μ Opioid Receptor Phosphorylation, Desensitization, and Ligand Efficacy

Abstract

μ opioid receptors are subject to phosphorylation and desensitization through actions of at least two distinct biochemical pathways: agonist-dependent μ receptor phosphorylation and desensitization induced by a biochemically distinct second pathway dependent on protein kinase C activation (1). To better understand the nature of the agonist-induced μ receptor phosphorylation events, we have investigated the effects of a variety of opiate ligands of varying potencies and intrinsic activities on μ receptor phosphorylation and desensitization. Exposure to the potent full agonists sufentanil, dihydroetorphine, etorphine, etonitazine, and [d-Ala2, MePhe4, Glyol5]enkephalin (DAMGO) led to strong receptor phosphorylation, while methadone,l-α-acetylmethadone (LAAM), morphine, meperidine, DADL, β-endorphin(1–31), enkephalins, and dynorphin A(1–17) produced intermediate effects. The partial agonist buprenorphine minimally enhanced receptor phosphorylation while antagonists failed to alter phosphorylation. Buprenorphine and full antagonists each antagonized the enhanced μ receptor phosphorylation induced by morphine or DAMGO. The rank order of opiate ligand efficacies in producing μ receptor-mediated functional desensitization generally paralleled their rank order of efficacies in producing receptor phosphorylation. Interestingly, the desensitization and phosphorylation mediated by methadone and LAAM were disproportionate to their efficacies in two distinct test systems. This generally good fit between the efficacies of opiates in μ receptor activation, phosphorylation, and desensitization supports the idea that activated receptor/agonist/G-protein complexes and/or receptor conformational changes induced by agonists are required for agonist-induced μ receptor phosphorylation. Data for methadone and LAAM suggest possible contribution from their enhanced desensitizing abilities to their therapeutic efficacies. μ opioid receptors are subject to phosphorylation and desensitization through actions of at least two distinct biochemical pathways: agonist-dependent μ receptor phosphorylation and desensitization induced by a biochemically distinct second pathway dependent on protein kinase C activation (1). To better understand the nature of the agonist-induced μ receptor phosphorylation events, we have investigated the effects of a variety of opiate ligands of varying potencies and intrinsic activities on μ receptor phosphorylation and desensitization. Exposure to the potent full agonists sufentanil, dihydroetorphine, etorphine, etonitazine, and [d-Ala2, MePhe4, Glyol5]enkephalin (DAMGO) led to strong receptor phosphorylation, while methadone,l-α-acetylmethadone (LAAM), morphine, meperidine, DADL, β-endorphin(1–31), enkephalins, and dynorphin A(1–17) produced intermediate effects. The partial agonist buprenorphine minimally enhanced receptor phosphorylation while antagonists failed to alter phosphorylation. Buprenorphine and full antagonists each antagonized the enhanced μ receptor phosphorylation induced by morphine or DAMGO. The rank order of opiate ligand efficacies in producing μ receptor-mediated functional desensitization generally paralleled their rank order of efficacies in producing receptor phosphorylation. Interestingly, the desensitization and phosphorylation mediated by methadone and LAAM were disproportionate to their efficacies in two distinct test systems. This generally good fit between the efficacies of opiates in μ receptor activation, phosphorylation, and desensitization supports the idea that activated receptor/agonist/G-protein complexes and/or receptor conformational changes induced by agonists are required for agonist-induced μ receptor phosphorylation. Data for methadone and LAAM suggest possible contribution from their enhanced desensitizing abilities to their therapeutic efficacies. Opioid receptors are G-protein coupled receptors that mediate the potent analgesic actions and addictive properties of morphine-derived compounds. Under physiological conditions, these receptors interact with endogenous opioid peptides to modulate pain-controlling pathways and circuits that modulate behaviors including mood and reward (2DiChiara G. North R.A. Trends Pharmacol. Sci. 1992; 13: 185-193Abstract Full Text PDF PubMed Scopus (478) Google Scholar). μ opioid receptors interact with rapidly acting opioid drugs, such as heroin, to produce marked euphoria and behavioral reward. They are also primary targets of the slower and longer acting opioids, such as methadone and LAAM, 1The abbreviations used are: LAAM,l-α-acetylmethadone; DAMGO, [d-Ala2, MePhe4, Glyol5]enkephalin; CHO, Chinese hamster ovary cells; hμCHO, CHO cells stably expressing human μ receptor; PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: LAAM,l-α-acetylmethadone; DAMGO, [d-Ala2, MePhe4, Glyol5]enkephalin; CHO, Chinese hamster ovary cells; hμCHO, CHO cells stably expressing human μ receptor; PAGE, polyacrylamide gel electrophoresis. that represent the best current substitution therapeutics for opiate addiction (3Ling W. Rawson R.A. Compton M.A. J. Psychoact. Drugs. 1994; 26: 119-128Crossref PubMed Scopus (109) Google Scholar). μ receptors desensitize after repeated stimulation by opioid agonists, in fashions that display similarities to desensitizing events noted for other G-protein coupled receptors. Agonist- induced μ receptor desensitization can be correlated with receptor phosphorylation. μ receptors display naloxone-reversible phosphorylation and desensitize after morphine or DAMGO treatments (1Zhang L. Yu Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Both of these agonist-induced events are insensitive to pretreatments with the protein kinase C inhibitor staurosporine, which inhibits phorbol ester-induced μ receptor phosphorylation and desensitization. Many opiates and opioid ligands can recognize μ receptors with high affinities. Plant-derived alkaloids, synthetic compounds of several classes, and endogenous opioid peptides can function as agonists, partial agonists, or antagonists with a range of abilities to induce or block induction of analgesia and euphoria. However, mutagenesis studies and studies with receptor chimeras support the idea that different receptor features could be involved in recognition of these different ligand classes (4Onogi T. Minami M. Katao Y. Nakagawa T. Aoki Y. Toya T. Katsumata S. Satoh M. FEBS Lett. 1995; 357: 93-97Crossref PubMed Scopus (79) Google Scholar, 5Reisine T. Neuropharmacology. 1995; 34: 463-472Crossref PubMed Scopus (137) Google Scholar, 6Surratt C.K. Johnson P.S. Moriwaki A. Seidleck B.K. Blaschak C.J. Wang J.B. Uhl G.R. J. Biol. Chem. 1994; 269: 20548-20553Abstract Full Text PDF PubMed Google Scholar). Mutations that change naloxone from a full antagonist to a partial agonist can leave the intrinsic activity of opioid peptides unchanged, for example see Claude et al.(7Claude P.A. Wotta D.R. Zhang X.H. Prather P.L. Mcginn T.M. Erickson L.J. Loh H.H. Law P.Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5715-5719Crossref PubMed Scopus (64) Google Scholar). These differences raise the possibility that μ receptor occupancies by opioid drugs of different classes could alter the conformation of the μ receptor in distinct fashions. Some of these differences could render the receptor an improved or a worse substrate for kinases and phosphatases and thus directly confer different susceptibilities to phosphorylation or dephosphorylation. Alternatively, selective activation of different G-protein classes by different μ agonists could trigger different μ receptor phosphorylation and desensitization events. The current study thus investigates the effects of opioid ligands of various classes and varying intrinsic activities on μ receptor activation, phosphorylation, and desensitization using human μ receptors expressed in CHO cells (hμCHO), and the receptors coexpressed in Xenopus oocytes with a G-protein linked K+ channel. The results support striking parallels between opioid efficacies in opening ion channels and inhibiting adenylyl cyclase activity and their efficacies in agonist-induced μ receptor phosphorylation and desensitization. The data aso reveal that methadone and LAAM provide phosphorylation and desensitization disproportionate to their efficacies in mediating μ receptor-mediated ion channel activation or adenylyl cyclase inhibition, differences that could conceivably contribute to their efficacies as principal current agonist-substitution antiaddiction therapeutics. Opioid peptides and naloxone were purchased from Research Biochemicals Inc. (Natick, MA); morphine and buprenorphine were purchased from Mallinckrodt Chemical Co (St. Louis, MO); and dihydroetorphine was a gift from Dr. Xiongqi Gong (China). Other opioid ligands were kindly provided by Dr. Richard Rothman and Dr. Heng Xu, (National Institute on Drug Abuse-IRP, Baltimore). All other chemicals and reagents were purchased from Sigma or as indicated in methods specifically. Phosphorylation of the μ opioid receptor in hμCHO (8Wang J.B. Johnson P.S. Persico A.M. Hawkins A.L. Griffin C.A. Uhl G.R. FEBS Lett. 1994; 338: 217-222Crossref PubMed Scopus (288) Google Scholar) was described as (1Zhang L. Yu Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Briefly, hμCHO and non-transfected Chinese hamster ovary (CHO) cells were plated at 80% confluence in 6-well