• Archives of Pharmacal Research
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• v.18 no.2
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• pp.113-117
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• 1995

We re-cloned mouse ${\delt}-Opioid$receptor from NG108-15 cells using RT-PCR, and confirmed it by restriction analysis and by sequencing the beginning and end part of the amplified DNA. When transiently expressed in COS-7 cells, cloned ${\delt}-Opioid$ receptor showed saturable and specific binding to $[^3H]$naloxone with very similar binding parameters to originally reported ones. To make antibodies specific for the ${\delt}-Opioid$ receptor, the carboxy tail of the receptor, which is unique to the ${\delt}-Opioid$ receptor compared with other opioid receptors, was expressed in bacteria as a ufsion proteinwith glutathione S-transferase. Purified fusion protein selective for ${\delt}-Opioid$ receptor when tested by western blotting using membrane proteins prepared from transfected COS-7 cells. Cloned ${\delt}-Opioid$ receptor andl antibodies specific for ${\delt}-Opioid$ receptor are going to be valuable tools for studying pharmacological actions of the ${\delt}-Opioid$ receptor and morphine dependence.

• The Korean Journal of Pharmacology
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• v.32 no.2
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• pp.151-158
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• 1996

Following the subcutaneous administration of $R(-)N^6-(2-phenylisopropyl)adenosine(600\;nmol/kg/hr)$ to rats for 1 week using t$Alzet^{\circledR}$ mini-osmotic pumps, $A_1$ adenosine receptor functions were determined using $[^3H]DPCPX$ binding, $[^{35}S]GTP_{\gamma}S$ binding, and adenylyl cyclase assays. $A_1$ adenosine receptor binding and the inhibition of adenylyl cyclase activity by PIA was not altered in cerebrocortical membranes prepared from PIA-treated rats. However, there was a significant decrease in the $A_1$ adenosine receptor-mediated stimulation of $[^{35}S]GTP_{\gamma}S$ binding to cerebrocortical membranes prepared from PIA-treated rats(22.0% decrease in basal activity; 19.7% decrease in maximal activity). These results suggest that the desensitization of $A_1$ adenosine receptors following chronic administration involves agonist-induced uncoupling of the receptors from G proteins rather than alteration of $A_1$ adenosine receptor molecules. It is also suggested that the determination of stimulation of $[^{35}S]GTP_{\gamma}S$ binding to G proteins is a suitable tool in studying the receptor regulation including desensitization

• Journal of Life Science
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• v.19 no.11
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• pp.1506-1513
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• 2009

RGS proteins have been identified as negative regulators of G protein signalling pathways and attenuate the activity of GPCR receptors. However, information on the regulatory effects of RGS proteins in the activity of cannabinoid receptors is limited. In this study, the role of RGS proteins on the signal transduction of the CB2 cannabinoid receptor was investigated in HEK293 cells co-transfected with CB2-receptors and plasmids encoding RGS2, RGS3, RGS4 and RGS5. Treatment of cells with WIN55, 212-2, a CB2 receptor agonist, inhibited forskolin-induced cAMP response element (CRE) activity in CB2-transfected HEK293 (CB2-HEK293) cells. This inhibitory effect of WIN 55, 212-2 on CRE activity was reversed by co-transfection of CB2-HEK293 cells with RGS3, but not with RGS2, RGS4 and RGS5. However, endogenous RGS3 protein knocked down by a small interfering siRNA targeting RGS3 gene enhanced inhibition of forskolin induced CRE activity via agonist induced CB2 receptor signal transduction. These results indicate the functional role of endogenous RGS protein in cannabinoid signaling pathways and define receptor-selective roles of endogenous RGS3 in modulating CRE transcriptional responses to agonist induced CB2 receptor activity.

• Journal of Periodontal and Implant Science
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• v.39 no.sup2
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• pp.231-237
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• 2009

Purpose: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite $AB_2$ toxin (CdtB: the enzymatic A subunit; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for $GM_1$ ganglioside. Methods: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to $GM_1$ ganglioside was checked through ELISA. Results: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to $GM_1$ ganglioside, while CdtA alone did not. Conclusions: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.

• Molecules and Cells
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• v.20 no.3
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• pp.378-384
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• 2005

