posted on 2018-09-18, 13:04authored byM. F. Bird, R. Guerrini, J. M. Willets, J. P. Thompson, G. Caló, D. G. Lambert
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
Funding
Funded by Biotechnology and Biological Sciences Research Council (BB/N000188/1)
History
Citation
British Journal of Pharmacology, 2018
Author affiliation
/Organisation/COLLEGE OF LIFE SCIENCES/School of Medicine/Department of Cardiovascular Sciences