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

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 NOP_GFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca²⁺ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN).

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 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.

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.