Apoptozole

Dopamine Transporter is Downregulated and its Association with Chaperone Protein Hsc70 is Enhanced by Activation of Dopamine D3 Receptor

Pi-Kai Chang1,3 Kun-Yi Chien2 and Jin-Chung Chen1,3,4,5
1Graduate Institute of Biomedical Sciences, Department of Physiology and Pharmacology, School of Medicine, Chang-Gung University
2Graduate Institute of Biomedical Sciences, Department of Biochemistry and Molecular Biology, School of Medicine, Chang Gung University
3 Healthy Ageing Research Center, School of Medicine, Chang-Gung University 4Neuroscience Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan 5Chang Gung Memorial Hospital, Keelung, Taiwan
Address: No. 259, Wenhua 1st Road, Guishan Dist, Taoyuan City, Taiwan, R.O.C.33302
Tel: 011-886-3-2118800 ext 5282 Fax: 011-886-3-2118700
E-mail: [email protected]

Abstract
Synaptic dopamine (DA) concentrations are largely determined by the activities of presynaptic D2 and D3 autoreceptors (D2R and D3R) and DA transporter (DAT). Furthermore, the activity of DAT is regulated by phosphorylation events and protein interactions that affect its surface expression. Because DA autoreceptors and DAT coordinately maintain synaptic DA homeostasis, we hypothesized that D3R might crosstalk with DAT to fine-tune synaptic DA concentrations. To test this hypothesis,we established [3H]DA uptake and DAT surface expression assays in hD3/rDAT- double-transfected HEK-293 cells or limbic forebrain synaptosomal preparations. Ropinirole, a preferential D3R agonist, reduced [3H]DA uptake in HEK-hD3/rDAT cells in a dose-dependent manner, an effect which could be blocked by the D2R/D3R antagonist, raclopride. Furthermore, ropinirole also reduced DAT surface expression in limbic forebrain synaptosomes, and this effect could be blocked by raclopride or the internalization inhibitor, concanavalin A. To identify potential mediators of this apparent D3R-DAT crosstalk, DAT-associated proteins were co-immunoprecipitated from limbic forebrain synaptosomes after D3R activation and identified by MALDI- TOF. From this analysis, the Hsc70 chaperone was identified as a DAT-associated protein. Interestingly, ropinirole induced the association of Hsc70/Hsp70 with DAT, and the Hsc70/Hsp70 inhibitor, apoptozole, prevented the ropinirole-induced reduction of DAT surface expression. Together, these results suggest that D3R negatively regulates DAT activity by promoting the association of DAT and Hsc70/Hsp70.
Abbreviation:
Dopamine, DA; Dopamine D3 receptor, D3R; heat shock cognate protein 70, Hsc70; heat shock protein 70, Hsp70; concanavalin A, ConA; attention deficit with hyperactivity disorder, ADHD; norepinephrine transporter, NET; serotonin transporter, SERT; γ-aminobutyric acid transporter, GAT; glutamate transporter, GluT; transmembrane domains, TMs; protein kinase C, PKC; cAMP-dependent protein kinase, PKA; mitogen-activated protein kinases, MAPKs; cyclin-dependent kinases, CDKs; Ca2+/calmodulin-dependent protein kinase II, CaMKII; phosphoinositide 3-kinase, PI3K; extracellular signal- regulated kinases 1/2, ERK1/2; protein interacting with C kinase, PICK-1; receptor of activated protein C kinase 1, RACK-1; knock-down, KD; novel object recognition task, NORT; hydrphobic polyvinylidene difluoride, PVDF; vesicular monoamine transporter-2, VMAT-2; tyrosine hydroxylase, TH
Keywords: D3 autoreceptor; dopamine transporter; heat shock cognate protein 70; endocytosis; uptake

1.Introduction
Dopamine (DA) signaling is tightly controlled by presynaptic autoreceptors and transporters, which are crucial for proper functioning of a wide variety of physiological processes, including learning and memory, locomotion, cognition, and attention (Ford, 2014). Thus, it is unsurprising that abnormal DA regulation is a feature of many neurological or psychiatric disorders, such as Parkinson disease, psychosis and depression (Beaulieu and Gainetdinov, 2011). Regulation of DA neurotransmission is multifaceted. For instance, activation of presynaptic DA autoreceptors is known to induce auto-inhibition of dopaminergic neurons by inhibiting either tyrosine hydroxylase-mediated DA synthesis or vesicular DA release. In addition, other regulatory mechanisms fine-tune the DA signal in the synapse and are mainly induced by DA D2 receptor (D2R) and D3 receptor (D3R) subtypes (Bello et al., 2011; Chen et al., 2009; Ford, 2014). Importantly, most uptake of synaptic DA occurs via the DA transporter (DAT), which belongs to the SLC6 family of Na+/Cl– dependent neuronal membrane transporters (Gainetdinov and Caron, 2003). For these transporters, the rate of substrate translocation is principally determined by two factors: the protein surface expression level and the electrochemical gradient across the plasma membrane (Torres et al., 2003). Surface expression can be quickly modulated, as these transporters are constitutively internalized and recycled/trafficked back into the plasma membrane at relatively rapid rates. Numerous factors have been shown to acutely regulate DAT function by altering its surface expression or uptake efficiency via signaling through protein kinases or associated proteins (Foster and Vaughan, 2017; Torres, 2006). As such, protein kinase C (PKC), cAMP-dependent protein kinase (PKA), mitogen-activated protein kinases (MAPKs), cyclin-dependent kinases (CDKs), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phosphoinositide 3-kinase (PI3K) can all phosphorylate DAT at different amino acid residues, affecting transporter trafficking and/or internalization, and consequently, the synaptic DA clearance rate (Batchelor and Schenk, 1998; Carvelli et al., 2002; Speed et al., 2010; Vaughan and Foster, 2013; Vaughan et al., 1997).
