ChemiSCREEN™ Membrane Preparation Recombinant Human NK₂ Tachykinin Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human NK₂ Tachykinin Receptor

Chemicon (Millipore)

200 units

Full-length human TACR2 cDNA encoding NK₂

The tachykinin peptide family in mammals comprises three peptides, substance P, neurokinin A and neurokinin B, which bind to the tachykinin receptor family of GPCRs, NK₁, NK₂ and NK₃ (Severini et al., 2002). Tachykinins have prominent activity in the GI system, in which they stimulate intestinal contraction and salivation; these effects are mediated by NK₁ and NK₂. Experiments with selective antagonists of NK₂, SR 48,968 and MEN 10,376 indicate that NK₂ mediates neurokinin- induced constriction of pulmonary artery and bronchus (Maggi et al., 1993). NK₂ antagonists display efficacy in treatment of irritable bowel disorder (Lecci et al., 2004). Millipore's NK₂ 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 NK₂ receptor interactions with its ligand. The membrane preparations exhibit a Kd of 0.76nM for [¹²⁵I]-NKA. With 10.0 ug/well NK₂ Membrane Prep and 0.5nM [¹²⁵I]-NKA, a greater than 10-fold signal-to-background ratio was obtained.

Radioligand binding assay and GTPγS binding

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

This gene belongs to a family of genes that function as receptors for tachykinins. Receptor affinities are specified by variations in the 5'-end of the sequence. The receptors belonging to this family are characterized by interactions with G proteins and 7 hydrophobic transmembrane regions. This gene encodes the receptor for the tachykinin neuropeptide substance K, also referred to as neurokinin A.

FUNCTION: SwissProt: P21452 # This is a receptor for the tachykinin neuropeptide substance K (neurokinin A). It is associated with G proteins that activate a phosphatidylinositol-calcium second messenger system.
SIZE: 398 amino acids; 44439 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: P21452 ## Belongs to the G-protein coupled receptor 1 family.
MISCELLANEOUS: The rank order of affinity of this receptor to tachykinins is: substance K > neuromedin-K > substance P.

Human

Chemicon

NK2

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.

peptide

Tackykinin /neurokinin

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

A

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• TACR2
• SKR
• NKNAR
• NK-2R
• NK2R
• TAC2R

GPCR Membrane Preparations

ChemiSCREEN™ Membrane Preparation Recombinant Human NK₂ Tachykinin Receptor

Table 1. Signal:background and specific binding values obtained in a competition binding assay with 10ug/well of NK₂ Receptor membrane prep.

	10 µg/well
Signal:Background	16.5
Specific Binding (cpm)	19906

SPECIFICATIONS: 1 unit = 10.0 µg membrane preparation
Bmax: 2.21 pmol/mg
K_d: 0.76 nM

Chem-1

RECOMMENDED ASSAY CONDITIONS: 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: 50mM Tris, pH 7.4, 150mM NaCl, 3mM MnCl₂, 0.1% BSA, containing one Protease Inhibitor Cocktail Tablet (Roche Cat. No. 11 873 580 001) for each 50ml binding buffer.
Radioligand: [¹²⁵I]-NKA (NEX 252)
Wash Buffer: 50mM Tris, pH 7.4, 150mM NaCl, 3mM MnCl₂, 0.1% BSA

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 NKA at 0.5nM.

Chem-1

P21452

NM_001057.2

GPCR