Using the TRUPATH BRET platform to analyse GPCR biased agonism

The G protein-coupled receptor (GPCR) family is comprised of hundreds of receptors that respond to a variety of stimuli to modulate cellular activity. Their ubiquitous expression and cell membrane localization makes them attractive. In fact, GPCR represent the most highly targeted protein family by FDA-approved drugs. 

 

Biased agonism, the concept whereby ligands select for and stabilise receptor states that preferentially interact with certain effectors, could be used to enhance therapeutic effects but diminish side-effects. This approach, mostly limited to drugs that bias receptor signaling between G proteins versus beta-arrestins, has had mixed success so far. A potential reason is that GPCRs are capable of coupling to multiple G proteins and that it is the differences in G protein signaling that accounts for desired and unwanted effects. 

 

Monitoring discrete GPCR-G protein coupling would provide important biological information about signaling capabilities and facilitate the discovery of drugs biased towards desired G protein effectors. 

 

TRUPATH is a BRET-based platform that enables the 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, Gα-RLuc and GFP2-Gγ are in close association as part of an intact Gαβγ heterotrimer and produce a BRET signal. Upon receptor activation, dissociation of the heterotrimer results in a decrease in the BRET signal. 

 

Here, an overview of the TRUPATH platform is presented that includes the design and optimisation of its biosensor components, a general protocol for its use, and examples of its potential to discover drugs with inter-G protein bias.