The muscarinic acetylcholine type 2 receptor (M2 mAChR), a member of the GPCR superfamily, is found throughout the parasympathetic nervous system where it controls pulmonary, urinary, and cardiac function, and neurotransmission. The molecular mechanisms that regulate M2 mAChR availability at the cell surface are an important component in controlling these physiological events. Since beta-arrestin proteins are known to regulate the activity of other GPCRs, we sought to identify their role in regulating M2 mAChR activity, a topic that remains contentious in the field. To achieve this goal we utilized mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice lacking one or both isoforms (MEF KO1, KO2, or KO1/2 cells) in addition to exogenous expression of beta-arrestin mutants. This study demonstrates that agonist-induced internalization of M2 mAChR is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles suggesting it behaves as a class B receptor. Next, we sought to identify beta-arrestin s function in regulating the post-endocytic trafficking (down-regulation) of the M2 mAChR. MEF KO1/2 cells were unable to down-regulate M2 mAChRs whereas MEF KO1 or KO2 cells retained the ability to do so. In MEFwt cells, both M2 mAChR and beta-arrestin exhibited basal ubiquitination that increased following agonist stimulation. Receptor degradation appeared to be regulated by the ubiquitination status of beta-arrestin 2, since expression of a chimeric รข-arrestin 2 form fused to ubiquitin increased both constitutive and agonist-promoted down-regulation, whereas expression of a beta-arrestin 2 mutant lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, significantly blocked degradation while internalization and stable association remained intact. Upon further analysis, the beta-arrestin 2K18R, K107R, K108R, K207R, K296R mutant blocked delivery of M2 mAChR to the late endosome/lysosome, presumably where degradation occurs. Inhibition of proteasome-dependent recycling of ubiquitin blocked receptor down-regulation without affecting internalization or the ubiquitination state of the M2 mAChR while ubiquitination of beta-arrestin 2 diminished significantly. These results support a role for ubiquitinated beta-arrestin in mediating M2 mAChR sorting and degradation in the lysosome. Collectively, these studies give us new insight on the function of beta-arrestin in regulating the activity of the M2 mAChR.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/24815 |
| Date | 06 July 2007 |
| Creators | Jones, Kymry Thereasa |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
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