ChemiSCREEN Membrane Preparation Recombinant Human CXCR7 Chemokine Receptor

: Full-length human CXCR7 cDNA encoding CXCR7

The orphan receptor RDC1 is phylogenetically related to chemokine receptors, and its gene (CMKOR1, chemokine orphan receptor 1) occupies a chromosomal location near the genes for other CXC receptors. Several immune system cells, including T lymphocytes, mature dendritic cells and memory B cells, express RDC1 (Infantino et al., 2006). A subset of HIV and SIV strains use RDC1 as a coreceptor (Shimizu et al., 2000). The chemokine SDF-1/CXCL12, which binds to CXCR4, has been shown also to bind and activate RDC1 to promote chemotaxis of T lymphocytes; thus, CXCR7 has been proposed to be the new name for RDC1 (Balabanian et al., 2005). I-TAC, one of the chemokines thought to bind to CXCR3 only, has been reported to distinguish CXCR7 from CXCR4 in radioligand binding experiments (Burns et al. 2006). Chemicon′s CXCR7 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 CXCR7. The membrane preparations exhibit a Kd of 0.32 nM for [¹²⁵I]-SDF-1α. With 0.25 nM [125I]-SDF-1α, 10µg/well CXCR7 Membrane Prep typically yields greater than 4-fold signal-to-background ratio.

Radioligand binding assay and GTPγS binding

Target Sub-Family: Chemokine

GPCR Class: A

Protein Target: CXCR7 / RDC1

Inucbation Conditions
Membranes are mixed with radioactive ligand and unlabeled competitor (see Figures 1 and 2 for concentrations tested) in binding buffer in a nonbinding 96-well plate, and incubated for 1-2 h. Prior to filtration, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. Binding reaction is transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate is dried and counted.
Binding buffer: 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 0.2% BSA, filtered and stored at 4°C
Radioligand: [¹²⁵I] SDF-1α (Perkin Elmer#: NEX-346)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.

One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 4-fold signal:background with ¹²⁵I-labeled SDF-1α at 0.25 nM

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 1 mg/mL in packaging buffer, and dispensed at 1 mL/vial. Vials were rapidly frozen, and stored at -80°C.

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

Table 1. Signal:background and specific binding values obtained in a competition binding assay with CXCR7 membrane prep and unlabeled SDF-1α.

	10 µg/well
Signal:Background	4.92
Specific Binding (cpm)	15919

1 unit = 10 µg
Bmax for [¹²⁵I]- SDF-1α binding: 0.51 pmol/mg protein
Kd for [¹²⁵I]- SDF-1α binding: ~0.32 nM

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