title: Increased agonist affinity at the μ-opioid receptor induced by prolonged agonist exposure.
abstract: Prolonged exposure to high-efficacy agonists results in desensitization of the μ-opioid receptor (MOR). Desensitized receptors are thought to be unable to couple to G-proteins, preventing downstream signaling; however, the changes to the receptor itself are not well characterized. In the current study, confocal imaging was used to determine whether desensitizing conditions cause a change in agonist-receptor interactions. Using rapid solution exchange, the binding kinetics of fluorescently labeled opioid agonist, dermorphin Alexa594 (derm A594), to MORs was measured in live cells. The affinity of derm A594 binding increased after prolonged treatment of cells with multiple agonists that are known to cause receptor desensitization. In contrast, binding of a fluorescent antagonist, naltrexamine Alexa594, was unaffected by similar agonist pretreatment. The increased affinity of derm A594 for the receptor was long-lived and partially reversed after a 45 min wash. Treatment of the cells with pertussis toxin did not alter the increase in affinity of the derm A594 for MOR. Likewise, the affinity of derm A594 for MORs expressed in mouse embryonic fibroblasts derived from arrestin 1 and 2 knock-out animals increased after treatment of the cells with the desensitization protocol. Thus, opioid receptors were "imprinted" with a memory of prior agonist exposure that was independent of G-protein activation or arrestin binding that altered subsequent agonist-receptor interactions. The increased affinity suggests that acute desensitization results in a long-lasting but reversible conformational change in the receptor.
authors: Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, USA.
authors: William T
authors: WT
authors: Birdsong
authors: Seksiri
authors: S
authors: Arttamangkul
authors: Mary J
authors: MJ
authors: Clark
authors: Kejun
authors: K
authors: Cheng
authors: Kenner C
authors: KC
authors: Rice
authors: John R
authors: JR
authors: Traynor
authors: John T
authors: JT
authors: Williams
mesh_terms: D000701:Analgesics, Opioid / Q000493:pharmacokinetics; D000704:Analysis of Variance; D000818:Animals; D019393:Arrestin / Q000172:deficiency / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000493:pharmacokinetics; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D018345:Mice, Knockout; D009020:Morphine / Q000494:pharmacology; D009271:Naltrexone / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D009292:Narcotic Antagonists / Q000493:pharmacokinetics; D018847:Opioid Peptides / Q000493:pharmacokinetics; D009930:Organic Chemicals / Q000493:pharmacokinetics; D037342:Pertussis Toxin / Q000494:pharmacology; D000069437:Pharmacological Phenomena / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011869:Radioligand Assay; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics
chemical_list: C417664:Alexa594; D000701:Analgesics, Opioid; D019393:Arrestin; D008024:Ligands; D009292:Narcotic Antagonists; D018847:Opioid Peptides; D009930:Organic Chemicals; D017450:Receptors, Opioid, mu; D014316:Tritium; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C030058:dermorphin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009271:Naltrexone; C035438:beta-naltrexamine; D009020:Morphine; D037342:Pertussis Toxin