TRUPATH: An Open-Source Biosensor Platform for Interrogating the GPCR Transducerome

G protein-coupled receptors (GPCRs) are the most highly targeted protein family by United States Food and Drug Administration-approved drugs. Despite their historic and continued importance as drug targets, their therapeutic potential remains underexplored and underexploited. While it has been known for some time that GPCRs are able to engage multiple signaling pathways, the majority of drug research and development has followed the older dogma of a single primary pathway for each receptor. This has been due in part to historical reasons, or to a lack of appreciation of the potential to exploit specific pathways over others as a therapeutic modality. Additionally, only recently have technologies been developed to discern selective GPCR-G protein interactions. In this chapter, we introduce TRUPATH, a bioluminescence resonance energy transfer (BRET)-based platform that allows the unambiguous measurement of receptor-catalyzed dissociation or rearrangement of 14 Gα subunits from their respective Gβ and Gγ subunits. Specifically, we provide a detailed protocol for TRUPATH plasmid transfection, microplate preparation, assay implementation, and data analysis. In doing so, we create a template for using TRUPATH to answer basic biological questions, such as “To which G proteins does a given GPCR couple?”, and facilitate drug discovery efforts to identify ligands with intra- and inter-G protein family pathway selectivity.