Identification of a G protein-coupled receptor specifically responsive to beta-alanine.
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.
Shinohara
Tokuyuki
T
Discovery Research Laboratories, Pharmaceutical Research Division, Takeda Chemical Industries Ltd., 10 Wadai, Tsukuba, Ibaraki 300-4293, Japan.
Harada
Masataka
M
Ogi
Kazuhiro
K
Maruyama
Minoru
M
Fujii
Ryo
R
Tanaka
Hideyuki
H
Fukusumi
Shoji
S
Komatsu
Hidetoshi
H
Hosoya
Masaki
M
Noguchi
Yuko
Y
Watanabe
Takuya
T
Moriya
Takeo
T
Itoh
Yasuaki
Y
Hinuma
Shuji
S
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005727:Ganglia, Spinal / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D000882:Haplorhini; D006801:Humans; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010146:Pain / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D015091:beta-Alanine / Q000378:metabolism*
D018076:DNA, Complementary; D043562:Receptors, G-Protein-Coupled; D015091:beta-Alanine