Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M&lt;sub&gt;1&lt;/sub&gt; &lt;em&gt;vs&lt;/em&gt; M&lt;sub&gt;2&lt;/sub&gt; muscarinic acetylcholine receptors

Valerie A Mosser,Katie M Hoffman,Kymry T Jones,Darrell A Jackson,Nael A Mccarty

doi:10.1186/1750-2187-3-20

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

Valerie A Mosser, Katie M Hoffman + Show 3 more

Open Access

https://doi.org/10.1186/1750-2187-3-20

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Abstract

BackgroundSustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.ResultsHerein we report that agonist activation of M1 mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and β-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 β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-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 β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-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 β-arrestin lysine mutant.ConclusionThese findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.

Highlights

Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the G protein-coupled receptor (GPCR) – β-arrestin complex
Stimulation of M1 and M2 muscarinic acetylcholine receptor (mAChR) significantly increased ubiquitination of β-arrestin 2 (Figure 3). This increase is clearly visible at both the 15 and 30 min time point for both mAChR subtypes. These results demonstrate that stimulation of M1 or M2 mAChRs promotes a stable ubiquitination of β-arrestin 2
Since β-arrestin 2K18R, K107R, K108R, K207R, K296R appears to interfere with the agonist promoted down-regulation of the M2 mAChR and increased the constitutive down-regulation of the M1 mAChR we examined the ability of this mutant to co-localize with the M1/M2 mAChR compared to wt and β-arrestin 2K11R, K12R following agonist stimulation

Summary

Introduction

Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Muscarinic receptors regulate a variety of physiological responses ranging from cardiac homeostasis to cholinergic signaling in the brain [1]. Agonistpromoted trafficking of mAChRs, and most other GPCRs can be broken down into five distinct phases: agonistbinding promotes G protein dissociation (I) from the receptor which allows phosphorylation of specific serine and threonine residues (II) on internal loops of the receptor by G protein receptor kinases (GRKs). This phosphorylation allows the binding of β-arrestins (III) which promotes homologous desensitization and subsequent internalization of the receptor into clathrin coated pits. The receptor can either be dephosphorylated and recycled (IV) to the cell surface or targeted for degradation (V) in proteasomes or lysosomes [1]

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