scholar). The study of membrane rafts in intact cells is controversial, due in part to the limitations of the current methods used to study rafts (5Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1329) Google Scholar, 6Shogomori H. Brown D.A. Biol. Chem. 2003; 384: 1259-1263Crossref PubMed Scopus (170) Google Scholar). Regardless, the membrane environment formed in regions of high cholesterol and sphingolipids may be such that certain proteins have an affinity for these regions, especially proteins with a propensity to interact with cholesterol. Many G protein-coupled receptors and signaling proteins have been found to prefer cholesterol-enriched domains leading to the hypothesis that these domains can organize signaling molecules in the membrane to enhance or inhibit specific signaling events (7Allen J.A. Halverson-Tamboli R.A. Rasenick M.M. Nat. Rev. Neurosci. 2007; 8: 128-140Crossref PubMed Scopus (674) Google Scholar). This includes μ- (8Head B.P. Patel H.H. 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Mol. Pharmacol. 2002; 62: 983-992Crossref PubMed Scopus (122) Google Scholar), 5/6 (9Zhao H. Loh H. Law P. Mol. Pharmacol. 2006; 69: 1421-1432Crossref PubMed Scopus (67) Google Scholar, 18Ostrom R.S. Liu X. Head B.P. Gregorian C. Seasholtz T.M. Insel P.A. Mol. Pharmacol. 2002; 62: 983-992Crossref PubMed Scopus (122) Google Scholar, 19Fagan K.A. Smith K.E. Cooper D.M. J. Biol. Chem. 2000; 275: 26530-26537Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and 8 (20Smith K.E. Gu C. Fagan K.A. Hu B. Cooper D.M. J. Biol. Chem. 2002; 277: 6025-6031Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) have been found to associate with cholesterol and/or the cholesterol-binding protein caveolin. Activated opioid receptors couple to Gαi/o proteins and acutely inhibit the activity of adenylyl cyclase. Longer term exposure to opioid agonists causes sensitization of adenylyl cyclase and a rebound overshoot of cAMP production upon withdrawal of the agonist (21Watts V.J. J. Pharmacol. Exp. Ther. 2002; 302: 1-7Crossref PubMed Scopus (106) Google Scholar). Consequently, we sought to assess the role of cholesterol depletion on the ability of μ- and δ-opioid receptor agonists to inhibit and cause sensitization of adenylyl cyclase. There are conflicting data for the effect of changes in membrane cholesterol on opioid signaling. For example, an increase in plasma membrane microviscosity by addition of cholesteryl hemisuccinate to SH-SY5Y cell membranes increased μ-opioid receptor coupling to G proteins (22Emmerson P.J. Clark M.J. Medzihradsky F. Remmers A.E. J. Neurochem. 1999; 73: 289-300Crossref PubMed Scopus (27) Google Scholar). Conversely, removal of membrane cholesterol from Chinese hamster ovary cells has been shown to either decrease (23Gaibelet G. Millot C. Lebrun C. Ravault S. Sauliere A. Andre A. Lagane B. Lopez A. Mol. Membr. Biol. 2008; 25: 423-435Crossref PubMed Scopus (16) Google Scholar) or increase (24Huang P. Xu W. Yoon S.I. Chen C. Chong P.L. Unterwald E.M. Liu-Chen L.Y. Brain Res. 2007; 1184: 46-56Crossref PubMed Scopus (35) Google Scholar) the coupling of μ-opioid receptors to G proteins, as measured by [35S]GTPγS 3The abbreviations used are: GTPγSguanosine 5′-3-O-(thio)triphosphateSNC80(+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamideDPDPE[d-Pen2,5]-enkephalinDAMGO[d-Ala2,N-Me-Phe4,Gly5-ol]enkephalinDPNdiprenorphineCTAPd-Phe-[Cys-Tyr-d-Trp-Arg-Thr-Pen]Thr-NH2MβCDmethyl-β-cyclodextrinCTBcholera toxin B subunitACadenylyl cyclaseTfRtransferrin receptorANOVAanalysis of varianceHEKhuman embryonic kidney; FLAG epitope (DYKDDDDK)HEK FLAG-δHEK cells stably expressing FLAG-δreceptorHEK FLAG-μHEK cells stably expressing FLAG-μreceptorDMEMDulbecco舗s modified Eagle舗s mediumFBSfetal bovine serumMBSMES-buffered salineMES2-(N-morpholino)ethanesulfonic acidPBSphosphate-buffered saline. binding stimulated by the μ-opioid agonist DAMGO. Furthermore, the effect of cholesterol removal on δ-opioid agonist-stimulated [35S]GTPγS binding varies by cell type (10Huang P. Xu W. Yoon S.I. Chen C. Chong P.L. Liu-Chen L.Y. Biochem. Pharmacol. 