INTERNALIZATION AND DOWN-REGULATION OF HUMAN MUSCARINIC ACETYLCHOLINE-RECEPTOR M2 SUBTYPES - ROLE OF 3RD INTRACELLULAR M2 LOOP AND G-PROTEIN-COUPLED RECEPTOR KINASE-2

Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a centr al part of the third intracellular loop (I3-del m2 receptor) were exam ined in Chinese hamster ovary (CHO-K1) cells stably expressing these r eceptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induc ed internalization of up to 80-90% of hm2 receptors was demonstrated b y measuring loss of [H-3]N-methylscopolamine binding sites from the ce ll surface, and transfer of [H-3]quinuclidinyl benzilate binding sites from the plasma membrane into the light-vesicle fractions separated b y sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunoflu orescence confocal microscopy. Agonist-induced down-regulation of up t o 60-70% of hm2 receptors was demonstrated by determining the loss of [H-3]quinuclidinyl benzilate binding sites in the cells. The half-time (t(1/2)) of internalization and down-regulation in the presence of 10 (-4) nn carbamylcholine was estimated to be 9.5 min and 2.3 h, respect ively. The rates of both internalization and down-regulation of hm2 re ceptors in the presence of 10(-6) M or lower concentrations of carbamy lcholine were markedly increased by coexpression of GRK2. Agonist-indu ced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10(-4) M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t(1/2) = 9.9 versus 2.3 h). T hese results indicate that rapid internalization of hm2 receptors is f acilitated by their phosphorylation with GRK2 and does not occur in th e absence of the third intracellular loop, but downregulation of hm2 r eceptors may occur through both GRK2-facilitating pathway and third in tracellular loop-independent pathways.