Novel GαGTP Sensors Reveal Endogenous and Subcellular G Protein Signaling Dynamics

G protein-coupled receptors (GPCRs) perceive spatially and temporally diverse stimuli and activate G protein heterotrimers comprising , {beta}, and {gamma} subunits, which broadcast signals through a broad range of effectors at various subcellular compartments. Therefore, understanding endogenous G protein activity dynamics at the subcellular level, thereby recapitulating in vivo signaling paradigms, will facilitate the identification of pathological signaling pathways. However, the lack of sensors for endogenous G proteins has been an obstacle. Here, we demonstrate the engineering of sensors to probe endogenous GiGTP and GqGTP. Compared to examining overexpressed and fluorescently tagged G, our sensors capture the magnitude and kinetics of endogenous GGTP dynamics, including their generation, equilibrium signaling, and hydrolysis, with native fidelity. Using the translocation-based GiGTP sensor, we show that heterotrimer dissociation upon Gi-GPCR activation is G{gamma}-subtype dependent. Confirming our previous findings, the GqGTP sensor showed that Gq expression is low and tightly regulated in most cells. Using optogenetic tools, we demonstrate that our sensors detect GGTP generation and hydrolysis during asymmetric GPCR-G protein activation, a capability that will be particularly useful in morphologically diverse cells such as neurons. Therefore, our engineered novel GGTP sensors can be highly beneficial in decoding subcellularly resolved endogenous G protein signaling dynamics.