beta-2-Adrenergic Receptor Activation Redistribution Assay

• Designed to assay compounds for their ability to activate a cAMP response through beta2-Adrenergic Receptor
• 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

beta-2-adrenergic receptor has its predominant function in lung where receptor agonists are used for relief of bronchoconstriction in treatment of asthma and chronic obstructive pulmonary disease. In this assay the beta-2-adrenergic receptor has been stably transfected into the GPCR Reporter Assay for Gs-coupled Receptors. Translocation of protein kinase A (PKA), caused by changes in the cytoplasmic cAMP concentration, is used as a reporter for activation of beta-2-adrenergic receptor. Binding of an agonist to the extracellular parts of beta-2-adrenergic receptor causes a conformational change in the receptor. This leads to conformational changes in heterotrimeric G proteins at the intracellular face of the receptor, exchange of GDP for GTP on the alpha subunit (Gαs) and subsequent release of Gαs from the beta-gamma subunit. GTP-bound Gαs diffuses into the cytoplasm where it activates adenylate cyclase, which then catalyzes the formation of cAMP from ATP. In turn, cAMP activates PKA.

Figure 1. Cytoplasmic foci dispersion of PKAcat-GFP. Cells expressing the beta-2-adrenergic receptor were treated with 300 nM isoproterenol for 30 min (right panel). Activation of the receptor causes an increase in intracellular cAMP levels, resulting in dispersion of PKAcat-GFP aggregates. Arrows indicate the cytoplasmic foci detected by the image analysis algorithm (DMSO control, left panel).

Figure 2. Isoproterenol concentration response in the beta2-AR:PKA assay. Concentration response was measured in 9 point half log dilution series (n=8). The EC₅₀ of isoproterenol is ~2 nM. Cells were treated with isoproterenol for 30 min. Cells were then fixed and cytoplasmic spot formation was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (300 nM isoproterenol) and negative control (0.25% DMSO).