Project Summary We propose to elucidate fine molecular mechanism of arrestin-1 interaction with rhodopsin, focusing on the aspects of this mechanism that ensure remarkable selectivity of arrestin-1 for light-activated phosphorylated rhodopsin and much lower binding to all other functional forms of rhodopsin (inactive phosphorylated, light-activated unphosphorylated, inactive unphosphorylated, opsin, and phosphorylated opsin). We propose to design “enhanced” arrestin-1 mutants with high binding to unphosphorylated light- activated rhodopsin. We expect these mutants to compensate for defects in rhodopsin phosphorylation in vivo. Mutants of this kind have therapeutic potential in human visual disorders caused by absent or insufficient rhodopsin phosphorylation. Based on the discoveries of non-rhodopsin binding partners of arrestin-1 we propose to use in vivo proximity labeling to identify novel arrestin-1 interaction partners in rod photoreceptors. These partners will be biotinylated by AirID fused to arrestin-1 (we validated both functions of the two fusions we propose to use), purified via biotin binding to avidin, digested into peptides, which will be identified by mass spectrometry (LC-MS/MS).