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GRK-mediated phosphorylation of mu-opioid receptor at serine 375 induced by a range of opioid agonists Mu-opioid receptor (MOPr) desensitisation and internalisation mechanisms have been implicated in the development of tolerance to opioid drugs such as morphine. We have recently shown that for many agonists, internalisation of MOPr is closely correlated with agonist efficacy for G-protein activation, except for the opioid agonists endomorphin 1 and 2 (McPherson et al, 2010). Upon agonist binding MOPr can undergo phosphorylation by G-protein coupled receptor kinase (GRK) and subsequent arrestin binding, which would drive receptor internalisation. In this work, we first studied whether the phosphorylation of MOPr at serine 375 (pSer375), considered a key step in MOPr regulation, is mediated by GRK2. Second, we evaluated the relative abilities of a range of MOPr agonists to induce pSer375. HEK293 cells stably expressing the HA-tagged MOPr were seeded into 96-well plates. In order to suppress GRK2 protein activity, cells were transfected with a dominant negative mutant of the GRK2 protein (K440R). Subsequently, transfected and untransfected cells were exposed to increasing concentrations of DAMGO and morphine (10 min, 37°C). For the evaluation of protein kinase C (PKC)-mediated pSer375, cells were incubated with the PKC inhibitor chelerythrine (5 µM) prior to exposure to DAMGO and morphine. To study the pSer375 induced by a range of different opioid agonists, cells were incubated with increasing concentrations of: buprenorphine, morphine, endomorphin 2, alfentanil, methadone, DAMGO, met-enkephalin or etorphine for 10 min at 37°C. Cells were then fixed, permeabilised and exposed to an antibody that recognises the pSer375 MOPr. After incubation with Alexa488-conjugated secondary antibody, cells were imaged by fluorescence microscopy using an automated system for image acquisition (IN Cell Analyzer 1000) and validated algorithms for image analysis (IN Cell Analyzer version 1.0). Data were normalised to the pSer375 signal obtained by DAMGO 100 µM. Concentration-response curves were analysed by Graphpad®. After transfection with GRK2 dominant negative mutant, DAMGO and morphine potency to induce pSer375 was reduced, suggesting that GRK2 is partly responsible for phosphorylation of MOPr at serine 375. However, pre-incubation with chelerythrine did not alter DAMGO and morphine-induced pSer375 ruling out the PKC involvement. Evaluation of the different opioid agonists revealed that the ability of those agonists normally regarded as full agonists to induce pSer375 was similar to that induced by the prototypical agonist DAMGO (Emax (%): alfentanil, 127±29; etorphine, 96±9). Partial agonists produced less pSer375 than DAMGO (Emax (%): buprenorphine, 12±6). However, endomorphin 2 produced more pSer375 (Emax (%): 83±11) than would be expected from its efficacy for G-protein activation. These data indicate that agonist-induced pSer375 is mediated by GRK2, which might be one of the initial steps in the MOPr desensitisation and internalisation cascades. For the majority of MOPr agonists the ability to induce pSer375 can be predicted from its efficacy for G-protein activation. Furthermore, the ability of MOPr agonists to induce pSer375 correlates well with their ability to induce MOPr internalisation (McPherson et al, 2010). However, endomorphin 2 is able to phosphorylate Ser375 better than would be predicted from its efficacy for G protein activation, which may underlie its apparent activity as a biased agonist with regard to arrestin recruitment.
McPherson J, Rivero G, Baptist M, Llorente J, Al-Sabah S, Krasel C, Dewey WL, Bailey CP, Rosethorne EM, Charlton SJ, Henderson G, Kelly E. 2010. μ-Opioid receptors: correlation of agonist efficacy for signalling with ability to activate internalization. Molecular Pharmacology; 78: 756-66. Funded by MRC and the Basque Government.
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