Human ADRB2 Receptor Membrane Preparation
Technical Manual No. TM0514
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Version 03092011
Introduction ….…………………………………………………………………………….
Background ………..……………………………………………………………………….
Representative Data.………………………………………………………………………
Brief Competition Binding Protocol ………………………………………………………
References ……………………………………………………………………………….
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Introduction
Catalog Number: M00406
Cell Line Name: CHO-K1/ADRB2/Gα15
Gene Synonyms: BAR; B2AR; ADRBR; ADRB2R; BETA2AR; ADRB2
Expressed Gene: Genbank Accession Number NM_000024; no expressed tags
Host cell: CHO-K1/Gα15
Quantity: 1 vial (1 ml per vial)
Protein Concentration: 1 mg/ml
Storage Buffer: 50 mM HEPES, 0.1 mM EDTA, 10 % glycerol
Application: Binding assay for ADRB2 receptor
Storage: Store at -80°C
II.
Background:
β-Adrenergic receptors (β-ARs) are members of the superfamily G-protein-coupled receptors that are stimulated by
naturally occurring catecholamines, epinephrine, and norepinephrine. As part of the sympathetic nervous system,
β-ARs have been shown to have important roles in cardiovascular, respiratory, metabolic, central nervous system,
and reproductive functions. Three distinct β-AR subtypes have been identified (β1-AR, β2-AR, and β3-AR). All
three of these β-AR subtypes are believed to signal by coupling to the stimulatory G-protein Gsα which leads to the
activation of adenylyl cyclase and accumulation of the second messenger cAMP. β1-ARs mediate cardiac
stimulation, β2-ARs mediate smooth muscle relaxation in the peripheral vasculature and respiratory system, and
β3-AR has been shown to have important roles in adipose tissue and gastrointestinal tract. In studies using
subtype-selective agonists and antagonists in the human heart, β2-AR stimulation leads to the activation of
adenylyl cyclase and contributes to both inotropic and chronotropic responses.
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Representative Data
Saturation Binding for ADRB2 Receptor
Total Binding
pmol/mg protein
30
Specific Binding
NSB
20
Bmax = 31.1 pmol/mg protein
Kd = 0.46 nM
10
0
0
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4
6
8
10
3
[ H]CGP-12177 (nM)
Figure 1 0.5 μg of membranes prepared from CHO-K1 cells stably expressing ADRB2 receptors were incubated
with indicated concentrations of [3H]CGP-12177 in the absence (total binding) or presence of 1000-fold excess
unlabeled ICI 118551 (nonspecific binding, NSB). Binding was terminated by rapid filtration. Specific binding was
defined by subtracting NSB from total binding. Data were fit to one-site binding equation using a non-linear
regression method.
Copetition Binding for ADRB2 Receptor
Inhibition %
100
80
60
40
20
Ki = 1.0 nM
IC50 = 3.2 nM
0
-13 -12 -11 -10
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Log[ICI 118551] M
Figure 2 0.5 μg of membranes prepared from CHO-K1 cells stably expressing ADRB2 receptors were incubated
with indicated concentrations of ICI 118551 in the presence of 1 nM [3H]CGP-12177. Binding was terminated by
rapid filtration. Data were fit to one-site competition equation using a non-linear regression method.
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IV.
Brief Competition Binding Protocol
1.
Incubated 0.5 μg membranes with radio labeled ligand and unlabeled competitor (see Figures 1 and 2 for
concentrations tested) in binding buffer in a nonbinding 96-well plate. Incubated for 60 min at 37°C.
2.
Prior to filtration, coat a GF/C 96-well filter plate with 0.5% polyethyleneimine (PEI) for 30 min at 4°C, then
washed the plate with 1 ml/well 50mM HEPES, 0.5% BSA (pH 7.4).
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Transfer the binding mixtures then to the filter plate. After quick filtration, wash the plate for 3 times (3 ml per
well totally) with Wash Buffer.
4.
Dry the plate for 0.5 h and then add 50 μl scintillation cocktail (Microscint20). Stay for 1min and count on
TopCount NXT for 1 min/well.
5.
Binding buffer: 50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA, filtered and stored at 4°C
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Wash Buffer: 50 mM HEPES, pH 7.4, 500 mM NaCl, 0.1% BSA, filtered and stored at 4°C.
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References
1.
James A. (1984) ICI 118,551: an effective ocular hypotensive agent with selectivity for the ciliary process 132adrenoceptor and with minimal cardiac side effects. Br. J. Pharmac. 83,821- 829.
2.
Staehelin M, et. al. (1983) CGP-12177. A hydrophilic beta-adrenergic receptor radioligand reveals high affinity
binding of agonists to intact cells. J. Biol. Chem. Mar 25; 258(6):3496-502.
3.
Koike K. et. al. (1997) Characteristics of [3H]CGP12177 binding sites at beta 2- and beta 3-adrenoceptors in
the guinea pig taenia caecum. Gen. Pharmacol. Jan;28(1):73-6.
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