M3 muscarinic acetylcholine receptor facilitates the endocytosis of mu opioid receptor mediated by morphine independently of the formation of heteromeric complexes

Lopez-Gimenez, J., Alvarez-Curto, E. and Milligan, G. (2017) M3 muscarinic acetylcholine receptor facilitates the endocytosis of mu opioid receptor mediated by morphine independently of the formation of heteromeric complexes. Cellular Signalling, 35, pp. 208-222. (doi: 10.1016/j.cellsig.2017.04.006) (PMID:28411124)

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Morphine inefficiency to induce the internalization of mu opioid (MOP) receptors observed in numerous experimental models constitutes a paradigm of G-protein coupled receptor (GPCR) functional selectivity. We recently described that activation of Gαq/11 proteins through 5-HT2A serotonin receptors co-expressed in the same cells facilitates MOP receptor endocytosis promoted by morphine. In order to explore whether a different Gαq/11 coupled GPCR would emulate this effect, a double stable Flp-In T-REx HEK293 cell line permanently expressing MOP-YFP receptors along with FLAG-M3-Cerulean receptors expressed in an inducible manner was generated. Fluorescence microscopy examination of these cells revealed a co-distribution of both receptors mainly compartmentalized in plasma membrane. Concurrent stimulation with carbachol and morphine promoted MOP receptor internalization, desensitization and down-regulation and this facilitation was not dependent on PKC activation. Co-immunoprecipitation experiments demonstrated that FLAG-M3-Cerulean/MOP-YFP receptors interact forming heteromeric complexes in a time depending manner, i.e. the strongest interaction was detected after 96h of FLAG-M3-Cerulean induced expression. Under these experimental conditions, treatment of cells with carbachol plus morphine resulted in the internalization of both receptors within separated endocytic vesicles as visualized by confocal microscopy. This trafficking segregation observed for FLAG-M3-Cerulean and MOP-YFP receptors upon agonist stimulation suggests that this protein-protein interaction presents temporal and dynamic properties. Moreover, MOP-YFP receptor internalization facilitated by FLAG-M3-Cerulean receptors is independent of the constitution of heteromeric complexes. [Abstract copyright: Copyright © 2017. Published by Elsevier Inc.]

Item Type:Articles
Additional Information:This study was supported by Biotechnology and Bioscience Research Council Grant BB/G0011200/1, Consejo Superior de Investigaciones Científicas, CSIC, Grant 20098I110 and Grant SAF2010-15663 from the Spanish Government (MINECO).
Keywords:Endocytosis, GPCR, heteromerization, M3 receptor, MOP receptor, morphine.
Glasgow Author(s) Enlighten ID:Alvarez-Curto, Dr Elisa and Milligan, Professor Graeme
Authors: Lopez-Gimenez, J., Alvarez-Curto, E., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Cellular Signalling
ISSN (Online):1873-3913
Published Online:11 April 2017
Copyright Holders:Copyright © 2017 Elsevier Inc.
First Published:First published in Cell Signalling 35: 208-222
Publisher Policy:Reproduced in accordance with the publisher copyright policy

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