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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

Identification of the DJ-1 domain involved in the DAT/DJ-1 interaction.(A) Schematic illustration of the different segments of DJ-1 that were used to generate GST fusion peptides and the nomenclature used for each of the various different regions. (B) Association of DJ-1,3 region with DAT. Various GST fusion peptides of DJ-1 were used to affinity purify the DAT from lysates prepared from HEK-293T cells transfected with DAT. 50 μg of HEK-293T lysate was used as a positive control. Western blots reveal the ability of the DJ-1,3 (G108-D189) region to affinity purify the DAT, while none of the other peptides were capable of pulling down DAT. Lower panel shows Ponceau S stain of blots to index the relative amounts of GST fusion peptide. GST fusion peptide levels were equivalent to or greater than the amount of GST-DJ-1,3 peptide. (C) The DJ-1,3A region mediates the interaction between DJ-1 and DAT. GST fusion peptides of DJ-1,3A and DJ-1,3B regions were used to affinity purify the DAT from lysates prepared from HEK293T cells transfected with DAT. 1 μg of HEK293T lysate was used as a positive control. Western blots show the ability of the DJ-1,3A (S161-K175) region to affinity purify DAT, while none of the other peptides were capable of pulling down DAT. Lower panel shows Ponceau S staining of blots to index the relative amounts of GST fusion peptide. GST fusion peptide levels were equivalent to or greater than the amount of GST-DJ-1,3A peptide. (D) Direct association of the intracellular loop 4 of DAT (DAT-IL4) with DJ-1. His-tagged full-length DJ-1 protein (10 μg) was used to affinity purify 0.5 μg various GST fusion peptides of DAT. GST protein alone (0.1 μg) was used as a positive control. Western blots reveal the ability of DJ-1 to affinity purify DAT-IL4, while none of the other DAT GST fusion peptides were pulled down by DJ-1. Lower panel shows Ponceau S stain of blots to index the relative amounts of His-tagged DJ-1 used in each sample.
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pone.0136641.g004: Identification of the DJ-1 domain involved in the DAT/DJ-1 interaction.(A) Schematic illustration of the different segments of DJ-1 that were used to generate GST fusion peptides and the nomenclature used for each of the various different regions. (B) Association of DJ-1,3 region with DAT. Various GST fusion peptides of DJ-1 were used to affinity purify the DAT from lysates prepared from HEK-293T cells transfected with DAT. 50 μg of HEK-293T lysate was used as a positive control. Western blots reveal the ability of the DJ-1,3 (G108-D189) region to affinity purify the DAT, while none of the other peptides were capable of pulling down DAT. Lower panel shows Ponceau S stain of blots to index the relative amounts of GST fusion peptide. GST fusion peptide levels were equivalent to or greater than the amount of GST-DJ-1,3 peptide. (C) The DJ-1,3A region mediates the interaction between DJ-1 and DAT. GST fusion peptides of DJ-1,3A and DJ-1,3B regions were used to affinity purify the DAT from lysates prepared from HEK293T cells transfected with DAT. 1 μg of HEK293T lysate was used as a positive control. Western blots show the ability of the DJ-1,3A (S161-K175) region to affinity purify DAT, while none of the other peptides were capable of pulling down DAT. Lower panel shows Ponceau S staining of blots to index the relative amounts of GST fusion peptide. GST fusion peptide levels were equivalent to or greater than the amount of GST-DJ-1,3A peptide. (D) Direct association of the intracellular loop 4 of DAT (DAT-IL4) with DJ-1. His-tagged full-length DJ-1 protein (10 μg) was used to affinity purify 0.5 μg various GST fusion peptides of DAT. GST protein alone (0.1 μg) was used as a positive control. Western blots reveal the ability of DJ-1 to affinity purify DAT-IL4, while none of the other DAT GST fusion peptides were pulled down by DJ-1. Lower panel shows Ponceau S stain of blots to index the relative amounts of His-tagged DJ-1 used in each sample.

