Modulation of A_2a receptor antagonist on D₂ receptor internalization and ERK phosphorylation

Abstract

Aim:

To explore the effects of heterodimerization of D₂ receptor/A_2a receptor (D₂R/A_2aR) on D₂R internalization and D₂R downstream signaling in primary cultured striatal neurons and HEK293 cells co-expressing A_2aR and D₂R in vitro.

Methods:

Primary cultured rat striatal neurons and HEK293 cells co-expressing A_2aR and D₂R were treated with A_2aR- or D₂R-specific agonists. D₂R internalization was detected using a biotinylation assay and confocal microscopy. ERK, Src kinase and β-arrestin were measured using Western blotting. The interaction between A_2aR and D₂R was detected using bioluminescence resonance energy transfer (BRET) and immunoprecipitation.

Results:

D₂R and A_2aR were co-localized and formed complexes in striatal neurons, while both the receptors formed heterodimers in the HEK293 cells. In striatal neurons and the HEK293 cells, the D₂R agonist quinpirole (1 μmol/L) marked increased Src phosphorylation and β-arrestin recruitment, thereby D₂R internalization. Co-treatment with the A_2aR antagonist ZM241385 (100 nmol/L) significantly attenuated these D₂R-mediated changes. Furthermore, both ZM241385 (100 nmol/L) and the specific Src kinase inhibitor PP2 (5 μmol/L) blocked D₂R-mediated ERK phosphorylation. Moreover, expression of the mutant β-arrestin (319-418) significantly attenuated D₂R-mediated ERK phosphorylation in HEK293 cells expressing both D₂R and A_2aR, but not in those expressing D₂R alone.

Conclusion:

A_2aR antagonist ZM241385 significantly attenuates D₂R internalization and D₂R-mediated ERK phosphorylation in striatal neurons, involving Src kinase and β-arrestin. Thus, A_2aR/D₂R heterodimerization plays important roles in D₂R downstream signaling.

Introduction

Dopamine receptors are G protein-coupled receptors (GPCRs). These receptors can be divided into D₁-like and D₂-like families. D₂ receptors belong to the D₂-like family. The activation of D₂R reduces cAMP expression¹, extracellular signal-regulated kinase1/2 (ERK) phosphorylation², phospholipase C activation³, and receptor internalization^4,5,6.

Adenosine receptors are also GPCRs⁷. Dopamine D₂ and Adenosine A_2a receptors form heterodimers^8,9,10. In D₂R-cotransfected neuroblastoma cells, long-term exposure to A_2aR and D₂R agonists resulted in the co-aggregation, co-internalization, and co-desensitization of A_2aR and D₂R⁹. However, the modulation of D₂R downstream signaling and internalization through A_2aR has not been fully demonstrated.

The A_2aR antagonist has been demonstrated to improve the effects of L-dopa and to reduce “off” time in clinical trials^11,12,13. Because dopamine receptors play a key role in Parkinson's disease¹⁴, characterizing the modulation of D₂R downstream signaling through A_2aR would increase our understanding of the role of D₂R/A_2aR heterodimers in Parkinson's disease.

In the present study, we explored the ability of the A_2aR antagonist ZM241385 in A_2aR/D₂R cotransfected cells and striatal neurons to attenuate the receptor internalization and ERK phosphorylation induced by the D₂R agonist quinpirole; we also demonstrated the participation of Src kinase and β-arrestin in D₂R internalization and ERK phosphorylation.

Materials and methods

Primary striatal neurons culture

Dissociated primary cultures of striatal neurons from embryonic d 18 (E18) rats were prepared from timed-pregnant Sprague Dawley rats as described previously, with minor modifications¹⁵. Briefly, the fetuses were removed under sterile conditions and maintained on iced D-Hank's solution for microscopic dissection of the striatum. The meninges were removed. The tissue was briefly minced and triturated using a fire-polished Pasteur pipette. The cells were counted and plated on six-well culture plates in DMEM containing 10% fetal bovine serum (FBS). After 5 h, the medium was replaced with serum-free B27/neurobasal medium supplemented with 0.5 mmol/L glutamine and antibiotics. The cultures were maintained at 37 °C in a humidified 5% CO₂ atmosphere.

