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Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO 594: a novel fluorescent probe for the N/OFQ (NOP) receptor
Author(s) -
Bird M F,
Guerrini R,
Willets J M,
Thompson J P,
Caló G,
Lambert D G
Publication year - 2018
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.14504
Subject(s) - nop , nociceptin receptor , receptor , chemistry , opioid receptor , biophysics , microbiology and biotechnology , hek 293 cells , biochemistry , biology , opioid peptide , opioid
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.