Akt is essential for insulin modulation of amphetamine-induced human dopamine transporter cell-surface redistribution.
Uptake by the dopamine transporter (DAT) is the primary pathway for the clearance of extracellular dopamine (DA) and consequently for regulating the magnitude and duration of dopaminergic signaling. Amphetamine (AMPH) has been shown to decrease simultaneously DAT cell-surface expression and [(3)H]DA uptake. We have shown that insulin and its subsequent signaling through the phosphatidylinositol 3-kinase (PI3K)-dependent pathway oppose this effect of AMPH by promoting increased cell-surface expression. Here, we used human embryonic kidney 293 cells stably expressing the human DAT (hDAT cells) to investigate the downstream cellular components important for this effect of insulin. Akt is a protein kinase effector immediately downstream of PI3K. Both overexpression of a dominant-negative mutant of Akt (K179R) and the addition of 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML9), a pharmacological inhibitor of Akt, decreased cell-surface expression of DAT, suggesting a role of basal Akt signaling in the homoeostasis of DAT. Moreover, expression of a constitutively active Akt mutant reduced the ability of AMPH to decrease hDAT cell-surface expression as well as [(3)H]DA uptake. In contrast, overexpression of K179R blocked the ability of insulin to oppose AMPH-induced reduction of hDAT cell-surface expression and [(3)H]DA uptake, as did ML9. Our data demonstrate that hDAT cell-surface expression is regulated by the insulin signaling pathway and that Akt plays a key role in the hormonal modulation of AMPH-induced hDAT trafficking and in the regulation of basal hDAT cell-surface expression.
Garcia
B G
BG
Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Vanderbilt University, 465 21st Avenue South, Nashville, TN 37232-8548, USA.
Wei
Y
Y
Moron
J A
JA
Lin
R Z
RZ
Javitch
J A
JA
Galli
A
A
D000661:Amphetamine / Q000494:pharmacology*; D001381:Azepines / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D007328:Insulin / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt
D001381:Azepines; D050483:Dopamine Plasma Membrane Transport Proteins; D007328:Insulin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; C493214:SLC6A3 protein, human; C056218:ML 9; D000661:Amphetamine; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D004298:Dopamine