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

Background and Purpose: The nociception / 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 have conjugated a red flurophore-ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594) for the NOP receptor and explored NOP function at high (in recombinant systems) and low (on immune cells) expression. Experimental Approach: We have 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, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). Key Results: N/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 visualise NOP at low expression on PMN cells. In NOP-GFP tagged receptors, N/OFQATTO594 was used in a Fluorescence Resonance Energy Transfer (FRET) protocol where GFP emission activated ATTO; visualising ligand-receptor interaction. When NOPGFP is activated by N/OFQATTO594 movement of ligand and receptor from the cell surface to the cytosol can be measured. Conclusion and Implications: 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/OFQATTO594 to study a wide range of N/OFQ driven cellular responses

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