• 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.

• Journal of the Korea Society of Computer and Information
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• v.14 no.1
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• pp.73-80
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• 2009

G protein-coupled receptors(GPCRs) family is a cell membrane protein, and plays an important role in a signaling mechanism which transmits external signals through cell membranes into cells. But, GPCRs each are known to have various complex control mechanisms and very unique signaling mechanisms. Structural features, and family and subfamily of GPCRs are well known by function. and accordingly, the most fundamental work in studies identifying the previous GPCRs is to classify the GPCRs with given protein sequences. Studies for classifying previously identified GPCRs more easily with mathematical models have been mainly going on. In this paper Considering that functions of proteins are determined by their stereoscopic structures, the present paper proposes a method to compare secondary structures of two GPCRs having different amino acid sequences, and then detect an unknown GPCRs assumed to have a same function in databases of previously identified GPCRs.

• 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.

• 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.

• Biomolecules & Therapeutics
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• v.25 no.3
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• pp.239-248
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• 2017

Desensitization and acute tolerance are terms used to describe the attenuation of receptor responsiveness by prolonged or intermittent exposure to an agonist. Unlike desensitization of G protein-coupled receptors (GPCRs), which is commonly explained by steric hindrance caused by the ${\beta}$-arrestins that are translocated to the activated receptors, molecular mechanisms involved in the acute tolerance of GPCRs remain unclear. Our studies with several GPCRs and related mutants showed that the acute tolerance of GPCRs could occur independently of agonist-induced ${\beta}$-arrestin translocation. A series of co-immunoprecipitation experiments revealed a correlation between receptor tolerance and interactions among receptors, ${\beta}$-arrestin2, and $G{\beta}{\gamma}$. $G{\beta}{\gamma}$ displayed a stable interaction with receptors and ${\beta}$-arrestin2 in cells expressing GPCRs that were prone to undergo tolerance compared to the GPCRs that were resistant to acute tolerance. Strengthening the interaction between $G{\beta}{\gamma}$ and ${\beta}$-arrestin rendered the GPCRs to acquire the tendency of acute tolerance. Overall, stable interaction between the receptor and $G{\beta}{\gamma}$ complex is required for the formation of a complex with ${\beta}$-arrestin, and determines the potential of a particular GPCR to undergo acute tolerance. Rather than turning off the signal, ${\beta}$-arrestins seem to contribute on continuous signaling when they are in the context of complex with receptor and $G{\beta}{\gamma}$.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.6
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• pp.383-388
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• 2011

Regulators of G-protein signaling (RGS) proteins are regulators of $Ca^{2+}$ signaling that accelerate the GTPase activity of the G-protein ${\alpha}$ -subunit. RGS1, RGS2, RGS4, and RGS16 are expressed in the pancreas, and RGS2 regulates G-protein coupled receptor (GPCR)-induced $Ca^{2+}$ oscillations. However, the role of RGS4 in $Ca^{2+}$ signaling in pancreatic acinar cells is unknown. In this study, we investigated the mechanism of GPCR-induced $Ca^{2+}$ signaling in pancreatic acinar cells derived from $RGS4^{-/-}$ mice. $RGS4^{-/-}$ acinar cells showed an enhanced stimulus intensity response to a muscarinic receptor agonist in pancreatic acinar cells. Moreover, deletion of RGS4 increased the frequency of $Ca^{2+}$ oscillations. $RGS4^{-/-}$ cells also showed increased expression of sarco/endoplasmic reticulum $Ca^{2+}$ ATPase type 2. However, there were no significant alterations, such as $Ca^{2+}$ signaling in treated high dose of agonist and its related amylase secretion activity, in acinar cells from $RGS4^{-/-}$ mice. These results indicate that RGS4 protein regulates $Ca^{2+}$ signaling in mouse pancreatic acinar cells.

• Genomics & Informatics
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• v.10 no.3
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• pp.153-166
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• 2012

A variety of ligands differ in their capacity to bind the receptor, elicit gene expression, and modulate physiological responses. Such receptors include Toll-like receptors (TLRs), which recognize various patterns of pathogens and lead to primary innate immune activation against invaders, and G-protein coupled receptors (GPCRs), whose interaction with their cognate ligands activates heterotrimeric G proteins and regulates specific downstream effectors, including immuno-stimulating molecules. Once TLRs are activated, they lead to the expression of hundreds of genes together and bridge the arm of innate and adaptive immune responses. We characterized the gene expression profile of Toll-like receptor 4 (TLR4) in RAW 264.7 cells when it bound with its ligand, 2-keto-3-deoxyoctonate (KDO), the active part of lipopolysaccharide. In addition, to determine the network communications among the TLR, Janus kinase (JAK)/signal transducer and activator of transcription (STAT), and GPCR, we tested RAW 264.7 cells with KDO, interferon-${\beta}$, or cAMP analog 8-Br. The ligands were also administered as a pair of double and triple combinations.

