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Open Access Research article

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

Kymry T Jones1, Maria Echeverry2, Valerie A Mosser4, Alicia Gates3 and Darrell A Jackson4*

Author Affiliations

1 School of Biology, Georgia Institute of Technology, Atlanta, GA 30332 USA

2 Laboratorio de Parasitologia (301), Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Columbia

3 Department of Anatomy and Neurobiology, Neuroscience Institute, Morehouse School of Medicine, Atlanta, GA 30310 USA

4 Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812 USA

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Journal of Molecular Signaling 2006, 1:7  doi:10.1186/1750-2187-1-7

Published: 5 December 2006



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 over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted 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).


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 agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged 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 carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells.


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.