Co-expression of mu and delta opioid receptors as receptor-G protein fusions enhances both mu and delta signalling via distinct mechanisms

Abstract Mu and delta opioid receptors were co-expressed as fusion proteins between a receptor and a pertussis insensitive mutant Galphai/o protein in HEK 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Galphai/o proteins by pertussis toxin. Co-expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonist CTAP. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic-deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonist N,N(CH3)2-Dmt-Tic-NH2. When a mu opioid receptor fused to a non-functional Galpha subunit was co-expressed with the delta opioid receptor-Galpha protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced delta opioid receptor coupling to its tethered Galpha subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the delta opioid receptor and also results in activation of the Galpha subunit fused the delta opioid recepto