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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

Mini-gene DJ-1,3A (DJ-1 CT161-175) blocks the interaction between DJ-1 and DAT.(A) Co-expression of the DJ-1,3A mini-gene blocks the physical interaction between DJ-1 and DAT. Co-immunoprecipitation of DAT with DJ-1 was performed by incubating 500 μg of solubilized HEK-293T cells that were co-transfected with DAT, DJ-1, and either pcDNA3 or DJ-1,3A. Western blot reveals that the DAT/DJ-1 interaction is blocked by the addition of DJ-1,3A mini-gene. (B) 2.5 μg of HEK-293T lysates were run on SDS-PAGE and immunoblotted with DAT, DJ-1 or β-tubulin. (C) Quantification of co-immunoprecipitation of DAT with DJ-1 in HEK-293T cells co-transfected with pcDNA3 or the DJ-1,3A mini-gene* P<0.05, t-test. n = 7. (D) Mini-gene DJ-1,3A blocks the functional interaction between DJ-1 and DAT. [3H] DA uptake was measured in HEK-293T cells that were co-expressing the DJ-1,3A mini-gene. DJ-1-mediated increase in DAT-mediated [3H] DA uptake was blocked with the co-expression of mini-gene encoding DJ-1,3A but does not alter the uptake in cells only transfected with DAT. (* P<0.05, significantly different from DAT/pcD group; # P<0.05, significantly different from DAT/DJ-1 group; one-way ANOVA post hoc Tukey test, n = 3). (E) Quantification of DAT cell surface localization using a cell-based ELISA. HEK-293T cells co-expressing DAT and DJ-1 exhibit an approximate 30% increase in DAT cell surface localization compared with cells co-transfected with DAT and pcDNA3, an effect blocked by the co-expression of the DJ-1,3A mini-gene (** P<0.01 compared to DAT/p3 group; one-way ANOVA, post hoc Tukey test, n = 6).
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pone.0136641.g005: Mini-gene DJ-1,3A (DJ-1 CT161-175) blocks the interaction between DJ-1 and DAT.(A) Co-expression of the DJ-1,3A mini-gene blocks the physical interaction between DJ-1 and DAT. Co-immunoprecipitation of DAT with DJ-1 was performed by incubating 500 μg of solubilized HEK-293T cells that were co-transfected with DAT, DJ-1, and either pcDNA3 or DJ-1,3A. Western blot reveals that the DAT/DJ-1 interaction is blocked by the addition of DJ-1,3A mini-gene. (B) 2.5 μg of HEK-293T lysates were run on SDS-PAGE and immunoblotted with DAT, DJ-1 or β-tubulin. (C) Quantification of co-immunoprecipitation of DAT with DJ-1 in HEK-293T cells co-transfected with pcDNA3 or the DJ-1,3A mini-gene* P<0.05, t-test. n = 7. (D) Mini-gene DJ-1,3A blocks the functional interaction between DJ-1 and DAT. [3H] DA uptake was measured in HEK-293T cells that were co-expressing the DJ-1,3A mini-gene. DJ-1-mediated increase in DAT-mediated [3H] DA uptake was blocked with the co-expression of mini-gene encoding DJ-1,3A but does not alter the uptake in cells only transfected with DAT. (* P<0.05, significantly different from DAT/pcD group; # P<0.05, significantly different from DAT/DJ-1 group; one-way ANOVA post hoc Tukey test, n = 3). (E) Quantification of DAT cell surface localization using a cell-based ELISA. HEK-293T cells co-expressing DAT and DJ-1 exhibit an approximate 30% increase in DAT cell surface localization compared with cells co-transfected with DAT and pcDNA3, an effect blocked by the co-expression of the DJ-1,3A mini-gene (** P<0.01 compared to DAT/p3 group; one-way ANOVA, post hoc Tukey test, n = 6).

