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Agonist mediated internalization of M(2 )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 over-expression of wild type or dominant-neg...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2006
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1769497/ https://www.ncbi.nlm.nih.gov/pubmed/17224084 http://dx.doi.org/10.1186/1750-2187-1-7 |
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author | Jones, Kymry T Echeverry, Maria Mosser, Valerie A Gates, Alicia Jackson, Darrell A |
author_facet | Jones, Kymry T Echeverry, Maria Mosser, Valerie A Gates, Alicia Jackson, Darrell A |
author_sort | Jones, Kymry T |
collection | PubMed |
description | 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 over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M(2 )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 M(2 )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 M(2 )mAChRs, 40% of surface M(2 )mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M(2 )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 M(2 )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 M(2 )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 M(2 )mAChRs in wild type MEF cells. CONCLUSION: In summary, this study demonstrates that agonist-promoted internalization of M(2 )mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles. |
format | Text |
id | pubmed-1769497 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-17694972007-01-16 Agonist mediated internalization of M(2 )mAChR is β-arrestin-dependent Jones, Kymry T Echeverry, Maria Mosser, Valerie A Gates, Alicia Jackson, Darrell A J Mol Signal Research Article 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 over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M(2 )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 M(2 )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 M(2 )mAChRs, 40% of surface M(2 )mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M(2 )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 M(2 )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 M(2 )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 M(2 )mAChRs in wild type MEF cells. CONCLUSION: In summary, this study demonstrates that agonist-promoted internalization of M(2 )mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles. BioMed Central 2006-12-05 /pmc/articles/PMC1769497/ /pubmed/17224084 http://dx.doi.org/10.1186/1750-2187-1-7 Text en Copyright © 2006 Jones et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Jones, Kymry T Echeverry, Maria Mosser, Valerie A Gates, Alicia Jackson, Darrell A Agonist mediated internalization of M(2 )mAChR is β-arrestin-dependent |
title | Agonist mediated internalization of M(2 )mAChR is β-arrestin-dependent |
title_full | Agonist mediated internalization of M(2 )mAChR is β-arrestin-dependent |
title_fullStr | Agonist mediated internalization of M(2 )mAChR is β-arrestin-dependent |
title_full_unstemmed | Agonist mediated internalization of M(2 )mAChR is β-arrestin-dependent |
title_short | Agonist mediated internalization of M(2 )mAChR is β-arrestin-dependent |
title_sort | agonist mediated internalization of m(2 )machr is β-arrestin-dependent |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1769497/ https://www.ncbi.nlm.nih.gov/pubmed/17224084 http://dx.doi.org/10.1186/1750-2187-1-7 |
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