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

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.

Biased Opioid Ligands

“…The endocytotic activity of NOP receptor agonists is associated with their ability to induce receptor phosphorylation at Ser 346 , Ser 351 , and Thr 362 /Ser 363 ; a direct positive linear correlation was observed between the phosphorylation at Thr 362 /Ser 363 and receptor internalization as well as between phosphorylation at Thr 362 /Ser 363 and GIRK channel activation. This phosphorylation pattern in the C-terminal domain proved to be agonist selective [ 129 ].…”

“…After central administration of cebranopadol, antihyperalgesic efficacy is reached at doses that are not yet antinociceptive [ 142 ]. Cebranopadol was also shown to not induce either phosphorylation, or NOP receptor internalization [ 129 ].…”

“…MCOPPB shows a concentration-dependent or potency bias, as the ligand is a full agonist in both signaling pathways but distinguishes itself in its potency at G-protein versus arrestin signaling [ 127 ]. MCOPPB is a very potent agonist to activate the NOP receptor GIRK channels with an EC 50 of 0.06 nM compared to N/OFQ (EC 50 of 1.5 nM) for GIRK activation [ 129 ]. MCOPPB has an anxiolytic activity comparable to that of the benzodiazepine diazepam, but did not affect motor activity or memory function nor did it interact with alcohol at an anxiolytic dose in mice [ 143 ].…”

“…Besides its a NOP receptor selectivity, its arrestin recruitment could not be measured in HEK cells using BRET, which makes it a G protein-biased agonist exhibiting partial agonist activity with an efficacy of 71% and relatively low potency as indicated by a 130 fold shift in the value of EC 50 in comparison with N/OFQ in cAMP inhibition assay in HEK cells [ 127 ]. NNC 63-0532 was not able to induce multisite phosphorylation of the NOP receptor [ 129 ].…”

Compartment-specific opioid receptor signaling is selectively modulated by Dynorphin subtypes

“…At present it is not clear what could provide such a conformational memory. It is possible that different Dynorphins could cause agonist-specific post-translational modifications, which is a general emerging theme for opioid receptors (21)(22). To the best of our knowledge, however, this is the first example of different physiological agonists driving spatial localization and trafficking of a GPCR -a process that has been studied largely using receptor mutants or by depleting key components of the trafficking machinery so far (9,11,23).…”