MultiBacMam Technology for Studying the Downstream cAMP Signaling Pathway of M2 Muscarinic Acetylcholine Receptor

Measuring the inhibition of adenylate cyclase upon Gαi-coupled receptor activation has been challenging as it usually requires prior activation of the enzyme. Herewith, we propose an assay system that avoids this activation step for Gαi-coupled receptors, therefore simplifying the measurements. This is achieved by using chimeric Gαsi proteins that leads to elevation of cAMP levels in cells upon activation of Gαi coupled receptors. In this case, the cAMP concentration is monitored by a genetically encoded FRET-based biosensor, but this assay only works if it is expressed in the same cells as the chimeric Gαsi proteins. MultiBacMam technology enables to combine genes of different proteins into a single-plasmid system, utilizing a modified insect virus (baculovirus) as a vehicle to transport and efficiently express them in mammalian cells. We provide here a detailed protocol for establishing and carrying out the cAMP assay for detecting receptor activation for M2 receptors. Using this novel biosensor system, we can distinguish between agonists, superagonists, and antagonists when measuring the activation of the M2 receptor. The assay is well suited for screening ligands targeting various G protein-coupled receptors, allowing the examination of activation kinetics alongside the assessment of drug potencies and efficacies.