ChemiSCREEN Membrane Preparation Recombinant Rehsus Macaque CCR5 Chemokine Receptor

CCR5 is the receptor for CC chemokines MIP-1α, MIP-1β, and RANTES (Raport et al., 1996), and is preferentially expressed on Th1 lymphocytes (Loetscher et al., 1998). CCR5 is a coreceptor for macrophage-tropic HIV, and its ligands potently inhibit HIV replication in human leukocytes (Cocchi et al., 1995). In addition, HIV-infected patients with the nonfunctional CCR5Δ32 allele exhibit delayed onset of AIDS symptoms (Samson et al., 1996), and pharmacological antagonism of CCR5 inhibits HIV-1 infection (Strizki et al., 2001). Preclinical testing of small molecule antagonists of CCR5 has been hampered by low affinity of the compounds to rodent and dog CCR5, but two such compounds, maraviroc and AD101, have been shown to have potent antagonist activity at rhesus macaque CCR5, which differs from human CCR5 by 8 amino acids (Napier et al., 2005; Billick et al., 2004). One antagonist of human CCR5, SCH-C, does not block HIV entry through rhesus macaque CCR5, and one amino acid difference is responsible for the functional difference (Billick et al., 2004). Millipore′s rhesus macaque CCR5 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 of rCCR5. The membrane preparations exhibit a Kd of 0.32 nM for [¹²⁵I]-MIP-1α. With 0.25 nM [¹²⁵I]-MIP-1α, 5 μg/well rCCR5 Membrane Prep typically yields greater than 7-fold signal-to-background ratio.

Full-length rhesus macaque CCR5 cDNA encoding CCR5

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: CCR5

Target Sub-Family: Chemokine

Inucbation Conditions
RECOMMENDED ASSAY 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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) 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]-MIP-1α (Perkin Elmer#: NEX-298 )

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 7-fold signal:background with ¹²⁵I labeled MIP-1α at 0.15 nM

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml packaging buffer, 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 varying amounts of CCR5 Receptor membrane prep.

	10 µg/well	5 µg/well
Signal:Background	13.6	12.1
Specific Binding (cpm)	5869	4538

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
B_max for [¹²⁵I]-MIP-1α binding: 1.9 pmol/mg protein
K_d for [¹²⁵I]-MIP-1α binding: ~0.35 nM

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