2007; 73: 534-549Crossref PubMed Scopus (70) Google Scholar, 25André A. Gaibelet G. Le Guyader L. Welby M. Lopez A. Lebrun C. Biochim. Biophys. Acta. 2008; 1778: 1483-1492Crossref PubMed Scopus (17) Google Scholar). In these previous studies, the variety of cell types utilized and the conflicting results make comparisons between opioid receptor types difficult. The objective of this study was to directly compare the role of membrane cholesterol in modulating acute and chronic μ- and δ-opioid signaling in the same cell systems using identical methods, including the following: 1) depletion of cholesterol by the cholesterol-sequestering agent methyl-β-cyclodextrin (MβCD); 2) separation of cholesterol-enriched membranes by sucrose gradient ultracentrifugation; and 3) clustering of lipid raft patches in whole cells with cholera toxin B subunit. guanosine 5′-3-O-(thio)triphosphate (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide [d-Pen2,5]-enkephalin [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin diprenorphine d-Phe-[Cys-Tyr-d-Trp-Arg-Thr-Pen]Thr-NH2 methyl-β-cyclodextrin cholera toxin B subunit adenylyl cyclase transferrin receptor analysis of variance human embryonic kidney; FLAG epitope (DYKDDDDK) HEK cells stably expressing FLAG-δreceptor HEK cells stably expressing FLAG-μreceptor Dulbecco舗s modified Eagle舗s medium fetal bovine serum MES-buffered saline 2-(N-morpholino)ethanesulfonic acid phosphate-buffered saline. In initial experiments using human embryonic kidney (HEK) cells heterologously expressing μ- or δ-opioid receptors, we found that δ-opioid receptors were located in caveolin-poor fractions following 1% Triton X-100 homogenization and sucrose gradient ultracentrifugation. This differs from studies using a detergent-free method to identify lipid raft fractions (10Huang P. Xu W. Yoon S.I. Chen C. Chong P.L. Liu-Chen L.Y. Biochem. Pharmacol. 2007; 73: 534-549Crossref PubMed Scopus (70) Google Scholar, 11Patel H.H. Head B.P. Petersen H.N. Niesman I.R. Huang D. Gross G.J. Insel P.A. Roth D.M. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H344-350Crossref PubMed Scopus (92) Google Scholar). In contrast, we found that the μ-opioid receptor was found in both caveolin-poor and caveolin-rich fractions, in accordance with previous literature (8Head B.P. Patel H.H. Roth D.M. Lai N.C. Niesman I.R. Farquhar M.G. Insel P.A. J. Biol. Chem. 2005; 280: 31036-31044Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 9Zhao H. Loh H. Law P. Mol. Pharmacol. 2006; 69: 1421-1432Crossref PubMed Scopus (67) Google Scholar). This differential localization of opioid receptors led us to test the hypothesis that, in contrast to the μ-opioid receptor, the δ-opioid receptor would not be dependent on cholesterol for signaling. The results show that μ- but not δ-opioid receptors have a dependence on cholesterol for signaling to adenylyl cyclase and that this effect is much more pronounced following chronic exposure to opioids. SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), DPDPE ([d-Pen2,5]-enkephalin), and naltrindole hydrochloride were obtained from the Narcotic Drug and Opioid Peptide Basic Research Center at the University of Michigan (Ann Arbor, MI). Lovastatin hydroxy acid was obtained from Cayman Chemical (Ann Arbor, MI). [3H]Diprenorphine, [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin), and [35S]GTPγS were obtained from PerkinElmer Life Sciences. Tissue culture media, geneticin, fetal bovine serum, and trypsin were from Invitrogen. All other chemicals were obtained from Sigma unless otherwise stated. Human embryonic kidney 293 cells stably transfected with the N-terminal FLAG-tagged δ- (HEK FLAG-δ) or μ-opioid receptor (HEK FLAG-μ) were grown in Dulbecco舗s modified Eagle舗s medium (DMEM) containing 0.8 mg/ml geneticin and 10% fetal bovine serum at 37 °C in 5% CO2. Receptor expression in HEK FLAG-δ cells (8.4 ± 1.5 pmol/mg protein; n = 5) and HEK FLAG-μ cells (9.7 ± 1.3 pmol/mg protein; n = 5) was similar (p > 0.05). Receptor expression was measured by saturation binding of the opioid antagonist [3H]diprenorphine as described previously (22Emmerson P.J. Clark M.J. Medzihradsky F. Remmers A.E. J. Neurochem. 