ChemiSCREEN™ Membrane Preparation Recombinant Human AT₂ Angiotensin II Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human AT₂ Angiotensin II Receptor

Full-length human AGTR2 cDNA encoding AT₂

Angiotensin II (Ang II), an octapeptide produced by cleavage of angiotensinogen by angiotensin-converting enzyme, plays a fundamental role in cardiovascular homeostasis. Two GPCRs, AT₁ and AT₂, mediate the effects of AngII. Most of the known actions of angiotensin II are mediated through the AT₁ receptor and serve to maintain blood pressure and glomerular filtration rate in the face of extracellular volume depletion (Griendling et al. 1996). In vitro and in vivo studies indicated that the AT₂ receptor counterbalances the effect of the AT₁ receptor (Horiuchi et al. 1999). Expression of the AT₂ receptor is developmentally regulated: it is highly expressed in various fetal tissues and at a lower density in adult adrenal medulla, brain, and reproductive tissues (Griendling et al. 1996). AT₂ expression appears to be re-expressed or up-regulated after vascular injury, myocardial infarction, cardiac failure or wound healing (Matsubara 1998). Millipore's AT₂ 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 agonists and antagonists of AT₂. The membrane preparations exhibit a Kd of 0.32 nM for [¹²⁵I]-CGP42112. With 0.25 nM [¹²⁵I]-CGP42112, 5µg/well AT2 Membrane Prep typically yields greater than 50-fold signal-to-background ratio.

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.

GPCR

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

Chem-1

• AGTR2
• MRX88
• ATGR2
• AT2

Membranes

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C

Table 1. Signal:background and specific binding values obtained in a competition binding assay with AT₂ Receptor membrane prep.

	5 µg/well
Signal:Background	148.1
Specific Binding (cpm)	17301

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
B_max for [¹²⁵I]-CGP42112 binding: 3.9 pmol/mg protein
K_d for [¹²⁵I]-CGP42112 binding: ~0.32 nM

GPCR Membrane Preparations

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 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.

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]-CGP42112. (Perkin Elmer # NEX-324 )

Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.

One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 50-fold signal:background with ¹²⁵I labeled CGP42112 at 0.25 nM

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

Human