Phorbol ester induced trafficking-independent regulation and enhanced phosphorylation of the dopamine transporter associated with membrane rafts and cholesterol

Authors

  • James D. Foster,

    1. Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, North Dakota, USA
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  • Steven D. Adkins,

    1. Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, North Dakota, USA
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  • John R. Lever,

    1. Departments of Radiology, and Medical Pharmacology and Physiology, University of Missouri-Columbia and Harry S. Truman Veterans Administration Medical Center, Columbia, Missouri, USA
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  • Roxanne A. Vaughan

    1. Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, North Dakota, USA
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Address correspondence and reprints requests to Roxanne A. Vaughan, Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202. E-mail: rvaughan@medicine.nodak.edu

Abstract

We examined the mechanisms involved in protein kinase C (PKC)-dependent down-regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin-mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl-β-cyclodextrin (MβC) prior to treatment with the PKC activator phorbol 12-myristate, 13-acetate (PMA). Con A blocked PMA-induced surface reductions of DAT but only partially inhibited down-regulation, while MβC partially blocked down-regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC-induced DAT down-regulation occurs by a combination of trafficking and non-trafficking processes. Using density-gradient centrifugation, we found that DATs are distributed approximately equally between Triton-insoluble, cholesterol-rich membrane rafts and Triton-soluble non-raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA-induced loss of cell surface DAT occurred only from non-raft populations, demonstrating that non-raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol-dependent non-trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft-non-raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non-rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.

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