• 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.

• The Korean Journal of Food And Nutrition
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• v.35 no.6
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• pp.531-536
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• 2022

Umami taste-yielding foods, such as, Joseonganjang, dried anchovies, dried shiitake, dried Konbu (kelp), and Yukjeot, are widely used in the Korean cuisine as soup base. While Umami taste enhancement related to Kokumi taste substances has been proposed in human sensory studies, the potential action of Kokumi taste substances has not been explored on calcium-sensing receptors (CaSR), here referred to as Kokumi taste receptors. In this study, we investigated the effect of Umami taste-yielding foods on Kokumi taste receptors using cells expressing human CaSR. We monitored the temporal changes in intracellular Ca²⁺ in HEK293T cells expressing CaSR in response to aqueous extract of Joseonganjang, dried anchovies, dried shiitake, dried Konbu, and Yukjeot. Kokumi substances tested-glutathione and γ-Glu-Val-Gly- evoked intracellular Ca²⁺ influx in a concentration-dependent manner. A similar increment of intracellular Ca²⁺ influx was induced by Joseonganjang, Yukjeot, and dried anchovies, but not by dried shiitake and dried Konbu. Only Joseonganjang- and Yukjeot-evoked intracellular Ca²⁺ influx was significantly reduced by NPS 2143, a CaSR-specific antagonist. These data indicated that some Umami substances/Umami-yielding materials could activate CaSR, but this property was not observed for all the Umami tasting substances.