1999; 73: 289-300Crossref PubMed Scopus (27) Google Scholar). SH-SY5Y cells were grown as above but without geneticin. SH-SY5Y cells were differentiated by adding 10 μm retinoic acid (Calbiochem) 3–5 days prior to assay. HEK FLAG-μ or HEK FLAG-δ cells were grown to confluence in DMEM + 10% FBS. Media were replaced with serum-free DMEM with or without 2% (15 mm) MβCD (Sigma) for 1 h at 37 °C. SH-SY5Y cells were treated with 5 mm MβCD for 10 min. For cholesterol replenishment, cells were incubated with or without a 2% MβCD-cholesterol complex (MβCD-CH) in serum-free DMEM for 2 h following cholesterol depletion. MβCD-cholesterol complexes were formed in an 8:1 molar ratio as described previously (26Christian A.E. Haynes M.P. Phillips M.C. Rothblat G.H. J. Lipid Res. 1997; 38: 2264-2272Abstract Full Text PDF PubMed Google Scholar). Briefly, cholesterol was dissolved in a 1:1 ratio by volume of chloroform/methanol in a glass tube. Following evaporation of the solvent, the dried cholesterol was reconstituted with a suitable volume of serum-free DMEM containing 2% MβCD, vortexed, sonicated for 30 s, and incubated overnight at 37 °C with shaking. For lovastatin experiments, cells were treated with serum-free Opti-MEM containing 10 μm lovastatin hydroxy acid or DMSO vehicle for 48 h. Cholesterol content from cell lysates was determined using the Amplex Red cholesterol assay kit (Invitrogen) following the manufacturer舗s instructions. Cholesterol content was normalized to protein content, as determined by the method of Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). Membranes were prepared from HEK FLAG-δ or FLAG-μ cells following treatment with or without 2% MβCD as described previously (28Clark M.J. Harrison C. Zhong H. Neubig R.R. Traynor J.R. J. Biol. Chem. 2003; 278: 9418-9425Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Final membrane pellets were resuspended in 50 mm Tris-HCl buffer, aliquoted, and stored at −80 °C. Protein concentration was measured using the Bradford assay (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). Membranes (30 μg of protein) were incubated with 0.1 nm [35S]GTPγS for 60 min at 25 °C with or without various concentrations of the δ-opioid agonist SNC80 or the μ-opioid agonist DAMGO in [35S]GTPγS binding buffer (50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 100 mm NaCl, 1 mm EDTA, 2 mm dithiothreitol, and 30 μm GDP). Membranes with bound [35S]GTPγS were collected on GF/C filters (Whatman) using a Brandel harvester (MLR-24, Gaithersburg, MD) and rinsed three times with cold wash buffer (50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 100 mm NaCl). Dried filters were saturated with EcoLume liquid scintillation mixture (MP Biomedicals, Solon, OH), and radioactivity was counted in a Wallec 1450 MicroBeta (PerkinElmer Life Sciences). Inhibition of adenylyl cyclase activity was measured in HEK FLAG-δ or FLAG-μ cells grown to confluence in 24-well poly-d-lysine-coated plates. Cells were washed with serum-free DMEM and incubated with various concentrations of the δ-opioid agonist SNC80 or the μ-opioid agonist DAMGO in the presence of 5 μm forskolin and 1 mm 3-isobutyl-1-methylxanthine in serum-free media for 10 min at 37 °C. The assay was stopped by replacing the media with 1 ml of ice-cold 3% perchloric acid. After at least 30 min at 4 °C, a 