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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

Wild-type DJ-1 up-regulates DAT activity.(A) Bar plot of [3H]DA uptake in HEK-293T cells co-transfected with DAT/pcDNA3 (pcD), DAT/wild-type (wt) DJ-1, DAT/M26I DJ-1, or DAT/D149A DJ-1. Cells co-expressing DAT and wt DJ-1 have significantly up-regulated DAT activity as indexed by [3H]DA uptake (** P<0.01, significantly different from DAT/pcD group; one-way ANOVA post hoc SNK test, n = 11). (B) Representative saturation plot of [3H]DA uptake in HEK-293T cells co-expressing DAT with pcDNA3, wt DJ-1, M26I DJ-1, or D149A DJ-1. Cells co-expressing DAT and wt DJ-1 exhibit an increase in Vmax for [3H]DA uptake accumulation without any significant alteration in estimated affinity values (Km = 3.77–8.73 μM, n = 11, one-way ANOVA, P = 0.2988). Western blots of samples that were transfected with DAT and pcDNA3, wt DJ-1, M26I DJ-1, or D149A DJ-1 were analyzed for semi-quantitative measurement of either total DAT levels (C) or DJ-1 levels (D) expressed in cells (** P<0.01, *** P<0.001 significantly different from DAT/pcDNA3 control group, n = 3).
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pone.0136641.g001: Wild-type DJ-1 up-regulates DAT activity.(A) Bar plot of [3H]DA uptake in HEK-293T cells co-transfected with DAT/pcDNA3 (pcD), DAT/wild-type (wt) DJ-1, DAT/M26I DJ-1, or DAT/D149A DJ-1. Cells co-expressing DAT and wt DJ-1 have significantly up-regulated DAT activity as indexed by [3H]DA uptake (** P<0.01, significantly different from DAT/pcD group; one-way ANOVA post hoc SNK test, n = 11). (B) Representative saturation plot of [3H]DA uptake in HEK-293T cells co-expressing DAT with pcDNA3, wt DJ-1, M26I DJ-1, or D149A DJ-1. Cells co-expressing DAT and wt DJ-1 exhibit an increase in Vmax for [3H]DA uptake accumulation without any significant alteration in estimated affinity values (Km = 3.77–8.73 μM, n = 11, one-way ANOVA, P = 0.2988). Western blots of samples that were transfected with DAT and pcDNA3, wt DJ-1, M26I DJ-1, or D149A DJ-1 were analyzed for semi-quantitative measurement of either total DAT levels (C) or DJ-1 levels (D) expressed in cells (** P<0.01, *** P<0.001 significantly different from DAT/pcDNA3 control group, n = 3).

Mentions: To first determine if DJ-1 can modulate DAT function, we measured [3H]DA uptake activity in cells that were co-expressing DAT with either wild-type DJ-1 or the empty expression plasmid pcDNA3. As shown in Fig 1A, there is a significant increase in DAT-mediated [3H]DA uptake in cells co-expressing DJ-1 compared to cells co-transfected with the empty expression plasmid. Furthermore, this increase in DAT activity appears to be specific to wild-type DJ-1 as co-expression of M26I or D149A DJ-1 mutants did not enhance DAT-mediated [3H]DA uptake (Fig 1A and 1B). Vmax values (pmol/105 cells/min) for DAT/p3, DAT/DJ-1, DAT/M26I and DAT/D149A were calculated to be 2.48 ± 0.88, 4.72 ± 1.47, 2.37 ± 0.94 and 2.65 ± 1.65, respectively. Homozygous M26I and heterozygous D149A DJ-1 mutations have been associated with early onset PD [51]. While it is unclear how exactly these mutations affect DJ-1 function, several studies suggest that the mutations can have potential effects on protein stability, aggregation dynamics and antioxidant function [66–72]. Examining the kinetics of [3H]DA uptake revealed that there was no significant change in the affinity for DA exhibited by the DAT upon co-expression of DJ-1 (Fig 1B). Km values (μM) of 6.29 ± 1.59, 8.73 ± 2.55, 3.77 ± 1.05 and 5.37 ± 2.53 were calculated for DAT/p3, DAT/DJ-1, DAT/M26I and DAT/D149A, respectively. In addition, the increase in [3H]DA uptake does not appear to be due to an increase in DAT protein levels as determined by western blots that were used to quantify the level of total DAT protein in cells co-transfected with DAT and DJ-1. As shown in Fig 1C, there was no significant change in DAT levels upon co-expression of either wild-type or mutant DJ-1 and suggests that the increase in DAT activity is due to another mechanism such as increased cell surface localization. In addition, DJ-1 expression levels were not significantly different between wildtype DJ-1 and the various mutants (Fig 1D). Western blots for both DAT and DJ-1 are shown in S1 Fig.


