ChemiSCREEN™ Human 5-HT_2C Receptor Membrane Preparation

ChemiSCREEN™ Human 5-HT_2C Receptor Membrane Preparation

• ChemiScreen
• Chemicon (Millipore)

200 units

Human 5-HT_2C

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.

Radioligand binding assay and GTPγS binding.

Human

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Serotonin (5-hydroxytryptamine, 5-HT), a neurotransmitter, elicits a wide array of physiological effects by binding to several receptor subtypes, including the 5-HT2 family of seven-transmembrane-spanning, G-protein-coupled receptors, which activate phospholipase C and D signaling pathways. This gene encodes the 2C subtype of serotonin receptor and its mRNA is subject to multiple RNA editing events, where genomically encoded adenosine residues are converted to inosines. RNA editing is predicted to alter amino acids within the second intracellular loop of the 5-HT2C receptor and generate receptor isoforms that differ in their ability to interact with G proteins and the activation of phospholipase C and D signaling cascades, thus modulating serotonergic neurotransmission in the central nervous system. Studies in humans have reported abnormalities in patterns of 5-HT2C editing in depressed suicide victims.

FUNCTION: SwissProt: P28335 # This is one of the several different receptors for 5- hydroxytryptamine (serotonin), a biogenic hormone that functions as a neurotransmitter, a hormone, and a mitogen. This receptor mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system.
SIZE: 458 amino acids; 51821 Da
SUBUNIT: Interacts with MPDZ.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
DOMAIN: SwissProt: P28335 The PDZ domain-binding motif is involved in the interaction with MPDZ.
PTM: N-glycosylated.
SIMILARITY: Belongs to the G-protein coupled receptor 1 family.

Human

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

Chem-1

Chemicon

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.

Chem-1

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.

5-HT2C

P28335

non-peptide

Serotonin

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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A

• HTR2C
• 5-HT-2C
• 5-HT2C
• HTR1C
• 5HT-1C
• 5-HTR2C

GPCR Membrane Preparations

ChemiSCREEN™ Human 5-HT_2C Receptor Membrane Preparation

NM_000868.1

GPCR