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M3 muscarinic receptor coupling to Gaq/11 protein facilitates mu opioid receptor desensitisation, internalisation

Autor López-Giménez, Juan F. ; Milligan, Graeme
Fecha de publicación 2011
EditorAmerican Society for Pharmacology and Experimental Therapeutics
ResumenWe recently reported that co-activation of serotonin 5-HT2A receptor facilitates morphine desensitisation, internalisation and down regulation of the mu opioid receptor (MOP) when both are co-expressed in a heterologous/inducible cell system (Lopez-Gimenez et al. Mol. Pharmacol. 2008; 74(5):1278-91). Additionally, it was observed that this facilitation is prevented by inhibition of either 5-HT2A receptor coupling to G¿q/11 protein or PKC activity. In order to investigate whether a different receptor coupled to Gaq/11 proteins promotes similar effects on MOP receptors activated by morphine, we generated a double stable cell line harbouring in the inducible locus a FLAG-M3Cerulean muscarinic receptor and constitutively expressing a MOP-YFP construct. The inducible expression of FLAG-M3Cerulean receptors was doxycycline dose-dependent and increased with time (up to 96 hours). Epifluorescence microscopy examinations showed the cellular distribution of M3 receptors to be mainly in the plasma membrane, where they co-localised with MOP-YFP receptors. As observed previously in this cell system, both fluorescence microscopy and biotinylation protection assays demonstrated that morphine failed to internalise MOP-YFP receptors whilst, in contrast, DAMGO promoted robust receptor endocytosis. However, after co-expression of FLAG-M3Cerulean receptors, concomitant treatment of cells with morphine+carbachol resulted in a significant internalisation of MOPYFP receptors. MOPYFP receptor internalisation was not accompanied by substantial FLAG-M3Cerulean receptor internalisation after treatment with carbachol, carbachol+morphine or carbachol+DAMGO. The endocytosis of MOPYFP receptors induced by carbachol+morphine was accompanied by desensitisation and down regulation of these receptors after prolonged treatment of cells with the combination of agonist drugs. Finally, pre-treatment of cells with the receptor-Gaq/11 protein coupling inhibitor YM254890 resulted in a lack of MOPYFP receptor endocytosis promoted by morphine+carbachol, indicating that coupling of the M3 receptors to Gzq/11 proteins is necessary to accomplish MOP receptor internalisation by morphine in this experimental system.
URI http://hdl.handle.net/10261/79242
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