HEK293 cell culture

HEK293 cells were cultured at 37 °C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS.

Co-immunoprecipitation and immunoblotting

Cell lysates were prepared after incubation in modified radioimmunoprecipitation assay (RIPA) lysis buffer (Tris-HCl 50 mmol/L, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.25% Na-deoxycholate, 1% NP-40, 1 mmol/L PMSF, 1 mmol/L Na₃VO₄, 1 mmol/L NaF, 10 μg/mL aprotinin, 5 μg/mL leupeptin, and 5 μg/mL pepstatin). The clarified lysates were subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes as described previously¹⁵. The blots were incubated overnight at 4 °C with primary antibodies. After probing with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, the immunoreactive proteins were visualized using an enhanced chemiluminescence kit (Pierce, Rockford, Illinois, USA). In some cases, the blots were stripped and reprobed with other antibodies.

Interactions between D₂R and β-arrestin or A_2aR were examined by coimmunoprecipitation. Briefly, the cells were prepared after incubation in Tx/G lysis buffer (300 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 1.5 mmol/L MgCl₂, 1 mmol/L CaCl₂, 10 mmol/L EDTA and 50 mmol/L Tris, pH 7.4) containing 100 mmol/L iodoacetamide, 10 μg/mL aprotinin and 5 μg/mL leupeptin for 45 min on ice¹⁶. The clarified lysates were immunoprecipitated through overnight incubation at 4 °C using anti-D₂R (Millipore, Bedford, MA, USA) or anti-A_2aR antibodies (Millipore, Bedford, MA, USA), followed by incubation with Protein A/G-Sepharose beads (Santa Cruz, CA, USA). Equivalent amounts of protein were analyzed for each condition. The beads were washed three times with lysis buffer, and the immune complexes were boiled in SDS sample buffer and loaded onto SDS-PAGE gels for immunoblot analysis. The immunoreactive protein bands were detected using the enhanced chemiluminescence kit (Pierce, Rockford, Illinois, USA).

Biotinylation assays

Living striatal neurons or HEK293 cells were labeled for 20 min at 37 °C with EZ-link NHS-SS-biotin (300 μg/mL, Pierce, Rockford, Illinois, USA) to biotinylate the surface proteins¹⁷. After washing in PBS+MC, the cells were incubated in medium alone or medium containing different compounds for various times. Trafficking was terminated after rapid cooling to 4 °C. The biotinylated proteins remaining on the cell surface were stripped of biotin using the non-permeant reducing agent glutathione (150 mmol/L glutathione and 150 mmol/L NaCl, pH 8.75). Glutathione was subsequently neutralized with 50 mmol/L iodoacetamide in PBS+MC. The cells were incubated in RIPA buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% SDS) for 30 min at 4 °C. After centrifugation at 16 000×g, supernatants containing equal amounts of total protein were incubated with streptavidin beads overnight at 4 °C to capture biotinylated proteins. After washing in extraction buffer, the samples were boiled in sample buffer to elute the biotinylated proteins from the streptavidin beads. The proteins were separated by SDS-PAGE and immunoblotted using antibodies against D₂R (1:600), A_2aR (1:4000, Millipore Bedford, MA, USA) or Myc (1:1000 Cell Signaling Technology, Beverly, MA, USA). The immunoblots were processed using a standard chemiluminescence protocol. Equal amount of proteins untreated with streptavidin beads were used for normalization.

Fluorescence internalization assay

HEK293 cells co-transfected with D₂GFP/A_2aRFP were first treated for 1 h with the indicated compounds, after which they were fixed with 4% paraformaldehyde (PFA). Fluorescence images were acquired using confocal microscopy (Olympus, Lake Success, NY, USA) and quantified with Image Pro Plus. Total fluorescence intensities (a) and cytoplasmic fluorescence intensities (b) were quantified separately. The percentage of cell surface protein was quantified as (a−b)/a×100%.

