Construction of non‐visual arrestins with enhanced specificity for individual G protein‐coupled receptors

Abstract

We identified elements that drive arrestin‐3 (Arr3) binding to G protein coupled receptors. Based on the identification of residues that determine arrestin‐1 and ‐2 selectivity for cognate receptors and the analysis of the evolution in the arrestin family, we mutated “receptor discriminator” residues in Arr3. The recruitment of these mutants to different receptors was assessed using BRET‐based assays in cells. D260K and Q262P showed enhanced preference for M2 muscarinic (M2R), D1 (D1R), and D2 (D2R) dopamine receptors, over β2‐adrenergic receptors (β2AR). The specificity was further enhanced by combining D260K and Q262P. Conversely, Y239T enhanced Arr3 binding to β2AR, while reducing the binding to M2R, D1R, and D2R. Mutation Q257Y reduced arrestin recruitment to D2R. The Y239T+Q257Y combination virtually eliminated binding to β2AR, reduced D2R binding, and enhanced Arr3 recruitment to D1R. Thus, Y239T+Q257Y had opposite effects on Arr3 binding to D1R and D2R. Thus, manipulation of key residues on the receptor‐binding surface modifies receptor preference, allowing for the construction of non‐visual arrestins specific for particular GPCR subtypes. These findings pave the way to the construction of signaling‐biased arrestins targeting the receptor of choice for research or therapeutic purposes.NIH grants GM081756, GM077561 and EY011500 (VVG).