Identification of a Basolateral Sorting Signal for the M3 Muscarinic Acetylcholine Receptor in Madin-Darby Canine Kidney Cells

Abstract

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 M 2 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 M 2 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 α-chain caused significant basolateral targeting of this heterologous apical protein. The results indicate that the M 3 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.