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Originally published In Press as doi:10.1074/jbc.M007190200 on January 2, 2001

J. Biol. Chem., Vol. 276, Issue 13, 10539-10547, March 30, 2001
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Identification of a Basolateral Sorting Signal for the M3 Muscarinic Acetylcholine Receptor in Madin-Darby Canine Kidney Cells*

Laurie S. Nadler, Geetha Kumar, and Neil M. NathansonDagger

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

Muscarinic acetylcholine receptors (mAChRs) can be differentially localized in polarized cells. To identify potential sorting signals that mediate mAChR targeting, we examined the sorting of mAChRs in Madin-Darby canine kidney cells, a widely used model system. Expression of FLAG-tagged mAChRs in polarized Madin-Darby canine kidney cells demonstrated that the M2 subtype is sorted apically, whereas M3 is targeted basolaterally. Expression of M2/M3 receptor chimeras revealed that a 21-residue sequence, Ser271-Ser291, from the M3 third intracellular loop contains a basolateral sorting signal. Substitution of sequences containing the M3 sorting signal into the homologous regions of M2 was sufficient to confer basolateral localization to this apical receptor. Sequences containing the M3 sorting signal also conferred basolateral targeting to M2 when added to either the third intracellular loop or the C-terminal cytoplasmic tail. Furthermore, addition of a sequence containing the M3 basolateral sorting signal to the cytoplasmic tail of the interleukin-2 receptor alpha -chain caused significant basolateral targeting of this heterologous apical protein. The results indicate that the M3 basolateral sorting signal is dominant over apical signals in M2 and acts in a position-independent manner. The M3 sorting signal represents a novel basolateral targeting motif for G protein-coupled receptors.

* This work was supported by National Institutes of Health Grant NS26920 (to N. M. N.) and by a postdoctoral fellowship in pharmacology/morphology from the Pharmaceutical Research and Manufacturers of America Foundation (to L. S. N.).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.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, P. O. Box 357750, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230; E-mail: nathanso@u.washington.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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