Other than phosphorylation-dependent effects on trafficking, protein-protein interactions have been shown to affect the surface expression and uptake efficiency of DAT (Torres, 2006). Several cytosolic proteins are known to associate with DAT, including Vav2 (Zhu et al., 2015), nuclear protein Ctr9 (De Gois et al., 2015), - synuclein (Sidhu et al., 2004) and small G-protein Rin (Navaroli et al., 2011a).
Additionally, protein interacting with C kinase (PICK-1) and the receptor of activated protein C kinase 1 (RACK-1) functionally associate with PKC, through protein interactions, to stimulate PKC-mediated promotion of DAT uptake activity (Lee et al., 2004; Madsen et al., 2012). Furthermore, it is known that some membrane receptors interact directly with transporters to modulate the transporter uptake activity ( Lee et al., 2004; Madsen et al., 2012). For instance, kappa opioid receptor activation triggers its interaction with DAT to enhance DA uptake (Kivell et al., 2014), and D2R is similarly functionally coupled with DAT to stimulate DAT activity ( Lee et al., 2007). Thus, DAT activity and surface expression represent dynamic points of control for synaptic DA clearance and modulation of DA signaling (Torres et al., 2003).
In addition to its effects on DAT activity, PICK-1 is known to cluster D3R and increase its surface expression (Zheng et al., 2016), suggesting D3R and DAT may exhibit some crosstalk. While D3R has been reported to exert biphasic effects on DAT function and trafficking regulation (Zapata et al., 2007), the mechanisms of D3R- mediated regulation of DAT function are not clear. In this report, we found that activation of D3R decreased [3H]DA uptake and reduced DAT surface expression in both HEK-hD3/rDAT cells and striatal synaptosomes. Furthermore, systemic administration of ropinirole, a D3R preferential agonist, promoted the association of DAT and Hsc70/Hsp70, a heat shock cognate protein, in the limbic forebrain. Treatment with the Hsc70/Hsp70 inhibitor, apoptozole, effectively reversed the ropinirole-mediated suppression of DAT surface expression. The current findings suggest that D3R autoreceptor activation may stimulate the association of Hsc70/Hsp70 with DAT, leading to reduced DAT function at the plasma membrane.

2.Methods
2.1.Reagents
Antibodies were purchased from following sources: rabbit polyclonal antibody to D3R from Santa Cruz Biotechnology (Dallas, TX, USA); monoclonal rat antibody against the N-terminus of DAT from Merck Millipore (Burlington, MA, USA); rabbit polyclonal antibody to Hsc70 from Enzo Life Sciences (Farmindale, NY, USA); mouse monoclonal antibody to -actin from Sigma-Aldrich (St. Louis, MO, USA);
goat anti-rat antibody conjugated with FITC from Thermo Fisher Scientific (Waltham, MA, USA). Ropinirole was purchased from Tocris Bioscience (Avonmouth, Bristol, UK). 7-OH DPAT, raclopride, apoptozole, GBR12,909 and concanavalin A were purchased from Sigma Aldrich. FAUC365 was purchased from Glixx Laboratories (Hopkinton, MA, USA). Most drugs were dissolved in PBS buffer, except apoptozole and FAUC365, which were dissolved in DMSO. Dissolved drugs were passed
through a syringe filter before use (Merk Millipore). [3H]7-OH-DPAT and [3H] DA were purchased from Perkin-Elmer (Boston, MA, USA). Protein G agarose beads were purchased from Merck Millipore. Sulfo-NHS-SS-biotin and monomeric avidin beads were purchased from Thermo Fisher Scientific.
