ChemiSCREEN Membrane Preparation Recombinant Human GABA_B1b/GABA_B2 Receptor

Full-length human GABBR1b and GPR51 cDNAs encoding GABA_B1b/GABA_B2

The neurotransmitter γ-aminobutyric acid (GABA) exerts its effects through an ion channel, GABA_A, and a GPCR, GABA_B. Functional GABA_B is a heterodimer composed of the GABA_B1 and GABA_B2 subunits, which share 35% sequence identity and belong to the class 3 family of GPCRs. The GABA_B1 subunit, which exists as splice variants GABA_B1a and GABA_B1b, binds directly to GABA and is required for agonist activation. The GABA_B2 and GABA_B1 subunits associate by formation of a coiled coil by their C-terminal tails; this association masks an ER retention sequence in GABA_B1 to permit export from the ER and trafficking to the cell surface. In addition to its chaperone function, GABA_B2 is the component that couples to Gi to reduce intracellular cAMP. Agonists of GABA_B, such as baclofen, are used clinically for treatment of muscle spasticity, migraine headache and musculoskeletal pain (Bowery et al., 2002). Chemicon′s GABA_B1b 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 GABA_B1b receptor interactions with its ligand. The membrane preparations exhibit a Kd of 1.15nM for [³H]-CGP 54626A. With 5.0 ug/well GABA_B1b Membrane Prep and 7.5nM [³H]-CGP 54626A, a greater than 5-fold signal-to-background ratio was obtained.

Radioligand binding assay and GTPγS binding

GPCR Class: C

Protein Target: GABAB1b

Target Sub-Family: GABAB

Table 1. Signal:background and specific binding values obtained in a competition binding assay with 5ug/well of GABA_B1b Receptor membrane prep.

	5 µg/well
Signal:Background	6.4
Specific Binding (cpm)	2879

SPECIFICATIONS: 1 unit = 5.0 µg membrane preparation
Bmax: 8.49 pmol/mg
Kd: 1.15 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 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: 1X Hanks′ Balanced Salt Solution (HBSS): GIBCO Cat. No. 14175
Radioligand: [³H]-CGP 54626A (American Radiolabeled Chemicals Inc., ART 517)
Wash Buffer: 1X Hanks′ Balanced Salt Solution (HBSS): GIBCO Cat. No. 14175

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 5-fold signal:background with ³H-labeled CGP 54626A at 7.5nM.

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 -80°C.

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

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