ChemiSCREEN Membrane Preparation Recombinant Human mGLU₂ Metabotropic Glutamate Receptor

Glutamate is a main excitatory neurotransmitter in the central nervous system, and it plays a role in learning, memory and neurotoxicity. The biological actions of glutamate are mediated by ionotropic and metabotropic glutamate receptors, which are ion channels and GPCRs respectively. Metabotropic glutamate receptors (mGluRs) are members of the class 3 G-protein coupled receptor family, which are characterized by a large extracellular domain. They are further classified into group I, II, and III mGluRs on the basis of their sequence identity, pharmacology, and signal transduction mechanism. Group I (mGlu₁ and mGlu₅) couple to the phospholipase C pathway through G_αq, whereas group II (mGlu₂ and mGlu₃) and group III (mGlu₄, mGlu₆, mGlu₇, and mGlu₈) negatively couple to the adenylyl cyclase pathway though G_αi (Conn and Pin, 1997). Agonists of the Group II metabotropic glutamate receptors, mGlu₂ and mGlu₃, display efficacy in animal models of anxiety and psychosis. A key role for mGlu₂ in mediating these effects is indicated by the observation that selective allosteric potentiator of mGlu₂ also retains antipsychotic-like activities in mice (Galici et al., 2005). In addition, mGlu_2/3 agonists display analgesic activity in animal models (Jones et al., 2005). Chemicon′s mGlu₂ 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 mGlu₂ interactions with its ligands. The cell line exhibits a calcium response with EC50s of 0.51uM, 5.6uM, and 8.3uM for DCG IV, (2R4R) APDC, and glutamate. The membrane preparations exhibit EC50s of of 0.72uM, 5.07uM, and 6.29uM for DCG IV, (2R4R) APDC, and glutamate in a GTPγS binding assay.

human GRM2 cDNA encoding mGlu₂

Radioligand binding assay and GTPγS binding

GPCR Class: C

Protein Target: mGlu2

Target Sub-Family: Glutamate (metabotropic)

Inucbation Conditions
Membranes are permeabilized by addition of saponin to an equal concentration by mass, then mixed with [35S]-GTPγS (final concentration of 0.1 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 DCG IV, (2R4R) APDC, and glutamate are 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, and washed 3 times (1 mL per well per wash) with cold 10 mM sodium phosphate, pH 7.4. The plate is 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 DCG IV, (2R4R) APDC, or glutamate -stimulated [³⁵S]-GTPγS binding.
The mGlu₂ 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.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to 1 mg/ml in packaging buffer, rapidly frozen, and stored at -80°C.

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

EC50 in GTPγS binding assay by Glutamate: ~ 6.29 μM
EC50 in GTPγS binding assay by (2R4R) APDC: ~ 5.07 μM
EC50 in GTPγS binding assay by DCG IV: ~ 0.72 μM

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