Regulation of dopamine D1 receptor trafficking by protein kinase A-dependent phosphorylation

John N. Mason, Laura B. Kozell, Kim Neve

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Abstract

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 D1 receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D1 receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D1-EGFP). D1-EGFP was similar to the untagged D1 receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D1-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 D1 receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D1 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.

Original languageEnglish (US)
Pages (from-to)806-816
Number of pages11
JournalMolecular Pharmacology
Volume61
Issue number4
DOIs
StatePublished - 2002

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Dopamine D1 Receptors
Cyclic AMP-Dependent Protein Kinases
Phosphorylation
Cyclic AMP
Protein Kinase Inhibitors
Neuroblastoma
GTP-Binding Proteins
Pharmacology
Ligands
Mutation

ASJC Scopus subject areas

  • Pharmacology

Cite this

Regulation of dopamine D1 receptor trafficking by protein kinase A-dependent phosphorylation. / Mason, John N.; Kozell, Laura B.; Neve, Kim.

In: Molecular Pharmacology, Vol. 61, No. 4, 2002, p. 806-816.

Research output: Contribution to journalArticle

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