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Comparative Study
. 2011 Sep 28;31(39):13758-70.
doi: 10.1523/JNEUROSCI.2649-11.2011.

The Plasma Membrane-Associated GTPase Rin Interacts With the Dopamine Transporter and Is Required for Protein Kinase C-regulated Dopamine Transporter Trafficking

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Free PMC article
Comparative Study

The Plasma Membrane-Associated GTPase Rin Interacts With the Dopamine Transporter and Is Required for Protein Kinase C-regulated Dopamine Transporter Trafficking

Deanna M Navaroli et al. J Neurosci. .
Free PMC article

Abstract

Dopaminergic signaling and plasticity are essential to numerous CNS functions and pathologies, including movement, cognition, and addiction. The amphetamine- and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT C terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (for Ras-like in neurons) (Rit2), as a protein that interacts with the DAT C-terminal endocytic signal. Yeast two-hybrid, GST pull down and FRET studies establish that DAT and Rin directly interact, and colocalization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Coimmunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis.

Figures

Figure 1.
Figure 1.
Rin is expressed in PC12 cells and rat striatum and specifically coimmunoprecipitates with DAT. A, Rin expression in catecholaminergic tissue and cell lines. Twenty micrograms of PC12 and SK-N-MC lysate and 150 μg of rat striatal lysate were resolved by SDS-PAGE and immunoblotted with mouse anti-Rin antibodies. B, Coimmunoprecipitations. DAT was immunoprecipitated from equivalent amounts lysate from PC12 cells transfected with either vector (−) or DAT (+). Control immunoprecipitations were performed using anti-TfR antibodies or with Protein A/G beads alone. Bead eluents (Bead) and 110th supernatant volumes (Sup) were resolved by SDS-PAGE and immunoblotted with an anti-Rin antibody. Representative blots are shown (n = 3). C, Coimmunoprecipitations from rat striatal synaptosomes. Immunoprecipitations were performed with solubilized rat striatal synaptosomes using Protein A beads coated with rabbit anti-DAT antibodies or rabbit IgG alone. Eluents were resolved by SDS-PAGE, and immunoreactive bands were detected with the indicated antibodies. A representative blot is shown (n = 2).
Figure 2.
Figure 2.
DAT and Rin colocalization at the plasma membrane is enriched in lipid raft microdomains. Immunofluorescence. PC12 cells were cotransfected with DAT and CFP–Rin and were fixed and stained as follows. A, Cells were stained with anti-DAT and anti-GFP antibodies and imaged as described in Materials and Methods. Boxes indicate an enlargement of the areas indicated with white boxes in the merged images. B, Cells were labeled with Alexa Fluor 594–CTX and were subsequently stained with anti-DAT and anti-GFP antibodies and imaged as described in Materials and Methods. C, Enlargement of the boxed areas from images in B. Arrows indicate DAT-immunoreactive pixels that colocalize with neither Rin (blue) nor CTX (red). Arrowheads indicate foci of DAT/Rin colocalization that also colocalize with CTX. D, Average data. DAT/Rin colocalization in CTX-positive (+CTX) and CTX-negative (−CTX) cell regions was measured at 10 independent thresholds per channel, as described in Materials and Methods. Average data at the median threshold are expressed as percentage ± SEM DAT/Rin colocalization. *p < 0.0001, significantly different from −CTX, paired t test; n = 16.
Figure 3.
Figure 3.
Rin and DAT oligomerization as demonstrated by FRET microscopy in intact cells. HEK293 cells were transiently transfected with cDNAs encoding CFP- or YFP-tagged proteins as indicated, and epifluorescence microscopy was performed 2 d after transfection. A, The first and second columns show images obtained with CFP and YFP filter sets, respectively; the third column displays a corrected and normalized FRET image (NFRET) established with PIXFRET. A look-up table of the color code used is presented in the last column. All images shown are representative of three to seven experiments. In all images, background fluorescence was subtracted. B, Averaged data. Normalized FRET efficiencies (NFRET values) are given for cells expressing the following constructs: C-Rin and membrane-bound YFP (membr-YFP; n = 15), Y-DAT and membr-CFP (n = 25), C-TfR and Y-Rin (n = 20), C-Rin and Y-Rin (n = 16), C-SERT and Y-Rin (n = 27), C-rGAT1 and Y-Rin (n = 30), C-DAT and Y-Rin (n = 24), C-Rin and Y-DAT (n = 20), C-DAT and Y-DAT (n = 23), and C-TfR and Y-TfR (n = 37). All values were analyzed by one-way ANOVA with Dunn's multiple comparison test. *p < 0.001, significantly different from (YFP–DAT and membr-CFP), (CFP–Rin and YFP–Rin), and (Rin and membr-YFP). **p < 0.001, significantly different from (CFP–TfR and YFP–Rin), (CFP–Rin and YFP–Rin), and (Rin and membr-YFP). See Table 1 for statistical analysis of complete dataset.
Figure 4.
Figure 4.
Rin interacts with the DAT C terminus. GST pull-down assays. PC12 homogenate was incubated with either GST or GST–DAT 587–617, 1 h, 37°C. Complexes were isolated with glutathione agarose, and Rin was detected by immunoblot as described in Materials and Methods. A, Rin is isolated with the DAT C terminus but not GST alone. B, Concentration dependence. GST–DAT 587–617 was incubated with increasing concentrations of PC12 homogenate, 1 h, 37°C, followed by isolation of complexes with glutathione agarose. Top, Representative immunoblot probed for Rin. Bottom, Averaged data. Results are expressed as percentage maximal Rin signal versus log PC12 homogenate concentration (milligrams per milliliters) and were fit to a sigmoidal dose–response curve (r2 = 0.98, n = 3).
