ChemiScreen Membrane Preparation
Recombinant Human TBXA2R (TP) Thromboxane Receptor

Human Full-length human TBXA2R cDNA encoding the TP short form (TPα)

TP receptors (Thromboxane A2 receptors) are widely distributed among different organ systems and have been localized on both cell membranes and intracellular structures. TP receptors belong to G protein-coupled receptor family (Hirata et al., 1991). Activation of TP receptors induces platelet aggregation, vascular and respiratory smooth muscle constriction, and enhances mitogenic responses of vascular smooth muscle cells that are stimulated by growth factors (Ali et al, 1993; Hanasaki et al., 1990). The human TP receptor is encoded by a single gene that is alternatively spliced at the carboxyl terminus, resulting in two isoforms, TPα (343 residues) and TPβ (407 residues). Both isoforms couple to Gq pathway, but couple oppositely to adenylate cyclase. The cDNA encoding human TPα has been stablely expressed in the Chem-1 host, which supports high levels of recombinant receptor expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. The membrane preparations exhibit a Kd of 9.19 nM for [3H]-SQ 29548. With 10 nM [3H]-SQ 29548, 10 ug/well TP Membrane Prep yields greater than 5-fold signal-to-background ratio.

Radioligand binding assay, and GTPγS binding.

GPCR Class: A

Protein Target: TP / TXA2 / PGH2

Target Sub-Family: Prostanoid

200 units

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of Y₂ membrane prep:

	10 µg/well	5 µg/well
Signal:Background	6.6	4.6
Specific Binding (cpm)	2090	963

1 unit = 10 μg membrane preparation
Bmax: 13.9 pmol/mg
Kd: 14.6 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, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50 mM 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]-SQ 29548 (Perkin Elmer # NET936)
Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM NaCl , 0.1% BSA, 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 5-fold signal:background with 3H-labeled SQ 29548 at 15 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.

Store 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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