Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M1 vs M2 muscarinic acetylcholine receptors.

BACKGROUND: Sustained agonist-promoted ubiquitination of beta-arrestin has been correlated with increased stability of the GPCR - beta-arrestin complex. Moreover, abrogation of beta-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation. RESULTS: Herein we report that agonist activation of M1 mAChRs produces a sustained beta-arrestin ubiquitination but no stable co-localization with beta-arrestin. In contrast, sustained ubiquitination of beta-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and beta-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of beta-arrestin following agonist treatment, we sought to determine the effects of beta-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated beta-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of beta-arrestin 2 (YFP-beta-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M1 and M2 mAChRs, with the effect substantially higher on the M2 mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the beta-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of beta-arrestin lysine mutants lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this beta-arrestin lysine mutant were significantly reduced. Interestingly, expression of beta-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this beta-arrestin lysine mutant. CONCLUSION: These findings indicate that ubiquitination of beta-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.

Transient hypoxia induces sequestration of M1 and M2 muscarinic acetylcholine receptors.

Oxidative stress has been implicated in impairing muscarinic acetylcholine receptor (mAChR) signaling activity. It remains unclear, however, whether alterations in the cell surface distribution of mAChRs following oxidative stress contribute to the diminished mAChR signaling activity. We report here that M1 and M2 mAChRs, stably expressed in Chinese hamster ovary cells, undergo sequestration following transient hypoxic-induced oxidative stress (2% O2). Sequestration of M1 and M2 mAChRs following transient hypoxia was associated with an increase in phosphorylation of these receptors. Over-expression of a catalytically inactive G protein-coupled receptor kinase 2 (GRK2 K220R) blocked the increased phosphorylation and sequestration of the M2, but not M1, mAChRs following transient hypoxia. Hypoxia induced phosphorylation and sequestration of the M1 mAChR was, however, blocked by over-expression of a catalytically inactive casein kinase 1 alpha (CK1alpha K46R). These results are the first demonstration that M1 and M2 mAChRs undergo sequestration following transient hypoxia. The data suggest that increased phosphorylation of M1 and M2 mAChRs underlies the mechanism responsible for sequestration of these receptors following transient hypoxia. We report here that distinct pathways involving CK1alpha and GRK2 mediated sequestration of M1 and M2 mAChRs following transient hypoxic-induced oxidative stress.

Agonist mediated internalization of M2 mAChR is beta-arrestin-dependent.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is beta-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of beta-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a beta-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous beta-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M2 mAChR in mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice that lack expression of either one or both isoforms of beta-arrestin (beta-arrestin 1 and 2). RESULTS: In wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF beta-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either beta-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged beta-arrestin within endocytic structures in multiple cell lines; the compartment to which beta-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF beta-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (beta-arrestin 2 DeltaLIELD), AP-2 (beta-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of beta-arrestin 1 (319-418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells. CONCLUSION: In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles.

Kinetic analysis of M2 muscarinic receptor activation of Gi in Sf9 insect cell membranes.

A steady-state kinetic mechanism describing the interaction of M(2) muscarinic acetylcholine receptors and the guanine nucleotide-binding protein G(i)alpha(2)beta(1)gamma(3) are presented. Data are consistent with two parallel pathways of agonist-promoted GTPase activity arising from receptor coupled to a single or multiple guanine nucleotide-binding proteins. An aspartate 103 to asparagine receptor mutation resulted in a receptor lacking the ability to catalyze the binding of guanosine-5'-O-(3-thiotriphosphate) or guanosine triphosphate hydrolysis by the G protein. An aspartate 69 to asparagine receptor mutant was able to catalyze agonist-specific guanine nucleotide exchange and GTPase activity. A threonine 187 to alanine receptor mutation resulted in a receptor that catalyzed guanine nucleotide exchange comparable with wild-type receptors but had reduced ability to stimulate GTP hydrolysis. A tyrosine 403 to phenylalanine receptor mutation resulted in an increase in agonist-promoted GTPAse activity compared with wild type. The observation that the threonine 187 and tyrosine 403 mutants promote guanine nucleotide exchange similarly to wild type but alter GTPase activity compared with wild type suggests that the effects of the mutations arise downstream from guanine nucleotide exchange and may result from changes in receptor-G protein dissociation.