Figure 5.
Figure 5.
DAT/Rin interactions are regulated by PKC activation and are sensitive to DAT C-terminal residues 587–590. A–C, Coimmunoprecipitations. PC12 cells stably expressing either wild-type DAT (A) or DAT 587–590(4A) (B) were pretreated with either 1 μm BIM or vehicle, 20 min, 37°C, followed by treatment with either vehicle or 1 μm PMA, 30 min, 37°C. Cells were lysed, and DAT was immunoprecipitated from equivalent amounts of cellular protein. Immunoprecipitants were resolved by SDS-PAGE and immunoblotted for both DAT and Rin. Left, Representative immunoblots. Right, Averaged data. Rin signals from nonsaturating bands were normalized to DAT signals and are expressed as percentage vehicle signal ± SEM. *p < 0.002, significantly different from vehicle control (one-way ANOVA with Tukey's multiple comparison test; n = 4). C, Basal Rin interaction with wild-type versus 587–590(4A) DAT. Left, Representative immunoblot. Right, Averaged data. Rin signals from nonsaturating bands were normalized to DAT signals and expressed as percentage wild-type signal ± SEM. *p < 0.0005, significantly different from wild-type DAT (Student's t test; n = 3). D, GST pull downs. GST fused to either wild-type or DAT 587–590(4A) C termini were induced and isolated on glutathione sepharose as described in Materials and Methods. Equivalent amounts of GST–DAT fusion proteins were incubated with PC12 homogenates, and bound proteins were resolved by SDS-PAGE and immunoblotted for both Rin and GST. Left, Representative immunoblot. Right, Averaged data. Rin densities were normalized to total GST pulled down for each sample and are expressed as percentage ± SEM Rin isolated compared with wild-type GST–DAT. *p < 0.04, significantly different from wild-type GST–DAT (Student's t test; n = 5).
Figure 6.
Figure 6.
PKC-induced DAT internalization requires Rin activity. Internalization assays. PC12 cells were cotransfected with DAT and the indicated Rin or control cDNAs and were assayed 48–72 h after transfection. Relative DAT internalization rates over 10 min were measured by reversible biotinylation during treatment with or without 1 μm PMA, 37°C as described in Materials and Methods. Top, Representative immunoblots showing total surface DAT at time 0 (T), strip controls (S), and internalized DAT under vehicle-treated (V) and PMA-treated (P) conditions. Bottom, Average data. veh, Vehicle. Data are expressed as percentage ± SEM DAT internalized/10 min compared with total surface DAT at t = 0. *p < 0.001, **p < 0.05, significantly different from vehicle-treated control (Student's t test; n = 4).
Figure 7.
Figure 7.
Guanyl nucleotide exchange is required for PKC-mediated Rin dissociation from DAT. Immunofluorescence. PC12 cells were cotransfected with DAT and the indicated Rin or control cDNAs and were assayed 48 h after transfection. Cells were treated with or without 1 μm PMA, 30 min, 37°C and were fixed, stained with anti-DAT or anti-GFP (to detect CFP–Rin) antibodies, and imaged as described in Materials and Methods. Planes through the cell center are shown and are representative of 12–16 cells imaged per condition in instances in which PMA-induced internalization was apparent (n = 4).
Figure 8.
Figure 8.
Rin is required for PKC-mediated DAT sequestration. hRin knockdown and antibody internalization assay. A, hRin knockdown. hRin-directed shRNAs were screened in HEK293 cells, 72 h after transfection. Top, Representative immunoblot showing Rin levels in vector-transfected, scrambled shRNA-transfected (scr), and hRin228-transfected cells, with actin probed as a loading control. Bottom, Averaged data. Data are expressed as percentage Rin levels compared with vector-transfected cells (normalized to actin loading control). *p < 0.05, significantly different from vector control (one-way ANOVA with Dunnett's post hoc analysis; n = 5). B, C, Antibody internalization assay. SK-N-MC cells cotransfected with EL2–HA–DAT and the indicated constructs were assayed 72 h after transfection. Cells were labeled with HA antibody, and DAT internalization was assessed for 15 min, 37°C in the presence of 1 μm PMA as described in Materials and Methods. Cells expressing shRNA constructs were identified by GFP coexpression. Optical z-stacks were collected and deconvolved as described in Materials and Methods. B, Averaged data. Data are expressed as percentage ± SEM cells exhibiting PMA-induced intracellular DAT puncta for each of the indicated transfection conditions. *p < 0.05, significantly different from vector-transfected control (one-way ANOVA with Dunnett's multiple comparison test; n = 3). C, Representative images. Planes through the cell center are shown for two cells per condition and are representative of 30–34 cells imaged per condition over three independent experiments.
Figure 9.
Figure 9.
Rin is required for PKC-induced DAT functional downregulation. [3H]DA uptake assays. SK-N-MC cells were cotransfected with DAT and the indicated constructs, and [3H]DA uptake was assessed 72 h after transfection. Cells were treated with or without 1 μm PMA, 30 min, 37°C, followed by addition of [3H]DA as described in Materials and Methods. Averaged data are shown and are expressed as percentage ± SEM uptake of vehicle-treated cells. *p < 0.03, significantly different from vehicle-treated control (Student's t test; n = 4).

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