Identification of a Region Required for Subtype-specific Agonist-induced Sequestration of the m2 Muscarinic Acetylcholine Receptor

doi:10.1074/jbc.271.8.4215

Identification of a Region Required for Subtype-specific Agonist-induced Sequestration of the m2 Muscarinic Acetylcholine Receptor (*)

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750

** To whom correspondence should be addressed:
Dept. of Pharmacology, K536A HSB, University of Washington, Box 357750, Seattle, WA 98195-7750
; Tel.: 206-543-9457; Fax: 206-616-4230.

↵^§ Present address: Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR 97201-3098.
↵^¶ Present address: Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Ave., Seattle, WA 98121.

Abstract

When the m1 and m2 muscarinic acetylcholine receptors are transiently expressed in JEG-3 cells, the m2, but not the m1, receptor undergoes agonist-induced sequestration. Both receptors exhibit internalization when expressed in Y1 cells. These results suggest that the m1 and m2 receptors use distinct cellular mechanisms or pathways for agonist-induced internalization and that JEG-3 cells are deficient in the mechanism or pathway used by the m1 receptor. Transfection experiments with chimeric receptors indicate that the specificity for agonist-induced internalization for the m2 receptor lies in the carboxyl-terminal fifth of the receptor. The intracellular carboxyl-terminal tail of the m2 receptor is neither sufficient nor required for the m2-specific sequestration. Site-directed mutagenesis demonstrates that two amino acids in the carboxyl-terminal end of the third cytoplasmic loop of the m2 receptor are required for sequestration in JEG-3 cells. In addition, the sixth transmembrane domain, which is adjacent to this cytoplasmic domain, is also required. Thus, m2-specific agonist-induced sequestration requires sequences both in the carboxyl-terminal end of the third cytoplasmic loop and the adjacent transmembrane domain.

Footnotes

↵* This work was supported by National Institutes of Health Grant HL44948 and Training Grants GM07750 and GM07270. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

mAChR

muscarinic acetylcholine receptor

PCR

polymerase chain reaction

QNB

quinuclidinyl benzilate

NMS

N-methylscopolamine

CRE

cAMP response element.
↵²P. S. Goldman and N. M. Nathanson, unpublished observations.
- Received November 8, 1995.