Immunofluorescence

Primary cultured striatal neurons were fixed with acetone, and after washing twice with PBS, the cells were blocked for 1 h in blocking buffer (5% normal goat serum and 1% BSA in PBS) before incubation with anti-D₂R and anti-A_2aR antibodies diluted in blocking buffer at 4 °C overnight. The cells were washed three times with PBS and incubated with Alexa Fluor 488 conjugated goat anti-rabbit IgG and Alexa Fluor 555 conjugated goat anti-mouse IgG (1:100) (Molecular probes, Eugene, OR, USA) for 1.5 h at room temperature. The images were captured using a confocal microscope (Olympus, Lake Success, NY, USA).

BRET assay

The plasmids were transfected into HEK293 cells. At 48 h after transfection, the culture medium was removed, and the cells were washed twice with PBS and subsequently resuspended in DPBS at a density of 3.2×10⁵/mL. The cells were added into 384-well plate at 25 μL/well, and 25 μL of DeepBlueC solution was added to obtain a final concentration of 5 μmol/L DeepBlueC (Biotium, Hayworth, CA, USA). The plate was immediately read on a POLARstar Omega Microplate Reader (BMG GmbH, Offenburg, Germany) at EM 460 nm and 510/20 nm. The BRET ratio was calculated using the formula below (cells co-transfected with A_2aRluc/pGFP were used as a blank):

BRET ratio=[(emission at 510/20 nm)−(emission at EM 460 nm)×Cf]/(emission at EM 460 nm)

(Cf=emission at 510 nm of blank/emission at 460 nm of blank).

Plasmid construction

Prof Jerrey L BENOVIC (Thomas Jefferson University) kindly provided β-arrestin (319–418). A₁R-pcDNA3.1 was generated in our lab¹⁸. The other constructs were produced by PCR using specific primers. For D₂GFP, the D₂R fragments were generated by PCR, ligated into the expression vector pCDNA3.1 (Invitrogen, Carlsbad, CA, USA), and sequenced. The primers used were forward 5′-CCAAGCTTGCCACCATGGATCCACTGAATCTGTCCTGGT-3′ and reverse 5′-CCGGAATTCTCAGCAGTGGAGGATCTTCAGGA-3′.

The amplified fragments were fluorescently tagged at the carboxyl terminus using GFP. For D₂Myc, the same D₂R fragment was generated by PCR using the same primers and inserted into pcDNA3.1/myc-His A (Invitrogen, Carlsbad, CA, USA). For A_2aRFP, the A_2aR fragment was generated by PCR, ligated into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), and sequenced. The primers used were forward 5′-CGGAATTCATGCCCATCATGGGCTCCTC-3′ and reverse 5′-GCTCTAGACTACAGCTCGTCCATGCCGA-3′. The amplified fragments were fluorescently tagged at the carboxyl terminus using RFP. Prof Jing-gen LIU (Shanghai Institute of Materia Medica, Chinese Academic of Science) kindly provided pGFP2-N3 and pRluc-N3. For D2pGFP2-N3, the D₂R fragment was generated by PCR, ligated into the expression vector pGFP2-N3, and sequenced. The primers used were forward 5′-CCGGAATTCATGGATCCACTGAATCTGTCCTGGT-3′ and reverse 5′-CCAAGCTTGCAGTGGAGGATCTTCAGGAAGGCCTTG-3′. For A_2aRluc-N3, the A_2aR fragment was generated by PCR, ligated into the expression vector pRluc-N3, and sequenced. The forward and reverse primers were 5′-CCCTCGAGATGCCCATCATGGGCTCCTC-3′ and 5′-CCAAGCTTGGACACTCCTGCTCCATCCT-3′, respectively.

Statistics

The data were presented as the means±SEM and compared with one-way ANOVA followed by the Bonferroni post hoc test using the GraphPad InStat statistical program. The level of statistical significance was set at P<0.01 and P<0.05. The data were obtained from at least three separate experiments.

