Agonist-Induced Endocytosis and Receptor Phosphorylation Mediate Resensitization of Dopamine D2 Receptors
J. H Park
K. M. Kim
2010, 24(3), 574-586. DOI: 10.1210/me.2009-0369
The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D2 receptors (D2Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for β-arrestins was altered. Agonist-induced internalization of D2Rs 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 D2R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D2R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D2R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D2R-intracellular loop 2/3 provoked desensitization as in wild-type D2R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D2R. When dissociation between D2R and β-arrestin was inhibited or when the expression of cellular β-arrestins was decreased, agonist-induced desensitization of D2R did not occur, suggesting that dissociation from β-arrestin is the main cellular process required for resensitization of D2R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with β-arrestin plays a major role in agonist-induced desensitization.