Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor.

M F Bird,G Caló,J P Thompson,J M Willets,D G Lambert,R Guerrini

doi:10.1111/bph.14504

Abstract

Background and PurposeThe nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore‐ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression.Experimental ApproachWe assessed N/OFQATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN).Key ResultsN/OFQATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPGαqi5 cells. N/OFQATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP‐GFP‐tagged receptors, N/OFQATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand–receptor interaction. When the NOPGFP receptor is activated by N/OFQATTO594, movement of ligand and receptor from the cell surface to the cytosol can be measured.Conclusions and ImplicationsIn the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQATTO594 to study a wide range of N/OFQ‐driven cellular responses.

Highlights

The nociceptin/orphanin FQ (N/OFQ) receptor (NOP receptor) is the newest member of the opioid receptor family (Lambert, 2008)
Selectivity for N/OFQATTO594 was measured in CHO cells expressing human μ, δ, κ and NOP receptors
A range of concentrations of N/OFQATTO594 were added to HEK cells expressing the NOP receptor and binding measured using 594 nm laser on a confocal microscope (Figure 2)

Summary

Introduction

The nociceptin/orphanin FQ (N/OFQ) receptor (NOP receptor) is the newest member of the opioid receptor family (Lambert, 2008). Its own endogenous ligand, N/OFQ, is highly selective for the NOP receptor, displaying little or no affinity for the classical opioid receptors. Like the classical opioid receptors, NOP receptor expression has been demonstrated throughout the pain pathway, with antinociceptive actions ascribed to activation (Mollereau and Mouledous, 2000; Schroder et al, 2014; Ding et al, 2015). The nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. We conjugated a red fluorophore-ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN)

Methods

Results

Discussion

Conclusion