Quantification of Arrestin–Rhodopsin Binding Stoichiometry

Abstract

We have developed several methods to quantify arrestin-1 binding to rhodopsin in the native rod disk membrane. These methods can be applied to study arrestin interactions with all functional forms of rhodopsin, including dark-state rhodopsin, light-activated metarhodopsin II (Meta II), and the products of Meta II decay, opsin and all-trans-retinal. When used in parallel, these methods report both the actual amount of arrestin bound to the membrane surface and the functional aspects of arrestin binding, such as which arrestin loops are engaged and whether Meta II is stabilized. Most of these methods can also be applied to recombinant receptor reconstituted into liposomes, bicelles, and nanodisks.