title: Calmodulin regulation of basal and agonist-stimulated G protein coupling by the mu-opioid receptor (OP(3)) in morphine-pretreated cell.
abstract: Calmodulin (CaM) has been shown to suppress basal G protein coupling and attenuate agonist-stimulated G protein coupling of the mu-opioid receptor (OP(3)) through direct interaction with the third intracellular (i3) loop of the receptor. Here we have investigated the role of CaM in regulating changes in OP(3)-G protein coupling during morphine treatment, shown to result in CaM release from plasma membranes. Basal and agonist-stimulated G protein coupling by OP(3) was measured before and after morphine pretreatment by incorporation of guanosine 5'-O-(3-[(35)S]thiotriphosphate) into membranes, obtained from HEK 293 cells transfected with human OP(3) cDNA. The opioid antagonist beta-chlornaltrexamine fully suppressed basal G protein coupling of OP(3), providing a direct measure of basal signaling. Pretreatment of the cells with morphine enhanced basal G protein coupling (sensitization). In contrast, agonist-stimulated coupling was diminished (desensitization), resulting in a substantially flattened morphine dose-response curve. To test whether CaM is involved in these changes, we constructed OP(3)-i3 loop mutants with reduced affinity for CaM (K273A, R275A, and K273A/R275A). Basal signaling of these mutant OP(3) receptors was higher than that of the wild-type receptor and, moreover, unaffected by morphine pretreatment, whereas desensitization to agonist stimulation was only slightly attenuated. Therefore, CaM-OP(3) interactions appear to play only a minor role in the desensitization of OP(3). In contrast, release of CaM from the plasma membrane appears to enhance the inherent basal G protein coupling of OP(3), thereby resolving the paradox that OP(3) displays both desensitization and sensitization during morphine treatment.
authors.affiliation: Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California at San Francisco, San Francisco, California, USA.
authors.forename: D
authors.initials: D
authors.lastname: Wang
authors.forename: C K
authors.initials: CK
authors.lastname: Surratt
authors.forename: W
authors.initials: W
authors.lastname: Sadée
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002147:Calmodulin / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002645:Chickens; D004533:Egtazic Acid / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000493:pharmacokinetics; D006801:Humans; D007668:Kidney; D007700:Kinetics; D009020:Morphine / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009270:Naloxone / Q000493:pharmacokinetics; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D017450:Receptors, Opioid, mu / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D002147:Calmodulin; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D009270:Naloxone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D009271:Naltrexone; C017120:chlornaltrexamine; D009020:Morphine; D019204:GTP-Binding Proteins