PROJECT SUMMARY/ABSTRACT Heart failure is a leading cause of morbidity and mortality in western countries and represents the final common clinical event of numerous cardiovascular diseases. Cardiac β2 adrenergic signaling determines cardiac output and has been extensively studied as treatments directed toward β2 adrenergic signaling have proven effective at improving cardiac performance and heart failure outcomes. The β2 adrenergic receptor (β2AR) is a G-protein- coupled receptor (GPCR), a family of membrane proteins representing the largest and most versatile group of cell surface receptors. They are ubiquitous membrane proteins that facilitate the initial role in the transduction of information into the cell via a wide array of extracellular signal-receptor interactions. Although much progress has been made in elucidating conformational insights into ligand-mediated signaling of GPCRs, there has not been commensurate investigation into endogenous modulators of GPCR signaling. Endogenous modulators include but are not limited to protein-lipid interactions, post-translational modifications, and accessory protein interactions. The β2AR has a site for S-palmitoylation, the covalent attachment of palmitate to a cysteine, in the C-terminal helix 8 (H8). Interestingly, H8 of the β2AR is implicated in β-arrestin recruitment, resulting in a non- canonical signaling pathway, yet little is known of the effect that palmitoylation of H8 has on intracellular signaling and structural dynamics of the receptor. I hypothesize that S-palmitoylation of the β2AR has an allosteric effect on the conformational plasticity therein modulating functional transducer coupling in the context of ligand mediated protein activation. The goal of this project is to determine the effect S-palmitoylation has on β2AR signaling in the following two specific aims. Aim 1. To define the impact of s-palmitoylation on the conformational dynamics at the cytoplasmic surface of the β2AR and the effect it has on the interaction with transducers to facilitate intracellular signaling using both electron paramagnetic resonance (EPR) techniques and signaling assays. In preliminary experiments, we performed site-directed spin-labeling to generate a set of nitroxide spin labeled receptor mutants to perform EPR spectroscopy experiments. Aim 2. To determine the impact of S-palmitoylation on H8 and tail dynamics of the β2AR using EPR techniques. These experiments will help us better understand β2AR signaling as a whole and provide new insight into novel ways of GPCR signaling regulation via endogenous modulators. Further, the experiments will provide new insight into the effects of S- palmitoylation on the conformational plasticity and, ultimately, the signaling of the β2AR. These results will provide a greater clarity on β2AR signaling and regulation within the context of a physiologically relevant cellular environment, which will help with the design of effective treatments targeting cardiac β2 adr...