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-adrenoceptors (Bylund et al., 1994). The three members of the α1 subclass of adrenoceptors, α1A, α1B and α1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue. The α1D adrenergic receptor mediates smooth muscle contraction in several tissues. In the vasculature, activation of α1D increases blood pressure (Tanoue et al., 2002; Hosoda et al., 2005). In the urinary tract, α1D promotes bladder contraction. Antagonists of α1 receptors are used to treat bladder outlet obstruction, and this effect is thought to be mediated by α1D (Chen et al., 2005). The α1D adrenergic receptors has a relatively long N-terminal extracellular domain, and truncation of this domain has been shown to increase expression of the receptor at the cell surface (Pupo et al., 2003). Millipore′s α1D membrane preparations, which contain a version of α1D lacking residues 2-79, 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 α1D. The membrane preparations exhibit a Kd of 0.4 nM for [3H]-prazosin. With 0.5 nM [3H]-prazosin, 5 µg/well α1D (Δ2-79) Membrane Prep typically yields greater than 5-fold signal-to-background ratio.

Truncated human ADRA1D cDNA encoding α1D lacking residues 2-79

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: alpha1D

Target Sub-Family: Adrenergic

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of α1D (Δ2-79) receptor membrane prep.

	10 µg/well	5 µg/well
Signal:Background	15.4	11.2
Specific Binding (cpm)	778.8	670.5

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
B_max for [3H]-prazosin binding: 4.23 pmol/mg protein
K_d for [3H]-prazosin binding: ~0.4 nM

Inucbation Conditions
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: [3H]-prazosin. (PerkinElmer NET-823 )

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 5-fold signal:background with 3H labeled prazosin at 0.5 nM

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

Maintain frozen at -70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.

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