Estrogen metabolites are carcinogenic. The comparative mitogenic activities of $17{\beta}$-estradiol (E2) and four metabolites, 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), $16{\alpha}$-hydroxyestrone ($16{\alpha}$-OHE1) and 2-methoxyestradiol (2-ME), were determined in estrogen receptor(ER)-positive MCF-7 human breast cancer cells. Each of the E2 metabolites caused proliferation of the MCF-7 cells, but only E2 and $16{\alpha}$-OHE1 induced a greater than 20-fold increases in transcripts of the progesterone receptor (PR) gene, a classical ER-mediated gene. This suggests that the mitogenic action of E2 and $16{\alpha}$-OHE1 could result from their effects on gene expression via the ER. E2 metabolites altered the expression of E2-regulated proteins including heat shock proteins (Hsps). $16{\alpha}$-OHE1 and 2-ME as well as E2 increased levels of Hsp56, Hsp60, $Hsp90{\alpha}$ and Hsp110 transcripts, and the patterns of these inductions resembled that of PR. Hsp56 and Hsp60 protein levels were increased by all the E2 metabolites. Levels of the transcripts of 3 E2-upregulated proteins (XTP3-transactivated protein A, protein disulfide isomerase-associated 4 protein and stathmin 1) and an E2-downregulated protein (aminoacylase 1) were also affected by the E2 metabolites. These results suggest that the altered expression of Hsps (especially Hsp56 and Hsp60) by E2 metabolites such as E2, $16{\alpha}$-OHE1 and 2-ME could be closely linked to their mitogenic action.

• Biomolecules & Therapeutics
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• v.25 no.1
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• pp.26-43
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• 2017

Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with ${\beta}$-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.3
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• pp.139-142
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• 2002

To examine intracellular signaling of human $S1P_3\;(hS1P_3),$ a sphingosine 1-phosphate (S1P) receptor in plasma membrane, $hS1P_3$ DNA was transfected into RH7777 rat hepatoma cell line, and the inhibition of forskolin-induced cAMP accumulation and activation of MAP kinases by S1P were tested. In $hS1P_3$ transformants, S1P inhibited forskolin-induced activation of adenylyl cyclase activity by about 80% and activated MAP kinases in dose-dependent and pertussis-toxin (PTX) sensitive manners. In oocytes expressing $hS1P_3$ receptor, S1P evoked $Cl^-$ conductance. These data suggested that PTX-sensitive G proteins are involved in $hS1P_3-mediated$ signaling, especially the positive action of S1P in cell proliferation. The potential advantages of rat hepatoma cells for the research of sphingosine 1-phosphate receptor are discussed.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.270.1-270.1
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• 2002

The signaling pathway of D2 dopamine receptor was studied using yeaslt two-hybrid system.. The 3rd cytoplasmic loop of rat D2 dopamine receptor was used to screen the cDNA library of mouse brain. and ALY was found to interact with it. The interaction in the yeast was observed only with the 3rd cytoplasmic loop of D2 dopamine receptor but not with that of D3 or D4 dopamine receptor. The interaction between two proteins was also confirmed by GST pull-down assay. (omitted)

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.70-70
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• 2003

Estrogen receptor is a ligand-activated transcription factor. Its action depends on the receptor, its ligand, and its coactivator proteins. As a consequence, the concentration of the receptor is a major component that governs the magnitude of the estrogen response. Despite the extensive knowledge on mechanism of estrogen receptor action, regulation of estrogen receptor itself is not very well understood. Estrogen receptor is known to be downregulated under hypoxia leading to inhibition of estrogen receptor mediated transcription activation. We have studied mechanism of loss of estrogen responsiveness under hypoxia. We found that Hif-l${\alpha}$, a major transcription factor regulating hypoxic response, inhibited transcription of estrogen response element driven luciferase gene by expression of HIF-l${\alpha}$/vp16 construct designed to contain transcription activity under normoxia. This loss of estrogen responsiveness appears to be the result of ER${\alpha}$ downregulation. ER${\alpha}$was downregulated at the levels of ligand-biding and protein within l2-24h, and the response was blocked by the proteasome inhibitor MG132, protein synthesis inhibitor cyclohexamide, and tyrosine kinase inhibitor Genistein. These results demonstrate that Hif-l${\alpha}$ downregulates ER${\alpha}$ by proteasome dependent pathway.

• Biomolecules & Therapeutics
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• v.25 no.1
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• pp.12-25
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• 2017

G protein-coupled receptors (GPCRs) are a family of cell-surface proteins that play critical roles in regulating a variety of pathophysiological processes and thus are targeted by almost a third of currently available therapeutics. It was originally thought that GPCRs convert extracellular stimuli into intracellular signals through activating G proteins, whereas ${\beta}$-arrestins have important roles in internalization and desensitization of the receptor. Over the past decade, several novel functional aspects of ${\beta}$-arrestins in regulating GPCR signaling have been discovered. These previously unanticipated roles of ${\beta}$-arrestins to act as signal transducers and mediators of G protein-independent signaling have led to the concept of biased agonism. Biased GPCR ligands are able to engage with their target receptors in a manner that preferentially activates only G protein- or ${\beta}$-arrestin-mediated downstream signaling. This offers the potential for next generation drugs with high selectivity to therapeutically relevant GPCR signaling pathways. In this review, we provide a summary of the recent studies highlighting G protein- or ${\beta}$-arrestin-biased GPCR signaling and the effects of biased ligands on disease pathogenesis and regulation.