Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M(1 )vs M(2 )muscarinic acetylcholine receptors

BACKGROUND: Sustained 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. RESULTS: Herein we report that agonist activation of M(1 )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 M(2 )mAChRs does result in stable co-localization between the M(2 )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 M(1 )and M(2 )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 M(1 )and M(2 )mAChRs, with the effect substantially higher on the M(2 )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 M(2 )mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2(K18R, 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 2(K18R, K107R, K108R, K207R, K296R )increased the agonist-promoted down-regulation of the M(1 )mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant. CONCLUSION: These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M(1 )and M(2 )mAChRs.