Agonist-induced endocytosis and receptor phosphorylation mediate resensitization of dopamine D(2) receptors.
The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D(2) receptors (D(2)Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for beta-arrestins was altered. Agonist-induced internalization of D(2)Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D(2)R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D(2)R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D(2)R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D(2)R-intracellular loop 2/3 provoked desensitization as in wild-type D(2)R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D(2)R. When dissociation between D(2)R and beta-arrestin was inhibited or when the expression of cellular beta-arrestins was decreased, agonist-induced desensitization of D(2)R did not occur, suggesting that dissociation from beta-arrestin is the main cellular process required for resensitization of D(2)R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with beta-arrestin plays a major role in agonist-induced desensitization.
Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Korea.
Dongim
D
Cho
Mei
M
Zheng
Chengchun
C
Min
Lan
L
Ma
Hitoshi
H
Kurose
Jae H
JH
Park
Kyeong-Man
KM
Kim
D000595:Amino Acid Sequence; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D007545:Isoproterenol / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering; D017448:Receptors, Dopamine D2 / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071557:beta-Arrestins
D019390:Arrestins; D034741:RNA, Small Interfering; D017448:Receptors, Dopamine D2; D000071557:beta-Arrestins; D000242:Cyclic AMP; D007545:Isoproterenol; D004298:Dopamine