An Intracellular Loop 2 Amino Acid Residue Determines Differential Binding of Arrestin to the Dopamine D2 and D3 Receptors

Dopamine D-2 and D-3 receptors are similar subtypes with distinct interactions with arrestins; the D-3 receptor mediates less agonist-induced translocation of arrestins than the D-2 receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D-2 and D-3 receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin3 bound to glutathione transferase (GST) fusion proteins of the D-2 receptor IC2 more avidly than to the D-3 receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin3 to D-2-IC2; arrestin binding to D-2-IC2-K149C was greatly decreased compared with wild-type D-2-IC2, whereas binding to the reciprocal mutant D-3-IC2-C147K was enhanced compared with wild-type D-3-IC2. Mutating this lysine in the full-length D-2 receptor to cysteine decreased the ability of the D-2 receptor to mediate agonist-induced arrestin3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D-3 receptor increased receptor-mediated translocation of arrestin3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D-2 receptor over the D-3 receptor is due in part to Lys149, which could be exposed as a result of receptor activation.