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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

Association of DJ-1 with DAT.(A) Co-immunoprecipitation of DAT with DJ-1 was performed by incubating 500 μg of solubilized HEK-293T cells that were co-transfected with DAT and DJ-1 with 2 μg of DJ-1 antibodies or with 2 μg of mouse IgG controls. 500 ng of HEK-293T lysate was used as a positive control. The top panel shown co-immunoprecipitation of DAT with DJ-1 and the lower panel confirms immunoprecipitation of DJ-1. (B) Co-immunopreciptation of DJ-1 with DAT. 500 μg of lysates from HEK-293T co-expressing DAT and DJ-1 was incubated with 2 μg of DAT antibodies or with 2 μg of rat IgG control antibody. Five μg of HEK-293T lysate was used as a positive control. Although we use antibodies from different species for the immunoprecipitation and the western blot, there is still some crossreactivity that leads to the detection of IgG bands in the resulting immunoblots (IgG HC—heavy chain; IgG LC–light chain). (C) Co-immunoprecipitation of DAT with DJ-1 from solubilized rat striatal tissue. 750 μg of striatal tissue was immunoprecipitated with DJ-1 antibody. Resulting immunoprecipitates were run on SDS-PAGE, transferred to PVDF membranes, and blotted with DAT monoclonal antibodies.
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pone.0136641.g003: Association of DJ-1 with DAT.(A) Co-immunoprecipitation of DAT with DJ-1 was performed by incubating 500 μg of solubilized HEK-293T cells that were co-transfected with DAT and DJ-1 with 2 μg of DJ-1 antibodies or with 2 μg of mouse IgG controls. 500 ng of HEK-293T lysate was used as a positive control. The top panel shown co-immunoprecipitation of DAT with DJ-1 and the lower panel confirms immunoprecipitation of DJ-1. (B) Co-immunopreciptation of DJ-1 with DAT. 500 μg of lysates from HEK-293T co-expressing DAT and DJ-1 was incubated with 2 μg of DAT antibodies or with 2 μg of rat IgG control antibody. Five μg of HEK-293T lysate was used as a positive control. Although we use antibodies from different species for the immunoprecipitation and the western blot, there is still some crossreactivity that leads to the detection of IgG bands in the resulting immunoblots (IgG HC—heavy chain; IgG LC–light chain). (C) Co-immunoprecipitation of DAT with DJ-1 from solubilized rat striatal tissue. 750 μg of striatal tissue was immunoprecipitated with DJ-1 antibody. Resulting immunoprecipitates were run on SDS-PAGE, transferred to PVDF membranes, and blotted with DAT monoclonal antibodies.

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)

Association of DJ-1 with DAT.(A) Co-immunoprecipitation of DAT with DJ-1 was performed by incubating 500 μg of solubilized HEK-293T cells that were co-transfected with DAT and DJ-1 with 2 μg of DJ-1 antibodies or with 2 μg of mouse IgG controls. 500 ng of HEK-293T lysate was used as a positive control. The top panel shown co-immunoprecipitation of DAT with DJ-1 and the lower panel confirms immunoprecipitation of DJ-1. (B) Co-immunopreciptation of DJ-1 with DAT. 500 μg of lysates from HEK-293T co-expressing DAT and DJ-1 was incubated with 2 μg of DAT antibodies or with 2 μg of rat IgG control antibody. Five μg of HEK-293T lysate was used as a positive control. Although we use antibodies from different species for the immunoprecipitation and the western blot, there is still some crossreactivity that leads to the detection of IgG bands in the resulting immunoblots (IgG HC—heavy chain; IgG LC–light chain). (C) Co-immunoprecipitation of DAT with DJ-1 from solubilized rat striatal tissue. 750 μg of striatal tissue was immunoprecipitated with DJ-1 antibody. Resulting immunoprecipitates were run on SDS-PAGE, transferred to PVDF membranes, and blotted with DAT monoclonal antibodies.
© Copyright Policy
Related In: Results  -  Collection

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pone.0136641.g003: Association of DJ-1 with DAT.(A) Co-immunoprecipitation of DAT with DJ-1 was performed by incubating 500 μg of solubilized HEK-293T cells that were co-transfected with DAT and DJ-1 with 2 μg of DJ-1 antibodies or with 2 μg of mouse IgG controls. 500 ng of HEK-293T lysate was used as a positive control. The top panel shown co-immunoprecipitation of DAT with DJ-1 and the lower panel confirms immunoprecipitation of DJ-1. (B) Co-immunopreciptation of DJ-1 with DAT. 500 μg of lysates from HEK-293T co-expressing DAT and DJ-1 was incubated with 2 μg of DAT antibodies or with 2 μg of rat IgG control antibody. Five μg of HEK-293T lysate was used as a positive control. Although we use antibodies from different species for the immunoprecipitation and the western blot, there is still some crossreactivity that leads to the detection of IgG bands in the resulting immunoblots (IgG HC—heavy chain; IgG LC–light chain). (C) Co-immunoprecipitation of DAT with DJ-1 from solubilized rat striatal tissue. 750 μg of striatal tissue was immunoprecipitated with DJ-1 antibody. Resulting immunoprecipitates were run on SDS-PAGE, transferred to PVDF membranes, and blotted with DAT monoclonal antibodies.
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