ChemiScreen™ CCR4 Calcium-Optimized FLIPR Cell Line

ChemiScreen™ CCR4 Calcium-Optimized FLIPR Cell Line

• ChemiScreen
• Chemicon (Millipore)

2 vials

Full-length human CCR4 cDNA

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 cloned human CCR4-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CCR4 expression on the cell surface and contains high levels of the promiscuous G protein Ga15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between CCR4 and its ligands.

Calcium flux assay, ligand binding assays

Human

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The protein encoded by this gene belongs to the G-protein-coupled receptor family . It is a receptor for the CC chemokine - MIP-1, RANTES, TARC and MCP-1. Chemokines are a group of small polypeptide, structurally related molecules that regulate cell trafficking of various types of leukocytes. The chemokines also play fundamental roles in the development, homeostasis, and function of the immune system, and they have effects on cells of the central nervous system as well as on endothelial cells involved in angiogenesis or angiostasis.

FUNCTION: SwissProt: P51679 # High affinity receptor for the C-C type chemokines CCL17/TARC and CCL22/MDC. The activity of this receptor is mediated by G(i) proteins which activate a phosphatidylinositol- calcium second messenger system. Can function as a chemoattractant homing receptor on circulating memory lymphocytes and as a coreceptor for some primary HIV-2 isolates. In the CNS, could mediate hippocampal-neuron survival.
SIZE: 360 amino acids; 41403 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Predominantly expressed in the thymus, in peripheral blood leukocytes, including T-cells, mostly CD4+ cells, and basophils, and in platelets; at lower levels, in the spleen and in monocytes. Detected also in macrophages, IL-2-activated natural killer cells and skin-homing memory T-cells, mostly the ones expressing the cutaneous lymphocyte antigen (CLA). Expressed in brain microvascular and coronary artery endothelial cells.
PTM: In natural killer cells, CCL22 binding induces phosphorylation on yet undefined Ser/Thr residues, most probably by beta-adrenergic receptor kinases 1 and 2.
SIMILARITY: SwissProt: P51679 ## Belongs to the G-protein coupled receptor 1 family.

Human

Chemicon

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CCR4

Cells are frozen at 2 x 10(e6) cells/mL in DMEM/20% fetal bovine serum/100U/mL each penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

peptide

Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media, and replace media after 8-24 h. Cells are typically passaged 1:10 with 0.05% Trypsin/0.2 g/L EDTA every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.

Chemokine

A

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

GPCRs

ChemiScreen™ CCR4 Calcium-Optimized FLIPR Cell Line

DMEM containing 4.5 g/L glucose/10% heat inactivated fetal bovine serum/1x nonessential amino acids/10 mM HEPES/0.25 mg/mL Geneticin (G418) /100 U/mL each penicillin and streptomycin

2 x 10⁶ cells/mL

SPECIFICATIONS: EC50 for calcium mobilization by TARC: 0.59 nM
Signal/noise at TARC E_max: 524
Z' = 0.85 with TARC at 2x EC50

Chem-5

GPCR Cell Lines

Chem-5

Cell Line

P51679

2 x 10⁶ cells/mL

NM_005508.4