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Sci Signal. 2019 Mar 26;12(574). pii: eaau8072. doi: 10.1126/scisignal.aau8072.

Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists.

Author information

1
Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Drackendorfer Str. 1, Jena 07747, Germany. anika.mann@med.uni-jena.de stefan.schulz@med.uni-jena.de.
2
Research Center on Animal Cognition, Center for Integrative Biology, Toulouse University, CNRS, UPS, 31062 Toulouse Cedex 09, France.
3
Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, 31077 Toulouse Cedex 04, France.
4
Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
5
Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Drackendorfer Str. 1, Jena 07747, Germany.
6
Biomedical Science Department, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL 33431, USA.
7
Douglas Research Center, Department of Psychiatry, Faculty of Medicine, McGill University, Montreal, QC H3A 1A1, Canada.
8
Center for the Neurobiology of Addiction, Pain, and Emotion, Departments of Anesthesiology and Pharmacology, University of Washington, Seattle, WA 98195, USA.
9
Astraea Therapeutics LLC, Mountain View, CA 94043, USA.

Abstract

Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely, Ser346, Ser351, Thr362, and Ser363, and proceeded with a temporal hierarchy, with Ser346 as the first site of phosphorylation. G protein-coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which, in turn, facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein-dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.

PMID:
30914485
DOI:
10.1126/scisignal.aau8072

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