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

Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is beta-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of beta-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a beta-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous beta-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 beta-arrestin knockout mice that lack expression of either one or both isoforms of beta-arrestin (beta-arrestin 1 and 2).

In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles.

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 beta-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either beta-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 beta-arrestin within endocytic structures in multiple cell lines; the compartment to which beta-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF beta-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (beta-arrestin 2 DeltaLIELD), AP-2 (beta-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of beta-arrestin 1 (319-418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells.