MOP and NOP receptor interaction: Studies with a dual expression system and bivalent peptide ligands

Opioids targeting mu;μ (MOP) receptors produce analgesia in the peri-operative period and palliative care. They also produce side effects including respiratory depression, tolerance/dependence and addiction. The N/OFQ opioid receptor (NOP) also produces analgesia but is devoid of the major MOP side effects. Evidence exists for MOP-NOP interaction and mixed MOP-NOP ligands produce analgesia with reduced side effects. We have generated a HEK(MOP/NOP) human expression system and used bivalent MOP-NOP and fluorescent ligands to (i) probe for receptor interaction and (ii) consequences of that interaction. We used HEK(MOP/NOP) cells and two bivalent ligands; Dermorphin-N/OFQ (MOP agonist-NOP agonist; DeNO) and Dermorphin-UFP101 (MOP agonist-NOP antagonist; De101). We have determined receptor binding profiles, GTPγ[(35)S] binding, cAMP formation and ERK1/2 activation. We have also probed MOP and NOP receptor interactions in HEK cells and hippocampal neurones using the novel MOP fluorescent ligand, Dermorphin(ATTO488) and the NOP fluorescent ligand N/OFQ(ATTO594). In HEK(MOP/NOP) MOP ligands displaced NOP binding and NOP ligands displaced MOP binding. Using fluorescent probes in HEK(MOP/NOP) cells we demonstrated MOP-NOP probe overlap and a FRET signal indicating co-localisation. MOP-NOP were also co-localised in hippocampal tissue. In GTPγ[(35)S] and cAMP assays NOP stimulation shifted the response to MOP rightwards. At ERK1/2 the response to bivalent ligands generally peaked later. We provide evidence for MOP-NOP interaction in recombinant and native tissue. NOP activation reduces responsiveness of MOP activation; this was shown with conventional and bivalent ligands.