ChemiSCREEN™ Membrane Preparation Recombinant Human NPBW₂ Neuropeptide B/W Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human NPBW₂ Neuropeptide B/W Receptor

Chemicon (Millipore)

200 units

Full length human GPR8 cDNA encoding NPBW2

Neuropeptide B (NPB) and neuropeptide W (NPW) are members of a recently identified neuropeptide family that are ligands for NPBW₁ and NPBW₂ receptors, both of which are coupled to Gi/o protein to inhibit intracellular cAMP production and share 64% sequence homology (Singh and Davenport, 2006). NPBW₁ recognizes both NPB and NPW with similar nanomolar affinities (with a slight preference for NPB), whereas NPBW₂ is moderately selective for NPW (Tanaka et al., 2003). NPBW₂ is one of a few GPCRs that have no rat or mouse orthologue, although gene encoding NPBW₂ has been discovered in other mammalian species such as rabbit (Lee et al., 1999). NPBW₂ mRNA is known to be expressed in the frontal cortex, parietal cortex hippocampus, caudate nucleus, thalamus, pituitary, adrenal gland, and lymph node (Brezillon et al., 2003). Functions of NPBW₂ may be involved in feeding, weight regulation, and pain response through direct or indirect actions in the central nervous system. Millipore's NPBW₂ 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 NPBW₂ receptor interactions with its ligand. The membrane preparations exhibit a Kd of 2nM for [¹²⁵I]-Neuropeptide W23. With 10 ug/well NPBW₂ 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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The protein encoded by this gene is an integral membrane protein and G protein-coupled receptor. The encoded protein is similar in sequence to another G protein-coupled receptor (GPR7), and it is structurally similar to opioid and somatostatin receptors. This protein binds neuropeptides B and W. This gene is intronless and is expressed primarily in the frontal cortex of the brain.

FUNCTION: SwissProt: P48146 # 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.
SIZE: 333 amino acids; 36861 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Detected at high levels in caudate nucleus, hyppocampus and amygdala; at moderate levels in the adult brain, thalamus, parietal cortex, pituitary gland, adrenal gland and lymph nodes.
SIMILARITY: SwissProt: P48146 ## 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 10 or 20 µg/well of NPBW₂ Receptor membrane prep.

	20 µg/well	10 µg/well
Signal:Background	26.3	30.2
Specific Binding (cpm)	20148	19282

SPECIFICATIONS: 1 unit = 10.0 µg membrane preparation
Bmax: 11.34 pmol/mg
Kd: 2 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.

NPBW2 / GPR8

P48146

peptide

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

Neuropeptide B/W

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A

• NPBWR2
• GPR8

GPCR Membrane Preparations

ChemiSCREEN™ Membrane Preparation Recombinant Human NPBW₂ Neuropeptide B/W Receptor

NM_005286.2

GPCR