Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the lone form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled rec eptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [M-3] sulpiride-binding activity from the cell surface and by trans fer of [H-3] spiperone-binding activity from the membrane fraction to the l ight vesicle fraction in sucrose-density gradients. In COS-7 cells expressi ng D2 receptors alone, virtually no sequestration was observed with or with out dopamine (< 4%). When GRK2 was coexpressed 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h. whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S rec eptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting tha t the sequestration follows the clathrin pathway. internalization of D2S re ceptors was also assessed by immunofluorescence confocal microscopy. Transl ocation of D2 receptors from the cell membrane to intracellular vesicles wa s observed following the treatment with dopamine from HEK 293 cells only wh en GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were show n to be phosphorylated by GRK2 in an agonist-dependent manner. These result s indicate that the sequestration of D2 receptors occurs only through a GRK -mediated pathway.