2.2.Cell culture and transfection
HEK-293 cells were cultured in MEM supplemented with 10% fetal bovine serum (Biological Inductries, Cromwell, CT, USA) and 1% penicillin/streptomycin (Biological Inductries), and maintained in incubators at 37℃, 5% CO2. Stable clones of human DA D3 receptor (hD3R)-expressing HEK-293 cells were established. The HEK-hD3 cells were maintained in 400 μg/ml G418 (Biological Inductries). The plasmid expressing the hD3R was a gift from Dr. Pierre Sokoloff (Department of Neurobiology and Molecular Pharmacology, Cantre Paul Broca, Paris, France). The rDAT expression vector (pCEP4 /CMV/rDAT) was transfected into HEK-hD3 and HEK-293 cells by Metafectene (Biontex, Munich, Germany). Two micrograms DNA and 6 l Metafectene mixture were added to cells in 6-well plates at a density of 5 × 105 cells per well. The rDAT cDNA plasmids were amplified in E. coli and the plasmid DNA was extracted. Restriction enzyme digestion was used to confirm the fragment size. Clones were picked and amplified from plates treated with 100 μg/ml
hygromycin B (Invitrogen, San Diego, CA, USA) selection. The appropriate cell clones were screened and validated with a receptor binding assay and a [3H]DA uptake assay.

2.3.D3R binding
Membrane fractions of the HEK-D3/DAT cells were prepared by homogenizing the cells in 50 mM Tris buffer (pH 7.4) containing 1 mM EDTA and 5 mM MgCl2. After centrifugation at 34,000 ×g for 30 min, the pellets were re-suspended in the original buffer and used for D3R binding. The amount of membrane proteins in each well of a 96-well plate was approximately 60-80 μg. Proteins were incubated for 30 min at 37℃. The binding assay was performed in triplicate with 8 nM [3H]7-OH-DPAT (144 Ci/mmol; Perkin-Elmer) in a final volume of 200 μl. Specific binding was defined as the difference in signal between absence and presence of 4 μM 7-OH-DPAT. The incubation was terminated by filtering the membranes through a vacuum filtration manifold Cell Harvester (Merck Millipore). The filters were washed three times with
3.ml of Tris-HCl buffer (pH 7.4) at 4℃ and transferred to scintillation counting vials. Four milliliters of Beckman Ready Safe scintillation fluid (Fullerton, CA, USA) were then added into each vial. The radioactivity in each sample was determined with a Beckman-Coulter TM LS6500 Multi-purpose Scintillation Counter. The specific D3R binding of D3/DAT co-transfected cells was 85.6%.

2.4.[3H]DA uptake assay
Cells were seeded at a density of 1 × 105 per well onto polylysine pre-coated 24-well plates. After cells were allowed to adhere to the plate for 24 h, medium was removed and wells were washed once with 0.5 ml of KRH buffer (125 mM MgSO4, 1.2 mM KH2PO4, 10 mM HEPES, 5.6 mM glucose, pH 7.2; 1 mM ascorbic acid and 0.01 mM pargyline were added just prior to experiments). Total reuptake and nonspecific reuptake were tested by treating with 300 μl of KRH buffer or 10 μM GBR12,909, respectively in 37℃ for 10 min. Uptake was initiated by replacing buffer with 300 μl KRH buffer containing 10 nM [3H]DA (56.8 Ci/mmol; Perkin-Elmer) at 37℃ for 5 min. The reaction was terminated by removing solution and washing cells with ice cold KRH buffer. Then cells were dissolved in 0.5 N NaOH, and transferred to scintillation counting vials. Four milliliters of Beckman Ready Safe scintillation fluid (Fullerton, CA, USA) were then added into each vial. The accumulated radioactivity was determined by a Beckman-Coulter TM LS6500 Multi-purpose Scintillation Counter.

