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

Kymry T Jones, Maria Echeverry, Valerie A Mosser, Alicia Gates, Darrell A Jackson


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.

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How to cite: Jones, K.T., Echeverry, M, Mosser, V.A., Gates, A and Jackson, D.A. 2006. Agonist mediated internalization of M2 mAChR is β-arrestin-dependent. Journal of Molecular Signaling 1:7, DOI:

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Published on 5 December 2006.

ISSN: 1750-2187 | Published by Ubiquity Press | Creative Commons License This work is licensed under a Creative Commons License.