plates and grown for 24 h in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were incubated at 37 °C for 2 h with 300 μCi/ml of [32P]orthophosphate (8500 Ci/mmol; NEN Life Science Products) in phosphate-free Dulbecco's modified Eagle's medium. Labeled cells were then exposed to various opioid ligands at 1 μm for 20 min or to other treatment times and concentrations as indicated in the figure legends. Ligands and free32P were removed from cells by washing with ice-cold phosphate-buffered saline; subsequent procedures were carried out at 4 °C. Cells were solubilized for 60 min with 0.8 ml of RIPA+ buffer (1% IGEPAL CA-630, 0.5% Na2deoxycholate, 0.1% SDS, 5 mm EDTA, 10 mm NaF, 10 mm Na2 pyrophosphate, 1 μmokadaic acid, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml benzamidine, 10 μg/ml leupeptin, and 1 μg/ml pepstatin A in phosphate-buffered saline buffer). Supernatant from a 15 min, 150,000 × g centrifugation was preadsorbed by incubation with 100 μl of presoaked protein A-Sepharose beads (Pharmacia Biotech Inc.), followed by microcentrifugation. A sample of this supernatant was assayed for protein concentration by the Bradford method (Bio-Rad). For immunoprecipitation, 700 μl of the supernatant was incubated for 2 h with 100 μl of the protein A-Sepharose bead slurry and a 1:500 final dilution of an antiserum directed against the C-terminal 18 amino acids of the μ opioid receptor. Beads were washed three times by resuspension with 1 ml of RIPA+followed by microcentrifugation, and immunoprecipitated proteins then dissociated from beads by extraction with 60 μl of SDS-PAGE gel loading buffer (4% SDS, 25 mm Tris-HCl, pH 6.8, 5% glycerol, 0.5% 2-mercaptoethanol, and 0.005% bromphenol blue). 20 μl of the immunoprecipitated proteins were separated on 8% SDS-PAGE gels with prestained molecular mass standards (Amersham), and radiolabeled proteins were identified by autoradiography using Hyperfilm-MP (Amersham) with intensifying screens. Autoradiographic densities of bands of interest were quantified by scanning densitometry and normalized to the amounts of extracted cell protein subjected to immunoprecipitation. cDNAs encoding the human opioid μ receptor (8Wang J.B. Johnson P.S. Persico A.M. Hawkins A.L. Griffin C.A. Uhl G.R. FEBS Lett. 1994; 338: 217-222Crossref PubMed Scopus (288) Google Scholar) and GIRK1 (9Dascal N. Lin N.F. Schreibmayer W. Wang W. Davidson N. Lester H.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6596-6600Crossref PubMed Scopus (81) Google Scholar) were subcloned into the expression vector pcDNAI, plasmids linearized with XbaI, and capped mRNA-sense RNAs prepared by in vitrotranscription using T7 polymerase (mMACHIEN kit; Ambion). RNA quality and sizes were assessed after separations using 1.2% formaldehyde agarose gels, and full-length RNAs were stored in 75% ethanol at −70 °C. Oocytes were isolated from mature femaleXenopus laevis (Xenopus I, Ann Arbor, MI), defolliculated by treatment with 0.2% collagenase A, injected with 16–20 ng of RNAs encoding the μ opioid receptor and GIRK1 in molar ratios of 3:1, and incubated for 2–3 days at 19–20 °C in ND96 solution (96 mm NaCl, 2 mm KCl, 2.5 mm CaCl2, 1.0 mm MgSO4, and 5 mm HEPES, pH 7.5) supplemented with 2 mmsodium pyruvate, 10,000 units/liter penicillin, 10 mg/liter streptomycin, and 0.5 mm theophylline. Whole cell currents in expressing oocytes were measured at 22 °C under 2-electrode voltage clamped at −70 mV, using a GeneClamp 500 amplifier (Axon Instrument). Oocytes were placed on a nylon mesh in a 90-μl bath chamber and continuously superfused at 6 ml/min with either ND96 or “hK” medium (ND96 medium with 96 mm KCl and 2 mm NaCl). Oocytes were superfused with ND96 between applications of hK solution alone, hK solution during which opioid agonists were transiently applied with or without opioid antagonists, or ND96 containing phorbol esters (Sigma) in dimethyl sulfoxide (Me2SO) concentrations less than 0.01%. Values presented are mean ± standard error (S.E.). Concentration-response curves were obtained using the program NFIT by fitting data to the logistic equation, y = {(E max − E min)/(1 + [x/EC50]− n )} +E min, where × represents concentration, y represents response, E max represents the maximal response, E min represents the minimal response, EC50 represents the half-maximal concentration, andn represents the apparent Hill coefficient. hμCHO cells were cultured as described above, harvested, washed with 2.0 mm Tris·HCl, pH 7.4, 2.0 mmEDTA buffer, and suspended in the same Tris buffer. Cell suspension corresponding to 30 μg of protein/sample was added on ice to assay tubes containing 10 μm forskolin, assay buffer (80 mm Tris, pH 7.4, 10 mm theophylline, 1 mm MgSO4, 0.8 mm EGTA, 30 mm NaCl, 0.25 mm ATP, 0.01 mm GTP), and tested drugs. Triplicate samples for each treatment were incubated at 37 °C for 10 min, adenylyl cyclase activity was terminated by boiling for 2 min, and the amounts of cAMP formed were determined by a cAMP protein binding assay as described (10Brown B.L. Ekins R.P. Albano J.M.D. Greengard in Scholar, S. J. Pharmacol. 