Numerous G protein-coupled receptors (GPCRs) are glycosylated at extracellular regions. The regulatory roles of glycosylation on receptor function vary across receptor types. In this study, we used the dopamine D₂and D₃receptors as an experimental model to understand the underlying principles governing the functional roles of glycosylation. We used the pharmacological inhibitor, tunicamycin, to inhibit glycosylation, generated chimeric D₂and D₃receptors by swapping their respective N-termini, and produced the glycosylation site mutant D₂and D₃receptors to study the roles of glycosylation on receptor functions, including cell surface expression, signaling, and internalization through specific microdomains. Our results demonstrate that glycosylation on the N-terminus of the D₃ receptor is involved in the development of desensitization and proper cell surface expression. In addition, glycosylation on the N-terminus mediates the internalization of D₂and D₃receptors within the caveolae and clathrin-coated pit microdomains of the plasma membrane, respectively, by regulating receptor interactions with caveolin-1 and clathrin. In conclusion, this study shows for the first time that glycosylation on the N-terminus of GPCRs is involved in endocytic pathway selection through specific microdomains. These data suggest that changes in the cellular environment that influence posttranslational modification could be an important determinant of intracellular GPCR trafficking.
Classical G protein-coupled receptors (GPCRs) and canonical Wnt pathways were believed to use distinct signaling pathways. However, recent studies have shown that these two pathways interact each other by sharing several intermediate signaling components. Recent in vivo studies showed that antipsychotic drugs, which block dopamine D2-like receptors, increase the cellular levels of downstream signaling components of canonical Wnt pathways, such as dishevelled (Dvl), glycogen synthase kinase 3 (GSK3), and -catenin. These results suggest that some functional interactions might exist between Wnt pathway and D2-like receptors. In this study, we show that among five different dopamine receptor subtypes, D 2 receptor (D 2 R) selectively inhibited the Wnt signaling, which was measured by lymphoid enhancing factor-1 (LEF-1)-dependent transcriptional activities. D 2 R-mediated inhibition of Wnt signaling was agonist-and G protein-independent and did not require receptor phosphorylation or endocytosis. D 2 R inhibited the LEF-1-dependent transcriptional activities, and this inhibitory activity was not affected by the inhibition of GSK-3, suggesting that D 2 R inhibited the Wnt signaling by acting on the downstream of GSK3. D 2 R directly interacted with -catenin through the second and third loops, leading to a reduction of -catenin distribution in the nucleus, resulting in an inhibition of LEF-1-dependent transcription. This is a novel mechanism for the regulation of canonical Wnt signaling by GPCRs, in which receptor proteins recruit -catenin from cytosol to the plasma membrane, resulting in the decrement of the -catenin/LEF-1-dependent transcription in the nucleus.
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.
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