ChemiSCREEN CCR10 Membrane Preparation

EC₅₀ for CTACK/CCL27: ~5.6 nM in a GTPγS binding assay

GTPγS Binding Assay.

Protein Target: CCR10

Inucbation Conditions
GTPγS 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 ligand 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 an FB 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.

CCR10, previously known as GPR2, is a GPCR that is expressed in dermal endothelial cells, fibroblasts, melanocytes, and circulating T cells and PBMCs (Homey et al., 2000). Two chemokines, CTACK/CCL27 and MEC/CCL28, bind to CCR10 and stimulate migration and calcium flux (Pan et al., 2000; Wang et al., 2000). Expression of CTACK by keratinocytes, and CCR10 by lymphocytes from skin of patients with psoriasis and dermatitis, implicates this receptor ligand pair in skin inflammation. In addition, disruption of CCL27-CCR10 interaction reduces skin inflammation induced by allergen (Homey et al., 2002). CCR10 expressed on IgA antibody-secreting cells also mediates their migration to CCL28 expressed in the lactating mammary gland, and subsequent IgA accumulation in milk (Wilson and Butcher, 2004). Millipore′s CCR10 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 antagonists of CCR10 interactions with its ligands. The membrane preparations exhibit an EC50 of 47 nM for CTACK/CCL27 in a GTPγS binding assay.

Full length human CCR10 with a proprietary mutation in the cytoplasmic domain to enhance cell surface expression.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.

Packaging method: Membranes protein were adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80^oC.

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