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

Abstract

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 G proteins through 5-HT serotonin receptors co-expressed in the same cells facilitates MOP receptor endocytosis promoted by morphine. In order to explore whether a different G 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.

Cellular tolerance at the µ-opioid receptor is phosphorylation dependent

“…It is also possible that phosphorylation of other signaling proteins results in the inhibition ( Chu et al, 2010 ). Activation of muscarinic receptors has been shown to alter the trafficking of MORs heterologously expressed in HEK293 cells ( Lopez-Gimenez et al, 2017 ), however there was no change in internalization of FLAG-MORs in the mouse locus coeruleus ( Arttamangkul et al, 2015 ).…”

Interclass GPCR heteromerization affects localization and trafficking

“…The presence of multiple GPCRs in individual cells affects processes related to agonist-promoted GPCR endocytosis and down-regulation (19,37,80). For many GPCR heterocomplexes, such as -opioid- 2 -adrenergic (81),  2A -adrenergic- 1 -adrenergic (82), and adenosine A 2A -dopamine D 2 receptor (83) complexes, stimulation of only one of the components has been suggested to be sufficient to promote cotrafficking of the two protomers.…”