Glucagon Receptor Activation Redistribution Assay

• Designed to assay compounds for their ability to activate a cAMP response through Glucagon 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

Glucagon and glucagon receptor are involved in energy homeostasis through stimulation of gluconeogenic pathways in the liver, causing blood glucose levels to increase. In this assay the glucagon 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 glucagon receptor. Binding of an agonist to the extracellular parts of glucagon receptor causes a conformational change in the receptor. This leads to conformational changes in heterotrimeric Gs 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 glucagon receptor were treated with 10 nM glucagon for 30 min (right panel). Activation of the receptor causes an increase in intracellular cAMP levels, resulting in dispersion of PKAcat-GFP aggregates. Arrows indicated the cytoplasmic foci detected by the image analysis algorithm (DMSO control, left panel).

Figure 2. Glucagon concentration response in the GlucagonR:PKA assay. Concentration response was measured in 9 point half log dilution series (n=8). The EC₅₀ of glucagon is ~30 pM. Cells were treated with glucagon for 30 min. Cells were then fixed and cytoplasmic spot formation was measured using the IN Cell Analyzer 3000 (GE Healthcare). % activity was calculated relative to the positive (10 nM glucagon) and negative control (0.25% DMSO).