Cannabinoid Receptor 1 (CB1) Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of CB1
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Cannabinoid receptor 1 (CB1) is a Gi-coupled G protein-coupled receptor (GPCR/7TM receptor), which is activated by the endogenous ligands arachidonyl ethanolamine (anandamide, an eicosanoide), palmitoyl-ethanolamine (PEA), and oleamide. These ligands are called endocannabinoids. Cannabinoids, which are the primary psychoactive chemicals in marijuana, are agonists for the CB1 receptor, and CB1 mediates most of the effects of cannabinoids in the central nervous system. Both cannabinoids and endocannabinoids stimulate appetite, and recent studies indicate clinical effects of CB1 antagonists in the treatment of obesity, and of agonists in the treatment of eating disorders and body weight regulation. Rimonabant, a CB1 receptor antagonist developed by Sanofi-Aventis, has shown effect on weight loss in clinical trials. CB1 receptors are rapidly internalized following agonist binding and receptor activation. Agonists such as CP55,940, HU210, and WIN55,212-2 have been shown to cause rapid receptor internalization.

Figure 1. Internalization of CB1-EGFP. Cells were treated with 0.25% DMSO (control, left panel), 1 µM WIN55,212-2  (center panel) or 1 µM WIN55,212-2 + 100 nM AM251 (right panel). Arrows indicate WIN55,212-2-induced CB1 internalization detected by the image analysis algorithm.

Figure 2: Clotrimazole concentration response curve in the HKII Redistribution® assay. Concentration response was measured in 9 point half log dilution series of clotrimazole (n=8). Cells were then fixed and the mitochondria to cytoplasm translocation was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetector V3 BioApplication. % activity was calculated relative to the positive (100 µM clotrimazole) and negative control (0.25% DMSO). The EC₅₀ value of clotrimazole in the assay is approximately 15 µM.