Mentions: To explore the possibility that this increase in DAT function mediated by DJ-1 is facilitated, at least in part, by a direct physical interaction we examined whether DJ-1 forms a complex with DAT through co-immunoprecipitation experiments. Immunoprecipitation of DJ-1 with anti-DJ-1 antibody separated by protein A/G-agarose beads leads to the co-precipitation of DAT in cells co-transfected with both DAT and DJ-1 (Fig 3A). Heterologous DAT expression in HEK-293T cells often leads to the detection of an immature DAT immunoreactive band at ~55 kDa and a larger diffuse band that represents the mature DAT, which is heavily glycosylated and is visualized in a Western blot as a large smear centered at ~80 kDa [33,34,73]. No bands were detected in samples that were precipitated with mouse IgG or with samples incubated with only protein A/G-agarose beads. We also show the reverse co-immunoprecipitation whereby DJ-1 also precipitates with the DAT (Fig 3B). To provide evidence that the interaction can occur in neurons we also co-immunoprecipitated DAT with DJ-1 from solubilized rat striatal lysates (Fig 3C). Next, we delineated regions within DJ-1 that are critical in the formation of the DAT/DJ-1 complex by creating GST fusion proteins that were truncations of full length DJ-1 (Fig 4A). As shown in Fig 4B, affinity purifications using GST-DJ-1,3 (T108-D189) leads to the pull-down of DAT from solubilized HEK-293T lysates. Neither the parental GST fusion protein itself nor other truncations of DJ-1 GST fusion proteins led to the purification of DAT. This suggests that the carboxyl half of DJ-1 is important in the formation of the DAT/DJ-1 complex. To define a more discrete region within the DJ-1 carboxyl terminus that is involved in the DAT/DJ-1 interaction, we created smaller truncations of the DJ1-3 region, which were named DJ-1,3A [S161-K175] and DJ-1,3B [E176-D189] (Fig 4C). Using these GST proteins in affinity purification experiments with solubilized lysates of HEK-293T expressing DAT, we were able to define a 15 amino acid segment [DJ-1,3A: S161-K175] within DJ-1 that appears to be critical for the DAT/DJ-1 complex formation.


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)