2.5.Cell immunofluorescence
Cells were seeded at a density of 4 × 105 cells per well onto polylysine pre-coated glass coverslips and cultured in MEM supplemented with 1% fetal bovine serum and 1% penicillin/streptomycin solution and were treated with PBS or 1 μM ropinirole and incubated at 37℃, 5% CO2. After 40 min of ropinirole pretreatment, the cells were washed with PBS and fixed with freshly prepared 4% paraformaldehyde for 10 min at room temperature. The cells were then incubated in PBS containing 0.3% Triton X-100 at 4℃ for 24 h with primary antibodies against DAT (Merck Millipore, 1:500) and subsequently incubated for 1 h with secondary antibodies labeled with FITC (1:200) and 5 min with DAPI (1:1000). After staining, the coverslips were mounted in glycerol on a sealed slide. The images were collected using a confocal microscope (ZEISS LSM510) with a 63x oil immersion objective. Photos were taken
of a series confocal sections (1 m thickness) of immuno-stained cells at 0.14 m/pixel. The excitation and emission wavelengths for FITC and DAPI were 495/519 and 358/461 nm, respectively. Seven profiles were taken per cell, and at least 6 cells wree examined per group; three independent experiments were performed.

2.6.Crude synaptosomal preparation
Crude synaptosomes of the limbic forebrain were prepared according to a previous study with minor modifications (Maiya et al., 2007). Briefly, following decapitation, brains were rapidly removed from ten ICR mice (purchased from National Laboratory Animal Center, Taiwan), and the limbic forebrains (including the olfactory tubercle, ventral striatum and island of Callaja) were dissected out. Tissue was homogenized in ice-cold sucrose buffer (0.32 M sucrose and 10 mM HEPES, pH 7.4) with a glass/Teflon homogenizer. The homogenate was centrifuged at 1,000 ×g for 15 min at 4℃, and the resulting supernatant was centrifuged at 17,000 ×g for 30 min to isolate the P2 pellet (synaptosomes). The P2 pellet was re-suspended in KRH buffer (125 mM MgSO4, 1.2 mM KH2PO4, 10 mM HEPES, 5.6 mM glucose, pH 7.4) for DAT biotinylation and co-immunoprecipitation experiments. Crude synaptosomes were then pre-incubated at 37℃ with either KRH buffer (control) or drug. Following pre- incubation, samples were washed by centrifugation at 17,000 ×g for 10 min at 4℃. All experimental procedures using live animals were approved by the Animal Care and Use Committee at Chang-Gung University (CGU14-186).

2.7.DAT surface biotinylation in synaptosomes
After drug treatment, synaptosomes were incubated with 1 mg/ml sulfo-NHS-SS- biotin in PBS/Ca2+/Mg2+ buffer (137 mM sodium chloride, 10 mM phosphate buffer,2.7mM KCl, 1.0 mM MgCl2, 0.1 mM CaCl2, pH 7.4) for 60 min at 4℃ with continuous shaking. The free sulfo-NHS-SS-biotin was removed by centrifugation at 17,000 ×g for 10 min at 4℃, and the pellet was re-suspended in 0.1 mM glycine in PBS/Ca2+/Mg2+ buffer for 30 min on ice with continuous shaking. The samples were centrifuged at 17,000 ×g for 10 min at 4℃, and the pellets were re-suspended in RIPA buffer (10 mM Tris, 150 mM NaCl, 1.0 mM EDTA, 0.1% SDS, 1.0% Triton X-100, and 1% sodium deoxycholate, pH 7.4) with the protease inhibitors (3 μg/ml PMSF, 0.5 μg/ml aprotinin and 0.5 μg/ml leupeptin) for 30 min at 4℃ with continuous shaking. Total lysates were incubated with 30 μl monomeric avidin beads for 2 h at 4℃ with continuous shaking. Biotinylated proteins were prepared for western blot analysis by adding sample buffer (31.24 mM Tris, 1% SDS, 5% glycerol, 2.5% β- mecaptoethanol and trace bromophenol blue) and heating for 10 min at 100℃. Samples were stored at -20℃ until use.

2.8Co-immunoprecipitation of the DAT complex
After D3R drug treatment, synaptosomes were solubilized in ice-cold IP buffer (0.1% v/v Triton X-100, 10 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.4) containing protease inhibitors (3 μg/ml PMSF, 0.5 μg/ml aprotinin and 0.5 μg/ml leupeptin). The homogenate was incubated on ice for 1 h, and insoluble proteins were removed by centrifugation at 34,000 ×g for 30 min. The supernatant used for all IPs. The lysates (2 mg sample protein from supernatant) were pre-cleared for 90 min with protein G agarose beads (16-266, Merck Millipore) at 4℃ and used for IP. IP was carried out overnight at 4℃ using indicated antibodies, anti-DAT (1 μl of 500 μg sample protein; MAB369; Merck Millipore) followed by the addition of protein G Sepharose beads. Immunoprecipitated proteins were recovered by centrifugation at 3,800 ×g for 2 min and washed three times with IP buffer. Then, sample buffer was added, and the samples were heated for 10 min at 100℃ and stored at 20℃ until further use. For mass spectrometry analysis, IP samples were separated by SDS- PAGE (10% SDS) and stained with Coomassie blue solution. For western immunoblots, 500 μg sample protein was used for IP. IP was carried out overnight at 4℃ using indicated antibodies: anti-DAT (1 μl) or anti-Hsc70 (2 μl; SPA-757; Enzo Life Sciences), following the IP steps described above.

