The role of arrestin-1 N-edge in rhodopsin binding.

Arrestin-1, in contrast to other subtypes, demonstrates exquisite selectivity for the active phosphorylated form of its cognate receptor, rhodopsin. The loop between β-strands IX and X, termed N-edge because it is located on the distal tip of the N-domain in the folded arrestin molecule, was implicated in the binding of arrestin-1 and -2 to their cognate receptors. We performed alanine scanning and charge reversal mutagenesis of all twelve residues in this element of bovine arrestin-1. The mutants were tested for the binding to phosphorylated and unphosphorylated light-activated rhodopsin in the context of wild type and "enhanced" in terms of receptor binding C-terminally truncated arrestin-1-(1-378). The data identified two phosphate-binding lysines and seven other residues enhancing arrestin-1 preference for phosphorylated rhodopsin over unphosphorylated. We deleted three of these that are absent in the other mammalian arrestins and found that this insert is generally important for rhodopsin binding, not for enhanced selectivity. Eleven out of nineteen mutations differentially affected the binding of wild type arrestin-1 and its enhanced mutant, suggesting that the prevalent form of the complex of these two arrstin-1 variants with rhodopsin is different.