Agonist mediated internalization of M2 mAChR is β-arrestin-dependent

Background: Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous overexpression of wild type or dominant-negative forms of β-arrestins have reported that agonistpromoted internalization of M₂ mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-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 β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-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 β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonistpromoted internalization. Stimulation of M₂ mAChRs led to a stable co-localization with GFPtagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β- arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M₂ mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxyterminal region of β-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 β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β- arrestin in early endosomal vesicles.