The role of individual residues in arrestin-3 finger loop in its binding to β2-adrenergic receptor

Arrestins preferentially bind active phosphorylated forms of their cognate G-protein-coupled receptors (GPCRs). The finger loop inserts into the interhelical cytoplasmic cavity of activated receptors and has been proposed to serve as an activation sensor. We mutagenized the finger loop (residues 63-78) in bovine arrestin-3 and measured the binding to the β2-adrenergic receptor (β2AR) in living cells. Only one substitution in arrestin-3 reduced its binding to β2AR, whereas three others increased it. To eliminate the effect of receptor-attached phosphates, the same mutations were introduced on the background of phosphorylation-independent arrestin-3 mutant, and its in-cell binding to phosphorylation-deficient β2AR-15AG was tested. Three residues in the finger loop were found to be critical for the recognition of the active receptor. Interestingly, Phe76Ala mutation increased the binding in both cases, suggesting that Phe76 constrains rather than promotes the β2AR engagement. Lys78Ala mutation increased the binding, but Lys78Glu had no effect, suggesting that Lys78 imposes a conformational rather than electrostatic constraint. The residues where mutations had functional effects are conserved in the four vertebrate arrestin subtypes. Thus, the mechanism of activation recognition appears to be conserved in the arrestin family.