M2 Receptor Activation Redistribution Assay

• Designed to assay compounds for their ability to inhibit a cAMP response through the M2 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

Muscarinic acetylcholine receptor 2 (M2) belongs to a family of G protein-coupled receptors (GPCRs) which consists of five members. The odd-numbered members (M1, M3, M5) couple to Gαq whereas the even-numbered members (M2, M4) couple to Gαi. Muscarinic acetylcholine receptors mediate the effect of acetylcholine in synapses, and they are involved several indications related to the central nervous system.

In this assay, the M2 receptor is stably transfected into the PKAcat GPCR Reporter Assay for Gs/Gi-coupled Receptors (Product ID 045_02). This assay system measures translocation of protein kinase A (PKA) in reponse to changes in the cytoplasmic cAMP concentration. Binding of an agonist to the extracellular parts of the M2 receptor causes activation of the Gi complex at the intracellular face of the receptor. This leads to inhibition of adenylate cyclase, which catalyzes the formation of cAMP from ATP. In cells stimulated with forskolin that induces high levels of cAMP, PKAcat-GFP is found in a diffuse localization in the cytoplasm. M2 activation leads to inhibition of adenylate cyclase and thereby less cAMP. This, in turn, leads to aggregation of PKAcat-GFP in cytoplasmic foci.

Figure 1. Formation of cytoplasmic foci of PKAcat-GFP. Cells expressing the M2 receptor were treated with 40 μM forskolin for 5 min in the presence (right panel) or absence (left panel) of 10 μM carbachol. Activation of the receptor inhibits the forskolin-induced dispersion of PKAcat-GFP aggregates. Arrows indicated the cytoplasmic foci detected by the image analysis algorithm.

Figure 2. Carbachol concentration response in the M2:PKA assay. Concentration response was measured in 9 point half log dilution series (n=24). The EC₅₀ of carbachol is ~1 μM. Cells were treated with carbachol in the presence of forskolin for 5 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 (10 μM carbachol) and negative control (0.25% DMSO).