Regulation of muscarinic acetylcholine receptor sequestration and function by beta-arrestin.
After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic beta-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the beta2-adrenergic receptor have demonstrated that beta-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of beta-arrestin in mAChR sequestration, we determined the effect of overexpressing beta-arrestin-1 and the dominant-negative inhibitor of beta-arrestin-mediated receptor sequestration, beta-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing beta-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of beta-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a beta-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.
Vögler
O
O
Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany.
Nolte
B
B
Voss
M
M
Schmidt
M
M
Jakobs
K H
KH
van Koppen
C J
CJ
D019390:Arrestins / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D004789:Enzyme Activation; D006801:Humans; D011976:Receptors, Muscarinic / Q000378:metabolism*; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; D019390:Arrestins; D002966:Clathrin; D011976:Receptors, Muscarinic; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D017871:Calcium-Calmodulin-Dependent Protein Kinases