ChemiSCREEN™ Membrane Preparation Recombinant Human NPBW₁ Neuropeptide B/W Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human NPBW₁ Neuropeptide B/W Receptor

Chemicon (Millipore)

200 units

Full length human GPR7 cDNA encoding NPBW₁

Neuropeptide B (NPB) and neuropeptide W (NPW) are members of a recently identified neuropeptide family that are ligands for two highly similar receptors NPBW₁ and NPBW₂, both of which are coupled to Gi/o protein to inhibit intracellular cAMP production. Highest expression of NPBW₁ mRNA and protein was identified in the amygdala and hypothalamic nuclei. Physiological studies demonstrate that intracerebroventricular infusion of NPBW₁ ligands modulates feeding behavior, regulates the release of corticosterone, prolactin and growth hormone while also modulating pain pathway (Singh and Davenport, 2006). NPBW₁ knock out male mice have shown mild adult-onset obesity and decreased locomotor activity. They become progressively hyperglycaemic and hyperinsulinaemic (Ishii et al., 2003). Millipore's NPBW₁ 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 NPBW₁ receptor interactions with its ligand. The membrane preparations exhibit a Kd of 0.53nM for [¹²⁵I]-Neuropeptide W23. With 5 ug/well NPBW1 Membrane Prep and 0.35nM [¹²⁵I]-Neuropeptide W23, a greater than 10-fold signal-to-background ratio was obtained.

Radioligand binding assay and GTPγS binding

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FUNCTION: SwissProt: P48145 # Interacts specifically with a number of opioid ligands. Receptor for neuropeptides B and W, which may be involved in neuroendocrine system regulation, food intake and the organization of other signals. Has a higher affinity for neuropeptide B.
SIZE: 328 amino acids; 36117 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Found in cerebellum and frontal cortex. Detected at high levels in hipppocampus, amygdala and trachea; at moderate levels in fetal brain, pituitary gland and prostate. Not in caudate, accumbens, kidney or liver. Also detected at high levels in lung carcinoma.
SIMILARITY: SwissProt: P48145 ## Belongs to the G-protein coupled receptor 1 family.

Human

Table 1. Signal:background and specific binding values obtained in a competition binding assay with 5 µg/well of NPBW₁ Receptor membrane prep.

	5µg/well
Signal:Background	31.8
Specific Binding (cpm)	21932

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax: 8.74pmol/mg
Kd: 0.53 nM

Chem-1

Chemicon

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 Tris, pH 7.4. 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, filtered and stored at 4°C.
Radioligand: [¹²⁵I]-neuropeptide W23 (Perkin Elmer#: NET-403)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl, filtered and stored at 4°C.

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 10-fold signal:background with ¹²⁵I-labeled neuropeptide W23 at 0.35nM.

Chem-1

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

NPBW1 / GPR7

P48145

peptide

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

Neuropeptide B/W

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A

• NPBWR1
• GPR7
• MGC129755

GPCR Membrane Preparations

ChemiSCREEN™ Membrane Preparation Recombinant Human NPBW₁ Neuropeptide B/W Receptor

NM_005285.3

GPCR