2.9Western immunoblots
Proteins were separated by SDS-PAGE (10% SDS) and blotted onto hydrophobic polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated for 1 h at room temperature in TBS-T (Tris-buffered saline plus 0.1% Tween-20) containing 5% nonfat milk. Designated proteins were detected using specific primary antibodies, including anti-DAT (1:1k; MAB369; Merck Millipore), anti-Hsc70 (1:2k; SPA-757; Enzo Life Sciences), anti-D3R (1:200, sc-9114, Santa Cruz), anti--actin (1:5000, A2228, Sigma Aldrich), followed by polyclonal secondary antibody conjugated with horseradish peroxidase (GE Healthcare; 1:2k). Blots were then visualized using an ECL detection kit (GE Healthcare ECL kit or Perkin-Elmer Lightning-ECL kit), according to the manufacturer’s instructions. Protein immunoreactive signals were detected with Hyperfilm (MIDSCI, St Louis, Missouri, USA) and quantified using ImageJ software. For reprobing, the membrane was stripped with stripping buffer (62.5 mM Tris–HCl, 2% v/v SDS, 100 mM - mecaptoethanol, pH 6.7) at 50℃ for 30 min and then reblotted with primary antibody.

2.10Proteomic Analysis
Protein bands of interest were excised from Coomassie Brilliant blue-stained gels and further subjected to in-gel digestion according to a protocol described previously (Chen et al., 2013; Ni et al., 2008). The resulting tryptic peptides were analyzed by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometer using an UltraflexTM MALDI-TOF MS (Bruker-Daltonik, Inc., Billerica, MA). Monoisotopic peptide masses were identified and applied for database searches using the MASCOT search engine (http://www.matrixscience.com) (Matrix Science, London, UK).

2.11Statistical analyses
Data were analyzed with the computer program GraphPad PrismTM. Results are expressed as mean ± SEM. The data were analyzed with a one-way ANOVA followed by Dunnett’s or Tukey’s post-hoc multiple comparison test. The level of statistical significance was set at p < 0.05. 3.Results 3.1.hD3R activation on suppresses DA uptake in D3R/DAT co-expressing HEK cells. In order to test whether D3R activation can affect DAT-mediated DA uptake, we stably co-expressed DA hD3 and rDAT in HEK-293 cells and treated the cells with the D3R agonist, ropinierole. [3H]DA uptake (added to the media at 10 nM) was measured at different time-points after ropinirole (1 M) or vehicle treatment. One- way ANOVA showed that ropinirole treatment significantly reduced [3H]DA uptake (F5,20 = 8.86, p < 0.001; Figure 1A). Dunnett’s post hoc testing revealed that DA uptake was decreased at 5, 10, 20, 40 and 60 min (p < 0.01) after ropinirole treatment compared to the control. In HEK-DAT cells (stably transfected with DAT only), there was no change (p > 0.05) in [3H]DA uptake at the same time-points after ropinirole treatment (Fig. 1A). A range of ropinirole concentrations (10 nM to 10 M) was then added to HEK-D3/DAT cells, and decreased [3H]DA uptake was observed at 100 nM, 1 M and 10 M (F4,24 = 27.05, p < 0.001; Figure.1B). HEK-DAT cells showed no change (p > 0.05) in [3H]DA uptake after ropinirole treatment (Figure 1B). A comparable decrease in [3H]DA uptake in HEK-D3/DAT cells was also observed following treatment with another D3R agonist, 7-OH-DPAT (ki for D3R: 0.892 nM; Ropinirole ki for D3R: 36.8 nM; Tadori et al. 2011) (Tadori et al., 2011), with significant effects of time (F5,15 = 16.97, p < 0.001; Figure 1C) and concentration (F4,16 = 37.2, p < 0.001; Figure 1D). [3H]DA uptake was then measured in HEK- D3/DAT cells treated with raclopride (10 M for 10 min) before the addition of ropinirole or 7-OH-DPAT (1 M for 10 min), to confirm that the effects of the inhibitors on D3R were specific (Figure 1E & 1F). Raclopride prevented the decrease in [3H]DA uptake induced by ropinirole (1 M) (F3,15 = 10.06, p < 0.001), according to Tukey’s post hoc comparison (p < 0.05). Similar results were found with 7-OH- DPAT (F3,9 = 63.49, p < 0.001). No change in [3H]DA uptake was observed with raclopride alone (p > 0.05).
