INTERNALIZATION AND DOWN-REGULATION OF HUMAN MUSCARINIC ACETYLCHOLINE-RECEPTOR M2 SUBTYPES - ROLE OF 3RD INTRACELLULAR M2 LOOP AND G-PROTEIN-COUPLED RECEPTOR KINASE-2
H. Tsuga et al., INTERNALIZATION AND DOWN-REGULATION OF HUMAN MUSCARINIC ACETYLCHOLINE-RECEPTOR M2 SUBTYPES - ROLE OF 3RD INTRACELLULAR M2 LOOP AND G-PROTEIN-COUPLED RECEPTOR KINASE-2, The Journal of biological chemistry, 273(9), 1998, pp. 5323-5330
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.