• Biomolecules & Therapeutics
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• v.25 no.1
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• pp.4-11
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• 2017

Heterotrimeric G proteins are key intracellular coordinators that receive signals from cells through activation of cognate G protein-coupled receptors (GPCRs). The details of their atomic interactions and structural mechanisms have been described by many biochemical and biophysical studies. Specifically, a framework for understanding conformational changes in the receptor upon ligand binding and associated G protein activation was provided by description of the crystal structure of the ${\beta}2$-adrenoceptor-Gs complex in 2011. This review focused on recent findings in the conformational dynamics of G proteins and GPCRs during activation processes.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.96-96
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• 2002

We examined the role of SR 144528 (N-[-(1S-endo-1,3,,3-trimethyl-bicycle[2, 2, 1 ] heptan-2-y1]-5-(-4-chloro-3-mothyl-phenyl)-(4-methylbenzyl)-pyrazole-3- carboxamide) in the modulation of certain AC isoforms in transiently transfected COS-7 cells. We found that CB2 in COS cells has a constitutive activity, and thus leading to inhibition of AC-V activity even in the absence of agonist. In addition, this constitutive modulation of AC is reversed by SR144528. It is now well established that several G protein-coupled receptors can signal without agonist stimulation(constitutive receptors). Inverse agonists have been shown to inhibit the activity of such constitutive G protein-coupled receptor signaling. Agonist activation of the G$\_$i/o/-coupled peripheral cannabinoid receptor CB2 normally inhibits adenylyl cyclase type V and stimulates adenylyl cyclase type II. Using transfected COS cells, we show here that application of SR144528, an inverse agonist of CB2, leads to a reverse action (stimulation of adenylyl cyclase V and inhibition of adenylyl cyclase II). This inverse agonism of SR144528 is dependent on the temperature, as well as on the concentration of the cDNA of CB2 transfected. Pertussis toxin blocked the regulation of adenylyl cyclase activity by SR 144528.

• Journal of Applied Biological Chemistry
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• v.44 no.3
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• pp.125-130
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• 2001

A putative protein encoded by Arabidopsis AtMTN3 gene, a homologue of Medicago truncatula MTN3, consists of 285 amino acid residues, and has a predicted molecular mass of 31.5 kDa and a calculated pI of 9.1. Primary amino acid sequence analyses have revealed that the protein contains seven putative transmembrane regions with N-terminus oriented to the outside of the membrane. The AtMTN3 protein shows overall 16.4% of amino acid identity with the rat GALR3 protein, known to be a G-protein-coupled receptor. The gene is present as a single copy in the Arabidopsis genome, and expressed in aerial parts but not in roots of Arabidopsis. Therefore, AtMTN3 appears not to be specifically involved in Rhizobium-induced nodule development, as was predicted for the MTN3 gene. These proteins possibly mediate signal transmission through G-protein-coupled pathways during general interactions between plants and symbiotic or pathogenic microbes.

• Biomolecules & Therapeutics
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• v.27 no.6
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• pp.514-521
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• 2019

G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished ${\beta}$-arrestin-mediated desensitization, resulting in increased Gs signaling.

• Molecules and Cells
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• v.38 no.2
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• pp.105-111
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• 2015

The beta2-adrenergic receptor (${\beta}2AR$) belongs to the G protein coupled receptor (GPCR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound ${\beta}2AR$ in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, ${\beta}2AR$ is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of ${\beta}2AR$. In this review, structural features of inactive and active states of ${\beta}2AR$, the interaction of ${\beta}2AR$ with heterotrimeric G protein, and the comparison with ${\beta}1AR$ will be discussed.

• Journal of Ginseng Research
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• v.43 no.4
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• pp.527-538
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• 2019

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.

• Journal of Integrative Natural Science
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• v.6 no.1
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• pp.16-20
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• 2013

G-protein-coupled receptor (GPCR) superfamily is the largest known receptor family, characterized by seven transmembrane domains and considered to be an important drug target. In this study we focused on an orphan GPCR termed as GPR18. As there is no X-ray crystal structure has been reported for this receptor, we report on a homology model of GPR18. Template structure with high homology was used for modeling and ten models were developed. A model was selected and refined by energy minimization. The selected model was further validated using various parameters. Our results could be a starting point for further structure based drug design.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.17 no.11
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• pp.640-647
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• 2016

By minimizing fluorescence interference phenomena, aequorin-based luminescence technology can provide a relatively sensitive detection platform with integration of $G{\alpha}16$ 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 $G{\alpha}16$ 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 $G{\alpha}16$ 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-$G{\alpha}16$ stable cells. For the transient transfection cells, stable expression of $G{\alpha}16$ 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 $G{\alpha}16$ protein.

• Toxicological Research
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• v.29 no.3
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• pp.149-155
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• 2013

G protein-coupled receptors (GPCRs) are membrane receptors; approximately 40% of drugs on the market target GPCRs. A precise understanding of the activation mechanism of GPCRs would facilitate the development of more effective and less toxic drugs. Heterotrimeric G proteins are important molecular switches in GPCR-mediated signal transduction. An agonist-activated receptor interacts with specific sites on G proteins and promotes the release of GDP from the $G{\alpha}$ subunit. Because of the important biological role of the GPCR-G protein coupling, conformational changes in the G protein upon receptor coupling have been of great interest. One of the most important questions was the interface between the GPCR and G proteins and the structural mechanism of GPCR-induced G protein activation. A number of biochemical and biophysical studies have been performed since the late 80s to address these questions; there was a significant breakthrough in 2011 when the crystal structure of a GPCR-G protein complex was solved. This review discusses the structural aspects of GPCR-G protein coupling by comparing the results of previous biochemical and biophysical studies to the GPCR-G protein crystal structure.

• Biomolecules & Therapeutics
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• v.24 no.5
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• pp.517-522
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• 2016

${\beta}$-Arrestins are one of the protein families that interact with G protein-coupled receptors (GPCRs). The roles of ${\beta}$-arrestins are multifaceted, as they mediate different processes including receptor desensitization, endocytosis, and G protein-independent signaling. Thus, determining the GPCR regions involved in the interactions with ${\beta}$-arrestins would be a preliminary step in understanding the molecular mechanisms involved in the selective direction of each function. In the current study, we determined the roles of the N-terminus, intracellular loops, and C-terminal tail of a representative GPCR in the interaction with ${\beta}$-arrestin2. For this, we employed dopamine $D_2$ and $D_3$ receptors ($D_2R$ and $D_3R$, respectively), since they display distinct agonist-induced interactions with ${\beta}$-arrestins. Our results showed that the second and third intracellular loops of $D_2R$ are involved in the agonist-induced translocation of ${\beta}$-arrestins toward plasma membranes. In contrast, the N- and C-termini of $D_2R$ exerted negative effects on the basal interaction with ${\beta}$-arrestins.