Dopamine is a catecholamine neurotransmitter that functions in the CNS to control locomotor, cognitive, emotional and neurendocrine processes, and in the periphery to modulate cardiovascular, renal and gastrointestinal processes. The biological activities of dopamine are mediated by a family of five GPCRs. The D₁ and D₅ subtypes couple to Gs to increase intracellular cAMP, whereas the D₂, D₃ and D₄ subtypes couple to G_i to reduce cAMP (Missale et al., 1998). The hypertensive phenotype of mice with a targeted deletion of D₅ indicates that D₅ regulates central control of sympathetic vascular tone (Hollon et al., 2002). In addition, D₅ modulates locomotion and corticostriatal long-term depression (Centonze et al., 2003). Chemicon′s cloned human D₅-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant D₅ expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between D₅ and its ligands. The membrane preparations exhibit a Kd of 2.25 nM for [³H]-SCH 23390. With 0.5 nM [³H]-SCH 23390, 10 µg/well D₅ Membrane Prep yields greater than 3-fold signal-to-background ratio.

Full-length human DRD5 cDNA encoding D₅

Protein Target: D5

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 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]-SCH 23390. (Perkin Elmer#:NET-930 )
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 3-fold signal:background with ³H labeled SCH 23390 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 were adjusted to 1 mg/mL in 1 mL packaging buffer, rapidly frozen, and stored at -80%deg;C.

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of D₅ membrane prep:

	10 µg/well
Signal:Background	4.06
Specific Binding (cpm)	1952

SPECIFICATIONS: 1 unit = 10 µg
Bmax for [³H] SCH 23390 binding: 11.42 pmol/mg protein;
Kd for [³H] SCH 23390 binding: ~ 2.25 nM

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