ChemiSCREEN™ Membrane Preparation Recombinant Human CCR4 Chemokine Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human CCR4 Chemokine Receptor

Full-length human CCR4 cDNA encoding CCR4

CCR4 is a GPCR that is activated by chemokines TARC (thymus and activation regulated chemokine) and MDC (macrophage-derived chemokine) (Olsen and Ley, 2001). NK cells, Th2 cells, cutaneous memory T cells, macrophages and platelets express CCR4 and respond to TARC and MDC (Andrew et al., 2001; Inngjerdingen et al., 2000; Gear et al., 2001; Soler et al., 2003). CCR4-null mice display reduced airway hyperresponsiveness to fungal spores, lipopolysaccharide-induced endotoxic shock, and cardiac allograft rejection (Chvatchko et al., 2000; Schuh et al., 2002; Huser et al., 2005). In addition, antibody -mediated antagonism of MDC inhibits development of insulitis and diabetes in NOD mice (Kim et al., 2002). Chemicon's CCR4 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 agonists and antagonists at CCR4. The cell line exhibits a calcium response with EC50 of 3.31nM for TARC. The membrane preparations exhibit EC50 of 5.76 nM for TARC in a GTPγS binding assay.

Radioligand binding assay and GTPγS binding.

Human

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.

Chem-1, an adherent mammalian cell line with minimum amount of endogenous CCR4 expression.

• CCR4
• CMKBR4
• ChemR13
• K5-5
• k5-5
• CKR4
• HGCN:14099
• MGC88293
• CCR-4
• CC-CKR-4

Membranes

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.

1 unit = 5 µg
EC50 in GTPγS binding assay by TARC: ~ 5.76 nM

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 TARC is 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 to TARC-stimulated [³⁵S]-GTPγS binding.
The CCR4 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.

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