Full-length human CALCR (isoform 2) and RAMP1 cDNAs encoding AMY1

The calcitonin family of peptides, which includes calcitonin, two calcitonin-related gene peptides (CGRPs), adrenomedullin, and amylin, share a disulfide-bonded loop, an amphipathic a-helix, and an amidated C-terminus. The peptides influence a variety of physiological effects, including vascular tone, food intake and bone metabolism. The biological effects of the peptides are mediated by two class B GPCRs, the CT and CL receptors (Poyner et al., 2002). The pharmacology of the CT and CL receptors is strongly influenced by their association with a family of three receptor activity modifying proteins (RAMPs), which are 14-17 kD single pass transmembrane proteins. When expressed with no modifying proteins, the CT receptor binds with high affinity only to calcitonin. The CT receptor in complex with RAMP proteins binds amylin with high affinity, as well as calcitonin and αCGRP; the CT receptor complexes with RAMP1, RAMP2 and RAMP3 are termed AMY1, AMY2 and AMY3. Amylin (also known as islet amyloid polypeptide) is produced by pancreatic beta cells. Experiments with amylin-deficient mice indicate that amylin and its receptors control food intake and pain perception. Pramlintide, a synthetic analog of amylin, is used clinically for glucose control in diabetics. Chemicon′s AMY1 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 AMY1 interactions with hCGRP. The membrane preparations exhibit a Kd of 0.29 nM for [¹²⁵I]-hCGRP. With 10 ug/well AMY1 Membrane Prep and 0.2 nM [¹²⁵I]-hCGRP, a greater than 10-fold signal-to-background ratio was obtained.

Human AMY1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay, and GTPγS binding.

SPECIFICATIONS: 1 unit = 10 μg membrane preparation
Bmax: 0.45 pmol/mg
Kd: 0.29 nM

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: [¹²⁵I]-hCGRP (Perkin Elmer # NEX354)

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 10-fold signal:background with ¹²⁵I-labeled hCGRP at 0.2 nM. Liquid in packaging buffer: 50 mM Tris pH

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

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