ChemiSCREEN™ Human CCK₁ Cholecystokinin Receptor Membrane Preparation

ChemiSCREEN™ Human CCK₁ Cholecystokinin Receptor Membrane Preparation

• ChemiScreen
• Chemicon (Millipore)

Full-length human CCKAR cDNA encoding CCK₁.

Cholecystokinins are a series of peptides of heterogeneous length (5 to 58 amino acids) that are derived from preprocholecystokinin and are found in gastrointestinal tissues and the central nervous system. Gastrin is a related peptide with 5 C-terminal amino acids identical to those of cholecystokinin. Two GPCRs, CCK₁ (CCK_A) and CCK₂ (CCK_B), bind to CCK and/or gastrin to mediate the biological effects of the peptides. CCK₁ selectively binds sulfated CCK, whereas CCK₂ binds to CCK and gastrin with similar affinity. Binding of ligands to CCK₁ stimulates mobilization of intracellular calcium by activation of G_q/11. CCK₁ receptors in the periphery are primarily localized in the pancreas, gallbladder, pylorus, and intestine where they are responsible of the regulation of diverse digestive processes. They are also present in select areas of the peripheral nervous system (vagus nerve), and the CNS where they mediate the satiety effects of CCK, regulate an increase in dopamine release, and antagonize opiod analgesia (Noble et al., 1999). Chemicon's cloned human CCK₁-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CCK₁ expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between the CCK₁ and its ligands.

Calcium flux assay, ligand binding assays

Human

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CCKA

GPCR Cell Lines

Chem-1, an adherent cell line expressing the promiscuous G protein, Gα15.

2 x 10⁶ cells/vial

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.

Chem-1

• CCKAR
• CCK1-R
• CCK-AR
• CCKRA
• CCK-A

Cells are frozen at 2 x 10⁶ cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

EC50 for calcium mobilization by sulfated CCK-8: ~ 5.0 nM
EC50 for calcium mobilization by A-71623: ~5.1 nM
EC50 for calcium mobilization by gastrin I: >1 µM

GPCRs

Human

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 20 mL growth media, and place in a humidified 37°C incubator with 5% CO2. After 8-24 h, cells will adhere to the plate, at which time the media should be replaced to remove residual DMSO. Cells are passaged by washing with Ca⁺⁺ and Mg⁺⁺-free HBSS (10 mL/T75), incubating with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5-10 minutes at 37°C, and rapping the side of the flask to dislodge the cells. Neutralize the trypsin by addition of 4 volumes growth media. Cells are typically passaged 1:10 with every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.