Regulation of dopamine D(1) receptor trafficking by protein kinase A-dependent phosphorylation.
The aim of this study was to use pharmacological inhibition of protein kinase A and mutation of potential protein kinase A phosphorylation sites to determine the role of protein kinase A-catalyzed phosphorylation of the dopamine D(1) receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D(1) receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D(1)-EGFP). D(1)-EGFP was similar to the untagged D(1) receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D(1)-EGFP and two mutants in which either Thr268 or Ser380 was replaced with Ala were stably expressed in NS20Y neuroblastoma cells. Pretreatment with the protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) or substitution of Ala for Thr268 reduced agonist-stimulated phosphorylation of the receptor and resulted in diminished trafficking of the receptor to the perinuclear region of the cell. Substitution of Ala for Thr268 had no effect, however, on agonist-induced receptor sequestration or desensitization of cyclic AMP accumulation. Substitution of Ala for Ser380 had no effect on D(1) receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D(1) receptor on Thr268 regulates a late step in the sorting of the receptor to the perinuclear region of the cell, but that phosphorylation of Thr268 is not required for receptor sequestration or maximal desensitization of cyclic AMP accumulation.
Mason
John N
JN
Medical Research Service, Veterans Affairs Medical Center, Oregon Health and Science University, Portland, Oregon 97201, USA.
Kozell
Laura B
LB
Neve
Kim A
KA
D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D018491:Dopamine Agonists / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D017447:Receptors, Dopamine D1 / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018491:Dopamine Agonists; D008164:Luminescent Proteins; D017447:Receptors, Dopamine D1; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases