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

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 D&sub2; receptor were investigated. Experimental approach: All of the S/T residues located within the intracellular loops of D&sub2; 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 D&sub2; receptors were investigated in the transfected cells. Key

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 D&sub2; receptor were investigated. Experimental approach: All of the S/T residues located within the intracellular loops of D&sub2; 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 D&sub2; receptors were investigated in the transfected cells. Key

T134, T225/S228/S229 and S325 were involved in PKC-mediated D&sub2; receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D&sub2; 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 D&sub2; receptors, which induced receptor resensitization. ARF6 mediated the recycling of D&sub2; receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D&sub2; receptors internalized in a PKC-dependent manner. Conclusions and implications: GRK- and PKC-mediated internalizations of D&sub2; receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D&sub2; receptors and different sorting proteins are involved in the dissimilar regulation of D&sub2; receptors by GRK2 and PKC.