Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters.
The dopamine transporter plays an essential role in the modulation of dopaminergic neurotransmission by mediating the reuptake of dopamine into presynaptic neurons. In cells expressing the dopamine transporter, activation of protein kinase C by phorbol esters results in a significant reduction in dopamine uptake. This phorbol ester-mediated inhibition of dopamine transport is associated with a decrease in V(max), although the apparent affinity of the transporter for dopamine remains unchanged. Using a green fluorescent protein-tagged dopamine transporter stably expressed in Madin-Darby canine kidney cells, we show in live cells that the decrease in transporter activity is caused by the rapid internalization of carriers from the plasma membrane. This redistribution of the transporter is specific to phorbol ester activation and is unaffected by the presence of either substrates or inhibitors of the carrier. Upon the addition of phorbol esters, transporters at the cell surface are rapidly endocytosed through a clathrin-mediated and dynamin-dependent mechanism into early endosomes, where they colocalize with transferrin. The internalized carrier is targeted to the endosomal/lysosomal pathway and is completely degraded within 2 h of protein kinase C activation. Phorbol ester-mediated alterations in the trafficking of the dopamine transporter may serve as a mechanism for controlling extracellular dopamine levels in the central nervous system.
Daniels
G M
GM
Department of Cell Biology, Oregon Health Science University, Portland, Oregon 97201, USA.
Amara
S G
SG
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010704:Phorbols / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D002352:Carrier Proteins; D002966:Clathrin; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010704:Phorbols; D011994:Recombinant Proteins; C067713:lactacystin; D005576:Colforsin; D002738:Chloroquine; D003513:Cycloheximide; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D004298:Dopamine; D000111:Acetylcysteine; C033085:phorbol