효모 이중-중합 시스템을 이용한 G Protein-Coupled Receptor Kinase5(GRK5)의 단백 상호작용 연구

진병철,박태준,김은지,이지은,이정훈,문인규,박정랑,최동주,서봉관 대한순환기학회 2002 Korean Circulation Journal Vol.32 No.7

Ginsenoside Rg1 activates ligand-independent estrogenic effects via rapid estrogen receptor signaling pathway

Gao, Quan-Gui,Zhou, Li-Ping,Lee, Vien Hoi-Yi,Chan, Hoi-Yi,Man, Cornelia Wing-Yin,Wong, Man-Sau The Korean Society of Ginseng 2019 Journal of Ginseng Research Vol.43 No.4

Background: Ginsenoside Rg1 was shown to exert ligand-independent activation of estrogen receptor (ER) via mitogen-activated protein kinase-mediated pathway. Our study aimed to delineate the mechanisms by which Rg1 activates the rapid ER signaling pathways. Methods: ER-positive human breast cancer MCF-7 cells and ER-negative human embryonic kidney HEK293 cells were treated with Rg1 ($10^{-12}M$, $10^{-8}M$), $17{\beta}$-estradiol ($10^{-8}M$), or vehicle. Immunoprecipitation was conducted to investigate the interactions between signaling protein and ER in MCF-7 cells. To determine the roles of these signaling proteins in the actions of Rg1, small interfering RNA or their inhibitors were applied. Results: Rg1 rapidly induced $ER{\alpha}$ translocation to plasma membrane via caveolin-1 and the formation of signaling complex involving linker protein (Shc), insulin-like growth factor-I receptor, modulator of nongenomic activity of ER (MNAR), $ER{\alpha}$, and cellular nonreceptor tyrosine kinase (c-Src) in MCF-7 cells. The induction of extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase (MEK) phosphorylation in MCF-7 cells by Rg1 was suppressed by cotreatment with small interfering RNA against these signaling proteins. The stimulatory effects of Rg1 on MEK phosphorylation in these cells were suppressed by both PP2 (Src kinase inhibitor) and AG1478 [epidermal growth factor receptor (EGFR) inhibitor]. In addition, Rg1-induced estrogenic activities, EGFR and MEK phosphorylation in MCF-7 cells were abolished by cotreatment with G15 (G protein-coupled estrogen receptor-1 antagonist). The increase in intracellular cyclic AMP accumulation, but not Ca mobilization, in MCF-7 cells by Rg1 could be abolished by G15. Conclusion: Ginsenoside Rg1 exerted estrogenic actions by rapidly inducing the formation of ER containing signalosome in MCF-7 cells. Additionally, Rg1 could activate EGFR and c-Src ER-independently and exert estrogenic effects via rapid activation of membrane-associated ER and G protein-coupled estrogen receptor.

Platform Technologies for Research on the G Protein Coupled Receptor: Applications to Drug Discovery Research

( Sung Hou Lee ) 한국응용약물학회 2011 Biomolecules & Therapeutics(구 응용약물학회지) Vol.19 No.1

G-protein coupled receptors (GPCRs) constitute an important class of drug targets and are involved in every aspect of human physiology including sleep regulation, blood pressure, mood, food intake, perception of pain, control of cancer growth, and immune response. Radiometric assays have been the classic method used during the search for potential therapeutics acting at various GPCRs for most GPCR-based drug discovery research programs. An increasing number of diverse small molecules, together with novel GPCR targets identified from genomics efforts, necessitates the use of high-throughput assays with a good sensitivity and specificity. Currently, a wide array of high-throughput tools for research on GPCRs is available and can be used to study receptor-ligand interaction, receptor driven functional response, receptor-receptor interaction, and receptor internalization. Many of the assay technologies are based on luminescence or fluorescence and can be easily applied in cell based models toreduce gaps between in vitro and in vivo studies for drug discovery processes. Especially, cell based models for GPCR can be efficiently employed to deconvolute the integrated information concerning the ligand-receptor-function axis obtained from label free detection technology. This review covers various platform technologies used for the research of GPCRs, concentrating on the principal, non-radiometric homogeneous assay technologies. As current technology is rapidly advancing, the combination of probe chemistry, optical instruments, and GPCR biology will provide us with many new technologies to apply in the future.

