Dataset Information

Agonist Binding and Desensitization of the ?-Opioid Receptor Is Modulated by Phosphorylation of the C-Terminal Tail Domain.

ABSTRACT: Sustained activation of G protein-coupled receptors can lead to a rapid decline in signaling through acute receptor desensitization. In the case of the ?-opioid receptor (MOPr), this desensitization may play a role in the development of analgesic tolerance. It is understood that phosphorylation of MOPr promotes association with ?-arrestin proteins, which then facilitates desensitization and receptor internalization. Agonists that induce acute desensitization have been shown to induce a noncanonical high-affinity agonist binding state in MOPr, conferring a persistent memory of prior receptor activation. In the current study, live-cell confocal imaging was used to investigate the role of receptor phosphorylation in agonist binding to MOPr. A phosphorylation cluster in the C-terminal tail of MOPr was identified as a mediator of agonist-induced affinity changes in MOPr. This site is unique from the primary phosphorylation cluster responsible for ?-arrestin binding and internalization. Electrophysiologic measurements of receptor function suggest that both phosphorylation clusters may play a parallel role during acute receptor desensitization. Desensitization was unaffected by alanine mutation of either phosphorylation cluster, but was largely eliminated when both clusters were mutated. Overall, this work suggests that there are multiple effects of MOPr phosphorylation that appear to regulate MOPr function: one affecting ?-arrestin binding and a second affecting agonist binding.

SUBMITTER: Birdsong WT

PROVIDER: S-EPMC4576685 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Agonist Binding and Desensitization of the μ-Opioid Receptor Is Modulated by Phosphorylation of the C-Terminal Tail Domain.

Birdsong William T WT Arttamangkul Seksiri S Bunzow James R JR Williams John T JT

Molecular pharmacology 20150501 4

Sustained activation of G protein-coupled receptors can lead to a rapid decline in signaling through acute receptor desensitization. In the case of the μ-opioid receptor (MOPr), this desensitization may play a role in the development of analgesic tolerance. It is understood that phosphorylation of MOPr promotes association with β-arrestin proteins, which then facilitates desensitization and receptor internalization. Agonists that induce acute desensitization have been shown to induce a noncanoni ...[more]

PMID: 25934731

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Project description:The effects of phosphorylation of the tyrosine residue in the highly conserved DRY motif expressed in the putative second cytoplasmic loop of the mu-opioid receptor were assessed after expression in human embryonic kidney (HEK) 293 cells. Tyrosine kinase activation by epidermal growth factor (EGF) or hydrogen peroxide treatment effectively increased phosphorylation of the tyrosine-166 in the mu-opioid receptor (MOR-Tyr166p) as measured by a novel phosphoselective antibody. We were surprised to find that the increase in MOR-Tyr166p immunoreactivity (ir) required coactivation by the opioid agonist [D-Ala(2),methyl-Phe(4),Gly(5)-ol]enkephalin (DAMGO), as demonstrated by both Western blot imaging of membrane proteins and confocal microscopy of transfected cells; MOR-Tyr166p-ir did not significantly increase after either DAMGO, EGF, or H(2)O(2) treatment alone. The increase in MOR-Tyr166p-ir was blocked by pretreatment with the opioid antagonist naloxone or the Src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Consistent with these data, mutation of the tyrosine-166 to phenylalanine blocked the increased immunoreactivity, and untransfected HEK293 cells did not increase MOR-Tyr166p-ir after treatment. DAMGO increased guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding to membranes from cells expressing wild-type MOR or MOR-Y166F receptors in a dose-dependent manner. Pretreatment of the wild-type MOR-expressing cells with the combination of DAMGO and EGF completely blocked subsequent DAMGO stimulation of [(35)S]GTP gamma S binding membranes, whereas [(35)S]GTP gamma S binding to membranes from cells expressing mutated MOR(Y166F) was only partially inhibited. These results suggest that G-protein activation as measured by [(35)S]GTP gamma S binding can be regulated by DAMGO and EGF by convergent mechanisms and support the hypothesis that tyrosine phosphorylation within the DRY motif may reduce mu-opioid receptor-G-protein coupling efficiency.

Dataset Information

Agonist Binding and Desensitization of the ?-Opioid Receptor Is Modulated by Phosphorylation of the C-Terminal Tail Domain.

Publications

Agonist Binding and Desensitization of the μ-Opioid Receptor Is Modulated by Phosphorylation of the C-Terminal Tail Domain.

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