3.2.Activation of the hD3 receptor decreases rDAT surface expression in HEK- D3/DAT cells
Because suppression of [3H]DA uptake is likely to be a result of DAT internalization, we next used a cell immunofluorescence assay to test whether D3R activation affects subcellular DAT localization. Confocal fluorescence imaging of HEK-hD3/rDAT cells revealed that DAT was expressed diffusely throughout the cytoplasm, but mainly localized to the plasma membrane. Importantly, the DAT signal at the cell surface was decreased at 40 min after ropinirole (1 M) treatment (Figure 2A). This observation correlates with the data from the [3H]DA uptake assay, suggesting that D3R activation reduces DAT-dependent DA uptake via a decrease in DAT surface expression.
3.3.Ropinirole reduces DAT surface expression in mouse limbic forebrain synaptosomes
To validate our findings in an ex vivo system with endogenous DA autoreceptors and DAT, crude limbic forebrain synaptosomes were treated with ropinirole (1 μM) for 15, 30 or 60 min and surface DAT biotinylation was quantified to determine if D3R activation regulates DAT surface expression in the nervous system. Ropinierole treatment decreased the surface expression of DAT at 1 and 10 μM doses (F3,9=19.95,
p < 0.001; Figure 2B), and at 30 and 60 min (F3,9 = 12.83, p < 0.01; Figure 2C). Moreover, the ropinirole-induced reduction of surface DAT could be prevented by pretreatment with either D3R antagonist FAUC365 (F3,12 = 5, p < 0.05; Figure 2D) or concanavalin A (conA), an inhibitor of clathrin-mediated endocytosis (Supplementary Figure 1). These results suggested that D3R activation deceases surface expression of DAT via clathrin-mediated endocytosis in the limbic forebrain. 3.4.Ropinirole alters the proteins that associate with DAT in mouse limbic forebrain synaptosomes A proteomic analysis was performed on mouse limbic forebrain (a D3R enriched brain region) to determine if D3R activation affects the proteins that associate with DAT. Crude limbic forebrain synaptosomes from mice were treated with vehicle or ropinirole (1 M) for 5, 20 and 40 min. The DAT complex was then isolated by IP followed by SDS-PAGE (Figure 3A). A band of approximately 72-73 kDa increased progressively over time after ropinirole treatment, suggesting that D3R activation affects the association of this protein and DAT in the D3R-enriched limbic forebrain. The protein was identified by mass spectrometry and bioinformatics (Proteomics Center, CGU) as the 70-kDa heat shock cognate protein (Hsc70), a member of the 70- kDa family of heat shock proteins (Supplementary Figure 2). 3.5.Ropinirole increases the association of DAT and Hsc70 in the mouse limbic forebrain The DAT-Hsc70 association was confirmed in mouse brain tissue by using specific anti-Hsc70 and anti-DAT antibodies. Mouse limbic forebrain or cerebellum lysates were immunoprecipitated with the specific DAT antibody or the Hsp70/Hsc70 antibody, followed by immunoblot analyses with the other antibody. Cerebellum tissue served as a negative control because it does not express detectable levels of DAT. DAT and Hsc70 were co-immunoprecipitated with either DAT or Hsp70/Hsc70 antibodies (Figure 3B). A relatively stronger signal was seen for Hsc70 after pull- down with the anti-DAT antibody; a small, but considerably weaker, association was also seen between DAT and Hsp70 (Figure 3B). These results indicate that an association exists between DAT and Hsc70 in the DAT-enriched limbic forebrain region. Limbic forebrain synaptosomes treated with ropinirole (1 μM) for various times were then analyzed via co-immunoprecipitation and western immunoblot to determine if the preferential D3R agonist can affect the interaction between DAT and Hsc70 in the limbic forebrain. The association between DAT and Hsc70/Hsp70 (73 and 72 kDa) was increased by ropinirole (1 μM) at 60 min (F5,10 = 13.07, p < 0.01; Figure 3C),while no association between DAT and D3R was detected after ropinirole (1 μM for 30 min) treatment (Supplementary Figure 3). Furthermore, the ropinirole-suppressed surface DAT expression was effectively prevented by pretreatment with the Hsc70/Hsp70 inhibitor, apoptozole (F3,12=6.302, p < 0.01; Figure 3D). These results indicate that an association between DAT and Hsc70 could be regulated by the DA D3 autoreceptor. 