Reviews : Atypical Actions of G Protein-Coupled Receptor Kinases

( Hitoshi Kurose ) 한국응용약물학회 2011 Biomolecules & Therapeutics(구 응용약물학회지) Vol.19 No.4

G protein-coupled receptor kinases (GRKs) and β-arrestins have been known as regulators of G protein-coupled receptors. However, it has been recently reported that GRKs and β-arrestins mediate receptor-mediated cellular responses in a G proteinindependent manner. In this scheme, GRKs work as a mediator or a scaffold protein. Among 7 members of the GRK family (GRK1-GRK7), GRK2 is the most extensively studied in vitro and in vivo. GRK2 is involved in cellular migration, insulin signaling, and cardiovascular disease. GRK6 in concert with β-arrestin 2 mediates chemoattractant-stimulated chemotaxis of T and B lymphocytes. GRK5 shuttles between the cytosol and nucleus, and regulates the activities of transcription factors. GRK3 and GRK4 do not seem to have striking effects on cellular responses other than receptor regulation. GRK1 and GRK7 play specifi c roles in regulation of rhodopsin function. In this review, these newly discovered functions of GRKs are briefl y described.

Dysregulation of GPCR Signaling in Cardiovascular Diseases: A Potential Role for Exercise Training?

( Kwang-seok Hong ),( Sukho Lee ) 한국운동생리학회(구 한국운동과학회) 2017 운동과학 Vol.26 No.3

PURPOSE: The superfamily of G protein-coupled receptors (GPCR) are activated by biological molecules (e.g. neurotransmitters) and are involved in many physiological events. As exaggerated GPCR-mediated signaling may contribute to cardiovascular disorders, RGS proteins (regulators of G-protein signaling) are viewed as an important regulatory molecule for the negative modulation of this signaling pathway. To address the significance of RGS proteins and suggest potential effects of exercise training on this molecule, a literature review on RGS proteins was conducted. METHODS: A systemic search in PUBMED was performed to obtain previous studies investigating roles of RGS proteins in the cardio-vascular systems. RESULTS: RGS proteins directly bind to the Gα subunit of heterotrimeric G proteins to inhibit GPCR signaling pathways and terminate their activity. Using mice genetically lacking RGS2 and RGS5, these proteins have been shown to contribute to pressure overloadinduced cardiac remodeling. Further, it has been suggested that systemic knockout of RGS2 protein causes hypertension by potentiating G protein signaling-mediated vascular responses and impairing nitric oxide/cGMP-induced vasorelaxation. Thus, RGS proteins have been suggested as potential drug targets for cardiovascular disorders accompanied by dysregulation of RGS proteins and GPCR signaling. Although exercise training has also been well-documented to strengthen cardiovascular function and ameliorate circulatory diseases, cellular mechanisms underlying the contribution of exercise intervention to RGS-mediated GPCR signaling have not been explored. CONCLUSIONS: This brief review discusses roles played by RGS proteins in the cardiovascular system and suggests future studies for investigating the interaction between exercise training and RGS protein-mediated regulation of GPCR signaling.

