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Abstract: Most G protein coupled receptors (GPCR) regulate multiple cellular processes by coupling to more than one kind of G protein. Furthermore, recent studies have reported G protein-independent/β-arrestin-dependent signaling pathway for some GPCRs. Dopamine D2 and D3 receptors (D2R, D3R), the major targets of currently used antipsychotic drugs, are co-expressed in some of the same dopaminergic neurons and regulate the same overlapping effectors. However, the specific subunits of G proteins that regulate each signaling pathway are not clearly identified. In addition, the existence of β-arrestin-dependent/G protein-independent signaling is not clear for these receptors. In this study, we determined the G protein subtypes and β-arrestin dependency involved in the signaling of D2R and D3R, which was measured by inhibition of adenylyl cyclase and extracellular signal-regulated kinase (ERK) activation. For the inhibition of cAMP production in HEK-293 cells, D2R used the Gαo subunit but D3R used the βγ subunit of Gi family proteins. For the regulation of ERK activation, D2R used the α subunits of Gi/o proteins both in HEK-293 cells and COS-7 cells, but D3R used Gαo and Gβγ in HEK-293 cells and COS-7 cells, respectively. β-Arrestin-dependent/G protein-independent ERK activation was not observed for both D2R and D3R. Agonist-induced β-arrestin translocation was observed with D2R but not with D3R, and β-arrestins exerted inhibitory influences on G protein-dependent ERK activation by D2R, but not D3R. These results show that the D2R and D3R, which have overlapping cellular expressions and functional roles, employ distinct G protein subunits depending on the cell types and the effectors they control. [Copyright &y& Elsevier]

Abstract: Dopamine D2 receptor and D3 receptor (D2R and D3R) are the major targets for current antipsychotic drugs, and their proper regulation has pathological and pharmacological significance. This study was conducted to understand the functional roles and molecular mechanisms of RGS proteins (RGS2, RGS4, and RGS9-2) on the signaling of D2R and D3R. RGS proteins were co-expressed with D2R and D3R in HEK-293 cells. The protein interactions between RGS proteins and D2R/D3R, and effects of RGS proteins on the internalization, signaling, and desensitization of D2R/D3R were determined. In addition, the RGS4 proteins were subdivided into N-terminal region, RGS domain, and the C-terminal region, and the specific subdomain of RGS4 protein involved in the regulation of the signaling of D2R/D3R was determined. All of RGS proteins we tested interacted with D2R/D3R. RGS4 exerted potent inhibitory activities on the signaling of D2R/D3R. RGS9-2 exerted selective but moderate inhibitory activity on D3R and the internalization of D2R. RGS2 had no effect. The N-terminal domain of RGS4 was involved in its interaction with D2R and D3R and was required for the inhibitory activity of the RGS domain. The study for the first time showed that RGS4 is the major RGS protein which interacts through the N-terminal region and exerts potent inhibitory activities on the signaling of D2R and D3R. [Copyright &y& Elsevier]

Dopamine D2R and D3R (D2R, D3R) show very high sequence homology and employ virtually identical signaling pathways even though D2R is 2 ∼ 5 times more active. Among the structural motifs identified, a triplet sequence, Asp-Arg-Tyr (DRY motif), plays critical roles in the determination of receptor conformations for signaling and intracellular trafficking of G protein-coupled receptors by forming intramolecular interactions. Thus, it is possible that different signaling efficiencies of D2R and D3R might be caused by the receptor activation levels stabilized by their own DRY motifs. In this study, the Arg and Asp residues of D2R and D3R were mutated, and resulting changes in their signaling and intracellular trafficking properties were comparatively studied. Mutation of the Arg residues of D2R and D3R abolished their signaling but differently affected their intracellular localizations. The wildtype and R132H-D2R were expressed mainly on the plasma membrane. On the other hand, compared with the wildtype D3R, a substantial amount of R128H-D3R was localized intracellularly. The expression of receptor proteins on the plasma membrane and their signaling efficiencies were more drastically affected by the mutation of the Asp residue of D3R than D2R. Therefore, it was concluded that the different levels of conformational strain exerted by the DRY motif might partly determine the quantitative differences in the signaling efficiencies between D2R and D3R. [ABSTRACT FROM AUTHOR]

Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D 2 and D 3 receptors (D 2 R, D 3 R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D 2 R and D 3 R, comparative studies were conducted on the signaling and intracellular trafficking of the D 2 R and D 3 R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D 2 R and D 3 R. Lowering the level of cellular FLNA caused an elevation in RalA activity and resulted in selective interference with the normal intracellular trafficking and signaling of the D 2 R and D 3 R, through GRK2 and β-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D 2 R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D 2 R. Knockdown of FLNA or coexpression of active RalA prevented D 3 R from coupling with G protein. The selective involvement of GRK2- and β-arrestins in the RalA-mediated cellular processes of the D 2 R and D 3 R was achieved via their different modes of interactions with the receptor and their distinct functional roles in receptor regulation. Our results show that FLNA is a multi-functional protein that acts as a platform on which D 2 R and D 3 R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and β-arrestins. [ABSTRACT FROM AUTHOR]

PICK1, a PDZ domain-containing protein, is known to increase the reuptake activities of dopamine transporters by increasing their expressions on the cell surface. Here, we report a direct and functional interaction between PICK1 and dopamine D₃ receptors (D₃R), which act as autoreceptors to negatively regulate dopaminergic neurons. PICK1 colocalized with both dopamine D₂ receptor (D₂R) and D₃R in clusters but exerted different functional influences on them. The cell surface expression, agonist affinity, endocytosis, and signaling of D₂R were unaffected by the coexpression of PICK1. On the other hand, the surface expression and tolerance of D₃R were inhibited by the coexpression of PICK1. These findings show that PICK1 exerts multiple effects on D₃R functions.

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 3β (GSK3β), and β-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-3β, suggesting that D(2)R inhibited the Wnt signaling by acting on the downstream of GSK3β. D(2)R directly interacted with β-catenin through the second and third loops, leading to a reduction of β-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 β-catenin from cytosol to the plasma membrane, resulting in the decrement of the β-catenin/LEF-1-dependent transcription in the nucleus.