Trafficking of M2 Muscarinic Acetylcholine Receptors*
- From the Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611
Abstract
Internalization is an important mechanism regulating the agonist-dependent responses of G-protein-coupled receptors. The internalization of the M2 muscarinic cholinergic receptors (mAChR) in HEK293 cells has been demonstrated to occur by an unknown mechanism that is independent of arrestins and dynamin. In this study we examined various aspects of the trafficking of the M2mAChR in HEK293 cells to characterize this unknown pathway of internalization. Internalization of the M2 mAChR was rapid and extensive, but prolonged incubation with agonist did not lead to appreciable down-regulation (a decrease in total receptor number) of the receptors. Recovery of M2 mAChRs to the cell surface following agonist-mediated internalization was a very slow process that contained protein synthesis-dependent and -independent components. The protein synthesis-dependent component of the recovery of receptors to the cell surface did not appear to reflect a requirement for synthesis of new receptors, as no changes in total receptor number were observed either in the presence or absence of cycloheximide. Phosphorylation of the M2 mAChR did not appear to influence the rate or extent of the recovery of receptors to the cell surface, as the recovery of a phosphorylation-deficient mutant M2 mAChR, the N,CAla-8 mutant, was similar to the recovery of the wild type M2 mAChR. Finally, the constitutive, nonagonist-dependent internalization and recycling of the M2 mAChR was very slow and also contained protein synthesis-dependent and -independent components, suggesting that a similar pathway controls the recovery from agonist-dependent and -independent internalization. Overall, these data demonstrated a variety of previously unappreciated facets involved in the regulation of M2 mAChRs.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave. S215, Chicago, IL 60611. Tel.: (312) 503-3692; Fax: (312) 503-5349; E-mail: mhosey@nwu.edu.
- Abbreviations:
- GPCR
-
G-protein-coupled receptor
- β2AR
-
β2adrenergic receptor
- PBCM
-
propylbenzilylcholine mustard
- CCh
-
carbachol
- [3H]NMS
-
[3H]N-methylscopolamine
- [3H]QNB
-
[3H] quinuclidinyl benzilate
- PBS
-
phosphate-buffered saline
- mAChR
-
muscarinic cholinergic receptor
-
- Received June 14, 1999.
- Revision received August 27, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
