Phorbol ester induced trafficking-independent regulation and enhanced phosphorylation of the dopamine transporter associated with membrane rafts and cholesterol.
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-beta-cyclodextrin (MbetaC) 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 MbetaC 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.
Foster
James D
JD
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, North Dakota 58202, USA.
Adkins
Steven D
SD
Lever
John R
JR
Vaughan
Roxanne A
RA
D000818:Animals; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018374:LLC-PK1 Cells; D021962:Membrane Microdomains / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D013552:Swine
D050483:Dopamine Plasma Membrane Transport Proteins; D010703:Phorbol Esters; C119222:phorbol-12-myristate; D002784:Cholesterol