ChemiSCREEN Human 5-HT_2C Receptor Membrane Preparation

5-Hydroxytryptamine (5-HT, also commonly known as serotonin) is synthesized in enterochromaffin cells in the intestine and in serotonergic nerve terminals. In the periphery, 5-HT mediates gastrointestinal motility, platelet aggregation, and contraction of blood vessels. Many functions of the central nervous system are influenced by 5-HT, including sleep, motor activity, sensory perception, arousal and appetite. A family of 12 GPCRs and one ion channel mediate the biological effects of 5-HT (Hoyer et al., 1994). 5-HT_2C, which couples to G_q in most cells to stimulate intracellular calcium, is prominently expressed in brain and appears to modulate depression, anxiety and appetite (Miller, 2005; Serretti et al., 2004; Wood, 2003). The mRNA encoding 5-HT_2C undergoes selective RNA editing that changes 4 amino acids in the second intracellular loop; these changes result in alteration of efficiency of coupling to G proteins. Alterations in editing of 5-HT_2C have been detected in victims of suicidal depression and in mice treated with the SSRI, fluoxetine (Tohda et al., 2006). Millipore′′s 5-HT_2C 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 5-HT_2C interactions and its ligands. The membrane preparations exhibit a Kd of 5.9 nM for [³H]-mesulergine. With 6 nM [³H]- Mesulergine, 10 µg/well 5-HT_2C Membrane Prep yields greater than 5 fold signal-to-background ratio.

Human 5-HT_2C

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: 5-HT2C

Target Sub-Family: Serotonin

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of 5-HT_2C membrane prep:

	10 µg/well
Signal:Background	7.8
Specific Binding (cpm)	2433

SPECIFICATIONS: 1 unit = 10 µg membrane preparation
Bmax: 15.66 pmol/mg
Kd: 5.9 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 50mM HEPES, 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: 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, filtered and stored at 4°C
Radioligand: [³H]-Mesulergine (Amersham TRK 845)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl, filtered and stored at 4°C.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives. Packaging method: Membrane protein was 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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