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

Morphine induces terminal μ-opioid receptor desensitization by sustained phosphorylation of serine-375

Morphine is a poor inducer of μ-opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxy-terminal residue 375 (Ser(375)). Ser(375) phosphorylation was sufficient and required for morphine-induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO-desensitized receptors were rapidly dephosphorylated. In contrast, morphine-desensitized receptors remained at the plasma membrane in a Ser(375)-phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser(375)

Modulating μ-Opioid Receptor Phosphorylation Switches Agonist-dependent Signaling as Reflected in PKCϵ Activation and Dendritic Spine Stability*

A new role of G protein-coupled receptor (GPCR) phosphorylation was demonstrated in the current studies by using the μ-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKCϵ pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCϵ pathway. This was not due to the decreased recruitment of β-arrestin2 to the receptor signaling complex, because these agonists were unable to use the PKCϵ pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCϵ, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCϵ. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses