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

Background and Purpose Experimental Approach 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. In this study, we conjugated a red fluorophore-ATTO594 to the peptide ligand N/OFQ (N/OFQ(ATTO594)) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression. We assessed N/OFQ(ATTO594) receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQ(ATTO594) binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPG alpha qi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). Key Results Conclusions and Implications N/OFQ(ATTO594) bound to NOP receptor with nM affinity and high selectivity. N/OFQ(ATTO594) activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPG alpha qi5 cells. N/OFQ(ATTO594) was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP-GFP-tagged receptors, N/OFQ(ATTO594) was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand-receptor interaction. When the NOPGFP receptor is activated by N/OFQ(ATTO594), movement of ligand and receptor from the cell surface to the cytosol can be measured. In 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/OFQ(ATTO594) to study a wide range of N/OFQ-driven cellular responses.