Dong-Im Cho

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.

Molecular mechanisms of inhibitory activities of tanshinones on Lipopolysaccharide-lnduced nitric oxide generation in RAW 264.7 cells

The effects of four tanshinones isolated from Tanshen (the root ofSalvia miltiorrhiza Bunge, Labiatae) were tested for their inhibition of nitric oxide production in macrophage cells, and the underlying molecular mechanisms studied. Of the four tanshinones used, 15, 16-dihydrotanshinone-l, tanshinone-IIA and cryptotanshinone, but not tanshinone I, demonstrated significant inhibition of the LPS-induced nitric oxide production in RAW 264.7 cells, with calculated IC50 values of 5, 8, and 1.5 μM, respectively. Tanshinones exerted inhibitory activities on the LPS-induced nitric oxide production only when applied concurrently with LPS, and tanshinone-IIA and cryptotanshinone were found to inhibit LPS-inducedNF-kB mobilization and extracellular-regulated kinase (ERK) activation, respectively. These results suggest that tanshinones inhibit LPS-induced nitric oxide generation by interfering with the initial stage of LPS-induced expression of certain genes.NF-kB and ERK could be the molecular targets for tanshinones for the inhibition of LPS-induced nitric oxide production in macrophage cells.

An ARIA-interacting AP2 domain protein is a novel component of ABA signaling.

ADAP is an AP2-domain protein that interacts with ARIA, which, in turn, interacts with ABF2, a bZIP class transcription factor. ABF2 regulates various aspects of the abscisic acid (ABA) response by controlling the expression of a subset of ABA-responsive genes. Our expression analyses indicate that ADAP is expressed in roots, emerging young leaves, and flowers. We found that adap knockout mutant lines germinate more efficiently than wild-type plants and that the mutant seedlings grow faster. This suggests that ADAP is involved in the regulation of germination and seedling growth. Both germination and post-germination growth of the knockout mutants were partially insensitive to ABA, which indicates that ADAP is required for a full ABA response. The survival rates for mutants from which water was withheld were low compared with those for wild-type plants. The result shows that ADAP is necessary for the response to stress induced by water deprivation. Together, our data indicate that ADAP is a positive regulator of the ABA response and is also involved in regulating seedling growth. The role of ADAP is similar to that of ARIA, which is also a positive regulator of the ABA response. It appears that ADAP acts through the same ABA response pathway as ARIA.

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.

AtNEK6 interacts with ARIA and is involved in ABA response during seed germination.

ARIA is an ARM repeat protein that is a positive regulator of ABA response. To identify ARIA-interacting proteins, we conducted yeast two-hybrid screening. One of the positive clones obtained from the screen encoded a protein kinase, AtNEK6, which belongs to the NIMA (Never In Mitosis, gene A)-related kinase family. We analyzed AtNEK6 over-expression (OX) and knockout (KO) lines to investigate its in vivo function. The AtNEK6 OX lines grew slowly, whereas the KO line germinated and grew faster than wild type plants. AtNEK6 also affected ABA and stress responses. During seed germination, AtNEK6 OX lines were hypersensitive to ABA and high osmolarity, whereas its KO line was partially insensitive to ABA and high osmolarity. Previously, AtNEK6 was shown to be involved in epidermal cell morphogenesis. Our results indicate that AtNEK6 is also involved in plant growth regulation and responses to ABA and high osmolarity during the seed germination stage.

Direct and biochemical interaction between dopamine D3 receptor and elongation factor-1Bβγ

Novel signaling components of dopamine D3 receptor (D3R) were searched using yeast two-hybrid system, and the gamma subunit of elongation Factor-1B (eEF1Bgamma) was found to interact with D3R. This interaction was observed specifically between eEF1Bgamma and D3R but not with D2R or D4R. Immunocytochemical studies showed that D3R and eEF1Bgamma form clusters on the plasma membrane and their co-localization was evident in these clusters. The beta subunit of eEF1B (eEF1Bbeta), which forms a tight complex with eEF1Bgamma, was phosphorylated on serine residues in response to the stimulation of D3R. Phosphorylation of eEF1Bbeta was insensitive to pertussis toxin or wortmannin, however, stimulation of cellular protein kinase C (PKC) directly phosphorylated eEF1Bbeta and depletion of PKC abolished D3R-mediated phosphorylation of eEF1Bbeta. These results suggest the involvement of PKC, but not Gi/o proteins or phosphatidylinositol 3-kinase, in D3R-mediated phosphorylation of eEF1Bbeta. Stimulation of D3R did not activate PKC, but the activation of PKC resulted in the phosphorylation of D3R. These results show that PKC has a permissive role for the D3R-mediated phosphorylation of eEF1Bbeta, and suggest that PKC could modulate the mutual interaction between two protein by phosphorylating both D3R and eEF1Bbeta. Therefore, the cellular PKC level would be important for the D3R-mediated modulation of eEF1B, and for their cellular regulations such as protein synthesis or cellular proliferation.

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.

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.