title: An intracellular loop 2 amino acid residue determines differential binding of arrestin to the dopamine D2 and D3 receptors.
abstract: 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. Arrestin 3 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 arrestin 3 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 arrestin 3 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 arrestin 3 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.
authors.affiliation: Department of Physiology & Pharmacology, Oregon Health & Science University, Portland, Oregon, USA.
authors.forename: Hongxiang
authors.initials: H
authors.lastname: Lan
authors.forename: Martha M
authors.initials: MM
authors.lastname: Teeter
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
authors.forename: Kim A
authors.initials: KA
authors.lastname: Neve
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019393:Arrestin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D055592:Biophysical Phenomena; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007668:Kidney / Q000166:cytology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050637:Receptors, Dopamine D3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D019393:Arrestin; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D003545:Cysteine