The Human In. Opioid Receptor: Modulation of Functional Desensitization by Calcium/Calmodulin-Dependent Protein Kinase and Protein Kinase C
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The Journal of Neuroscience, March 1995, 75(3): 2396-2406 The Human in. Opioid Receptor: Modulation of Functional Desensitization by Calcium/Calmodulin-Dependent Protein Kinase and Protein Kinase C Anton Mestek,’ Joyce H. Hurley,’ Leighan S. Bye,’ Andrew D. Campbell,1~2 Yan Chen,’ Mingting Tian,’ Jian Liu,’ Howard Schulman,3 and Lei Yu’ ‘Department of Medical and Molecular Genetics and 21nstitute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202 and 3Depattment of Neurobiology, Stanford University School of Medicine, Stanford, California 94305 Opioids are some of the most efficacious analgesics used macological studies have defined three major types of opioid in humans. Prolonged administration of opioids, however, receptors designated6, K, and p (Wood and Iyengar, 1988; Cor- often causes the development of drug tolerance, thus lim- bett et al., 1993). Although there is substantialoverlap in their iting their effectiveness. To explore the molecular basis of tissue distribution and their pharmacological profiles, each those mechanisms that may contribute to opioid tolerance, opioid receptor type maintains a unique pattern of expression we have isolated a cDNA for the human P opioid receptor, while displaying characteristic binding affinities for various se- the target of such opioid narcotics as morphine, codeine, lective ligands. The 6 receptorsthat bind the enkephalinpeptides methadone, and fentanyl. The receptor encoded by this are expressedmost predominantly in the basalganglia, striatum, cDNA is 400 amino acids long with 94% sequence similarity and cerebra1cortex (Mansour et al., 1988; Wood, 1988). Al- to the rat (r. opioid receptor. Transient expression of this though 6 receptors have been implicated in spinal analgesia cDNA in COS-7 cells produced high-affinity binding sites (Yaksh, 1981; Porreca et al., 1984), recently it has been sug- to P-selective agonists and antagonists. This receptor dis- gested that specific 6 receptor subtypesmay also be involved in plays functional coupling to a recently cloned G-protein- supraspinalanalgesia (Pastemak, 1993). The K receptors are activated K+ channel. When both proteins were expressed most highly expressedin cortex, striatum, and hypothalamus in Xenopus oocytes, functional desensitization developed (Mansour et al., 1987), with various subtypesidentified by au- upon repeated stimulation of the P opioid receptor, as ob- toradiography using selective ligands (Neck et al., 1988; Unter- served by a reduction in K+ current induced by the second wald et al., 1991). With the development of highly selective P receptor activation relative to that induced by the first. ligands, thesereceptors have been shown to mediate both spinal The extent of desensitization was potentiated by both the and supraspinalanalgesia (Pasternak, 1993). multifunctional calcium/calmodulin-dependent protein ki- The third major class of opioid binding sites is represented nase and protein kinase C. These results demonstrate that by the p opioid receptor. It functions as the physiological target kinase modulation is a molecular mechanism by which the of such potent analgesicsas morphine and fentanyl, as well as desensitization of P receptor signaling may be regulated at the classof endogenousopioid peptidescomprised of B-endor- the cellular level, suggesting that this cellular mechanism phin, enkephalins, and dynorphins. The p. opioid receptor has may contribute to opioid tolerance in humans. also been implicated in analgesia(Wood and Iyengar, 1988). [Key words: opioid receptor, desensitization, G-protein- Opioid drugs with high abuse liability such as morphine and activated channel, inward rectifier, protein kinase C, calci- fentanyl, all bind selectively to the p, opioid receptor.In addition, umlcalmodulin-dependent protein kinase, Xenopus oo- heroin (diacetylmorphine), a semisynthetic derivative of mor- cvtesl phine, crossesthe blood-brain barrier more easily due to its increased hydrophobicity. Once in the brain, heroin is rapidly Opioid peptides and alkaloids affect a number of physiological hydrolyzed to morphine, which acts at the p opioid receptor and functions including hormone secretion,neurotransmitter release, results in an euphoric effect, thus conferring the reinforcing feeding, gastrointestinalmotility, and respiratory activity (Pas- properties of the drug and contributing to the development of ternak, 1988). Extensive physiological, behavioral, and phar- addiction. Becauseof the high affinity of theseopioid narcotics at the p receptor, it is consideredthe main cellular mediator in Received Aug. 15, 1994; revised Oct. 7, 1994; accepted Oct. 13, 1994. the