Regulation of Muscarinic Acetylcholine Receptor Sequestration and Function by β-Arrestin*

Abstract

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic β-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the β₂-adrenergic receptor have demonstrated that β-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 β-arrestin in mAChR sequestration, we determined the effect of overexpressing β-arrestin-1 and the dominant-negative inhibitor of β-arrestin-mediated receptor sequestration, β-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60–75% in cells overexpressing β-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of β-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 β-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.