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J. Biol. Chem., Vol. 281, Issue 46, 35381-35396, November 17, 2006

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Identification of a Novel Apical Sorting Motif and Mechanism of Targeting of the M₂ Muscarinic Acetylcholine Receptor^*

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

Previous studies have shown that the M₂ receptor is localized at steady state to the apical domain in Madin-Darby canine kidney (MDCK) epithelial cells. In this study, we identify the molecular determinants governing the localization and the route of apical delivery of the M₂ receptor. First, by confocal analysis of a transiently transfected glycosylation mutant in which the three putative glycosylation sites were mutated, we determined that N-glycans are not necessary for the apical targeting of the M₂ receptor. Next, using a chimeric receptor strategy, we found that two independent sequences within the M₂ third intracellular loop can confer apical targeting to the basolaterally targeted M₄ receptor, Val²⁷⁰-Lys²⁸⁰ and Lys²⁸⁰-Ser³⁵⁰. Experiments using Triton X-100 extraction followed by OptiPrep^TM density gradient centrifugation and cholera toxin -subunit-induced patching demonstrate that apical targeting is not because of association with lipid rafts. ³⁵S-Metabolic labeling experiments with domain-specific surface biotinylation as well as immunocytochemical analysis of the time course of surface appearance of newly transfected confluent MDCK cells expressing FLAG-M₂-GFP demonstrate that the M₂ receptor achieves its apical localization after first appearing on the basolateral domain. Domain-specific application of tannic acid of newly transfected cells indicates that initial basolateral plasma membrane expression is required for subsequent apical localization. This is the first demonstration that a G-protein-coupled receptor achieves its apical localization in MDCK cells via transcytosis.

Received for publication, June 21, 2006 , and in revised form, September 11, 2006.

^* This work was supported by Grant R01NS26920 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington School of Medicine, Box 357750, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230; E-mail: nathanso{at}u.washington.edu.

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