Identification of the DJ-1 domain involved in the DAT/DJ-1 interaction.(A) Schematic illustration of the different segments of DJ-1 that were used to generate GST fusion peptides and the nomenclature used for each of the various different regions. (B) Association of DJ-1,3 region with DAT. Various GST fusion peptides of DJ-1 were used to affinity purify the DAT from lysates prepared from HEK-293T cells transfected with DAT. 50 μg of HEK-293T lysate was used as a positive control. Western blots reveal the ability of the DJ-1,3 (G108-D189) region to affinity purify the DAT, while none of the other peptides were capable of pulling down DAT. Lower panel shows Ponceau S stain of blots to index the relative amounts of GST fusion peptide. GST fusion peptide levels were equivalent to or greater than the amount of GST-DJ-1,3 peptide. (C) The DJ-1,3A region mediates the interaction between DJ-1 and DAT. GST fusion peptides of DJ-1,3A and DJ-1,3B regions were used to affinity purify the DAT from lysates prepared from HEK293T cells transfected with DAT. 1 μg of HEK293T lysate was used as a positive control. Western blots show the ability of the DJ-1,3A (S161-K175) region to affinity purify DAT, while none of the other peptides were capable of pulling down DAT. Lower panel shows Ponceau S staining of blots to index the relative amounts of GST fusion peptide. GST fusion peptide levels were equivalent to or greater than the amount of GST-DJ-1,3A peptide. (D) Direct association of the intracellular loop 4 of DAT (DAT-IL4) with DJ-1. His-tagged full-length DJ-1 protein (10 μg) was used to affinity purify 0.5 μg various GST fusion peptides of DAT. GST protein alone (0.1 μg) was used as a positive control. Western blots reveal the ability of DJ-1 to affinity purify DAT-IL4, while none of the other DAT GST fusion peptides were pulled down by DJ-1. Lower panel shows Ponceau S stain of blots to index the relative amounts of His-tagged DJ-1 used in each sample.
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Show All Figures
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pone.0136641.g004: Identification of the DJ-1 domain involved in the DAT/DJ-1 interaction.(A) Schematic illustration of the different segments of DJ-1 that were used to generate GST fusion peptides and the nomenclature used for each of the various different regions. (B) Association of DJ-1,3 region with DAT. Various GST fusion peptides of DJ-1 were used to affinity purify the DAT from lysates prepared from HEK-293T cells transfected with DAT. 50 μg of HEK-293T lysate was used as a positive control. Western blots reveal the ability of the DJ-1,3 (G108-D189) region to affinity purify the DAT, while none of the other peptides were capable of pulling down DAT. Lower panel shows Ponceau S stain of blots to index the relative amounts of GST fusion peptide. GST fusion peptide levels were equivalent to or greater than the amount of GST-DJ-1,3 peptide. (C) The DJ-1,3A region mediates the interaction between DJ-1 and DAT. GST fusion peptides of DJ-1,3A and DJ-1,3B regions were used to affinity purify the DAT from lysates prepared from HEK293T cells transfected with DAT. 1 μg of HEK293T lysate was used as a positive control. Western blots show the ability of the DJ-1,3A (S161-K175) region to affinity purify DAT, while none of the other peptides were capable of pulling down DAT. Lower panel shows Ponceau S staining of blots to index the relative amounts of GST fusion peptide. GST fusion peptide levels were equivalent to or greater than the amount of GST-DJ-1,3A peptide. (D) Direct association of the intracellular loop 4 of DAT (DAT-IL4) with DJ-1. His-tagged full-length DJ-1 protein (10 μg) was used to affinity purify 0.5 μg various GST fusion peptides of DAT. GST protein alone (0.1 μg) was used as a positive control. Western blots reveal the ability of DJ-1 to affinity purify DAT-IL4, while none of the other DAT GST fusion peptides were pulled down by DJ-1. Lower panel shows Ponceau S stain of blots to index the relative amounts of His-tagged DJ-1 used in each sample.
Mentions: To explore the possibility that this increase in DAT function mediated by DJ-1 is facilitated, at least in part, by a direct physical interaction we examined whether DJ-1 forms a complex with DAT through co-immunoprecipitation experiments. Immunoprecipitation of DJ-1 with anti-DJ-1 antibody separated by protein A/G-agarose beads leads to the co-precipitation of DAT in cells co-transfected with both DAT and DJ-1 (Fig 3A). Heterologous DAT expression in HEK-293T cells often leads to the detection of an immature DAT immunoreactive band at ~55 kDa and a larger diffuse band that represents the mature DAT, which is heavily glycosylated and is visualized in a Western blot as a large smear centered at ~80 kDa [33,34,73]. No bands were detected in samples that were precipitated with mouse IgG or with samples incubated with only protein A/G-agarose beads. We also show the reverse co-immunoprecipitation whereby DJ-1 also precipitates with the DAT (Fig 3B). To provide evidence that the interaction can occur in neurons we also co-immunoprecipitated DAT with DJ-1 from solubilized rat striatal lysates (Fig 3C). Next, we delineated regions within DJ-1 that are critical in the formation of the DAT/DJ-1 complex by creating GST fusion proteins that were truncations of full length DJ-1 (Fig 4A). As shown in Fig 4B, affinity purifications using GST-DJ-1,3 (T108-D189) leads to the pull-down of DAT from solubilized HEK-293T lysates. Neither the parental GST fusion protein itself nor other truncations of DJ-1 GST fusion proteins led to the purification of DAT. This suggests that the carboxyl half of DJ-1 is important in the formation of the DAT/DJ-1 complex. To define a more discrete region within the DJ-1 carboxyl terminus that is involved in the DAT/DJ-1 interaction, we created smaller truncations of the DJ1-3 region, which were named DJ-1,3A [S161-K175] and DJ-1,3B [E176-D189] (Fig 4C). Using these GST proteins in affinity purification experiments with solubilized lysates of HEK-293T expressing DAT, we were able to define a 15 amino acid segment [DJ-1,3A: S161-K175] within DJ-1 that appears to be critical for the DAT/DJ-1 complex formation.

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