ChemiSCREEN™ Membrane Preparation Recombinant Human CCR3 Chemokine Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human CCR3 Chemokine Receptor

Chemicon (Millipore)

human CCR3 cDNA encoding CCR3

Eosinophils are major effector cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The chemokine receptor 3 (CCR3), a GPCR activated by chemokines eotaxin 1/2, MCP-3, MCP-4, and RANTES, mediates selective recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention (Fujisawa et al., 2000). It is widely expressed on cells involved in allergic inflammation, such as basophils, mast cells, airway epithelial cells, and potentially TH₂ T-lymphocytes. Allergen-induced eosinophil infiltration into airways is reduced or eliminated in CCR3 and eotaxin 1/2 knockout mice and in mice treated with antibodies directed against CCR3 (Grimaldi et al., 1999; Fulkerson et al., 2006). CCR3 antagonists are currently being developed for the treatment of asthma and other allergic disorders. Millipore's CCR3 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 CCR3 interactions with its ligands. The cell line exhibits a calcium response with EC50s of 6.5 nM for eotaxin. The membrane preparations exhibit EC50s of 5 nM for eotaxin in a GTPγS binding assay.

Radioligand binding assay

GPCR

Chem-1, an adherent cell line expressing the promiscuous G protein, Gα15.

Chem-1

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 eotaxin-stimulated [³⁵S]-GTPγS binding.

The CCR3 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.

SPECIFICATIONS: 1 unit = 5 ug
EC50 in GTPγS binding assay by Eotaxin: ~ 5 nM

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 uM GDP in a nonbinding 96-well plate. Unlabeled eotaxin was added to the final concentration indicated in Figure 1 (final volume 100 uL), 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 eotaxin-stimulated [³⁵S]-GTPγS binding.
The CCR3 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.

GPCR Membrane Preparations

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

Human