Constitutive and Protein Kinase C‐Induced Internalization of the Dopamine Transporter is Mediated by a Clathrin‐Dependent Mechanism
Open Access
- 22 December 2004
- Vol. 6 (2), 157-170
- https://doi.org/10.1111/j.1600-0854.2005.00259.x
Abstract
The amount of dopamine transporter (DAT) present at the cell surface is rapidly regulated by the rates of DAT internalization to endosomes and DAT recycling back to the plasma membrane. The re-distribution of the transporter from the cell surface to endosomes was induced by phorbol ester activation of protein kinase C in porcine aortic endothelial cells stably expressing the human DAT. Inhibition of DAT recycling with the carboxylic ionophore monensin also caused significant accumulation of DAT in early endosomes and a concomitant loss of DAT from the cell surface, due to protein kinase C-independent constitutive internalization of DAT in the absence of recycling. Such monensin-induced relocation of DAT to endosomes was therefore utilized as a measure of the constitutive internalization of DAT. Knock-down of clathrin heavy chain or dynamin II by small interfering RNAs dramatically inhibited both constitutive and protein kinase C-mediated internalization of DAT. In contrast, neither monensin-dependent nor phorbol ester-induced re-distribution of DAT were affected by inhibitors of endocytosis through cholesterol-rich membrane microdomains. Mutational analysis revealed the potential importance of amino acid residues 587–597 in DAT internalization. Altogether, the data suggest that both constitutive and protein kinase C-mediated internalization of DAT utilize the clathrin-dependent endocytic pathway, but likely involve unconventional mechanisms.Keywords
This publication has 42 references indexed in Scilit:
- Partitioning of the Serotonin Transporter into Lipid Microdomains Modulates Transport of SerotoninPublished by Elsevier BV ,2004
- Selective Stimulation of Caveolar Endocytosis by Glycosphingolipids and CholesterolMolecular Biology of the Cell, 2004
- Regulated Internalization and Phosphorylation of the Native Norepinephrine Transporter in Response to Phorbol EstersPublished by Elsevier BV ,2004
- Substrate-Induced Trafficking of the Dopamine Transporter in Heterologously Expressing Cells and in Rat Striatal Synaptosomal PreparationsJournal of Pharmacology and Experimental Therapeutics, 2003
- Clathrin-mediated endocytosis in AP-2–depleted cellsThe Journal of cell biology, 2003
- Oligomerization of Dopamine Transporters Visualized in Living Cells by Fluorescence Resonance Energy Transfer MicroscopyJournal of Biological Chemistry, 2003
- Which Form of Dopamine Is the Substrate for the Human Dopamine Transporter: the Cationic or the Uncharged Species?Published by Elsevier BV ,1999
- Dynamin-mediated Internalization of CaveolaeThe Journal of cell biology, 1998
- Dynamin at the Neck of Caveolae Mediates Their Budding to Form Transport Vesicles by GTP-driven Fission from the Plasma Membrane of EndotheliumThe Journal of cell biology, 1998
- Subcellular localization and molecular topology of the dopamine transporter in the striatum and substantia nigraJournal of Comparative Neurology, 1997