Mentions: To examine the effects of disrupting the physical interaction between DAT/DJ-1, we co-transfected mini-genes that encode the sequence within DJ-1,3A [S161-K175] that would compete with wild-type DJ-1 for binding to the DAT. As shown in Fig 5A, when indexed through co-immunoprecipitation assays there is a significant disruption in the DAT/DJ-1 interaction in cells that are co-expressing the DJ-1,3A mini-gene compared to cells co-transfected with the empty expression plasmid. To verify that the difference in co-immunoprecipitation is not due to changes in DAT or DJ-1 expression levels induced non-specifically by co-transfection of the DJ-1,3A mini-gene, we measured the levels of both DAT and DJ-1 in our samples. As seen in Fig 5B, there was no significant difference in expression levels of either DAT or DJ-1 with DJ-1,3A mini-gene co-transfection. Quantification of DAT/DJ-1 co-immunoprecipitation results reveals an approximate 30% decrease in DAT/DJ-1 co-immunoprecipitation levels as a result of DJ-1,3A mini-gene co-transfection (Fig 5C). As observed previously, cells co-transfected with DAT and DJ-1 exhibited a significant increase in [3H]DA uptake compared to cells transfected with DAT alone. This increase in DA uptake was blocked in cells that were co-transfected with the DJ-1,3A mini-gene. Moreover, this effect appears to be specific to the DAT/DJ-1 interaction as the DJ-1,3A mini-gene had no effect on cells expressing DAT alone (Fig 5D). Therefore, the data appears to confirm that the DAT/DJ-1 direct interaction facilitates the increase in DAT activity. We have previously shown an increase in DAT cell surface localization with co-expression of DJ-1 (Fig 2). To quantify these results and to examine the effects of the DJ-1,3A mini-gene we examined DAT cell surface levels in HEK-293T cells co-transfected with DAT and either pcDNA3, DJ-1 or DAT and the DJ-1,3A mini-gene using a cell based ELISA assay. We measured cell surface localization using an antibody that recognizes an epitope on the 2nd extracellular loop of the DAT. As shown in Fig 5E, there is a 30% increase in DAT cell surface localization with co-expression with DJ-1. This increase in plasmalemmal DAT is blocked with coexpression of the DJ-1,3A mini-gene.


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)

