Chengchun Min

Agonist-Induced Endocytosis and Receptor Phosphorylation Mediate Resensitization of Dopamine D2 Receptors

The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D(2) receptors (D(2)Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for beta-arrestins was altered. Agonist-induced internalization of D(2)Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D(2)R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D(2)R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D(2)R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D(2)R-intracellular loop 2/3 provoked desensitization as in wild-type D(2)R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D(2)R. When dissociation between D(2)R and beta-arrestin was inhibited or when the expression of cellular beta-arrestins was decreased, agonist-induced desensitization of D(2)R did not occur, suggesting that dissociation from beta-arrestin is the main cellular process required for resensitization of D(2)R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with beta-arrestin plays a major role in agonist-induced desensitization.

β-Arrestin2 Plays Permissive Roles in the Inhibitory Activities of RGS9-2 on G Protein-Coupled Receptors by Maintaining RGS9-2 in the Open Conformation

ABSTRACT 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 D3 receptor (D3R) 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 D3R. The DEP domain of RGS9-2, under the permission of β-arrestin2, inhibited the signaling of D3R 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.

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 D2 receptor were investigated.

Novel Regulatory Mechanism of Canonical Wnt Signaling by Dopamine D2 Receptor through Direct Interaction with β-catenin

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, D2 receptor (D2R) selectively inhibited the Wnt signaling, which was measured by lymphoid enhancing factor-1 (LEF-1)-dependent transcriptional activities. D2R-mediated inhibition of Wnt signaling was agonist- and G protein-independent and did not require receptor phosphorylation or endocytosis. D2R inhibited the LEF-1-dependent transcriptional activities, and this inhibitory activity was not affected by the inhibition of GSK-3β, suggesting that D2R inhibited the Wnt signaling by acting on the downstream of GSK3β. D2R 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.

Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors.

Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

Novel roles for β‐arrestins in the regulation of pharmacological sequestration to predict agonist‐induced desensitization of dopamine D3 receptors

In addition to typical GPCR kinase (GRK)‐/β‐arrestin‐dependent internalization, dopamine D3 receptor employed an additional GRK‐independent sequestration pathway. In this study, we investigated the molecular mechanism of this novel sequestration pathway.

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

Functional roles of the N‐terminal region of rhodopsin‐like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N‐terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N‐terminal region.

Characterization of functional roles of DRY motif in the 2nd intracellular loop of dopamine D2 and D3 receptors

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.

RGS4 exerts inhibitory activities on the signaling of dopamine D2 receptor and D3 receptor through the N-terminal region.

Dopamine D(2) receptor and D(3) receptor (D(2)R and D(3)R) 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 D(2)R and D(3)R. RGS proteins were co-expressed with D(2)R and D(3)R in HEK-293 cells. The protein interactions between RGS proteins and D(2)R/D(3)R, and effects of RGS proteins on the internalization, signaling, and desensitization of D(2)R/D(3)R 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 D(2)R/D(3)R was determined. All of RGS proteins we tested interacted with D(2)R/D(3)R. RGS4 exerted potent inhibitory activities on the signaling of D(2)R/D(3)R. RGS9-2 exerted selective but moderate inhibitory activity on D(3)R and the internalization of D(2)R. RGS2 had no effect. The N-terminal domain of RGS4 was involved in its interaction with D(2)R and D(3)R 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 D(2)R and D(3)R.

Agonist-induced changes in RalA activities allows the prediction of the endocytosis of G protein-coupled receptors.

GTP binding proteins are classified into two families: heterotrimeric large G proteins which are composed of three subunits, and one subunit of small G proteins. Roles of small G proteins in the intracellular trafficking of G protein-coupled receptors (GPCRs) were studied. Among various small G proteins tested, GTP-bound form (G23V) of RalA inhibited the internalization of dopamine D2 receptor independently of the previously reported downstream effectors of RalA, such as Ral-binding protein 1 and PLD. With high affinity for GRK2, active RalA inhibited the GPCR endocytosis by sequestering the GRK2 from receptors. When it was tested for several GPCRs including an endogenous GPCR, lysophosphatidic acid receptor 1, agonist-induced conversion of GTP-bound to GDP-bound RalA, which presumably releases the sequestered GRK2, was observed selectively with the GPCRs which have tendency to undergo endocytosis. Conversion of RalA from active to inactive state occurred by translocation of RGL, a guanine nucleotide exchange factor, from the plasma membrane to cytosol as a complex with Gβγ. These results suggest that agonist-induced Gβγ-mediated conversion of RalA from the GTP-bound form to the GDP-bound form could be a mechanism to facilitate agonist-induced internalization of GPCRs.