Shinohara Tokuyuki, Harada Masataka, Ogi Kazuhiro, Maruyama Minoru, Fujii Ryo, Tanaka Hideyuki, Fukusumi Shoji, Komatsu Hidetoshi, Hosoya Masaki, Noguchi Yuko, Watanabe Takuya, Moriya Takeo, Itoh Yasuaki, Hinuma Shuji
Discovery Research Laboratories, Pharmaceutical Research Division, Takeda Chemical Industries Ltd., 10 Wadai, Tsukuba, Ibaraki 300-4293, Japan.
J Biol Chem. 2004 May 28;279(22):23559-64. doi: 10.1074/jbc.M314240200. Epub 2004 Mar 22.
We isolated a cDNA encoding an orphan G protein-coupled receptor, TGR7, which has been recently reported to correspond to MrgD. To search for ligands for TGR7, we screened a series of small molecule compounds by detecting the Ca2+ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that beta-alanine at micromolar doses specifically evoked Ca2+ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, gamma-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, beta-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins Gq and Gi. In guanosine 5'-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, beta-alanine specifically increased the binding of guanosine 5'-O-3-thiotriphosphate. When a fusion protein composed of TGR7 and green fluorescent protein was expressed in cells, it localized at the plasma membrane but internalized into the cytoplasm after treatment with beta-alanine. In addition, we found that beta-[3H]alanine more efficiently bound to TGR7-expressing cells than to control cells. From these results, we concluded that TGR7 functioned as a specific membrane receptor for beta-alanine. Quantitative PCR analysis revealed that TGR7 mRNA was predominantly expressed in the dorsal root ganglia in rats. By in situ hybridization and immunostaining, we confirmed that TGR7 mRNA was co-expressed in the small diameter neurons with P2X3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain.
我们分离出了一个编码孤儿G蛋白偶联受体TGR7的cDNA，最近有报道称其与MrgD相对应。为了寻找TGR7的配体，我们通过检测表达TGR7的中国仓鼠卵巢细胞中的Ca2+内流，筛选了一系列小分子化合物。通过这次筛选，我们发现微摩尔剂量的β-丙氨酸能特异性地引起表达人、大鼠或小鼠TGR7的细胞中的Ca2+内流。一种结构类似物γ-氨基丁酸能微弱地刺激表达人或大鼠TGR7的细胞，但另一种类似物甘氨酸则不能。此外，β-丙氨酸降低了表达TGR7的细胞中福司可林刺激的cAMP产生，这表明TGR7与G蛋白Gq和Gi偶联。在使用表达TGR7的细胞的膜部分进行的鸟苷5'-O-3-硫代三磷酸结合试验中，β-丙氨酸特异性地增加了鸟苷5'-O-3-硫代三磷酸的结合。当由TGR7和绿色荧光蛋白组成的融合蛋白在细胞中表达时，它定位于质膜，但在用β-丙氨酸处理后内化到细胞质中。此外，我们发现β-[3H]丙氨酸与表达TGR7的细胞的结合比与对照细胞的结合更有效。从这些结果中，我们得出结论，TGR7作为β-丙氨酸的特异性膜受体发挥作用。定量PCR分析显示，TGR7 mRNA在大鼠背根神经节中主要表达。通过原位杂交和免疫染色，我们证实TGR7 mRNA在大鼠和猴背根神经节的小直径神经元中与P2X3和VR1共表达。我们的结果表明，TGR7参与神经性疼痛的调节。