Mini-gene DJ-1,3A (DJ-1 CT161-175) blocks the interaction between DJ-1 and DAT.(A) Co-expression of the DJ-1,3A mini-gene blocks the physical interaction between DJ-1 and DAT. Co-immunoprecipitation of DAT with DJ-1 was performed by incubating 500 μg of solubilized HEK-293T cells that were co-transfected with DAT, DJ-1, and either pcDNA3 or DJ-1,3A. Western blot reveals that the DAT/DJ-1 interaction is blocked by the addition of DJ-1,3A mini-gene. (B) 2.5 μg of HEK-293T lysates were run on SDS-PAGE and immunoblotted with DAT, DJ-1 or β-tubulin. (C) Quantification of co-immunoprecipitation of DAT with DJ-1 in HEK-293T cells co-transfected with pcDNA3 or the DJ-1,3A mini-gene* P<0.05, t-test. n = 7. (D) Mini-gene DJ-1,3A blocks the functional interaction between DJ-1 and DAT. [3H] DA uptake was measured in HEK-293T cells that were co-expressing the DJ-1,3A mini-gene. DJ-1-mediated increase in DAT-mediated [3H] DA uptake was blocked with the co-expression of mini-gene encoding DJ-1,3A but does not alter the uptake in cells only transfected with DAT. (* P<0.05, significantly different from DAT/pcD group; # P<0.05, significantly different from DAT/DJ-1 group; one-way ANOVA post hoc Tukey test, n = 3). (E) Quantification of DAT cell surface localization using a cell-based ELISA. HEK-293T cells co-expressing DAT and DJ-1 exhibit an approximate 30% increase in DAT cell surface localization compared with cells co-transfected with DAT and pcDNA3, an effect blocked by the co-expression of the DJ-1,3A mini-gene (** P<0.01 compared to DAT/p3 group; one-way ANOVA, post hoc Tukey test, n = 6).
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pone.0136641.g005: Mini-gene DJ-1,3A (DJ-1 CT161-175) blocks the interaction between DJ-1 and DAT.(A) Co-expression of the DJ-1,3A mini-gene blocks the physical interaction between DJ-1 and DAT. Co-immunoprecipitation of DAT with DJ-1 was performed by incubating 500 μg of solubilized HEK-293T cells that were co-transfected with DAT, DJ-1, and either pcDNA3 or DJ-1,3A. Western blot reveals that the DAT/DJ-1 interaction is blocked by the addition of DJ-1,3A mini-gene. (B) 2.5 μg of HEK-293T lysates were run on SDS-PAGE and immunoblotted with DAT, DJ-1 or β-tubulin. (C) Quantification of co-immunoprecipitation of DAT with DJ-1 in HEK-293T cells co-transfected with pcDNA3 or the DJ-1,3A mini-gene* P<0.05, t-test. n = 7. (D) Mini-gene DJ-1,3A blocks the functional interaction between DJ-1 and DAT. [3H] DA uptake was measured in HEK-293T cells that were co-expressing the DJ-1,3A mini-gene. DJ-1-mediated increase in DAT-mediated [3H] DA uptake was blocked with the co-expression of mini-gene encoding DJ-1,3A but does not alter the uptake in cells only transfected with DAT. (* P<0.05, significantly different from DAT/pcD group; # P<0.05, significantly different from DAT/DJ-1 group; one-way ANOVA post hoc Tukey test, n = 3). (E) Quantification of DAT cell surface localization using a cell-based ELISA. HEK-293T cells co-expressing DAT and DJ-1 exhibit an approximate 30% increase in DAT cell surface localization compared with cells co-transfected with DAT and pcDNA3, an effect blocked by the co-expression of the DJ-1,3A mini-gene (** P<0.01 compared to DAT/p3 group; one-way ANOVA, post hoc Tukey test, n = 6).
Mentions: To examine the effects of disrupting the physical interaction between DAT/DJ-1, we co-transfected mini-genes that encode the sequence within DJ-1,3A [S161-K175] that would compete with wild-type DJ-1 for binding to the DAT. As shown in Fig 5A, when indexed through co-immunoprecipitation assays there is a significant disruption in the DAT/DJ-1 interaction in cells that are co-expressing the DJ-1,3A mini-gene compared to cells co-transfected with the empty expression plasmid. To verify that the difference in co-immunoprecipitation is not due to changes in DAT or DJ-1 expression levels induced non-specifically by co-transfection of the DJ-1,3A mini-gene, we measured the levels of both DAT and DJ-1 in our samples. As seen in Fig 5B, there was no significant difference in expression levels of either DAT or DJ-1 with DJ-1,3A mini-gene co-transfection. Quantification of DAT/DJ-1 co-immunoprecipitation results reveals an approximate 30% decrease in DAT/DJ-1 co-immunoprecipitation levels as a result of DJ-1,3A mini-gene co-transfection (Fig 5C). As observed previously, cells co-transfected with DAT and DJ-1 exhibited a significant increase in [3H]DA uptake compared to cells transfected with DAT alone. This increase in DA uptake was blocked in cells that were co-transfected with the DJ-1,3A mini-gene. Moreover, this effect appears to be specific to the DAT/DJ-1 interaction as the DJ-1,3A mini-gene had no effect on cells expressing DAT alone (Fig 5D). Therefore, the data appears to confirm that the DAT/DJ-1 direct interaction facilitates the increase in DAT activity. We have previously shown an increase in DAT cell surface localization with co-expression of DJ-1 (Fig 2). To quantify these results and to examine the effects of the DJ-1,3A mini-gene we examined DAT cell surface levels in HEK-293T cells co-transfected with DAT and either pcDNA3, DJ-1 or DAT and the DJ-1,3A mini-gene using a cell based ELISA assay. We measured cell surface localization using an antibody that recognizes an epitope on the 2nd extracellular loop of the DAT. As shown in Fig 5E, there is a 30% increase in DAT cell surface localization with co-expression with DJ-1. This increase in plasmalemmal DAT is blocked with coexpression of the DJ-1,3A mini-gene.

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