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A Physical Interaction between the Dopamine Transporter and DJ-1 Facilitates Increased Dopamine Reuptake.

Luk B, Mohammed M, Liu F, Lee FJ - PLoS ONE (2015)

Bottom Line: Co-expression of DAT and DJ-1 in HEK-293T cells leads to an increase in [3H] dopamine uptake that does not appear to be mediated by increased total DAT expression but rather through an increase in DAT cell surface localization.Co-expression of a mini-gene that can disrupt the DAT/DJ-1 complex appears to block the increase in [3H] dopamine uptake by DJ-1.Our study suggests that DJ-1 may also play a role in regulating dopamine levels by modifying DAT activity.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.

ABSTRACT
The regulation of the dopamine transporter (DAT) impacts extracellular dopamine levels after release from dopaminergic neurons. Furthermore, a variety of protein partners have been identified that can interact with and modulate DAT function. In this study we show that DJ-1 can potentially modulate DAT function. Co-expression of DAT and DJ-1 in HEK-293T cells leads to an increase in [3H] dopamine uptake that does not appear to be mediated by increased total DAT expression but rather through an increase in DAT cell surface localization. In addition, through a series of GST affinity purifications and co-immunoprecipitations, we provide evidence that the DAT can be found in a complex with DJ-1, which involve distinct regions within both DAT and DJ-1. Using in vitro binding experiments we also show that this complex can be formed in part by a direct interaction between DAT and DJ-1. Co-expression of a mini-gene that can disrupt the DAT/DJ-1 complex appears to block the increase in [3H] dopamine uptake by DJ-1. Mutations in DJ-1 have been linked to familial forms of Parkinson's disease, yet the normal physiological function of DJ-1 remains unclear. Our study suggests that DJ-1 may also play a role in regulating dopamine levels by modifying DAT activity.

No MeSH data available.


Related in: MedlinePlus

Co-expression of DJ-1 increases DAT localization at the cell surface.HEK-293T cells were transfected with CFP-DAT and either pcDNA3 or DJ-1-YFP and fluorescence live cell imaging was performed on an inverted microscope 24 hours after transfection. In cells expressing CFP-DAT alone, DAT localization occurs both intracellularly and on the cell surface. However, co-expression of DJ-1-YFP leads to a significant increase in CFP-DAT localized at the cell surface. Merged images were generated using ImageJ Intensity Correlation Analysis plugin and displays the PDM value (Product of the Differences from the Mean) for each pixel with a corresponding scale bar. Images are representative of 4 independent experiments.
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pone.0136641.g002: Co-expression of DJ-1 increases DAT localization at the cell surface.HEK-293T cells were transfected with CFP-DAT and either pcDNA3 or DJ-1-YFP and fluorescence live cell imaging was performed on an inverted microscope 24 hours after transfection. In cells expressing CFP-DAT alone, DAT localization occurs both intracellularly and on the cell surface. However, co-expression of DJ-1-YFP leads to a significant increase in CFP-DAT localized at the cell surface. Merged images were generated using ImageJ Intensity Correlation Analysis plugin and displays the PDM value (Product of the Differences from the Mean) for each pixel with a corresponding scale bar. Images are representative of 4 independent experiments.

Mentions: To determine if the enhanced DAT mediated DA uptake upon co-expression of DJ-1 is due to increased DAT cell surface localization, we compared CFP-DAT cellular localization in cells transfected with CFP-DAT alone versus HEK-293T cells co-transfected with DJ-1-YFP. As shown in Fig 2, live imaging of cells that were transfected with CFP-DAT and the empty expression plasmid pcDNA3, 24 hours after transfection, revealed a significant population of DAT localized to intracellular compartments as well as a proportion of DAT localized at the cell surface. However, co-expression of DJ-1-YFP leads to an increase in cell surface localization of CFP-DAT, particularly in areas adjacent to other cells co-expressing both CFP-DAT and DJ-1-YFP. Although there is a significant amount of differential localization between DAT and DJ-1, co-localization of both proteins is evident throughout various regions of the cell including the cell surface. DJ-1 localization does not appear to be specific to any particular subcellular compartment but localized throughout the cell, with very low DJ-1-YFP fluorescence signal in the nucleus.