4.Discussion In this study, we found that D3R activation can act as a presynaptic autoreceptor to readily suppress DAT function, as evidenced by the ropinerole-induced inhibition of [3H]DA uptake. Furthermore, D3R stimulation reduced DAT surface expression, and pharmacological manipulations confirmed that this reduction occurs by a clathrin- mediated internalization-dependent mechanism and is a specific action of D3R. In addition, treatment of D3R agonist to striatal synaptosomes induced the association of chaperone Hsc70 with DAT, and pretreatment of an Hsc70/Hsp70 inhibitor prevented the ropinirole-suppressed DAT surface expression. Interestingly, we also did not find any evidence that D3R could form a protein complex with DAT upon receptor activation. Together, our findings reveal key participants in a novel D3R-mediated mechanism for regulating DA uptake. A biphasic effect of D3R activation on DAT function and trafficking has been reported, wherein acute stimulation of D3R (1 min) increases DAT surface expression, but prolonged receptor stimulation (30 min) decreases DAT function and surface expression (Zapata et al., 2007). The short-term effect was reported to occur in parallel with a fast increase in Akt and ERK phosphorylation, suggesting PI3K and MAPK signal pathways may be involved in D3R-induced DAT surface expression and DA uptake (Zapata et al., 2007). However, our observations in HEK-293 cells and synaptosomes are more consistent with the reported effects of prolonged D3R stimulation (30 min versus 5-60 min in our study) where [3H]DA uptake and DAT surface expression are suppressed. Although the underlying mechanism of this reduction in DA uptake is as yet unknown, it is reasonable to speculate that signal regulator(s) other than Akt or ERK could be triggered by long-term D3R activation. Transporters of neurotransmitters are key regulators of synaptic clearance, since more than 70% of extracellular neurotransmitters undergo reuptake into nerve terminals (Iversen, 1971). As such, the length of evoked neurotransmission is largely dependent on the abundance of transporters expressed on plasma membrane, rather than transporter-mediated signaling cascades (Torres et al., 2003). Nevertheless, several cellular kinases (PKC, PKA, PI-3K, MAPKs or CDKs) can modulate phosphorylation of upstream signaling components and transporter-associated proteins (PICK-1, PP2Ac, alpha-synuclein, Hic-5, syntaxin 1A, RACK or flottlin-1) affect protein trafficking to tightly control transporter uptake activity (for review, see Bermingham and Blakely 2016). For example, -PMA, a PKC activator, was shown to inhibit DAT activity, as the C-terminal region (amino acid residues of 587-596) of DAT encodes a PKC-sensitive internalization signal that sequesters phosphorylated DAT to the endosome (Boudanova et al., 2008). Additionally, Ras-like GTPase, Rin (Rit2), serves as a DAT-interacting protein that is critical for PKC-mediated DAT internalization (Navaroli et al., 2011b). Other DAT-interacting proteins (RACK1 and PICK-1) are also associated with PKC signaling, suggesting several candidate mechanisms that could mediate PKC-dependent DAT regulation (Lee et al., 2004; Madsen et al., 2012). Interestingly, DAT endocytosis can be induced by a wide variety of PKC subtypes and transporter internalization or recycling can be stimulated by several different PKC-dependent interacting proteins in response to diverse upstream regulators (Bermingham and Blakely, 2016). Synaptic transmitter homeostasis is achieved by the regulation of presynaptic autoreceptors, which are located mainly in nerve terminals but are also found in somatodendrites (Anzalone et al., 2012). Excess neurotransmitter release activates these autoreceptors to inhibit synthesis or release, in concert with transporter- mediated synaptic clearance (Raiteri, 2001). D2R was shown to regulate DAT activity via a direct protein-protein interaction (DAT N-terminal domain with the third loop in D2R) that increases DAT surface expression to enhance DA uptake (Lee et al., 2007). The D2R-DAT interaction appears to require the participation of PKC, since the absence of PKC abolishes the D2R-DAT association and reduces DAT surface expression (Chen et al., 2013). On the other hand, blockade of D3R (but not D2R) increases cocaine potency by inhibiting DAT-mediated DA uptake, suggesting that D3R also impacts DAT activity (McGinnis et al., 2016). In further support of this idea, prolonged administration of pramipexole (PPX), a preferential D3R agonist, decreases DA uptake in wild-type but not D3R knockout mice (Castro-Hernández et al., 2015). In this study, we found no evidence of a physical interaction between D3R and DAT after acute ropinirole treatment. However, our data from ropinirole and 7- OH-DPAT treated cells led us to a similar conclusion that D3R activation suppresses DAT-dependent [3H]DA uptake. It is still possible that a direct D3R-DAT interaction is induced by long-term stimulation of D3R (PPX; 0.1 mg/kg/d for 6 day), but our data do not support the existence of a direct interaction after short-term stimulation (ropinirole; 1 μM for 30 min). Moreover, our data showing