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.

Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with b-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis.

Background and Purpose: In addition to typical GPCR kinase (GRK)‐/β‐arrestin‐dependent internalization, dopamine D₃ receptor employed an additional GRK‐independent sequestration pathway. In this study, we investigated the molecular mechanism of this novel sequestration pathway.Experimental Approach: Radioligand binding, flow cytometry and cell surface biotinylation assay were used to characterize trafficking properties of D₂ and D₃ receptors. Serine/threonine and N‐linked glycosylation mutants of the D₃ receptor were utilized to locate receptor regions involved in pharmacological sequestration and desensitization. Various point mutants of the D₂ and D₃ receptors, whose sequestration and desensitization properties were altered, were combined with knockdown cells of GRKs or β‐arrestins to functionally correlate pharmacological sequestration and desensitization.Key Results: The D₃ receptor, but not the D₂ receptor, showed characteristic trafficking behaviour in which receptors were shifted towards the more hydrophobic domains within the plasma membrane without translocation into other intracellular compartments. Among various amino acid residues tested, S145/S146, C147 and N12/19 were involved in pharmacological sequestration and receptor desensitization. Both pharmacological sequestration and desensitization of D₃ receptor required β‐arrestins, and functional relationship was observed between two processes when it was tested for D₃ receptor variants and agonists.Conclusions and Implications: Pharmacological sequestration of D₃ receptor accompanies movement of cell surface receptors into a more hydrophobic fraction within the plasma membrane and renders D₃ receptor inaccessible to hydrophilic ligands. Pharmacological sequestration is correlated with desensitization of the D₃ receptor in a Gβγ‐ and β‐arrestin‐dependent manner. This study provides new insights into molecular mechanism governing GPCR trafficking and desensitization.