Internalization of the m2 Muscarinic Acetylcholine Receptor
ARRESTIN-INDEPENDENT AND -DEPENDENT PATHWAYS*
- Robin Pals-Rylaarsdam‡§,
- Vsevolod V. Gurevich¶,
- Katharine B. Lee‡,
- Judith A. Ptasienski‡,
- Jeffrey L. Benovic‖ and
- M. Marlene Hosey‡**
- From the ‡Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611, ¶Sun Health Research Institute, Sun City, Arizona 85372, and the ‖Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Abstract
Recent studies have identified agonist-dependent phosphorylation as a critical event in the rapid uncoupling of the m2 muscarinic cholinergic receptors (mAChR) from G-proteins and sequestration of the receptors away from the cell surface. However, mutant m2 mAChRs were identified that were phosphorylated but unable to desensitize in adenylyl cyclase assays, while they internalized like wild type (WT) mAChRs. We have tested whether these properties might stem from differences in the abilities of the WT and mutant mAChR to bind arrestins, proteins implicated in both receptor/G-protein uncoupling and internalization. We have determined that arrestin binding requires phosphorylation at a cluster of Ser/Thr residues in amino acids 307–311 in the m2 mAChR. A strong correlation was found between the ability of WT and mutant receptors to bind arrestins in vitro or in vivo and to desensitize in adenylyl cyclase assays. However, the phosphorylation-dependent internalization of the m2 mAChR in HEK-tsA201 cells did not require arrestins and did not proceed via clathrin-mediated endocytosis. While the m2 mAChR was able to enter a clathrin- and arrestin-dependent pathway when arrestin 2 or arrestin 3 was significantly overexpressed, the preferred pathway of internalization of WT and certain mutant m2 mAChR in HEK-tsA201 cells did not involve participation of arrestins. The results suggest that the phosphorylation-mediated regulation of the m2 mAChR may involve arrestin-dependent and -independent events.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants HL50121 (to M. M. H.), EY11500 (to V. V. G.), and GM4494 and GM47417 (to J. B. L.).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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↵§ Predoctoral fellow of the Howard Hughes Medical Institute.
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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 60614. Tel.: 312-503-3692; Fax: 312-503-5349; E-mail: mhosey{at}nwu.edu.
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↵1 The abbreviations used are: GPR, G-protein coupled receptor; R/G, receptor/G-protein; mAChR, muscarinic cholinergic receptor; WT, wild type; aa, amino acid(s); DMEM, Dulbecco’s modified Eagle’s medium; TBS, Tris-buffered saline; HA, hemagglutinin; HEK, hamster embryonic kidney; NMS,N-methylscopolamine.
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- Received July 2, 1997.
- Revision received July 17, 1997.
