DEVELOPMENT OF NEXT-GENERATION G PROTEIN BIOSENSORS FOR DECONVOLUTING THE COMPLEXITY OF GPCR SIGNALING

Despite the fact that G protein coupled receptors (GPCRs) represent the largest family of druggable targets in the human genome, only 14% are targeted by FDA approved medications. Identifying potential drug targets among the 100’s of potential GPCRs is difficult since the tissue expression patterns, functions, physiological roles and signaling pathways of many GPCRs are incompletely understood. GPCR signal transduction, in particular, is exceedingly complex as ligands can activate 16 distinct Gα subunits (18 if including visual transducin G proteins), each of which could produce distinctive signaling events. Unfortunately, the G protein transducers which interact with individual GPCRs is largely unknown and underexplored due to inefficient strategies to faithfully interrogate the G protein transducerome. Here I report my development strategy for creating optimal Bioluminescence Resonance Energy Transfer (BRET) sensors for 14 non-visual Gα transducers with optimized GαGβγ pairings dubbed TRUPATH (TRansdUcerome Profile Analysis of g protein paTHways). In addition to providing new BRET sensors for Gα15 and GαGustducin, TRUPATH enabled as much as a 100-fold enhancement of dynamic range over comparable sensors. TRUPATH also enabled the discovery of diverse patterns of signaling preferences for both prototypic and understudied GPCRs. Finally, I provide TRUPATH as an open-source resource to facilitate a comprehensive understanding of GPCR signaling by the scientific community. Notably, TRUPATH facilitates deconvolution of what is otherwise a very complex array of GPCR signal transduction events at single transducer resolution.Doctor of Philosoph