400-μl aliquot of sample was neutralized with 2.5 m KHCO3 and centrifuged at 13,000 × g. Cyclic AMP was measured from the supernatant using a [3H]cAMP assay system (GE Healthcare) following the manufacturer舗s instructions. Inhibition of cAMP formation was calculated as a percent of forskolin-stimulated cAMP accumulation in the absence of opioid agonist. For adenylyl cyclase sensitization experiments, HEK cells were rinsed with serum-free DMEM and incubated in the presence or absence of SNC80, DPDPE, or DAMGO in serum-free DMEM for 30 min at 37 °C. SH-SY5Y cells, plated in uncoated 24-well plates (5 × 105 cells/well) and differentiated with 10 μm retinoic acid for 3–5 days prior to sensitization experiments, were incubated in the presence or absence of 1 μm DAMGO for 60 min at 37 °C. The drug-containing media were then removed and replaced with serum-free media containing 5 μm forskolin, 1 mm 3-isobutyl-1-methylxanthine, and 10 μm of the opioid antagonist naloxone for HEK FLAG-μ cells and SH-SY5Y cells, or 10 μm of the δ-opioid antagonist naltrindole for HEK FLAG-δ cells, to precipitate cAMP overshoot. After 10 min at 37 °C, the assay was stopped with ice-cold 3% perchloric acid, and cAMP accumulation was quantified as described above. Overshoot was calculated as a percent of forskolin-stimulated cAMP accumulation in the absence of opioid agonist. In competition binding assays, membranes (5–12 μg of protein) from HEK FLAG-μ or -δ cells treated with or without 2% MβCD were incubated for 1 h with shaking at 25 °C with 0.2 nm [3H]diprenorphine and increasing concentrations of unlabeled ligand (DAMGO or SNC80) in 50 mm Tris-HCl, pH 7.4. Where indicated, a final concentration of 10 μm GTPγS and 100 mm NaCl was added to the incubation buffer. For saturation binding assays, membranes (20 μg of protein) from low expressing HEK FLAG-μ cells (1.6 ± 0.1 pmol/mg protein) were incubated with increasing concentrations of [3H]diprenorphine (0.08–5 nm) or [3H]DAMGO (0.06–12 nm) in 50 mm Tris buffer, pH 7.4, for 1 h with shaking at 25 °C. For whole cell binding, HEK FLAG-μ cells (1 × 105 cells/tube) were incubated for 1 h in a 37 °C shaking water bath with 4 nm [3H]diprenorphine ± 10 μm CTAP (d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2) in serum-free DMEM. Protein content from a representative aliquot was determined by the method of Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). For all binding assays, nonspecific binding was determined using 10 μm naloxone. All assays were stopped by rapid filtration through GF/C filters using a Brandel harvester (MLR-24, Gaithersburg, MD) and rinsed three times with ice-cold 50 mm Tris-HCl wash buffer, pH 7.4. Bound radioactivity was determined by liquid scintillation counting as described above. HEK FLAG-δ or FLAG-μ cells were grown to confluence in 10-cm2 dishes, washed, and resuspended in ice-cold phosphate-buffered saline (PBS). Cells were pelleted and homogenized with a Dounce homogenizer in 100 μl of MES-buffered saline (MBS) containing 1% Triton X-100. The homogenate was placed on the bottom of a 2.2-ml ultracentrifuge tube, adjusted to 40% by addition of 53.3% sucrose in MBS, and overlaid with 900 μl of 30% sucrose and 900 μl of 5% sucrose in MBS for a discontinuous gradient. The samples were centrifuged at 200,000 × g in a Beckman-Coulter (Fullerton, CA) Optima Max-E Ultracentrifuge using a swinging bucket rotor (TLS-55) for 16–24 h at 4 °C. Equal volume (183 μl) fractions were collected from the top. Equal volume aliquots were taken from each fraction, mixed with sample buffer (63 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.008% bromphenol blue, 50 mm dithiothreitol), separated by SDS-PAGE on a 10% polyacrylamide gel, and transferred to nitrocellulose membranes (Pierce) for Western blotting. Antibodies used were monoclonal anti-FLAG M1 (1:2000; Sigma), polyclonal