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)

Wild-type DJ-1 up-regulates DAT activity.(A) Bar plot of [3H]DA uptake in HEK-293T cells co-transfected with DAT/pcDNA3 (pcD), DAT/wild-type (wt) DJ-1, DAT/M26I DJ-1, or DAT/D149A DJ-1. Cells co-expressing DAT and wt DJ-1 have significantly up-regulated DAT activity as indexed by [3H]DA uptake (** P<0.01, significantly different from DAT/pcD group; one-way ANOVA post hoc SNK test, n = 11). (B) Representative saturation plot of [3H]DA uptake in HEK-293T cells co-expressing DAT with pcDNA3, wt DJ-1, M26I DJ-1, or D149A DJ-1. Cells co-expressing DAT and wt DJ-1 exhibit an increase in Vmax for [3H]DA uptake accumulation without any significant alteration in estimated affinity values (Km = 3.77–8.73 μM, n = 11, one-way ANOVA, P = 0.2988). Western blots of samples that were transfected with DAT and pcDNA3, wt DJ-1, M26I DJ-1, or D149A DJ-1 were analyzed for semi-quantitative measurement of either total DAT levels (C) or DJ-1 levels (D) expressed in cells (** P<0.01, *** P<0.001 significantly different from DAT/pcDNA3 control group, n = 3).
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Related In: Results  -  Collection

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pone.0136641.g001: Wild-type DJ-1 up-regulates DAT activity.(A) Bar plot of [3H]DA uptake in HEK-293T cells co-transfected with DAT/pcDNA3 (pcD), DAT/wild-type (wt) DJ-1, DAT/M26I DJ-1, or DAT/D149A DJ-1. Cells co-expressing DAT and wt DJ-1 have significantly up-regulated DAT activity as indexed by [3H]DA uptake (** P<0.01, significantly different from DAT/pcD group; one-way ANOVA post hoc SNK test, n = 11). (B) Representative saturation plot of [3H]DA uptake in HEK-293T cells co-expressing DAT with pcDNA3, wt DJ-1, M26I DJ-1, or D149A DJ-1. Cells co-expressing DAT and wt DJ-1 exhibit an increase in Vmax for [3H]DA uptake accumulation without any significant alteration in estimated affinity values (Km = 3.77–8.73 μM, n = 11, one-way ANOVA, P = 0.2988). Western blots of samples that were transfected with DAT and pcDNA3, wt DJ-1, M26I DJ-1, or D149A DJ-1 were analyzed for semi-quantitative measurement of either total DAT levels (C) or DJ-1 levels (D) expressed in cells (** P<0.01, *** P<0.001 significantly different from DAT/pcDNA3 control group, n = 3).
Mentions: To first determine if DJ-1 can modulate DAT function, we measured [3H]DA uptake activity in cells that were co-expressing DAT with either wild-type DJ-1 or the empty expression plasmid pcDNA3. As shown in Fig 1A, there is a significant increase in DAT-mediated [3H]DA uptake in cells co-expressing DJ-1 compared to cells co-transfected with the empty expression plasmid. Furthermore, this increase in DAT activity appears to be specific to wild-type DJ-1 as co-expression of M26I or D149A DJ-1 mutants did not enhance DAT-mediated [3H]DA uptake (Fig 1A and 1B). Vmax values (pmol/105 cells/min) for DAT/p3, DAT/DJ-1, DAT/M26I and DAT/D149A were calculated to be 2.48 ± 0.88, 4.72 ± 1.47, 2.37 ± 0.94 and 2.65 ± 1.65, respectively. Homozygous M26I and heterozygous D149A DJ-1 mutations have been associated with early onset PD [51]. While it is unclear how exactly these mutations affect DJ-1 function, several studies suggest that the mutations can have potential effects on protein stability, aggregation dynamics and antioxidant function [66–72]. Examining the kinetics of [3H]DA uptake revealed that there was no significant change in the affinity for DA exhibited by the DAT upon co-expression of DJ-1 (Fig 1B). Km values (μM) of 6.29 ± 1.59, 8.73 ± 2.55, 3.77 ± 1.05 and 5.37 ± 2.53 were calculated for DAT/p3, DAT/DJ-1, DAT/M26I and DAT/D149A, respectively. In addition, the increase in [3H]DA uptake does not appear to be due to an increase in DAT protein levels as determined by western blots that were used to quantify the level of total DAT protein in cells co-transfected with DAT and DJ-1. As shown in Fig 1C, there was no significant change in DAT levels upon co-expression of either wild-type or mutant DJ-1 and suggests that the increase in DAT activity is due to another mechanism such as increased cell surface localization. In addition, DJ-1 expression levels were not significantly different between wildtype DJ-1 and the various mutants (Fig 1D). Western blots for both DAT and DJ-1 are shown in S1 Fig.

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