ChemiSCREEN Membrane Preparation Recombinant Human β₁ Adrenoceptor Receptor

Full-length human ADRB1 cDNA encoding the β₁ adrenoceptor

The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenergic receptors (Bylund et al., 1994). The three members of the β-adrenergic receptor family, β₁, β₂ and β₃, couple to G_s to increase cAMP upon activation. In the heart, the β₁ receptor constitutes 70-80% of the β-adrenergic receptors. Activation of cardiac β-adrenergic receptors acutely increases heart rate, cardiac output, and cardiac automaticity, and chronically increases cardiac myocyte apoptosis. In failing hearts, the β₁ subtype is downregulated and desensitized, probably as a result of increased catecholamine levels. As a result, β-adrenergic receptor antagonists (β blockers) are effective in the treatment of congestive heart failure and arrhythmia (Lohse et al., 2003). Millipore′s β₁ adrenoceptor 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 β₁ adrenoceptor interactions. The membrane preparations exhibit a K_d of 2.6 nM for [¹²⁵I]-(-) Iodocyanopindolol (ICYP). With 5 µg/well β₁ Adrenoceptor Membrane Prep and 0.25 nM [¹²⁵I]-(-)ICYP, a greater than 15-fold signal-to-background ratio was obtained.

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: beta 1

Target Sub-Family: Adrenergic

	5 µg/well	10 µg/well
Signal:Background	28.5	34.6
Specific Binding (cpm)	27002	28467

SPECIFICATIONS: 1 unit = 5 μg membrane preparation
Bmax: 47.9 pmol/mg
Kd: ~2.6 nM

Inucbation 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. 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]-(-) Iodocyanopindolol (Perkin Elmer # NEX189)
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 15-fold signal:background with ¹²⁵I-labeled (-)Iodocyanopindolol at 0.25 nM.

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

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

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