Abstract
The m4 subtype of muscarinic acetylcholine receptor regulates many physiological processes and is a novel therapeutic target for neurologic and psychiatric disorders. However, little is known about m4 regulation because of the lack of pharmacologically selective ligands. A crucial component of G protein-coupled receptor regulation is intracellular trafficking. We thus used subtype-specific antibodies and quantitative immunocytochemistry to characterize the intracellular trafficking of m4. We show that following carbachol stimulation, m4 co-localizes with transferrin, and the selective marker of early endosomes, EEA1. In addition, m4 intracellular localization depends on Rab5 activity. The dominant negative Rab5S34N inhibits m4 endocytosis initially following carbachol stimulation, and reduces the size of m4 containing vesicles. The constitutively active Rab5Q79L enhances m4 intracellular distribution, even in unstimulated cells. Rab5Q79L also produces strikingly enlarged vacuoles, which by electron microscopy contain internal vesicles, suggesting that they are multivesicular bodies. m4 localizes both to the perimeter and interior of these vacuoles. In contrast, transferrin localizes only to the vacuole perimeter, demonstrating divergence of m4 trafficking from the pathway followed by constitutively endocytosed transferrin. We thus suggest a novel model by which multivesicular bodies sort G protein-coupled receptors from a transferrin-positive recycling pathway to a nonrecycling, possibly degradative pathway.
Highlights
Intracellular trafficking has recently emerged as a crucial component of G protein-coupled receptor (GPCR)1 regulation
We show that following carbachol stimulation, m4 co-localizes with transferrin, and the selective marker of early endosomes, EEA1
Because Muscarinic acetylcholine receptors (mAChRs) trafficking may depend on the cell line in which they are expressed (13), we first sought to characterize the extent of CCh induced loss of cell surface mAChRs in PC12 cells with previously established binding assays using membrane impermeant [3H]NMS
Summary
Intracellular trafficking has recently emerged as a crucial component of G protein-coupled receptor (GPCR) regulation. Recent cell biological studies have identified multiple distinct endocytic compartments involved in cell surface receptor trafficking These organelles include: 1) early sorting endosomes that contain both recycling proteins and proteins destined for degradation; 2) recycling endosomes; and 3) late endosomes and multivesicular bodies (MVBs) that target proteins for lysosomal degradation (2– 4). The constitutively active Rab enhances m4 intracellular localization and produces enlarged vacuoles to which m4 is targeted Ultrastructural analysis of these vacuoles reveals the presence of numerous internal vesicles, suggesting that these structures are MVBs. Interestingly, m4 shows a distinct distribution within the MVB compared with Tfn. Interestingly, m4 shows a distinct distribution within the MVB compared with Tfn These studies define the early endosomal trafficking of m4 and identify MVBs as a site of divergence between the m4 mAChR and constitutively recycled cell surface receptors
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