Using the TRUPATH Bioluminescence Resonance Energy Transfer Platform to Survey and Quantify G Protein-Coupled Receptor Coupling Preferences

The G protein-coupled receptor (GPCR) superfamily is comprised of hundreds of integral membrane proteins that collectively respond to a variety of stimuli to modulate cellular activity.
Their ubiquitous expression and cell membrane localization makes them attractive and accessible drug targets; correspondingly, they represent the most highly targeted protein family by United States Food and Drug Administration-approved drugs. In recent years, an emphasis has been placed in GPCR drug discovery on biased agonism, the concept whereby ligands select for and stabilize receptor states that preferentially interact with certain effectors but not others as a means to enhance therapeutic effects but diminish side-effects. This approach has been mostly limited to drugs that bias receptor signaling between G proteins versus beta-arrestins, which has had mixed success in clinical trials. A potential reason for this is that GPCRs are capable of coupling to and activating multiple G proteins, belonging to the same or different families, and that it is the differences in G protein signaling that account for desired and unwanted effects. Thus, inter-G protein bias may represent an opportunity to succeed in cases where G protein versus beta-arrestin bias has not. The ability to monitor discrete GPCR-G protein coupling would provide important biological information about GPCR signaling capabilities, and facilitate the discovery of drugs that are biased towards desired G protein effectors downstream of a given receptor. To facilitate such endeavors, we developed TRUPATH, a bioluminescence-resonance energy transfer (BRET)-based platform that enables the unambiguous monitoring of 14 Renilla lucerifase-fused Gα proteins with the GFP2-fused Gγ subunits of their cognate Gβγ heterodrimers. In the absence of receptor activation and productive G protein coupling, Gα-RLuc and GFP2-Gγ are in close association as part of an intact Gαβγ heterotrimer, and as a result of their spatial proximity and orientation, these components produce a BRET signal. Upon receptor activation and positive coupling, consequent dissociation of the heterotrimer results in a decrease in the BRET signal. The insertion sites of the BRET fusion proteins have been optimized through extensive empirical evaluation, providing improved dynamic range over previous iterations of heterotrimeric G protein-based biosensors. Additionally, TRUPATH shows low signal amplification with minimal sensitivity to receptor reserve, circumventing the convolution of pharmacological parameter estimates (i.e. efficacy, potency) that other functional assays are subject to. Here, an overview of the TRUPATH platform is presented that includes the design and optimization of its biosensor components, a general protocol for its use, and examples of its potential to discover drugs with inter-G protein bias.