Ginsenoside Rg1 activates ligand-independent estrogenic effects via rapid estrogen receptor signaling pathway

Quan-Gui Gao,Li-Ping Zhou,Vien Hoi-Yi Lee,Hoi-Yi Chan,Cornelia Wing-Yin Man,Man-Sau Wong 고려인삼학회 2019 Journal of Ginseng Research Vol.43 No.4

Background: Ginsenoside Rg1 was shown to exert ligand-independent activation of estrogen receptor(ER) via mitogen-activated protein kinaseemediated pathway. Our study aimed to delineate themechanisms by which Rg1 activates the rapid ER signaling pathways. Methods: ER-positive human breast cancer MCF-7 cells and ER-negative human embryonic kidneyHEK293 cells were treated with Rg1 (10 12M, 10 8M), 17ß-estradiol (10 8M), or vehicle. Immunoprecipitationwas conducted to investigate the interactions between signaling protein and ER in MCF-7 cells. To determine the roles of these signaling proteins in the actions of Rg1, small interfering RNA or theirinhibitors were applied. Results: Rg1 rapidly induced ERa translocation to plasma membrane via caveolin-1 and the formation ofsignaling complex involving linker protein (Shc), insulin-like growth factor-I receptor, modulator ofnongenomic activity of ER (MNAR), ERa, and cellular nonreceptor tyrosine kinase (c-Src) in MCF-7 cells. The induction of extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase(MEK) phosphorylation in MCF-7 cells by Rg1 was suppressed by cotreatment with small interferingRNA against these signaling proteins. The stimulatory effects of Rg1 on MEK phosphorylation in thesecells were suppressed by both PP2 (Src kinase inhibitor) and AG1478 [epidermal growth factor receptor(EGFR) inhibitor]. In addition, Rg1-induced estrogenic activities, EGFR and MEK phosphorylation in MCF-7 cells were abolished by cotreatment with G15 (G protein-coupled estrogen receptor-1 antagonist). Theincrease in intracellular cyclic AMP accumulation, but not Ca mobilization, in MCF-7 cells by Rg1 could beabolished by G15. Conclusion: Ginsenoside Rg1 exerted estrogenic actions by rapidly inducing the formation of ER containingsignalosome in MCF-7 cells. Additionally, Rg1 could activate EGFR and c-Src ER-independentlyand exert estrogenic effects via rapid activation of membrane-associated ER and G protein-coupled estrogenreceptor.

Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

( Xiaohan Zhang ),( Kyeong-man Kim ) 한국응용약물학회 2017 Biomolecules & Therapeutics(구 응용약물학회지) Vol.25 No.1

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 extracel-lular 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 β-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 en-docytosis, 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.

Brain Receptor Mosaics and Their Intramembrane Receptor-Receptor Interactions: Molecular Integration in Transmission and Novel Targets for Drug Development

Kjell Fuxe,Daniel Marcellino,Diego Guidolin,Amina S. Woods,Luigi Agnati 사단법인약침학회 2009 Journal of Acupuncture & Meridian Studies Vol.2 No.1