Results

D₂R and A_2aR form heterodimers

D₂R and A_2aR were highly expressed on striatopallidal neurons. To confirm the interaction between D₂R and A_2aR, immunofluorescence experiments were performed using primary cultured striatal neurons. The results showed that D₂R and A_2aR were co-localized on striatal neurons (Figure 1A). The co-immunoprecipitation results indicated that D₂R and A_2aR formed complexes on striatal neurons (Figure 1B). Furthermore, the BRET assay demonstrated that D₂R and A_2aR formed heterodimers on HEK293 cells (Figure 1C). These results suggested that D₂R and A_2aR co-localize and form heterodimers.

Figure 1

D₂R and A_2aR could form heterodimers. (A) Cells were processed for immunostaining using rabbit anti-D₂R and mouse anti-A_2aR antibodies and analyzed by confocal microscopy. The scale bar represents 40 μm. (B) Primary striatal neurons were lysed, and whole cell extracts were immunoprecipitated using an anti-A_2aR antibody or corresponding normal IgG and subsequently immunoblotted with antibodies to D₂R. (C) BRET ratios for HEK293 cells co-expressing A_2apRluc and D₂pGFP were measured as described in the Methods. A mixture of cells expressing A_2apRluc or D₂pGFP was used as a negative control. Cells co-expressing A_2apRluc and pGFP were used as a blank. Data are presented as the means±SEM of three experiments. ^cP<0.01 compared with the negative control.

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Quinpirole-induced internalization is attenuated upon combined treatment with ZM241385 and quinpirole

The long-term activation of D₂R could lead to D₂R internalization⁶. Internalization is important for regulating GPCR downstream signaling. A biotinylation assay and confocal microscopy were used to investigate the modulation of the A_2aR agonist or antagonist on D₂R internalization. DIV 9 striatal neurons were treated with the A_2aR agonist CGS21680 or the D₂R agonist quinpirole alone or in combination for 3 h, and the subsequent induction of A_2aR and D₂R co-internalization were observed (Figure 2A, 2B). Because A_2aR and D₂R form heterodimers, the activation of either the D₂ or A_2a receptor could induce the co-internalization of D₂R/A_2aR heterodimers, consistent with previously reported data⁹. The results also showed that the A_2a receptor antagonist ZM241385 reduced D₂ receptor internalization (Figure 2C).

Figure 2

Biotinylation assay of A_2aR and D₂R endocytosis. (A, B) Primary cultured striatal neurons were labeled with biotin, and the cells were subsequently treated with CGS21680 (1 μmol/L) or quinpirole (1 μmol/L) alone or in combination. After 3 h, the cells were analyzed as previously described. The samples were analyzed by SDS-PAGE and immunoblotting for D₂R (A) or A_2aR (B). (C) Neurons were treated with ZM241385 (100 nmol/L) or quinpirole (1 μmol/L) alone or in combination. After 3 h, the cells were analyzed as previously described. The samples were analyzed by SDS-PAGE and immunoblotting for D₂R (C). The data from C were quantitated, and the means±SEM are shown in the right panel (n=3). ^bP<0.05 compared with the untreated control. ^eP<0.05 compared with the quinpirole-treated group.

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To further confirm this result, the effects of A_2aR on D₂R internalization were also examined in HEK293 cells. Plasmids carrying D₂GFP and A_2aRFP were co-transfected into HEK293 cells. After treatment with different compounds, the cells were fixed with 4% paraformaldehyde, and images were captured using confocal microscopy (Olympus). The results showed that treatment with CGS21680 or quinpirole, separately or in combination, induced the co-internalization of the two receptors. In addition, ZM241385 reduced D₂R quinpirole-induced internalization (Figure 3), consistent with the result obtained using striatal neurons.

Figure 3

Endocytosis of D₂R on HEK293 cells. HEK293 cells were transiently transfected with D₂GFP and A_2aRFP. At 24–36 h after transfection, the cells were treated with CGS21680 (1 μmol/L), quinpirole (1 μmol/L), or ZM241385 (100 nmol/L) alone or in combination for 1 h, and subsequently, the cells were fixed and processed for confocal microscopy. The quantification of cell surface D₂ receptors is shown in the panel. ^bP<0.05 compared with the untreated control. ^eP<0.05 compared with the quinpirole-treated group. Data are presented as the mean±SEM of at least three independent experiments. The scale bar represents 10 μm.

