title: A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding.
abstract: Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.
authors.affiliation: Department of Physiology & Pharmacology, Oregon Health & Science University, Portland, Oregon, USA.
authors.forename: Hongxiang
authors.initials: H
authors.lastname: Lan
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Michal I
authors.initials: MI
authors.lastname: Bell
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
authors.forename: Kim A
authors.initials: KA
authors.lastname: Neve
mesh_terms: D000818:Animals; D019393:Arrestin / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D004298:Dopamine / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000276:immunology; D006801:Humans; D007668:Kidney / Q000166:cytology; D009154:Mutation; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D013469:Sulpiride / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D019393:Arrestin; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D013469:Sulpiride; D000242:Cyclic AMP; D006735:Horseradish Peroxidase; D005982:Glutathione Transferase; D048049:Extracellular Signal-Regulated MAP Kinases; D019204:GTP-Binding Proteins; D004298:Dopamine