Brudnick Neuropsychiatric Research Institute, Department of Psychiatry; Melikian Lab
Biochemistry, Biophysics, and Structural Biology | Neuroscience and Neurobiology
The dopamine transporter (DAT) removes dopamine from the extracellular milieu and is potently inhibited by number of psychoactive drugs, including cocaine, amphetamines, and methylphenidate (Ritalin). Multiple lines of evidence demonstrate that protein kinase C (PKC) down-regulates dopamine transport, primarily by redistributing DAT from the plasma membrane to endosomal compartments, although the mechanisms facilitating transporter sequestration are not defined. Here, we demonstrate that DAT constitutively internalizes and recycles in rat pheochromocytoma (PC12) cells. Temperature blockades demonstrated basal internalization and reliance on recycling to maintain DAT cell surface levels. In contrast, recycling blockade with bafilomycin A1 significantly decreased transferrin receptor (TfR) surface expression but had no effect on DAT surface levels, suggesting that DAT and TfR traffic via distinct endosomal mechanisms. Kinetic analyses reveal robust constitutive DAT cycling to and from the plasma membrane, independent of transporter expression levels. In contrast, phorbol ester-mediated PKC activation accelerated DAT endocytosis and attenuated transporter recycling in a manner sensitive to DAT expression levels. These data demonstrate constitutive DAT trafficking and that PKC-mediated DAT sequestration is achieved by a combination of accelerated internalization and reduced recycling. Additionally, the differential sensitivity to expression level exhibited by constitutive and regulated DAT trafficking suggests that these two processes are mediated by independent cellular mechanisms.
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© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Publisher PDF posted after 12 months as allowed by the publisher's author rights policy at http://www.jbc.org/site/misc/License%20to%20Publish_author%20retains%20ownership_v1_JBC.pdf.
DOI of Published Version
J Biol Chem. 2003 Jun 13;278(24):22168-74. Epub 2003 Apr 7. Link to article on publisher's site
The Journal of biological chemistry
Loder, Merewyn K. and Melikian, Haley, "The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines" (2003). University of Massachusetts Medical School Faculty Publications. 1457.