Identification of a novel apical sorting motif and mechanism of targeting of the M2 muscarinic acetylcholine receptor.
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 density gradient centrifugation and cholera toxin beta-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.
Chmelar
Renée S
RS
Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7750, USA.
Nathanson
Neil M
NM
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D004285:Dogs; D005786:Gene Expression Regulation; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D009154:Mutation; D021381:Protein Transport; D043585:Receptor, Muscarinic M2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D043585:Receptor, Muscarinic M2