Abstract

Dopamine D₂ and D₃ receptors are similar subtypes with distinct interactions with arrestins; the D₃ receptor mediates less agonist-induced translocation of arrestins than the D₂ receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D₂ and D₃ 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 D₂ receptor IC2 more avidly than to the D₃ 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 D₂-IC2; arrestin binding to D₂-IC2-K149C was greatly decreased compared with wild-type D₂-IC2, whereas binding to the reciprocal mutant D₃-IC2-C147K was enhanced compared with wild-type D₃-IC2. Mutating this lysine in the full-length D₂ receptor to cysteine decreased the ability of the D₂ 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 D₃ 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 D₂ receptor over the D₃ receptor is due in part to Lys149, which could be exposed as a result of receptor activation.