Dual‐color reporter switching system to discern dimer formations of G‐protein‐coupled receptors using Cre/<i>loxP</i> site‐specific recombination in yeast

Nakamura, Yasuyuki; Hashimoto, Takamichi; Ishii, Jun; Kondo, Akihiko

doi:10.1002/bit.25974

Cited by 5 publications

(4 citation statements)

References 39 publications

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“…Reporter gene expression was successfully switched in the engineered yeast cells and permitted the detection of the dimerized yeast endogenous pheromone receptor (Ste2p). The authors also validated the applicability of this system for monitoring the formation of human GPCRs homodimers and heterodimers, specifically human serotonin 1A receptor or β2-adrenergic receptor, and confirmed that this system had improved sensitivity when compared with the previous system [134]. Using a modified split-ubiquitin mY2H approach, Sokolina et al reported the systematic interactome analysis of 48 clinically important human GPCRs in their ligand-unoccupied state [135].…”

Section: Peripheral Membrane Proteinsmentioning

confidence: 75%

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Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

Nakamura¹,

Kondo²,

Ishii³

2018

Peripheral Membrane Proteins

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Guanine nucleotide-binding proteins (G-proteins) act as transducers of external stimuli for intracellular signaling, and control various cellular processes in cooperation with seven transmembrane G-protein-coupled receptors (GPCRs). Because GPCRs constitute the largest family of eukaryotic membrane proteins and enable the selective recognition of a diverse range of molecules (ligands), they are the major molecular targets in pharmaceutical and medicinal fields. In addition, GPCRs have been known to form heteromers as well as homomers, which may result in vast physiological diversity and provide opportunities for drug discovery. G-proteins and their signal transduction machinery are universally conserved in eukaryotes; thereby, the yeast Saccharomyces cerevisiae has been used to construct artificial in vivo GPCR biosensors. In this chapter, we focus on the yeast-based GPCR biosensors that can detect ligand stimulation and oligomer formation, and summarize their techniques using the G-protein signaling and protein-protein interaction assays.

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Section: Peripheral Membrane Proteinsmentioning

confidence: 75%

“…Furthermore, the authors subsequently designed a reporter switching system that can switch the expressions between two reporter genes (one from ON to OFF and the other from OFF to ON) in response to the Y2H readout (Figure 2C and D) [134]. Cre/loxP site-specific recombination was employed to induce reporter switching.…”

Section: Peripheral Membrane Proteinsmentioning

confidence: 99%

Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

Nakamura¹,

Kondo²,

Ishii³

2018

Peripheral Membrane Proteins

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“…The Cre recombinase and loxP sequence are both necessary for the initiation of the recombination reaction. Hence, this system can be applied in different ways such as fate mapping and gene regulation [ 23 , 24 , 25 , 26 ]. It was well known that Cre and loxP have a unique role in DNA recombination.…”

Section: Introductionmentioning

confidence: 99%

Differentiation Capacity of Bone Marrow-Derived Rat Mesenchymal Stem Cells from DsRed and Cre Transgenic Cre/loxP Models

Tseng

Lee

et al. 2022

Cells

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Cre/loxP recombination is a well-established technique increasingly used for modifying DNA both in vitro and in vivo. Nucleotide alterations can be edited in the genomes of mammalian cells, and genetic switches can be designed to target the expression or excision of a gene in any tissue at any time in animal models. In this study, we propose a system which worked via the Cre/loxP switch gene and DsRed/emGFP dual-color fluorescence imaging. Mesenchymal stem cells (MSCs) can be used to regenerate damaged tissue because of their differentiation capacity. Although previous studies have presented evidence of fusion of transplanted MSCs with recipient cells, the possibility of fusion in such cases remains debated. Moreover, the effects and biological implications of the fusion of MSCs at the tissue and organ level have not yet been elucidated. Thus, the method for determining this issue is significant and the models we proposed can illustrate the question. However, the transgenic rats exhibited growth slower than that of wild-type rats over several weeks. The effects on the stemness, proliferation, cell cycle, and differentiation ability of bone marrow–derived rat MSCs (BM-rMSCs) from the models were examined to ensure our design was appropriate for the in vivo application. We demonstrated that MSC surface markers were maintained in DsRed and Cre transgenic rMSCs (DsRed-rMSCs and Cre-rMSCs, respectively). A WST-8 assay revealed decreased proliferative activity in these DsRed-rMSCs and Cre-rMSCs; this result was validated through cell counting. Furthermore, cell cycle analysis indicated a decrease in the proportion of G1-phase cells and a concomitant increase in the proportion of S-phase cells. The levels of cell cycle–related proteins also decreased in the DsRed-rMSCs and Cre-rMSCs, implying decelerated phase transition. However, the BM-rMSCs collected from the transgenic rats did not exhibit altered adipogenesis, osteogenesis, or chondrogenesis. The specific markers of these types of differentiation were upregulated after induction. Therefore, BM-rMSCs from DsRed and Cre transgenic models can be used to investigate the behavior of MSCs and related mechanisms. Such application may further the development of stem cell therapy for tissue damage and other diseases.

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Section: Membrane Y2h Technology To Study Gpcr Oligomers In Yeast Cellsmentioning

confidence: 66%

Peripheral Membrane Proteins

Encyclopedia of Cancer

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Guanine nucleotide exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to physical and chemical stimuli and ultimately regulate numerous cellular responses such as polarized growth, morphogenesis, and movement. The GEF proteins are characterized by a Dbl-homology (DH) domain that contacts the Rho GTPases, to catalyzing nucleotide exchange, and an associated Pleckstrin homology to the binding of phosphoinositides. Most GEFs are divergent in regions outside the DH/ PH module and contain additional protein-protein or lipid-protein interaction domains that presumably dictate unique cellular functions. Fission yeast Rho1-GEFs act as a link between growth processes and the cell cycle machinery. In this chapter, we focus on the recent leaps in our understanding of how Rho1-GEFs control interphase and cytokinesis roles of Rho1-GEFs in the DNA damage response.

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Dual‐color reporter switching system to discern dimer formations of G‐protein‐coupled receptors using Cre/loxP site‐specific recombination in yeast

Cited by 5 publications

References 39 publications

Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

Differentiation Capacity of Bone Marrow-Derived Rat Mesenchymal Stem Cells from DsRed and Cre Transgenic Cre/loxP Models

Peripheral Membrane Proteins

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