Arrestin3 mediates D 2 dopamine receptor internalization

Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Synapse (Impact Factor: 2.43). 07/2009; 63(7):621-4. DOI: 10.1002/syn.20636
Source: PubMed
  • [Show abstract] [Hide abstract]
    ABSTRACT: A high-throughput screening campaign was conducted to interrogate a 380,000+ small molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and β-arrestin recruitment. While the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate β-arrestin recruitment. One such compound (MLS1547) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit β-arrestin, as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated β-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate β-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate β-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally-selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.
    Molecular pharmacology 04/2014; 86(1). DOI:10.1124/mol.113.090563 · 4.12 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Aim:To explore the effects of heterodimerization of D2 receptor/A2a receptor (D2R/A2aR) on D2R internalization and D2R downstream signaling in primary cultured striatal neurons and HEK293 cells co-expressing A2aR and D2R in vitro.Methods:Primary cultured rat striatal neurons and HEK293 cells co-expressing A2aR and D2R were treated with A2aR- or D2R-specific agonists. D2R internalization was detected using a biotinylation assay and confocal microscopy. ERK, Src kinase and β-arrestin were measured using Western blotting. The interaction between A2aR and D2R was detected using bioluminescence resonance energy transfer (BRET) and immunoprecipitation.Results:D2R and A2aR were co-localized and formed complexes in striatal neurons, while both the receptors formed heterodimers in the HEK293 cells. In striatal neurons and the HEK293 cells, the D2R agonist quinpirole (1 μmol/L) marked increased Src phosphorylation and β-arrestin recruitment, thereby D2R internalization. Co-treatment with the A2aR antagonist ZM241385 (100 nmol/L) significantly attenuated these D2R-mediated changes. Furthermore, both ZM241385 (100 nmol/L) and the specific Src kinase inhibitor PP2 (5 μmol/L) blocked D2R-mediated ERK phosphorylation. Moreover, expression of the mutant β-arrestin (319-418) significantly attenuated D2R-mediated ERK phosphorylation in HEK293 cells expressing both D2R and A2aR, but not in those expressing D2R alone.Conclusion:A2aR antagonist ZM241385 significantly attenuates D2R internalization and D2R-mediated ERK phosphorylation in striatal neurons, involving Src kinase and β-arrestin. Thus, A2aR/D2R heterodimerization plays important roles in D2R downstream signaling.
    Acta Pharmacologica Sinica 08/2013; 34(10). DOI:10.1038/aps.2013.87 · 2.50 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The activity of G protein-coupled receptors (GPCRs) is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs) and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R)- [(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK) 1/2. Additionally, an in vitro cellular model was also used to study PAOPA's effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA's development into a novel drug for the improved treatment of schizophrenia.
    PLoS ONE 08/2013; 8(8):e70736. DOI:10.1371/journal.pone.0070736 · 3.53 Impact Factor

Full-text (2 Sources)

Available from
May 15, 2014