• Journal of Life Science
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• v.17 no.6 s.86
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• pp.783-790
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• 2007

G protein coupled receptors (GPCRs) transmit various extracellular signals into the cells. Upon binding of the ligands, conformational changes in the extracellular and/or transmembrane (TM) domains of CPCRs were propagated into the cytoplasmic (CP) domain of the molecule leading to the activation of their cognate heterotrimeric C proteins and kinases. Constitutively active GPCR mutants causing the activation of C Protein signaling even in the absence of ligand binding are of interest for the study of activation mechanism of GPCRs. Two classes of constitutively active mutations, categorized by their effects on the salt bridge between Ell3 and K296, were found in the TM domain of rhodopsin. Opsin mutants containing combinations of the mutations were constructed to study the conformational changes required for the activation of rhodopsin. Rhodopsin chromophore regenerated with 11-cis-retinal showed a thermal stability inversely correlated with its constitutive activity. In contrast, rhodopsin mutants exhibited a binding affinity to an agonist, all-trans-retinal, in a constitutive activity-dependent manner. In order to test whether the conformational changes responsible for the activation of trans-ducin (Gt) are the same as the conformation required for the recognition of rhodopsin kinase, analysis of the mutants were carried out with phosphorylation by rhodopsin kinase. Rhodopsin mutants containing combinations of different classes of the mutations showed a strong synergistic effect on the phosphorylation of the mutants in the dark as similar to that of Gt activation. The results suggest that at least two or three kinds of segmental and independent conformational changes are required for the activation of rhodopsin and the conformational changes responsible for activating rhodopsin kinase and Gt are similar to each other.

• Korean Journal of Exercise Nutrition
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• v.25 no.3
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• pp.28-35
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• 2021

[Purpose] As Panax ginseng C. A. Meyer (ginseng) exhibits various physiological activities and is associated with exercise, we investigated the potential active components of ginseng and related target genes through network pharmacological analysis. Additionally, we analyzed the association between ginseng-related genes, such as the G-protein-coupled receptors (GPCRs), and improved exercise capacity. [Methods] Active compounds in ginseng and the related target genes were searched in the Traditional Chinese Medicine Database and Analysis Platform (TCMSP). Gene ontology functional analysis was performed to identify biological processes related to the collected genes, and a compound-target network was visualized using Cytoscape 3.7.2. [Results] A total of 21 ginseng active compounds were detected, and 110 targets regulated by 17 active substances were identified. We found that the active compound protein was involved in the biological process of adrenergic receptor activity in 80%, G-protein-coupled neurotransmitter in 10%, and leucocyte adhesion to arteries in 10%. Additionally, the biological response centered on adrenergic receptor activity showed a close relationship with G protein through the beta-1 adrenergic receptor gene reactivity. [Conclusion] According to bioavailability analysis, ginseng comprises 21 active compounds. Furthermore, we investigated the ginseng-stimulated gene activation using ontology analysis. GPCR, a gene upregulated by ginseng, is positively correlated to exercise. Therefore, if a study on this factor is conducted, it will provide useful basic data for improving exercise performance and health.

• Journal of Life Science
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• v.22 no.7
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• pp.863-870
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• 2012

Rhodopsin, a dim light photoreceptor, has been regarded as one of the model systems for the structural and functional study of G protein-coupled receptors (GPCRs). Constitutively active mutant GPCRs leading to the activation of heterotrimeric GDP/GTP-binding protein signaling in the absence of ligand binding are of interest for the study of the activation mechanism in GPCRs. The present study focused on the opsin mutant E134Q/M257Y, which showed a moderate level of constitutive activity and the formation of two distinct rhodopsin chromophores with absorption maxima of 500 nm and 380 nm, depending on the presence of an inverse agonist, 11-cis-retinal, and an agonist, all-trans-retinal, respectively. Reconstitution of the mutant rhodopsin upon incubation with different ratios of 11-cis-retinal and the all-trans-retinal, as well as upon sequential binding of the two retinals, indicated its preferential binding to 11-cis-retinal. The thermal stability of the 11-cis-retinal-bound form of the E134Q/M257Y mutant was lower than that of the mutants containing a single replacement but higher than that of the all-trans-retinal-bound forms. The mutant also showed a lower stability in its opsin state as compared with that of the wild-type opsin but had little effects on the binding affinity to 11-cis-retinal. Information obtained in this study will be helpful for analyzing the structural changes associated with the activation of rhodopsin and GPCRs.