Morphine-induced μ-opioid receptor rapid desensitization is independent of receptor phosphorylation and β-arrestins

Abstract

Receptor desensitization involving receptor phosphorylation and subsequent βArrestin (βArr) recruitment has been implicated in the tolerance development mediated by μ-opioid receptor (OPRM1). However, the roles of receptor phosphorylation and βArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca 2+ ([Ca 2+ ] i ) release to monitor receptor activation, as predicted, [D-Ala 2 , N-Me-Phe 4 , Gly 5 -ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a receptor phosphorylation-and βArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from βArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca 2+ ] i release to a greater extent than that of DAMGO and could induce the phosphorylation of Ser 375 residue, morphine-induced desensitization was not influenced by mutating the phosphorylation sites or in MEF cells lacking βArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of βArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of βArr.

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors

“…Our results show that, similarly to the MOP agonist DAMGO, the NPFF 2 agonist 1DMe induced the phosphorylation of the C-terminal tail of the human MOP receptor, in particular at a residue (Ser-377) known to play a key role in desensitization and internalization of the opioid receptor (35)(36)(37). This heterologous phosphorylation was specific to NPFF receptor activation because it did not occur when another G i/o -coupled receptor, the ␣ 2 -adrenergic receptor, was activated.…”

“…Previous studies have shown that Thr-370 and Ser-375 are the main targets of homologous regulation by G protein-coupled receptor kinases in the C-terminal tail of the rodent MOP receptor (35)(36)(37)(38), although a potential role for Thr-180 in the second intracellular loop has also been suggested in Xenopus oocytes (39). Here, we have studied the phosphorylation status of the human MOP receptor after homologous stimulation by its agonist (DAMGO) or after heterologous regulation by NPFF 2 receptors.…”

FK506-Binding Protein 12 Modulatesμ-Opioid Receptor Phosphorylation and Protein Kinase Cε–Dependent Signaling by Its Direct Interaction with the Receptor

“…FKBP12 Modulates the Morphine-Induced PKC« Activation and Recruitment. Morphine has been shown to increase the phosphorylation of OPRM1 at the Ser 375 residue only (Chu et al, 2008;Doll et al, 2011). Previous studies in our laboratory have shown that PKC« activation can be modulated by the phosphorylation state of the receptor (Zheng et al, 2008b;Chu et al, 2010).…”

“…The relative densities of immunoprecipitated PKC« were quantified by normalizing to the total amount of immunoprecipitated receptors (D). *P , 0.05. shown to be PKC«-dependent (Chu et al, 2008(Chu et al, , 2010Zheng et al, 2008b). Thus, to further demonstrate that FKBP12 can modulate morphine-mediated PKC« activation, the influence of FKBP12 on ERK1/2 activation was investigated.…”

Cholesterol level influences opioid signaling in cell models and analgesia in mice and humans

“…Cholesterol concentrations were determined in the fi rst 10 fractions using an Amplex Red Cholesterol Assay Kit (Invitrogen, Carlsbad, CA). The amount of OPRM1 in the same fractions was determined by using [ 3 H] diprenorphine binding as described previously ( 18 ).…”