Arrestin residues involved in the functional binding of arrestin to phosphorylated, photolyzed rhodopsin.

PURPOSE The purpose of our study was to determine whether arrestin residues previously predicted by computational modeling to interact with an aspartic acid substituted rhodopsin tail are actually involved in interactions with phospho-residues on the rhodopsin cytoplasmic tail. METHODS We generated arrestin mutants with altered charges at predicted positions. These mutants were then tested for the ability to inhibit rhodopsin using both direct binding assays, as well as functional assays involving transducin inhibition assays. RESULTS Our results demonstrate that the computer-predicted residues are indeed involved in both the ability of the low-affinity state of arrestin to bind to rhodopsin as well as the ability of arrestin to be induced into a higher-affinity state in a phospho-residue-dependent manner. CONCLUSIONS Our results also suggest that positions K14, K15, R29, H301, and K300 on arrestin interact with the phosphorylated carboxyl tail of rhodopsin and that this translates to the efficient activation of arrestin.