PROJECT SUMMARY/ABSTRACT The long term goal of our research program is to understand at an atomic level the mechanisms by which lipids regulate human G protein-coupled receptor (GPCR) activity, and in turn learn how GPCRs influence their surrounding membrane environment. GPCRs drive many physiological processes and represent the largest family of “druggable” protein targets. Drug binding and GPCR signaling are both allosterically regulated by the surrounding cellular environment through receptor-lipid interactions. Such membrane-protein interactions are ubiquitous within the cell and have well documented roles in physiology, however, little is known about the structural mechanisms by which membranes regulate GPCR function. The need to address this gap in knowledge is heightened by more recent studies associating GPCR-lipid interactions with cell-specific drug responses and revealing critical roles of GPCR-lipid interactions in a wide range of diseases, including Alzheimers’ disease, cancers, and heart disease. Our research will capture the different roles by which lipids regulate GPCR function, both as specific chemical partners and through the bulk physical and chemical properties of lipids, by integrating unique capabilities of solution and solid state nuclear magnetic resonance (NMR) with additional biophysical tools and correlative functional assays. Initial efforts are directed at two lines of investigation aimed at addressing the most immediate and important questions regarding receptor-lipid interactions. In the first, we will determine how lipids modulate drug binding and signal transduction for the A2A adenosine receptor (A2AAR), a representative model GPCR that shares structural and functional characteristics with many rhodopsin-like receptors. These studies will reveal how lipids impact protein dynamics, alter activation “hotspots”, and regulate formation of signaling complexes. Integrating this new data with available pharmacology and crystal structures will provide a new conceptual framework for interpreting cell- specific drug responses. In the second direction, we will determine the structural mechanisms by which membrane lipids allosterically modulate signaling of the smoothened receptor (SMO), a hedgehog signaling protein and validated cancer target. SMO functions in primary cilia, specialized organelles packed with sensory proteins that act as cellular “antenna”. An emerging concept is that ciliary membrane composition is fine-tuned for receptor function, yet little is actually known about the properties of receptor-lipid interactions in ciliary membranes. Our work will reveal for the first time how both specific lipids and bulk lipid properties regulate SMO signaling complexes. Together, these lines of investigation will reveal basic principles of lipid-mediated allostery and set the stage for long term efforts to apply these principles to design drugs targeting specific receptor-lipid interactions.