ChemiSCREEN Membrane Preparation Recombinant Human sst₂ Somatostatin Receptor

Full-length human SSTR2 cDNA encoding sst₂

Somatostatin (sst) is a multifunctional peptide with two biologically active forms, sst-14 and sst-28, which are synthesized in neurons throughout the brain as well as in peripheral tissues such as the pancreas and the gut (Gillies, 1997). SST exerts a diverse array of effects that include inhibition of endocrine secretion, modulation of neurotransmission, and regulation of cell proliferation by stimulating a family of five G-protein-coupled receptors. Somatostatin receptor sst₂ mRNA is predominantly expressed in central nervous system. Study using sst₂ knock-out mice has found the increased anxiety-related behaviour while locomotor and exploratory activity was decreased in stress-inducing situations (coupled with an increase in pituitary ACTH release, a regulator of the stress response) (Viollet et al., 2000) . In the periphery, inhibition of glucagon release by sst in mouse islets is primarily mediated via sst₂ (Strowski et al., 2000). In addition, endogenous sst functions through sst₂ to suppress gastric acid secretion through inhibition of gastrin activity (Martinez et al., 1998). Millipore′s sst₂ 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 agonists and antagonists of sst₂. The membrane preparations exhibit a Kd of 0.95 nM for [¹²⁵I]-Somatostatin 14. With 0.75 nM [¹²⁵I]-Somatostatin 14, 5 μg/well sst₂ Membrane Prep typically yields greater than 20-fold signal-to-background ratio.

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: sst2

Target Sub-Family: Somatostatin

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of sst₂ Receptor membrane prep.

	10 µg/well	5 µg/well
Signal:Background	16.2	36.2
Specific Binding (cpm)	58438	54632

SPECIFICATIONS: 1 unit = 5 μg
Bmax for [¹²⁵I]-Somatostatin binding: 18.9 pmol/mg protein
Kd for [¹²⁵I]-Somatostatin binding: ~0.95 nM

Inucbation Conditions
RECOMMENDED ASSAY 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 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: [¹²⁵I]-Somatostatin 14. (Perkin Elmer NEX-389 )

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 20-fold signal:background with 125I labeled somatostatin 14 at 0.75 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°C

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

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