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Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

Cremona ML, Matthies HJ, Pau K, Bowton E, Speed N, Lute BJ, Anderson M, Sen N, Robertson SD, Vaughan RA, Rothman JE, Galli A, Javitch JA, Yamamoto A - Nat. Neurosci. (2011)

Bottom Line: Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity.We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization.In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Columbia University, College of Physicians and Surgeons, New York, New York, USA.

ABSTRACT
Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

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Flot1 overexpression attenuates Gö6850-mediated inhibition of PKC-triggered endocytosisFlot1 overexpression in EM4-YFP-DAT cells attenuated Gö6850-mediated inhibition of PKC-regulated endocytosis. All experiments were performed in this stable cell line unless noted otherwise. a,b. Image based analysis. a. ‘% Cell with Internalization’ was calculated as cells considered positive for internalization (YFP-DAT co-localized to EEA1-positive endosomes) after 1 µM PMA for 30 min. PKC-triggered internalization of DAT was significantly inhibited by Gö6850 in a dose dependent manner (open circles; p < 0.0001). Flot1 overexpression significantly diminished the Gö6850-mediated inhibition (closed circles, p = 0.0078). For each dose, 150 to 200 cells were analyzed. Scale bar = 10 µm. Complete statistics can be found in Supporting Materials. b. Flot1 overexpression in the presence of inhibitor (Flot1+ Gö6850) blunted the effect of inhibitor alone, permitting internalization (white arrows). Scale bar = 10 µm. c. Cell surface biotinylation measuring YFP-DAT surface availability. “DAT surface expression (% vehicle)’ is calculated as described (Fig.1d). Values are normalized to no PMA. Data are plotted as Mean + SEM (n = 5). Ctrl and Gö6850 cells were transfected with mRFP alone as transfection control. Complete blots can be found in Figure. S10.
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Figure 2: Flot1 overexpression attenuates Gö6850-mediated inhibition of PKC-triggered endocytosisFlot1 overexpression in EM4-YFP-DAT cells attenuated Gö6850-mediated inhibition of PKC-regulated endocytosis. All experiments were performed in this stable cell line unless noted otherwise. a,b. Image based analysis. a. ‘% Cell with Internalization’ was calculated as cells considered positive for internalization (YFP-DAT co-localized to EEA1-positive endosomes) after 1 µM PMA for 30 min. PKC-triggered internalization of DAT was significantly inhibited by Gö6850 in a dose dependent manner (open circles; p < 0.0001). Flot1 overexpression significantly diminished the Gö6850-mediated inhibition (closed circles, p = 0.0078). For each dose, 150 to 200 cells were analyzed. Scale bar = 10 µm. Complete statistics can be found in Supporting Materials. b. Flot1 overexpression in the presence of inhibitor (Flot1+ Gö6850) blunted the effect of inhibitor alone, permitting internalization (white arrows). Scale bar = 10 µm. c. Cell surface biotinylation measuring YFP-DAT surface availability. “DAT surface expression (% vehicle)’ is calculated as described (Fig.1d). Values are normalized to no PMA. Data are plotted as Mean + SEM (n = 5). Ctrl and Gö6850 cells were transfected with mRFP alone as transfection control. Complete blots can be found in Figure. S10.

Mentions: How is PKC-triggered internalization achieved? Mutagenesis of canonical PKC phosphorylation sites in DAT did not affect its PKC-regulated endocytosis in heterologous systems25, 26, suggesting that rather than directly phosphorylating the transporter, PKC controls internalization by phosphorylating an unknown intermediary protein. Since structurally distinct NTTs can be internalized in response to PMA, and this PMA-triggered event is observed across a wide range of cells (Fig.1, S1), the intermediary protein may be a ubiquitous protein involved in fundamental cellular trafficking events. Pre-incubating EM4-YFP-DAT with the reversible PKC inhibitor Gö6850 (also known as Bisindolmaleimide I or GF 109203X) inhibited PMA-triggered internalization in a dose-dependent manner (Fig.2a–b). We hypothesized that overexpression of the requisite target of PKC for internalization would compete with the inhibitor for binding since Gö6850 reversibly binds the substrate binding site of PKC and consequently enhance internalization at low effective concentrations of the inhibitor (Fig.S2).


Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

Cremona ML, Matthies HJ, Pau K, Bowton E, Speed N, Lute BJ, Anderson M, Sen N, Robertson SD, Vaughan RA, Rothman JE, Galli A, Javitch JA, Yamamoto A - Nat. Neurosci. (2011)

Flot1 overexpression attenuates Gö6850-mediated inhibition of PKC-triggered endocytosisFlot1 overexpression in EM4-YFP-DAT cells attenuated Gö6850-mediated inhibition of PKC-regulated endocytosis. All experiments were performed in this stable cell line unless noted otherwise. a,b. Image based analysis. a. ‘% Cell with Internalization’ was calculated as cells considered positive for internalization (YFP-DAT co-localized to EEA1-positive endosomes) after 1 µM PMA for 30 min. PKC-triggered internalization of DAT was significantly inhibited by Gö6850 in a dose dependent manner (open circles; p < 0.0001). Flot1 overexpression significantly diminished the Gö6850-mediated inhibition (closed circles, p = 0.0078). For each dose, 150 to 200 cells were analyzed. Scale bar = 10 µm. Complete statistics can be found in Supporting Materials. b. Flot1 overexpression in the presence of inhibitor (Flot1+ Gö6850) blunted the effect of inhibitor alone, permitting internalization (white arrows). Scale bar = 10 µm. c. Cell surface biotinylation measuring YFP-DAT surface availability. “DAT surface expression (% vehicle)’ is calculated as described (Fig.1d). Values are normalized to no PMA. Data are plotted as Mean + SEM (n = 5). Ctrl and Gö6850 cells were transfected with mRFP alone as transfection control. Complete blots can be found in Figure. S10.
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Figure 2: Flot1 overexpression attenuates Gö6850-mediated inhibition of PKC-triggered endocytosisFlot1 overexpression in EM4-YFP-DAT cells attenuated Gö6850-mediated inhibition of PKC-regulated endocytosis. All experiments were performed in this stable cell line unless noted otherwise. a,b. Image based analysis. a. ‘% Cell with Internalization’ was calculated as cells considered positive for internalization (YFP-DAT co-localized to EEA1-positive endosomes) after 1 µM PMA for 30 min. PKC-triggered internalization of DAT was significantly inhibited by Gö6850 in a dose dependent manner (open circles; p < 0.0001). Flot1 overexpression significantly diminished the Gö6850-mediated inhibition (closed circles, p = 0.0078). For each dose, 150 to 200 cells were analyzed. Scale bar = 10 µm. Complete statistics can be found in Supporting Materials. b. Flot1 overexpression in the presence of inhibitor (Flot1+ Gö6850) blunted the effect of inhibitor alone, permitting internalization (white arrows). Scale bar = 10 µm. c. Cell surface biotinylation measuring YFP-DAT surface availability. “DAT surface expression (% vehicle)’ is calculated as described (Fig.1d). Values are normalized to no PMA. Data are plotted as Mean + SEM (n = 5). Ctrl and Gö6850 cells were transfected with mRFP alone as transfection control. Complete blots can be found in Figure. S10.
Mentions: How is PKC-triggered internalization achieved? Mutagenesis of canonical PKC phosphorylation sites in DAT did not affect its PKC-regulated endocytosis in heterologous systems25, 26, suggesting that rather than directly phosphorylating the transporter, PKC controls internalization by phosphorylating an unknown intermediary protein. Since structurally distinct NTTs can be internalized in response to PMA, and this PMA-triggered event is observed across a wide range of cells (Fig.1, S1), the intermediary protein may be a ubiquitous protein involved in fundamental cellular trafficking events. Pre-incubating EM4-YFP-DAT with the reversible PKC inhibitor Gö6850 (also known as Bisindolmaleimide I or GF 109203X) inhibited PMA-triggered internalization in a dose-dependent manner (Fig.2a–b). We hypothesized that overexpression of the requisite target of PKC for internalization would compete with the inhibitor for binding since Gö6850 reversibly binds the substrate binding site of PKC and consequently enhance internalization at low effective concentrations of the inhibitor (Fig.S2).

Bottom Line: Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity.We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization.In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Columbia University, College of Physicians and Surgeons, New York, New York, USA.

ABSTRACT
Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

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