The concept of intramembrane receptor-receptor interactions and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 suggesting the existence of heteromerization of receptors. In 1982, they proposed the existence of aggregates of multiple receptors in the plasma membrane and coined the term receptor mosaics (RM). In this way, cell signaling becomes a branched process beginning at the level of receptor recognition at the plasma membrane where receptors can directly modify the ligand recognition and signaling capacity of the receptors within a RM. Receptorreceptor interactions in RM are classified as operating either with classical cooperativity, when consisting of homomers or heteromers of similar receptor subtypes having the same transmitter, or non-classical cooperativity, when consisting of heteromers. It has been shown that information processing within a RM depends not only on its receptor composition, but also on the topology and the order of receptor activation determined by the concentrations of the ligands and the receptor properties. The general function of RM has also been demonstrated to depend on allosteric regulators (e.g., homocysteine) of the receptor subtypes present. RM as integrative nodes for receptor-receptor interactions in conjunction with membrane associated proteins may form horizontal molecular networks in the plasma membrane coordinating the activity of multiple effector systems modulating the excitability and gene expression of the cells. The key role of electrostatic epitope-epitope interactions will be discussed for the formation of the RM. These interactions probably represent a general molecular mechanism for receptor-receptor interactions and, without a doubt, indicate a role for phosphorylation-dephosphorylation events in these interactions. The novel therapeutic aspects given by the RMs will be discussed in the frame of molecular neurology and psychiatry and combined drug therapy appears as the future way to go. The concept of intramembrane receptor-receptor interactions and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 suggesting the existence of heteromerization of receptors. In 1982, they proposed the existence of aggregates of multiple receptors in the plasma membrane and coined the term receptor mosaics (RM). In this way, cell signaling becomes a branched process beginning at the level of receptor recognition at the plasma membrane where receptors can directly modify the ligand recognition and signaling capacity of the receptors within a RM. Receptorreceptor interactions in RM are classified as operating either with classical cooperativity, when consisting of homomers or heteromers of similar receptor subtypes having the same transmitter, or non-classical cooperativity, when consisting of heteromers. It has been shown that information processing within a RM depends not only on its receptor composition, but also on the topology and the order of receptor activation determined by the concentrations of the ligands and the receptor properties. The general function of RM has also been demonstrated to depend on allosteric regulators (e.g., homocysteine) of the receptor subtypes present. RM as integrative nodes for receptor-receptor interactions in conjunction with membrane associated proteins may form horizontal molecular networks in the plasma membrane coordinating the activity of multiple effector systems modulating the excitability and gene expression of the cells. The key role of electrostatic epitope-epitope interactions will be discussed for the formation of the RM. These interactions probably represent a general molecular mechanism for receptor-receptor interactions and, without a doubt, indicate a role for phosphorylation-dephosphorylation events in these interactions. The novel therapeutic aspects given by the RMs will be discussed in the frame of molecular neurology and psychiatry and combined drug therapy appears as the future way to go.

Biased G Protein-Coupled Receptor Signaling: New Player in Modulating Physiology and Pathology

( Zuzana Bologna ),( Jian-peng Teoh ),( Ahmed S. Bayoumi ),( Yaoliang Tang ),( Il-man Kim ) 한국응용약물학회 2017 Biomolecules & Therapeutics(구 응용약물학회지) Vol.25 No.1

G protein-coupled receptors (GPCRs) are a family of cell-surface proteins that play critical roles in regulating a variety of patho-physiological 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 β-arrestins have important roles in internalization and desensitization of the receptor. Over the past decade, several novel functional aspects of β-arrestins in regulating GPCR signaling have been discovered. These previously unanticipated roles of β-arrestins to act as signal transduc-ers 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 β-arrestin-mediated downstream signaling. This offers the potential for next generation drugs with high selectivity to therapeutically relevant GPCR signaling path-ways. In this review, we provide a summary of the recent studies highlighting G protein- or β-arrestin-biased GPCR signaling and the effects of biased ligands on disease pathogenesis and regulation.

Identification of G protein-coupled receptors in pheromone gland of Maruca vitrata

Wook Hyun Cha,So Young Yu,Jin Kyo Jung,Dae-Weon Lee 한국응용곤충학회 2018 한국응용곤충학회 학술대회논문집 Vol.2018 No.04

G protein-coupled receptors (GPCRs) belong to cell membrane protein family, which regulate various physiological process such as reproduction, behavior and immune etc. In other to identify the GPCRs in pheromone gland of Maruca vitrata, we carried out transcriptome analysis from both females. Transcriptome analysis in the pheromone gland yielded approximately 22Gb and 47,528 transcripts showed positive FPKM value. 48 Genes involved in GPCRs were identified such as pheromone biosynthesis activating neuropeptide receptor (PBANr), prostaglandin receptors, neuropeptide receptor, 5-hydroxytryptamine receptor, galanin receptor, calcitonin gene-related peptide receptor, diuretic hormone receptor, gonadotropin-releasing hormone receptor, frizzled and orphan receptors, etc. Various expression of GPCRs in the pheromone gland indicates the role of pheromone gland may not be limited to the production of pheromone.