RECEPTOR SUBTYPE-SPECIFIC REGULATION OF MUSCARINIC ACETYLCHOLINE-RECEPTOR SEQUESTRATION BY DYNAMIN - DISTINCT SEQUESTRATION OF M2 RECEPTORS

Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) a nd other G protein-coupled receptors usually leads to a loss of recept or binding sites from the plasma membrane, referred to as receptor seq uestration. Receptor sequestration can occur via endocytosis of clathr in-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Pr evious work has indicated that the monomeric GTPase dynamin controls t he endocytosis of plasma membrane receptors via clathrin-coated vesicl es. To investigate whether mAChRs sequester in a receptor subtype-spec ific 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, m 2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following ag onist exposure but displayed dissimilar sequestration pathways. Overex pression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestr ation, whereas m2 mAChR sequestration was not affected. Also, m4 mAChR s, which like m2 mAChRs preferentially couple to pertussis toxin-sensi tive 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 hams ter 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 r eceptor sequestration represents a new type of receptor subtype-specif ic regulation within the family of mAChRs.