ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D₂ receptors mediated by GRK and PKC in transfected cells.

BACKGROUND AND
PURPOSE: GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D₂ receptor were investigated. EXPERIMENTAL APPROACH: All of the S/T residues located within the intracellular loops of D₂ receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D₂ receptors were investigated in the transfected cells. KEY
RESULTS: T134, T225/S228/S229 and S325 were involved in PKC-mediated D₂ receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D₂ receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D₂ receptors, which induced receptor resensitization. ARF6 mediated the recycling of D₂ receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D₂ receptors internalized in a PKC-dependent manner. CONCLUSIONS AND IMPLICATIONS: GRK- and PKC-mediated internalizations of D₂ receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D₂ receptors and different sorting proteins are involved in the dissimilar regulation of D₂ receptors by GRK2 and PKC.