ChemiSCREEN™ Membrane Preparation Recombinant Human CCK₁ Cholecystokinin Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human CCK₁ Cholecystokinin Receptor

• ChemiScreen
• Chemicon (Millipore)

200 units

Human CCK₁ Receptor

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, intestine where they are responsible for 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 opioid analgesia (Noble et al., 1999). Chemicon's CCK₁ 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 CCK₁ interactions and its ligands. The membrane preparations exhibit a Kd of 0.5 nM for [¹²⁵I]-CCK-8 sulfated. With 10.0ug/well CCK1 membrane prep and 0.5 nM [¹²⁵I]-CCK-8 sulfated, a greater than 4-fold signal-to-background ratio was obtained.

Radioligand binding assay and GTPγS binding

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This gene encodes a G-protein coupled receptor that binds non-sulfated members of the cholecystokinin (CCK) family of peptide hormones. This receptor is a major physiologic mediator of pancreatic enzyme secretion and smooth muscle contraction of the gallbladder and stomach. In the central and peripheral nervous system this receptor regulates satiety and the release of beta-endorphin and dopamine.

FUNCTION: SwissProt: P32238 # Receptor for cholecystokinin. Mediates pancreatic growth and enzyme secretion, smooth muscle contraction of the gall bladder and stomach. Has a 1000-fold higher affinity for CCK rather than for gastrin. It modulates feeding and dopamine-induced behavior in the central and peripheral nervous system. This receptor mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system.
SIZE: 428 amino acids; 47841 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: P32238 ## Belongs to the G-protein coupled receptor 1 family.

Human

Chemicon

CCK1 / CCKa

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 4-fold signal:background with ¹²⁵I-labeled CCK-8 at 0.5 nM.

Liquid in packaging buffer: 50 mM Tris, pH 7.4, 10% glycerol with no preservatives.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

peptide

Cholecystokinin

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

A

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

GPCR Membrane Preparations

ChemiSCREEN™ Membrane Preparation Recombinant Human CCK₁ Cholecystokinin Receptor

CCK_A

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of CCK₁ Receptor membrane prep.

	10 µg/well	5 µg/well
Signal:Background	5.31	6.16
Specific Binding (cpm)	1274.9	1691.4

SPECIFICATIONS: 1 unit = 10 µg membrane preparation
Bmax: 0.15 pmol/mg
K_d: 0.5 nM

Chem-1

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 blocked 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.
Note: Due to the acidic property of the labeled sulfated CCK-8, filter plates coated with PEI bind the labeled ligand nonspecifically and result in elevated backgrounds.

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] -CCK-8 sulfated (Perkin Elmer # NEX203)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl, 0.1% BSA, filtered and stored at 4°C.

Chem-1

P32238

NM_000730.2

GPCR