Few residues within extensive binding interface determine receptor preference of arrestin proteins

Abstract

The “footprint” of light‐activated phosphorhodopsin (P‐Rh*) on rod arrestin covers much the concave surfaces of the two domains. We used site‐directed spin labeling EPR spectroscopy to map the receptor‐binding surface of non‐visual arrestin2. The mobility of 15 residues (out of 21 tested) was reduced upon receptor binding, indicating that these positions are at or near the interaction interface. The receptor‐binding site in arrestin2 is as extensive as the rhodopsin‐binding site of rod arrestin. Taking advantage of a 4‐5‐fold difference between visual arrestin and arrestin2 binding to P‐Rh*, we used a gain‐of‐function approach to mimic rod‐like arrestin on the background of arrestin2. The combination of 5 and 8 mutations of exposed residues in the N‐ and C‐domains, respectively, corresponding to those present in rod arrestin reverses receptor preference of arrestin2. The resulting mutant binds P‐Rh* as well as wild type rod arrestin. Subsequent analysis by “loss‐of‐acquired‐function” approach, in which we mutated individual residues back to those present in arrestin2, identified one position in the N‐domain and three in the C‐domain that are primarily responsible for receptor discrimination. Thus, out of >40 amino acids in the ∼20x60A interaction interface, four exposed residues largely determine receptor specificity of arrestin proteins. NIH grants EY11500, GM 77561 (VVG), AI58024, GM70642 (CSK).