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Synergistic Activation of PLC‐dependent Diacylglycerol (DAG) Production by Mu‐Opioid and Gq‐Coupled Receptor Signaling

The µ‐opioid receptor (MOR) is a Gi‐protein coupled receptor (GPCR) responsible for opioid‐induced analgesia and undesired effects such as constipation, respiratory depression, and addiction. Upon binding of an agonist, the Gαi and the Gβγ subunits dissociate to signal downstream effectors. One such effector is phospholipase‐Cβ3 (PLCβ3) that can be activated by both Gβγ subunits and Gαq. PLC enzymes hydrolyze phosphatidylinositol‐4,5‐bisphosphate (PIP2) to produce diacylglycerol (DAG) and inositol triphosphate (IP3). Knockout of PLCβ3 in mice, and small molecule inhibition of Gβγ‐PLC interactions potentiate opioid‐induced antinociception. Based on these data we hypothesized that MOR‐dependent activation of PLCβ3 opposes opioid‐induced analgesia. However, activation of PLC by MORs is difficult to observe in cellular models. Since PLCβ is synergistically activated by Gαq and Gβγ subunits, we hypothesized that MOR‐dependent PLC activation may require a coincident signal from a Gq‐signaling pathway. To test this, we established an assay for PLC activation using a fluorescent DAG sensor in HEK‐293 cells stably expressing MOR with or without co‐expression of exogenous M1 muscarinic receptors. In cells, coexpressing M1 receptors, saturating concentrations of carbachol strongly increased DAG production, but saturating concentrations of DAMGO or morphine gave no response. However, synergistic activation of PLC was observed upon simultaneous addition of subsaturating concentrations of the muscarinic agonist carbachol, and MOR agonists morphine and DAMGO. Strong synergy was also observed when activating endogenous muscarinic receptors (likely M3). When cells were treated with pertussis toxin (PTX), the synergy driven by the co‐activation of both types of GPCRs was lost demonstrating that Gβγ activation from MOR is necessary for PLC synergy. We used newly designed small molecule inhibitors of Gβγ‐PLC interaction to demonstrate that this synergistic activation is due to Gβγ and PLCβ activation. Physiologically, MOR containing neurons respond to multiple neurotransmitters including those coupled to Gq coupled receptors, suggesting that this mechanism may play a role in modulation of opioid‐induced analgesia. In the future, we will explore this mechanism using electrophysiological and in vivo models.