development of tolerance (Loh et al., 1988) and opioid ad- We thank Drs. Lily Jan and Henry Lester for their generous gifts of cDNAs diction (Di Chiara and North, 1992). encoding the G-protein-activated K’ channel, Dr. Leonard Adam for technical advice and Drs. Grant Nicol and Stanley Rane for reviewing the manuscript. The activation of all three opioid receptor types can inhibit This work was supported in part by grants from the National Institutes of adenylyl cyclase and modulate membraneconductances of Ca*+ Health (DA 09116, NS 28190, AA 07611, and GM 30179). L.Y. is a J. Alfred Prufrock investieator and a recioient of an NIH Research Career DeveIooment and K+ (Childers, 1993; North, 1993). The increasein K+ con- Award (NS 01557). ductance and the decreasein Ca2+ conductance both serve to Correspondence should be addressed to Lei Yu, Ph.D., Department of Med- reduce membraneexcitability and may account for the analgesic ical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut Street (IBI30), Indianapolis, IN 46202. propertiesof the opioids (North, 1993). Electrophysiologicalre- Copyright 0 1995 Society for Neuroscience 0270-6474/95/152396-l 1$05.00/O cordings from neuronslocated in the locus coeruleus(Williams The Journal of Neuroscience, March 1995, 75(3) 2397 et al., 1988) and hippocampus (Wimpey and Chavkin, 1991) and centrifuged as described before. Supernatants were combined and centrifuged for 10 min at 35.000 X .e. Membrane nellets were washed indicate that p opioid receptor stimulation causes membrane hy- in 50 n& Tris-HCl, pH 7.4, centrifuied again, ani resuspended in 50 perpolarization via an inwardly rectifying K+ conductance. The IILM Tris-HCl, pH 7.4. Protein concentrations were determined by the effect of the receptors upon the ion channels requires GTP but method of Bradford (Bradford, 1976). no diffusible cytosolic molecules. The inhibition of adenylyl cy- Binding analysis. Binding assays were performed essentially as de- clase, however, implicates a more complex mode involving scribed (Chen et al., 1993a). Binding mixtures containing at least 10 pg membrane protein were incubated at room temperature for 90 min opioid regulation of cellular mechanisms, which includes con- in 50 mu Tris-HCl, pH 7.4 containing 0.2% bovine serum albumin, trolling the levels of gene expression as well as modulating the various concentrations of unlabeled lig&ds, and 3H-diprenorphine (3H: activity of cellular phosphatases and kinases (Di Chiara and DPN) or 3H-I~-Ala2.N-MePhe4.G1v-o151-enkeohalin c3H-DAMGO). in a North, 1992; Guitart and Nestler, 1993). final ‘volume‘of 206 ~1. Reactions weie terkinated‘by quickly adding Analgesia and the development of opioid tolerance, depen- 3 ml of ice-cold binding buffer (50 mu Tris-HCl, pH 7.4) followed by vacuum filtration onto Whatman GF/B glass fiber filters, presoaked for dence, and addiction have been the subject of extensive studies 3 hr in 0.2% polyethylenimine to minimize nonspecific binding. Filters (Collin and Cesselin, 1991). Several schemes, including recep- were washed two times each with 3 ml of ice-cold binding buffer before tor-mediated modulation of membrane conductance, have been placed in vials containing 10 ml liquid scintillation cocktail (CytoScint). proposed for the acute and chronic actions of opioids in the CNS Radioactivity was determined using a Beckman LS-5801 scintillation (Johnson and Fleming, 1989). One scheme involves protein counter. Nonspecific binding was defined as the radioactivity bound in the presence of 10 FM unlabeled naloxone. Saturation analyses were phosphorylation by various kinases as a means to regulate performed as above with increasing concentrations of 3H-DPN (0.01 to opioid-induced cellular processes. The molecular mechanism of 2.5 nM) or )H-DAMGO (0.05 to 5 nM). such regulation, however, has not been clearly delineated. In the Data for all saturation binding experiments were analyzed by using study reported here, we expressed the human k opioid receptor the linear/nonlinear regression analysis program EBDA/LIGAND (Munson, and a G-protein-activated K+ channel in Xenopus oocytes, and 1983) to obtain estimates of equilibrium dissociation constant (K,,), Hill slope(n,), and bindingsite density (B,,,) values.Competition-curves examined the role of protein kinasesin modulating the p, recep- were obtained with 1.3 nM ?H-DAMGO incubated with concentrations tor-K+ channel coupling and in functional desensitization. of the indicated competitors from 0.01 nM to 5 FM. XC,, values were determined through nonlinear regression using InPlot, which utilizes the Materials and Methods Cheng-Pmsoff equation to determine the K, values (Cheng and Prusoff, Isolation of the human k opioid receptor cDNA. The open reading 1973). frame-containingfragment of the rat p. opioidreceptor cDNA was used Oocyte expression