PROJECT SUMMARY We propose to elucidate the molecular mechanisms of arrestin-1 interactions with rhodopsin, which significantly contribute to exquisitely regulated and precisely timed function of photoreceptor cells, using the combination of novel cutting-edge biophysical methods and in vivo experiments. We will compare the shape of the complex of arrestin-1 with rhodopsin using long-range distance measurements between selected points in arrestin-1 and rhodopsin with pulse EPR technique DEER. We will explore the conformation of arrestin-bound rhodopsin and compare it to the conformation of free light-activated rhodopsin and rhodopsin in complex with transducing to determine whether particular conformation states of rhodopsin (a model GPCR) predispose it to interact with G protein and arrestin. This is hypothesized in the field to be the basis of biased GPCR signaling. We will determine the biological role of arrestin-1 self-association in photoreceptors in vivo, which is currently unknown. Finally, we will test novel compensational approach to gene therapy of gain-of-function rhodopsin mutations using engineered enhanced arrestin-1 mutants capable of shutting off rhodopsin signaling independently of its phosphorylation. Proposed studies will significantly improve our understanding of photoreceptor physiology and advance our progress towards gene therapy of gain-of-function rhodopsin mutations. Due to high conservation of mechanics of GPCR regulation, mechanistic studies of arrestin-1 binding to rhodopsin will have broader implications, improving our understanding of the molecular basis of the function of other arrestin subtypes. Proposed studies will shed new light on many aspects of cell signaling. Our strategic goal is to gain sufficient understanding of protein-protein interactions that govern cell signaling to construct mutants with desired functional characteristics for research and therapeutic purposes.