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Together with G protein-coupled receptor (GPCR) kinases (GRKs) and β-arrestins, RGS proteins are the major family of molecules that control the signaling of GPCRs. The expression pattern of one of these RGS family members, RGS9-2, coincides with that of the dopamine D(3) receptor (D(3)R) in the brain, and in vivo studies have shown that RGS9-2 regulates the signaling of D2-like receptors. In this study, β-arrestin2 was found to be required for scaffolding of the intricate interactions among the dishevelled-EGL10-pleckstrin (DEP) domain of RGS9-2, Gβ5, R7-binding protein (R7BP), and D(3)R. The DEP domain of RGS9-2, under the permission of β-arrestin2, inhibited the signaling of D(3)R in collaboration with Gβ5. β-Arrestin2 competed with R7BP and Gβ5 so that RGS9-2 is placed in the cytosolic region in an open conformation which is able to inhibit the signaling of GPCRs. The affinity of the receptor protein for β-arrestin2 was a critical factor that determined the selectivity of RGS9-2 for the receptor it regulates. These results show that β-arrestins function not only as mediators of receptor-G protein uncoupling and initiators of receptor endocytosis but also as scaffolding proteins that control and coordinate the inhibitory effects of RGS proteins on the signaling of certain GPCRs.

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]

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]