Protein kinase-mediated bidirectional trafficking and functional regulation of the human dopamine transporter.
Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [3H]dopamine uptake kinetics, [3H] CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS-7 cells following modulation of protein kinase C (PKC)- and protein kinase A (PKA)-dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 microM), but not alphaPDD, reduced the Vmax (approximately 1 pmol/min/10(5) cells) for [3H]DA uptake by approximately 40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [3H]DA uptake velocity by approximately 30%, an effect mimicked by the potent PKA inhibitor Rp-cAMPS. Activation of PKA-dependent pathways with Sp-cAMPS did not significantly modify DA uptake. Neither the Km of [3H]DA uptake (approximately 200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA-dependent mechanisms, the estimated density of hDAT as indexed by whole-cell [3H] CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [3H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS-7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC- and PKA-dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function.
Department of Psychiatry, University of Toronto, Ontario, Canada.
Z B
ZB
Pristupa
F
F
McConkey
F
F
Liu
H Y
HY
Man
F J
FJ
Lee
Y T
YT
Wang
H B
HB
Niznik
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003042:Cocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010703:Phorbol Esters; D011994:Recombinant Proteins; C493214:SLC6A3 protein, human; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; C008876:phorbol-12,13-didecanoate; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; D003042:Cocaine; D013755:Tetradecanoylphorbol Acetate; D004298:Dopamine