A Physical Interaction between the Dopamine Transporter and DJ-1 Facilitates Increased Dopamine Reuptake.

Luk B, Mohammed M, Liu F, Lee FJ - PLoS ONE (2015)

Co-expression of DJ-1 increases DAT localization at the cell surface.HEK-293T cells were transfected with CFP-DAT and either pcDNA3 or DJ-1-YFP and fluorescence live cell imaging was performed on an inverted microscope 24 hours after transfection. In cells expressing CFP-DAT alone, DAT localization occurs both intracellularly and on the cell surface. However, co-expression of DJ-1-YFP leads to a significant increase in CFP-DAT localized at the cell surface. Merged images were generated using ImageJ Intensity Correlation Analysis plugin and displays the PDM value (Product of the Differences from the Mean) for each pixel with a corresponding scale bar. Images are representative of 4 independent experiments.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4549284&req=5

pone.0136641.g002: Co-expression of DJ-1 increases DAT localization at the cell surface.HEK-293T cells were transfected with CFP-DAT and either pcDNA3 or DJ-1-YFP and fluorescence live cell imaging was performed on an inverted microscope 24 hours after transfection. In cells expressing CFP-DAT alone, DAT localization occurs both intracellularly and on the cell surface. However, co-expression of DJ-1-YFP leads to a significant increase in CFP-DAT localized at the cell surface. Merged images were generated using ImageJ Intensity Correlation Analysis plugin and displays the PDM value (Product of the Differences from the Mean) for each pixel with a corresponding scale bar. Images are representative of 4 independent experiments.
Mentions: To determine if the enhanced DAT mediated DA uptake upon co-expression of DJ-1 is due to increased DAT cell surface localization, we compared CFP-DAT cellular localization in cells transfected with CFP-DAT alone versus HEK-293T cells co-transfected with DJ-1-YFP. As shown in Fig 2, live imaging of cells that were transfected with CFP-DAT and the empty expression plasmid pcDNA3, 24 hours after transfection, revealed a significant population of DAT localized to intracellular compartments as well as a proportion of DAT localized at the cell surface. However, co-expression of DJ-1-YFP leads to an increase in cell surface localization of CFP-DAT, particularly in areas adjacent to other cells co-expressing both CFP-DAT and DJ-1-YFP. Although there is a significant amount of differential localization between DAT and DJ-1, co-localization of both proteins is evident throughout various regions of the cell including the cell surface. DJ-1 localization does not appear to be specific to any particular subcellular compartment but localized throughout the cell, with very low DJ-1-YFP fluorescence signal in the nucleus.

Bottom Line: Co-expression of DAT and DJ-1 in HEK-293T cells leads to an increase in [3H] dopamine uptake that does not appear to be mediated by increased total DAT expression but rather through an increase in DAT cell surface localization.Co-expression of a mini-gene that can disrupt the DAT/DJ-1 complex appears to block the increase in [3H] dopamine uptake by DJ-1.Our study suggests that DJ-1 may also play a role in regulating dopamine levels by modifying DAT activity.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.

ABSTRACT
The regulation of the dopamine transporter (DAT) impacts extracellular dopamine levels after release from dopaminergic neurons. Furthermore, a variety of protein partners have been identified that can interact with and modulate DAT function. In this study we show that DJ-1 can potentially modulate DAT function. Co-expression of DAT and DJ-1 in HEK-293T cells leads to an increase in [3H] dopamine uptake that does not appear to be mediated by increased total DAT expression but rather through an increase in DAT cell surface localization. In addition, through a series of GST affinity purifications and co-immunoprecipitations, we provide evidence that the DAT can be found in a complex with DJ-1, which involve distinct regions within both DAT and DJ-1. Using in vitro binding experiments we also show that this complex can be formed in part by a direct interaction between DAT and DJ-1. Co-expression of a mini-gene that can disrupt the DAT/DJ-1 complex appears to block the increase in [3H] dopamine uptake by DJ-1. Mutations in DJ-1 have been linked to familial forms of Parkinson's disease, yet the normal physiological function of DJ-1 remains unclear. Our study suggests that DJ-1 may also play a role in regulating dopamine levels by modifying DAT activity.

No MeSH data available.


Related in: MedlinePlus