Abstract
BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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 NOP(GFP) receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca(2+) responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). KEY RESULTS: 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 Ca(2+) levels in CHO(hNOPGαqi5) 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 NOP(GFP) receptor is activated by N/OFQ(ATTO594) , movement of ligand and receptor from the cell surface to the cytosol can be measured. CONCLUSIONS 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/OFQ(ATTO594) to study a wide range of N/OFQ-driven cellular responses.