1993; Google Scholar). Briefly, 4 and binding protein were incubated with samples at 4 °C for were terminated by and and supernatant containing cAMP was assessed by curves were obtained by using Inc. 22 each of the opiate and opioid classes, produced different effects on μ receptor phosphorylation tested in receptor phosphorylation at concentrations that were their Wang J.B. Uhl G.R. 1995; PubMed Scopus Google Scholar). drugs were incubated with hμCHO cells at this concentration for 20 min, dihydroetorphine, sufentanil, etorphine, etonitazine, and DAMGO μ receptor phosphorylation producing morphine, meperidine, DADL, β-endorphin(1–31), and dynorphin A(1–17) produced intermediate the partial agonist the agonists and the the agonist the μ agonists and or the μ antagonists and on μ receptor phosphorylation. phosphorylation less than Phosphorylation was of naloxone to cells incubated with morphine μ receptor phosphorylation to to by 30 min The partial agonist properties of buprenorphine to effects on μ receptor phosphorylation. 1 μm buprenorphine μ receptor phosphorylation with 1 of the μ agonists morphine or DAMGO of adenylyl cyclase activity in hμCHO ± ± ± ± ± cells were with 10 μm and varying opiate concentrations for 10 Data represent mean ± from each in in a effects on μ receptor phosphorylation. hμCHO cells were without or with 1 μm morphine for 10 min at 37 °C. was then and the was terminated or 30 min The autoradiography results of two effects of buprenorphine on μ receptor phosphorylation induced by morphine and DAMGO. were added to as and were carried out as noted The autoradiography results of two hμCHO cells were with 10 μm and varying opiate concentrations for 10 Data represent mean ± from each in these results indicated that different opioid ligands could display on μ receptor phosphorylation, we opioid ligands for and efficacies of etorphine, sufentanil, DAMGO, LAAM, morphine, and were assessed in opening a K+ channel coexpressed with the μ receptor oocytes and in inhibiting adenylyl cyclase in hμCHO These studies provided of the efficacies of these with rank order DAMGO morphine methadone LAAM buprenorphine The desensitization by and to their potencies in cyclase of these opioid ligands also a on receptor phosphorylation agonist-induced μ receptor phosphorylation were at concentrations as as 10 for etorphine, 100 for sufentanil, than 10 for DAMGO, 100 methadone and LAAM and than 100 for of buprenorphine receptor phosphorylation were times than concentrations of etorphine, 100 μm treatments μ receptor phosphorylation. in the Xenopus and hμCHO 6 and was mediated by DAMGO methadone LAAM morphine buprenorphine or Xenopus The rank order of the desensitizing abilities of these opioid ligands was thus to their rank order for with two and LAAM each led to receptor desensitization than morphine, efficacies and potencies in producing μ receptor-mediated K+ channel opening and adenylyl cyclase that were than of Interestingly, LAAM and methadone treatments led to of μ receptor phosphorylation than by morphine at 1 This to represent a of μ receptor with these drugs, and to on test morphine to methadone and than LAAM in inhibiting adenylyl cyclase from the same hμCHO cell in which the μ receptor phosphorylation results were obtained desensitization of μ opioid receptor mediated in hμCHO hμCHO cells were for 20 min with indicated drugs in each concentration was 100 times of EC50 and then was tested for μ receptor mediated adenylyl cyclase as described under and of the ligands at the same changes in opioid receptor mediated cAMP as with are data represents mean ± of two each in The current data the effects of different opioid ligands on μ opioid receptor activation, phosphorylation, and desensitization (1Zhang L. Yu Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Wang J. W. J. 1995; PubMed Scopus Google Scholar, J. W. FEBS Lett. 1996; PubMed Scopus Google Scholar). The parallels between ligand and desensitization in the current data are with the between the properties of these opioid ligands in cell that opioid receptors Loh H.H. Pharmacol. Google Scholar). between the efficacies of these opioid ligands in receptor activation and their abilities to μ receptor phosphorylation, between receptor desensitization and phosphorylation, and parallels between and for μ receptor phosphorylation and desensitization (1Zhang L. Yu Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) each support the possibility that could agonist in ion channel adenylyl cyclase inhibition, receptor phosphorylation and desensitization. from several have desensitization in μ Xenopus oocytes by treatments that alter of kinases including protein kinase and protein kinase C (1Zhang L. Yu Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Y. Yu L. