SIGNIFICANCE: G protein-coupled receptors (GPCRs) are a large family of membrane proteins that initiate cellular responses to a wide range of extracellular signals, like neurotransmitters or hormones, by activating heterotrimeric G proteins inside cells. This makes GPCRs critical for many physiological processes and their dysregulation is frequently associated with disease. GPCRs are not only the family of proteins most widely targeted by clinically approved drugs, with an estimated market value of hundreds of billions of dollars per year, but also the subject of many ongoing campaigns towards novel therapeutics. Moreover, many GPCRs still remain understudied and “undrugged”, which has great untapped potential for future drug discovery. Despite the biomedical importance of these receptors in physiology and pharmacology, the methodologies currently used to measure GPCR signaling activity in cells have limitations that hinder progress. One significant limitation is the use of approaches that compromise the fidelity of the readout because they are too indirect and/or distort the natural stoichiometry of signaling components. Another significant limitation is that many of the approaches are only feasible in cells in which gene delivery is easy, limiting their implementation to a few cell lines instead of more physiologically relevant systems like primary cell cultures. GOAL: Here, we propose to develop technologies that will allow to measure GPCR signaling activity directly and in diverse, physiologically-relevant cell systems without introducing major perturbations on the natural stoichiometry of signaling components. The tools and resources generated in this project will be made available to other investigators without restrictions. SYNOPSIS OF AIMS: We will develop two complementary assay platforms based on optical biosensors that directly detect the activity of heterotrimeric G proteins. In Aim1, we will establish a suite of broadly applicable “compact” vectors compatible with simple transfection or with viral packaging for the expression of G protein activity biosensors. In Aim 2, we will validate mouse transgenic lines for the conditional expression of biosensors of endogenous G protein activity. Collectively, these two platforms will allow the direct assessment of GPCR signaling activity under native expression conditions, in physiologically relevant cellular systems, and in formats that enable increased throughput to permit the parallel interrogation of the druggable GPCR-ome. IMPACT: The achievement of our goal could transform how the large field of research interested in GPCR- mediated cell communication approaches something as fundamental as measuring signaling activity. The technologies to be developed here would advance the field by: (i) enabling the direct characterization of GPCR signaling in systems in which it is currently not possible, (ii) revealing mechanisms of GPCR signaling with unprecedented fidelity in diverse contexts, an...