Description
General description
Full-length human MRGPRD cDNA encoding MrgD
The Mas-related genes (Mrgs), also known as sensory neuron-specific G-protein coupled receptors (SNSRs), are selectively expressed in subpopulations of sensory neurons involved in the perceptor of pain (Dong et al., 2001). The Mrg family in rodents consists of 50 receptors in the MrgA-MrgH subfamilies, but humans contain fewer Mrg members. MrgD (also known as TGR7) is found across species from rodents to humans, and its expression is restricted to nonpeptidergic, small-diameter, IB4+ dorsal root ganglion (DRG) neurons. MrgD binds β-alanine, but not L-alanine, which results in increased intracellular calcium and the suppression of forskolin-induced cAMP production, thus suggesting coupling to Gq and Gi proteins (Shinohara et al., 2004). β-alanine-induced activation of MrgD inhibits KCNQ2/3-mediated M-currents in cells overexpressing MrgD and KCNQ2/3 and also in cultured DRG neurons, which indicates that MrgD enhances nociceptive signaling (Crozier et al., 2007). Millipore′s MrgD membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of MrgD interactions with its ligands. The cell line exhibits a calcium response with EC50s of 11 µM for β-alanine. The membrane preparations exhibit EC50s of 69 µM for β-alanine in a GTPS binding assay.
Application
Radioligand binding assay and GTPγS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: MRGPRD / MrgD
Target Sub-Family: Mas-related gene
Features and Benefits
Inucbation Conditions
ASSAY CONDITIONS: Membranes are permeabilized by addition of saponin to an equal concentration by mass, then mixed with [³⁵S]-GTPγS (final concentration of 0.3 nM) in 20 mM HEPES, pH 7.4/100 mM NaCl/10 mM MgCl₂/0.5 μM GDP in a nonbinding 96-well plate. Unlabeled β-alanine was added to the final concentration indicated in Figure 1 (final volume 100 μL), and incubated for 30 min at 30°C. The binding reaction is transferred to a GF/B filter plate (Millipore MAHF B1H) previously prewetted with water. The plate is washed 3 times (1 mL per well per wash) with cold 10 mM sodium phosphate, pH 7.4, then dried and counted.
One vial contains enough membranes for at least 200 assays (units), where one unit is the amount of membrane that will yield greater than 1000 cpm specific β-alanine -stimulated [³⁵S]-GTPγS binding.
The MrgD membrane preparation is expected to be functional in a radioligand binding assay; however, the end user will need to determine the optimal radiolabeled ligand for use with this product.
Physical form
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
SPECIFICATIONS: 1 unit = 5 µg membrane preparation
EC50 in GTPγS binding assay by β-alanine: ~ 69 μM
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