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, A. Y. M. J. Yu L. J. 1995; PubMed Google Scholar). (1Zhang L. Yu Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) separated a pathway from an agonist-induced et A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google data from a to suggest that desensitization receptor phosphorylation. the results from the current study for μ receptor phosphorylation the of opioid ligand efficacies in the Xenopus and the hμCHO are with the idea that the of receptor desensitization in these two distinct test are to be of phosphorylation and desensitization in receptor with for to to the strong current that μ receptor phosphorylation by agonist activation and to desensitization. The parallels between ligand and enhanced receptor phosphorylation that suggest that an activated receptor conformation μ receptor phosphorylation have also described for receptors J. Biol. Chem. Full Text PDF PubMed Google Scholar). The data from methadone and LAAM, on the other provide to this and LAAM represent of the current substitution (3Ling W. Rawson R.A. Compton M.A. J. Psychoact. Drugs. 1994; 26: 119-128Crossref PubMed Scopus (109) Google Scholar, N. A. Drug and Google Scholar). their human have for their therapeutic efficacies 1994; studies of these of have carried receptor phosphorylation and desensitization could the abilities of methadone and LAAM to block effects of subsequent of addictive to their properties and therapeutic efficacies. of ligand with μ receptors suggest that different ligand classes recognize amino acids in different μ receptor occupancies by opioid drugs of different classes thus alter μ receptor in distinct fashions. The differences in μ receptor phosphorylation in the studies could differences in that could or to as a substrate for activated kinases or methadone and LAAM occupancies produced a μ receptor conformation that in better to kinases without enhanced in G-protein activation, for differences from morphine could be receptor could also different activation of by μ receptors by methadone or LAAM could also conceivably enhanced efficacies in the kinases that to receptor phosphorylation than that ion channels or adenylyl for the differences described that test effects of μ receptor and chimeras on LAAM, and morphine and as as and for receptor provide to support the of each of these could represent the of μ receptors expressed on cell to agonist of μ receptors from cell to where can longer ligand recognition to G-protein activation could also contribute to the of desensitization. between desensitization results and the of receptors in expressing cell Y. A. M.A. J. Pharmacol. 1996; Google Scholar). opioid receptor S. P.A. L. M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). μ receptor in CHO cells can be mediated by opiate agonists in the rank order DAMGO methadone in However, morphine treatments at concentrations to 10 The for agonist-induced μ receptor phosphorylation are also than the of receptors (1Zhang L. Yu Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Y. A. M.A. J. Pharmacol. 1996; Google Scholar). and of agonist of these of differences between the results and the results obtained for phosphorylation and for desensitization. thus that to of the desensitizing in the current including using which to agonist-induced and the that phosphorylation in each G-protein receptor kinases the of several G-protein coupled receptors can or receptors J. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). of μ in including CHO Xenopus and (1Zhang L. Yu Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, S. J. W. J. PubMed Scopus Google Scholar, P.L. Law P.Y. J. Pharmacol. 1994; Google Scholar, J. 1992; PubMed Scopus Google Scholar, J. 1996; PubMed Google to a kinase of that a of the receptor kinase or could be for agonist-induced μ receptor phosphorylation. of could also a in the of receptor The of μ opioid receptor phosphorylation, and desensitization that these data that phosphorylation and desensitization are of μ opioid receptor by agonists that opioid to and endogenous opiates are thus to desensitize through biochemical these data also to the therapeutic efficacies of methadone and LAAM through enhanced abilities to and desensitize the μ opioid receptors that contribute to reward.