ropinirole-induced internalization of DAT are in contrast with the reported increase in DAT surface expression following D2R activation (Bolan et al., 2007). While D2R and D3R are both coupled to Go/Gi proteins, their downstream signal transduction pathways are quite different (Rangel-Barajas et al., 2015). D2R is coupled to the GIRK potassium channel and is dependent on the  subunit for downstream signaling, while D3R is not. The D2R protein also forms a complex with -arrestin to initiate the ERK/MAPK cascade, but this receptor internalization-dependent cellular event is not observed with D3R (Rangel-Barajas et al., 2015). Interestingly, while D3R activation reduces DA release, activation of the autoreceptor inhibits DAT activity in parallel, which would be expected to negate the effect of D3R-mediated autoinhibition (Chen et al., 2009). This counterintuitive situation suggests that D3R activation may stimulate a feedback mechanism of DAT internalization to fine-tune the level of synaptic DA neurotransmission. Treatment of D3R agonist to synaptosomes promoted an unexpected association between Hsc70/Hsp70 and DAT in the limbic forebrain. The experimental timing of this association (maximal at 60 min) appeared to differ from the timings at which D3R agonists (ropinirole and 7-OH-DPAT) maximally decreased [3H]DA uptake (~10 min) and reduced DAT surface expression (30 min). However, closer examination of the data reveals trends toward reduction of DAT surface expression and induced DAT- Hsc70 associations at early time-points (i.e., 15 min and 10 min, respectively). We therefore speculate that different detection sensitivities among the diverse bioassays could have caused this apparent discrepancy in timing of effects. Hsc70 encodes a constitutively expressed chaperone that belongs to the Hsp70 family (Liu et al., 2012). Hsc70 and Hsp70 exhibit structural and functional similarities and both participate in a wide variety of cellular activities, such as protein biogenesis, folding/unfolding, assembly/disassembly, autophagy, trafficking and ubiquitination (Liu et al., 2012; Stricher et al., 2014). Hsp70 and Hsc70 also both interact with the vesicular monoamine transporter-2 (VMAT-2) and exhibit a dose-dependent inhibition of VMAT-2 activity (German et al., 2015; Requena et al., 2009). An interaction between Hsc70 and TH was shown to enhance TH activity, and its association with synaptic vesicles suggests that Hsc70 may participate in synaptic DA homeostasis (Parra et al.,2016). Hsc70/Hsp70 was also previously shown to de-coat internalized and clathrin- coated vesicles, which are required for endocytosis and clathrin function (Conner and Schmid, 2003), and dominant-negative Hsc70 mutations inhibit endocytosis of membrane receptors (Chang et al., 2002). In light of these earlier findings and our current results, we suspect that Hsc70 is a crucial mediator of clathrin-mediated DAT internalization induced by presynaptic D3R. Proper neurotransmission requires tight regulation to fine-tune synaptic signals. This tight regulation is needed to control recruitment of autoreceptors and transporters to the synapse, in order to achieve a robust system of neurotransmission. As such, defects in DA biosynthesis, vesicular uptake and/or release in the presynaptic terminals may be rescued by modulation of autoreceptors and transporters (Chen et al., 2009; O'Hara et al., 1996). The current findings indicate that D3R negatively regulates DAT activity, likely through an association of DAT with Hsc70/Hsp70, revealing a tunable regulatory mechanism for synaptic DA homeostasis. Author contributions Pi-Kai Chang: Conceptualization, Methodology, Visualization, Investigation, Writing - Original Draft. Kun-Yi Chien: Resources of Proteomics Core, bioinformatics software. Jin-Chung Chen: Funding acquisition, Resources, Supervision, Writing - Review & Editing. Ethical Statement The authors have read the statement of ethical standards for manuscripts submitted to “Brain Research Bulletin”. All experimental procedures involving animals were approved by the University Animal Care Committee at Chang Gung University in accordance with the guidelines provided by the NIH guide for care and use of Laboratory animal (NIH Publications No. 8023). We confirm that this work is original and has not been submitted elsewhere for publication. The authors have declared no conflict of interest related to this work and no financial interests in the publication of these results. Conflict of interest statement The Authors declare no conflict of interest. Acknowledgements We thank Drs. Marcus Calkins (Academia Sinica, Taiwan) and Arnold Stern of New York University for English editing. 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