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

ABSTRACT: Agonists for 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 for other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands show differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (S) and threonine (T) 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 vivo, 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. Together, our study provides novel 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.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Carine Froment  

LAB HEAD: Stefan Schulz

PROVIDER: PXD012908 | Pride | 2020-05-26

REPOSITORIES: Pride