anti-caveolin (1:2000; BD Transduction Laboratories), and monoclonal anti-human TfR (1:2000; Zymed Laboratories Inc.). Secondary antibodies used were goat anti-mouse horseradish peroxidase or goat anti-rabbit horseradish peroxidase (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA). The above antibodies were diluted in 5% milk in Tris-buffered saline, 0.05% Tween 20 (+1 mm CaCl2 for FLAG M1 antibody). SuperSignal West Pico chemiluminescent substrate (Pierce) was used to detect immunoreactivity. For detection of adenylyl cyclase (AC) 5/6, samples were separated on a 6% polyacrylamide gel and transferred to an Immobilon-P polyvinylidene fluoride membrane (Millipore, Billerica, MA). AC 5/6 was detected by rabbit anti-AC 5/6 (Santa Cruz Biotechnology) diluted 1:200 in 1% bovine serum albumin in Tris-buffered saline, 0.05% Tween 20. HEK FLAG-δ or HEK FLAG-μ cells were plated on poly-d-lysine-coated coverslips in a 6-well plate (1 × 106 cells/well) 24 h prior to patching. Cells were incubated with AlexaFluor 488-conjugated cholera toxin B subunit (1 μg/μl in DMEM, 10% goat serum; Invitrogen) for 45 min at 4 °C to label endogenous ganglioside GM1. Lipid raft aggregation, or patching, was induced as described by Fra et al. (29Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar) by incubating with goat anti-CTB antibody (1:250 in DMEM, 10% goat serum; Calbiochem) for 30 min at 4 °C, followed by 20 min at 37 °C. Cells were fixed with 4% paraformaldehyde for 20 min and incubated in monoclonal anti-FLAG M1 antibody (1:1000 in PBS, 5% milk; Sigma) for 1 h followed by AlexaFluor 594-conjugated goat anti-mouse antibody (1:1000 in PBS, 5% milk; Invitrogen) to stain the FLAG-tagged μ- or δ-opioid receptor. For TfR and caveolin staining, cells were permeabilized with 0.1% Triton X-100 for 10 min and incubated with monoclonal anti-TfR antibody (1:200 in PBS, 5% milk; Zymed Laboratories Inc.) or polyclonal anti-caveolin antibody (1:200 in PBS, 5% milk; BD Transduction Laboratories). Coverslips were mounted on slides using ProLong Gold (Invitrogen). Fluorescent images of 0.5-μm Z planes were captured using an Olympus FV-500 confocal microscope. Quantification of colocalization was performed using the RG2B colocalization plug-in to ImageJ (version rsb.info.nih.gov). The minimum threshold pixel intensity was set to 50 for both channels, and the minimum ratio for pixel intensity between the channels was 50%. Colocalization pixels from individual Z planes were displayed as a stack of Z projections with maximum pixel intensity. Colocalization was reported as the average pixel density of colocalized pixels per cell. All data were analyzed using GraphPad Prism 4 software (San Diego, CA). All data points represent at least three separate experiments in duplicate and are presented as means ± S.E., unless otherwise noted. The effect of treatment on agonist responses at various concentrations was analyzed by two-way ANOVA with Bonferroni舗s post hoc test. EC50 values were calculated from individual concentration-effect curves using the fixed slope sigmoidal dose-response curve fit analysis in GraphPad Prism. Ki and Bmax/Kd values were calculated from individual binding experiments using one- or two-site competition or one-site hyperbola binding curve fit linear regression analysis, respectively. EC50, Ki, Bmax, and Kd values are expressed as mean ± S.E. and compared for statistical significance by unpaired, two-tailed Student舗s t test. Cholesterol repletion experiments were compared using one-way ANOVA with Bonferroni舗s post hoc test. All other statistical comparisons were made with unpaired, two-tailed Student舗s t test, unless otherwise indicated. For all tests significance was set at p < 0.05. HEK293 cells stably expressing either the FLAG-μ or FLAG-δ opioid receptor were treated with the cholesterol-sequestering agent MβCD (2%) for 1 h at 37 °C. This reduced cholesterol to 40 ± 5.6% of control, consistent with previously published results of 35–55% reductions (12Xu W. Yoon S.I. Huang P. Wang Y. Chen C. Chong P.L. Liu-Chen L.Y. J. Pharmacol. Exp. Ther. 2006; 317: 1295-1306Crossref PubMed Scopus (74) Google Scholar, 30Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Mol. Biol. Cell. 1999; 10: 961-974Crossref PubMed Scopus (825) Google Scholar, 31Pontier S.M. Percherancier Y. Galandrin S. Breit A. Galés C. Bouvier M. J. Biol. Chem. 2008; 283: 24659-24672Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and induced a rounder cell morphology, although cells were still viable by trypan blue exclusion. This treatment has been shown to eliminate caveolae as determined by electron microscopy (30Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Mol. Biol. Cell. 1999; 10: 961-974Crossref PubMed Scopus (825) Google Scholar), and it has been commonly used to disrupt lipid rafts to study effects on opioid signaling (9Zhao H. Loh H. Law P. Mol. Pharmacol. 2006; 69: 1421-1432Crossref PubMed Scopus (67) Google Scholar, 10Huang P. Xu W. Yoon S.I. Chen C. Chong P.L. Liu-Chen L.Y. Biochem. Pharmacol. 2007; 73: 534-549Crossref PubMed Scopus (70) Google Scholar, 11Patel H.H. Head B.P. Petersen H.N. Niesman I.R. Huang D. Gross G.J. Insel P.A. Roth D.M. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H344-350Crossref PubMed Scopus (92) Google Scholar, 12Xu W. Yoon S.I. Huang P. Wang Y. Chen C. Chong P.L. Liu-Chen L.Y. J. Pharmacol. Exp. Ther. 2006; 317: 1295-1306Crossref PubMed Scopus (74) Google Scholar). Therefore, we used this pharmacological tool to directly compare the effects of cholesterol depletion on μ- and δ-opioid receptor signaling. Agonist-activated opioid receptors couple to Gαi/o proteins and induce the exchange of GTP for GDP, which can be measured by the increase in binding of the guanine nucleotide analog [35S]GTPγS. Basal levels of [35S]GTPγS binding were similar in membranes from untransfected HEK293 cells and HEK cells expressing μ-opioid receptors but were higher in membranes from cells expressing δ-opioid receptors (Fig. 1A), which are thought to be tightly coupled to G proteins and show constitutive activity (32Costa T. Klinz F.J. Vachon L. Herz A. Mol. Pharmacol. 1988; 34: 744-754PubMed Google Scholar). Treatment of either HEK FLAG-μ cells or untransfected HEK293 cells with 2% MβCD for 1 h reduced basal [35S]GTPγS binding by 38 ± 5.5 or 39 ± 2.2%, respectively (Fig. 1A), suggesting an opioid receptor-independent effect. In contrast, treatment of HEK FLAG-δ cells with MβCD reduced basal levels of [35S]GTPγS binding by 61 ± 1.7%. However, the δ-opioid inverse agonist RTI-5989-25 (33Zaki P.A. Keith Jr., D.E. Thomas J.B. Carroll F.I. Evans C.J. J. Pharmacol. Exp. Ther. 2001; 298: 1015-1020PubMed Google Scholar) was able to reduce [35S]GTPγS binding by 18 ± 3.8% in control HEK FLAG-δ cells (data not shown). Therefore, in HEK FLAG-δ cells approximately one-third of the decrease in basal [35S]GTPγS binding caused by MβCD may be due to a loss of constitutively active receptors. The remaining decrease, which is similar to the decrease in HEK FLAG-μ or untransfected HEK293 cells, is likely because of a reduction in available, unoccupied Gα proteins themselves or a loss of constitutive activity of other G protein-coupled receptors endogenous to HEK293 cells. Because of this decrease in basal [35S]GTPγS binding, data were graphed as femtomoles of agonist-stimulated [35S]GTPγS bound/mg of protein rather than as percent change over basal, as has been reported previously (10Huang P. Xu W. Yoon S.I. Chen C. Chong P.L. Liu-Chen L.Y. Biochem. Pharmacol. 2007; 73: 534-549Crossref PubMed Scopus (70) Google Scholar, 24Huang P. Xu W. Yoon S.I. Chen C. Chong P.L. Unterwald E.M. Liu-Chen L.Y. Brain Res. 2007; 1184: 46-

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