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

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.
(© 2018 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)