The Functional Role of Gate Loop Residues in Arrestin Binding to GPCRs

In all arrestins, the gate loop is the central part of the lariat loop, which has an unusual shape and participates in maintaining the basal conformation. The gate loop supplies two out of five charges that constitute a stabilizing intramolecular interaction, aspartates in the polar core between the two domains. To elucidate the functional role of individual gate loop residues, we performed comprehensive site-directed mutagenesis and tested the effects of mutations on arrestin-1 binding to its preferred target, phosphorylated light-activated rhodopsin, and unphosphorylated activated form. Out of 34 mutations tested, 24 and 25 affected the binding to phosphorylated and unphosphorylated rhodopsin, respectively. Manipulation of residues following polar core aspartates reduced preference for phosphorylated over unphosphorylated light-activated rhodopsin as dramatically as replacing these negatively charged aspartates with positively charged arginine. The data show that numerous lariat loop residues play distinct roles in arrestin-1 binding and its exquisite preference for phosphorylated light-activated rhodopsin.