Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs.Muscarinic acetylcholine receptors (mAChRs) 1 belong to the superfamily of plasma membrane receptors that regulate a large number of signal transduction pathways via activation of heterotrimeric GTP-binding proteins (G proteins). The mAChR family consists of five subtypes of cloned mAChRs and can be subdivided into two functional groups: the m1, m3, and m5 subtypes, which preferentially couple to the G q family of G proteins, and the m2 and m4 subtypes, which effectively activate the G i family of G proteins. The m1, m2, m3, and m4 receptors are widely expressed in the central nervous system and peripheral tissues, whereas m5 receptors are present in only minute amounts in hippocampus and other areas of the brain (1). Prolonged exposure of mAChRs and other G proteincoupled receptors to agonists usually results in the attenuation of the cellular response. One molecular mechanism of attenuation involves the phosphorylation of the receptors by G protein-coupled receptor kinases (GRKs) and increased binding of the inhibitory protein -arrestin to the phosphorylated receptors, thereby inhibiting the coupling with G proteins (2). Another regulatory mechanism is the sequestration of receptors by which G protein-coupled receptors become inaccessible for hydrophil...
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