Identification of a G Protein-coupled Receptor Specifically Responsive to β-Alanine

Abstract

We isolated a cDNA encoding an orphan G proteincoupled 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 Ca 2؉ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that ␤-alanine at micromolar doses specifically evoked Ca 2؉ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, ␥-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, ␤-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins G q and G i . In guanosine 5-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, ␤-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 ␤-alanine. In addition, we found that ␤-[3 H]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 ␤-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 P2X 3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain.

The Drosophila black enigma: The molecular and behavioural characterization of the black1 mutant allele

“…Recently, a mammalian G-proteincoupled receptor specifically responsive to h-alanine has been isolated, the first such receptor identified (Shinohara et al, 2004). There have also been much earlier reports of a direct inhibitory role for h-alanine in the vertebrate visual system (see Sandberg and Jacobson, 1981).…”

MrgD Activation Inhibits KCNQ/M-Currents and Contributes to Enhanced Neuronal Excitability

“…MrgD is expressed across species from rodents to nonhuman primates and humans (Zhang et al, 2005). In addition, its expression is restricted to dorsal root ganglion (DRG) neurons (Dong et al, 2001;Shinohara et al, 2004;Milasta et al, 2006), specifically, nonpeptidergic, small-diameter, IB4 ϩ C-fiber neurons (Dong et al, 2001;Zylka et al, 2005). Together, these properties of MrgD provide for an experimentally tractable therapeutic target for pain.…”

“…The ability to probe for effects of MrgD activation was made possible by the identification of the putative ligand for MrgD, ␤-alanine (Shinohara et al, 2004). In recombinant cell lines, ␤-alanine-induced activation of MrgD resulted in elevated intracellular calcium and also suppression of forskolin-induced cAMP production, an effect that was inhibited by pertussis toxin (PTX) treatment, thus suggesting coupling to G q and G i proteins (Shinohara et al, 2004).…”

Prediction of the 3-D structure of rat MrgA G protein-coupled receptor and identification of its binding site

“…These receptors have been denoted as sensory neuron-specific receptors (SNSRs) or MAS-related gene (Mrg) receptors. Several studies have been carried out to identify the cognate ligands [5][6][7][8]. Most agonists are neuropeptides, such as FMRFa, or peptides similar to fragments of endogenous opioid peptides.…”

“…Indeed adenine activates rMrgA with a K i value of 18 nM, potentially identifying it as the endogenous ligand [4]. Another example is mouse MrgD that is activated by beta-alanine (EC50 = 44 μM) [6].…”