Molecular cloning and heterologous expression of GPR120 from the mouse taste tissue

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Abstract

The existence of fat taste along with the generally recognized taste modalities (sweet, bitter, umami, salty, and sour) is currently a subject of scientific debate and active research. Available data on the signaling cascade triggered by long-chain fatty acids (FAs) in the taste cell indicate its similarity to the transduction of sweet, bitter, and umami stimuli, but the initial stages of transduction of fatty stimuli remain unclear. A member of the G-protein-coupled receptor superfamily, GPR120, is considered as one of the candidates for the role of a long-chain FA receptor operating in the taste bud. At the same time, available reports implicating GPR120 in the FA perception by the peripheral taste system are highly contradictory. In order to create a platform for further study of the contribution of GPR120 to FA transduction, we cloned GPR120 from the mouse taste tissue and expressed it in HEK-293 cells. In contrast to the parental HEK-293 cells, GPR120-positive HEK-293 cells generated receptor-like Ca2+ transients in response to long-chain FA application, thus confirming the literature data on the coupling of the GPR120 receptor to the phosphoinositide cascade and intracellular Ca2+ mobilization. The HEK-293 cells expressing recombinant GPR120 receptor may present a useful cell model for screening natural and synthetic ligands of this receptor and analyzing its coupling to intracellular signaling pathways. Co-expression of GPR120 with other signaling proteins involved in the transduction of fatty stimuli in taste cells may be useful for interpreting taste cell responses to FAs.

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About the authors

A. P. Cherkashin

Pushchino scientific center for biological research of the Russian Academy of Sciences

Email: malehanova@mail.ru

Institute of Cell Biophysics

Russian Federation, Pushchino, 142290

N. P. Kovalenko

Pushchino scientific center for biological research of the Russian Academy of Sciences

Email: malehanova@mail.ru

Institute of Cell Biophysics

Russian Federation, Pushchino, 142290

Е. Е. Kopylova

Pushchino scientific center for biological research of the Russian Academy of Sciences

Email: malehanova@mail.ru

Institute of Cell Biophysics

Russian Federation, Pushchino, 142290

О. А. Rogachevskaja

Pushchino scientific center for biological research of the Russian Academy of Sciences

Email: malehanova@mail.ru

Institute of Cell Biophysics

Russian Federation, Pushchino, 142290

Е. А. Voronova

Pushchino scientific center for biological research of the Russian Academy of Sciences

Author for correspondence.
Email: malehanova@mail.ru

Institute of Cell Biophysics

Russian Federation, Pushchino, 142290

S. S. Kolesnikov

Pushchino scientific center for biological research of the Russian Academy of Sciences

Email: malehanova@mail.ru

Institute of Cell Biophysics

Russian Federation, Pushchino, 142290

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2. Fig. 1. Heterologous expression of the GPR120 receptor from mouse taste tissue in HEK-293 cells. a – Representative gel electrophoresis of the products of RT-PCR analysis of mouse taste tissue (n = 3) with primers specific for the Ffar1 and Ffar4 genes encoding the GPR40 and GPR120 receptors, respectively. The amplicon of the expected size corresponds to GPR120 (346 bp), the GPR40 transcript (303 bp) is undetectable. M – DNA marker GeneRuler 100 bp DNA Ladder (Fermentas). Agarose gel (1.2%) was stained with ethidium bromide. b – Visualization of HEK-293 cells expressing the mouse GPR120 receptor fused with the green fluorescent protein (GFP). Confocal images are shown in transmitted light (left panel), in the GFP fluorescence channel (central panel), and as their superposition (right panel). A Leica TCS SPE confocal microscope (Leica) equipped with a Plan Apochromat HCX PL APO Lambda Blue 63× 1.4 Oil UV oil immersion objective (Leica) and the Leica Application Suite Advanced Fluorescence software were used. Fluorescence was excited at 488 nm, emission was recorded in the range of 500–550 nm. Localization of fluorescence in the cell membrane indicates localization of the GPR120 receptor in the membrane. Scale bar is 20 μm. c, d – Representative monitoring of intracellular Ca2+ in a HEK-293 cell (c) and in a HEK-293 cell expressing the GPR120 receptor from mouse taste tissue (d). The moments and duration of substance applications are indicated by horizontal lines above the experimental curves. The change in intracellular Ca2+ was characterized by the relative fluorescence of the Fluo-8 probe using the parameter ΔF/F0, where F and F0 are the fluorescence intensities of Fluo-8 at the current time and at the beginning of recording, respectively, ΔF = F – F0. Ca2+ responses to acetylcholine are given to control cell functionality.

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