title: Novel roles for β-arrestins in the regulation of pharmacological sequestration to predict agonist-induced desensitization of dopamine D3 receptors.
abstract: OBJECTIVE
  In addition to typical GPCR kinase (GRK)-/β-arrestin-dependent internalization, dopamine D3 receptor employed an additional GRK-independent sequestration pathway. In this study, we investigated the molecular mechanism of this novel sequestration pathway.
  
  
  METHODS
  Radioligand binding, flow cytometry and cell surface biotinylation assay were used to characterize trafficking properties of D2 and D3 receptors. Serine/threonine and N-linked glycosylation mutants of the D3 receptor were utilized to locate receptor regions involved in pharmacological sequestration and desensitization. Various point mutants of the D2 and D3 receptors, whose sequestration and desensitization properties were altered, were combined with knockdown cells of GRKs or β-arrestins to functionally correlate pharmacological sequestration and desensitization.
  
  
  RESULTS
  The D3 receptor, but not the D2 receptor, showed characteristic trafficking behaviour in which receptors were shifted towards the more hydrophobic domains within the plasma membrane without translocation into other intracellular compartments. Among various amino acid residues tested, S145/S146, C147 and N12/19 were involved in pharmacological sequestration and receptor desensitization. Both pharmacological sequestration and desensitization of D3 receptor required β-arrestins, and functional relationship was observed between two processes when it was tested for D3 receptor variants and agonists.
  
  
  CONCLUSIONS
  Pharmacological sequestration of D3 receptor accompanies movement of cell surface receptors into a more hydrophobic fraction within the plasma membrane and renders D3 receptor inaccessible to hydrophilic ligands. Pharmacological sequestration is correlated with desensitization of the D3 receptor in a Gβγ- and β-arrestin-dependent manner. This study provides new insights into molecular mechanism governing GPCR trafficking and desensitization.
authors.affiliation: Department of Pharmacology, College of Pharmacy, Drug Development Research Institute, Chonnam National University, Gwang-Ju, 500-757, Korea.
authors.forename: C
authors.initials: C
authors.lastname: Min
authors.forename: M
authors.initials: M
authors.lastname: Zheng
authors.forename: X
authors.initials: X
authors.lastname: Zhang
authors.forename: M G
authors.initials: MG
authors.lastname: Caron
authors.forename: K M
authors.initials: KM
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D004298:Dopamine / Q000494:pharmacology; D018491:Dopamine Agonists / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004361:Drug Tolerance; D004705:Endocytosis / Q000187:drug effects*; D005434:Flow Cytometry; D054768:G-Protein-Coupled Receptor Kinases / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D021381:Protein Transport; D019257:Quinpirole / Q000494:pharmacology; D034622:RNA Interference; D011869:Radioligand Assay; D017448:Receptors, Dopamine D2 / Q000819:agonists / Q000378:metabolism; D050637:Receptors, Dopamine D3 / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; C581293:DRD2 protein, human; C493593:DRD3 protein, human; D018491:Dopamine Agonists; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D000071557:beta-Arrestins; D019257:Quinpirole; D054768:G-Protein-Coupled Receptor Kinases; D004298:Dopamine
keywords: Gβγ; glycosylation; phosphorylation; small hairpin RNA; vasopressin type 2 receptor