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

Mechanistic diversity involved in the desensitization of G protein-coupled receptors

Ningning Sun,김경만 대한약학회 2021 Archives of Pharmacal Research Vol.44 No.4

The desensitization of G protein-coupled receptors(GPCRs), which involves rapid loss of responsivenessdue to repeated or chronic exposure to agonists, can occurthrough various mechanisms at diff erent levels of signalingpathways. In this review, the mechanisms of GPCRdesensitization are classifi ed according to their occurrenceat the receptor level and downstream to the receptor. Thedesensitization at the receptor level occurs in a phosphorylation-dependent manner, wherein the activated receptors arephosphorylated by GPCR kinases (GRKs), thereby increasingtheir affi nities for arrestins. Arrestins bind to receptorsthrough the cavity on the cytoplasmic region of heptahelicaldomains and interfere with the binding and activation ofG-protein. Diverse mechanisms are involved in the desensitizationthat occurs downstream of the receptor. Some ofthese include the sequestration of G proteins, such as G qand G i/o by GRK2/3 and deubiquitinated arrestins, respectively. Mechanistically, GRK2/3 attenuates GPCR signalingby sequestering the G α subunits of the G q family and G βγ viaregulators of G protein signaling and pleckstrin homologydomains, respectively. Moreover, studies on G i/o -coupledD2-like receptors have reported that arrestins are deubiquitinatedunder desensitization condition and form a stablecomplex with G βγ , thereby preventing them from couplingwith G α and the receptor, eventually leading to receptor signalinginhibition. Notably, the desensitization mechanism thatinvolves arrestin deubiquitination is interesting; however,this is a new mechanism and needs to be explored further.

최적의 luminescence 신호 분석을 위한 유전자 전달 방법의 비교연구

박서현(Seohyun Park),이승호(Sunghou Lee) 한국산학기술학회 2016 한국산학기술학회논문지 Vol.17 No.11

형광 간섭 현상을 최소화시켜 상대적으로 민감한 측정이 가능한 aequorin기반 luminescence기술은 Gα16 단백질 도입을 통해 세포 내부의 칼슘 이동 신호를 감지하여 G 단백질 결합 수용체(G protein-coupled receptor, GPCR)의 기능 분석을 가능하게 하는 세포 기반 분석 기술로 수용체 및 G 단백질 유전자 전달의 최적화 과정이 필수적이다. 본 연구를 위해 corticotropin releasing factor receptor subtype 2(CRF2) 수용체를 모델 시스템으로 CRF2와 Gα16 단백질이 구축된 세 가지 안정화 세포주를 제작하였고, 이들을 이용한 서로 다른 세 가지 조건의 임시 발현 세포주에서 작용제(sauvagine)와 길항제(K41498)의 반응성을 분석하여 최적의 유전자 전달 방법을 도출하고자 하였다. 그 결과 sauvagine 및 K41498의 농도에 따른 반응에서 CRF2-Gα16 안정화 세포주가 임시 발현 세포주보다 10배 이상의 유효신호 비율을 나타내었고(z’=0.77) 임시 발현 세포주의 경우 Gα16의 안정화 발현 이후에 CRF2를 전달하는 경우가 다른 임시 발현 조건보다 2배 이상 높은 효율을 보였다(z’=0.84). 따라서 임시 유전자 전달 기술을 GPCR 세포 기능 분석 시스템에 활용할 경우 Gα16 단백질에 대한 안정화 세포주를 우선적으로 구축하고, 목표하는 다양한 수용체들을 단계적으로 발현시키는 것이 최선의 방법이라 판단된다. By minimizing fluorescence interference phenomena, aequorin-based luminescence technology can provide a relatively sensitive detection platform with integration of Gα16 protein in order to track internal calcium mobilization by G protein-coupled receptors (GPCR). In this type of cell-based functional assay format, it is essential to optimize the transfection process of a receptor and Gα16 protein. For this study, corticotropin releasing factor receptor subtype 2(CRF2) was set as a model system to generate three stable cells with CRF2 and Gα16 in addition to transiently transfected cells under three different conditions. Agonist (sauvagine) and antagonist (K41498) responses in those cells were analyzed to develop the optimum transfection process. As a result, the effective signal ratio in the dose response experiments of sauvagine and K41498 were at least 10-fold higher (z"=0.77) in CRF2-Gα16 stable cells. For the transient transfection cells, stable expression of Gα16 prior to the CRF2 represented a two-fold higher signal (z"=0.84) than the other cases of transient transfection. In conclusion, for the utilization of transient transfection processes to develop a cell-based GPCR functional assay system, it is suggested to introduce various target receptors after stable expression of Gα16 protein.

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.

Structural and Functional Analysis of a β₂-Adrenergic Receptor Complex with GRK5

Komolov, Konstantin E.,Du, Yang,Duc, Nguyen Minh,Betz, Robin M.,Rodrigues, Joã,o P.G.L.M.,Leib, Ryan D.,Patra, Dhabaleswar,Skiniotis, Georgios,Adams, Christopher M.,Dror, Ron O.,Chung, Ka Young Cell Press 2017 Cell Vol. No.

<P><B>Summary</B></P> <P>The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β<SUB>2</SUB>-adrenergic receptor (β<SUB>2</SUB>AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the β<SUB>2</SUB>AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs.</P> <P><B>PaperClip</B></P> <P>Display Omitted</P> <P><B>Highlights</B></P> <P> <UL> <LI> GRK5-β<SUB>2</SUB>AR binding is enhanced by receptor and kinase ligands and acidic lipids </LI> <LI> GRK5 binding to the β<SUB>2</SUB>AR involves a multi-site interaction </LI> <LI> Receptor binding triggers substantial conformational changes in GRK5 </LI> <LI> RH/catalytic domain separation in GRK5 is essential for receptor phosphorylation </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

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

Bologna, Zuzana,Teoh, Jian-peng,Bayoumi, Ahmed S.,Tang, Yaoliang,Kim, Il-man The Korean Society of Applied Pharmacology 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 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.

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.

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.

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.