Abstract
The Nociceptin/Orphanin FQ (N/OFQ) peptide NOP receptor is coupled to pertussis toxin (PTX)-sensitive G proteins (Gi/o) whose activation leads to the inhibition of both cAMP production and calcium channel activity, and to the stimulation of potassium currents. The label free dynamic mass redistribution (DMR) approach has been demonstrated useful for investigating the pharmacological profile of G protein-coupled receptors. Herein, we employ DMR technology to systematically characterize the pharmacology of a large panel of NOP receptor ligands. These are of peptide and non-peptide nature and display varying degrees of receptor efficacy, ranging from full agonism to pure antagonism. Using Chinese hamster ovary (CHO) cells expressing the human NOP receptor we provide rank orders of potency for full and partial agonists as well as apparent affinities for selective antagonists. We find the pharmacological profile of NOP receptor ligands to be similar but not identical to values reported in the literature using canonical assays for Gi/o-coupled receptors. Our data demonstrate that holistic label-free DMR detection can be successfully used to investigate the pharmacology of the NOP receptor and to characterize the cellular effects of novel NOP receptor ligands.
Highlights
Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino-acid (FGGFTGARKSARKLANQ) neuropeptide that binds with high affinity to the N/OFQ peptide (NOP) receptor [1, 2]
N/OFQ stimulated the dynamic mass redistribution (DMR) response in CHONOP cells but not in Chinese hamster ovary (CHO) cells demonstrating that this signal exclusively derives from the interaction of N/OFQ with the NOP receptor protein
The same is true for all the agonists evaluated since we selected their concentration range based on lack of DMR signal in CHO cells
Summary
Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino-acid (FGGFTGARKSARKLANQ) neuropeptide that binds with high affinity to the N/OFQ peptide (NOP) receptor [1, 2]. The NOP receptor mainly couples to pertussis toxin (PTX)-sensitive G proteins (Gi/o) whose activation leads to lowering of cAMP levels and inhibition of calcium channels, and to the stimulation of potassium currents [3]. Its pharmacology has been classically studied in vitro with bioassays such as the electrically stimulated mouse vas deferens, and biochemical assays based on [35S]GTPγS binding and inhibition of forskolin-stimulated cAMP production. Bioluminescence resonance energy transfer (BRET) based assays allowed the investigation of NOP/G protein and NOP/β-arrestin interactions demonstrating that several synthetic agonists. NOP receptor pharmacological profile - A DMR study
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