SIGNIFICANCE— Signal transduction mediated by G protein-coupled receptors (GPCR) and heterotrimeric G proteins (Gαβγ) is one of the most successful and versatile mechanisms of cellular communication used across eukaryotes and animal organ systems. In humans, these pathways regulate a myriad of physiological processes, from sensory perception to immune responses, and they are targets for about one-third of clinically used drugs. However, our current understanding of these receptors and proteins does not account for their remarkable flexibility in shaping cellular communication. Understanding the nuance, diversity, and context-dependent nature of GPCR and G protein signaling would have broad and deep implications for biology and medicine. The unifying goal of my NIGMS-funded research program is to understand the molecular mechanisms that impart plasticity to GPCR and G protein signaling and to leverage this information to imagine new ways to tackle human diseases. PAST ACCOMPLISHMENTS AND FUTURE PLANS— We are uniquely suited to lead research in this area based on our track record of perseverance and innovation: we have discovered new classes of G protein regulators that shape responses triggered by GPCRs or (surprisingly) by other receptor families, developed chemical and genetically-encoded probes to manipulate uncharted aspects of G protein signaling, and designed state-of-the-art tools for measuring G protein activity with high fidelity and precision. We will build on past experience and tools to advance in two areas related to G protein signaling plasticity. Area 1 will be focused on an extreme case G protein signaling plasticity— i.e., activation of G proteins independently of GPCRs. Here, we will define how widespread are the (patho)physiological implications of the best-characterized family of non-GPCR activators of G protein signaling in vivo. For this, we will use an innovative approach that combines multiple model organisms, including a novel transgenic mouse tool. Area 2 will be focused on the development and implementation of complementary optical biosensor platforms to measure GPCR signaling activity directly and with high spatiotemporal resolution in diverse, physiologically-relevant systems without introducing major perturbations on signaling. INNOVATION AND IMPACT— On one hand, this research program is anticipated to have a long term impact by (1) revealing new mechanisms underlying G protein signaling plasticity in the absence of GPCRs, (2) providing innovative tools for their investigation, and (3) establishing how their proper function is essential for physiological homeostasis. On the other hand, the biosensor technologies developed through this program could also transform how the large GPCR field approaches something as fundamental as recording signaling activity, thereby allowing to gain a more reliable picture of this process across diverse experimental models and accelerating translation of discoveries into biomedical applications.

PROJECT NARRATIVE Heterotrimeric G proteins mediate the action of many neurotransmitters, hormones and pharmacological agents involved in normal physiology, disease, or therapy. Here we propose studies to elucidate new molecular mechanisms by which G proteins are regulated and to develop technologies that enable their investigation with unprecedented detail and fidelity.