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D₂R internalization occurs in a β-arrestin/clathrin- and Src kinase-dependent manner

Src kinase and β-arrestin are important for GPCR internalization. We used a surface biotinylation assay to characterize the role of Src kinase in D₂R internalization. The results showed that striatal neurons treated with PP2, an Src kinase inhibitor, obviously inhibited quinpirole-induced D₂R internalization (Figure 4A), suggesting that D₂R internalization is Src kinase dependent.

Figure 4

Effects of Src kinase and β-arrestins on D₂R endocytosis. (A) Primary cultured striatal neurons were labeled with biotin, and the cells were pretreated with PP2 (5 μmol/L) for 30 min, followed by stimulation with quinpirole (1 μmol/L) for 3 h. Subsequently, the cells were analyzed as previously described, using SDS-PAGE and immunoblotting with a D₂R antibody (Millipore). (B) HEK293 cells were transfected with D₂Rmyc, HAA_2a, or β-arrestin (319–418) alone or in combination. At 48 h after transfection, the cells were treated with quinpirole (1 μmol/L) for 1 h after labeling with biotin and analyzed by SDS-PAGE and immunoblotting using the Myc antibody (Cell Signaling Technology). The data represent three independent experiments.

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Many GPCRs are internalized in a β-arrestin/clathrin-dependent manner. The dominant negative β-arrestin (319–418) mutant was used to determine the roles of β-arrestins in D₂R internalization. This plasmid inhibited β-arrestin/clathrin-dependent receptor internalization through blocking the formation of the β-arrestin/clathrin complex¹⁹. We treated β-arrestin (319–418)-transfected HEK293 cells with the D₂R agonist quinpirole to detect D₂R internalization using Western blotting. The results showed that the expression of β-arrestin (319–418) in HEK293 cells inhibited quinpirole-induced D₂R internalization, thereby demonstrating that β-arrestin was involved in D₂R internalization (Figure 4B). These results were consistent with previously reported data^2,4,6.

ZM241385 reduced D₂R-induced Src kinase phosphorylation and β-arrestin 2 recruitment

Because Src kinase is involved in D₂R internalization, this protein might also participate in the ZM241385-mediated modulation of D₂R internalization. We used Western blotting to determine whether Src plays a role in the internalization of A_2aR and D₂R. The results indicated that the activation of D₂R induces Src kinase phosphorylation in striatal neurons. ZM241385 also attenuated D₂R-induced Src kinase phosphorylation (Figure 5A).

Figure 5

Effects of ZM241385 on Src kinase phosphorylation and recruitment of β-arrestin 2 induced by D₂R activation. (A) Neurons were treated with ZM241385 at the indicated concentrations and with the D₂R agonist quinpirole (1 μmol/L) for 10 min. The cell lysates were analyzed by SDS-PAGE and immunoblotting to detect Src phosphorylation using a phospho-specific (Tyr416) antibody. (B) The neurons were treated with ZM241385 (100 nmol/L) or quinpirole (1 μmol/L) alone or in combination for 10 min. The cells were lysed with Tx/G buffer, and the lysates were immunoprecipitated using a D₂R antibody (Millipore). The immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with anti-D₂ or anti-β-arrestin2 antibodies. The data were quantitated, and the means±SEM are shown in the lower panel (n=3). ^bP<0.05 compared with the untreated control. ^eP<0.05 compared with the quinpirole-treated group.

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Moreover, the co-immunoprecipitation data showed that activated D₂R recruited β-arrestin 2 to striatal neurons and that ZM241385 reduced D₂R and β-arrestin 2 interactions (Figure 5B).

These results demonstrated that both Src kinase and β-arrestin are involved in D₂R internalization and that ZM241385 reduces D₂R-induced Src kinase phosphorylation and β-arrestin recruitment, resulting in the eventual reduction of D₂R internalization.

ZM241385 reduced D₂R-induced ERK phosphorylation

Extracellular regulated protein kinases (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family, which are involved in many important events, such as neuron survival and cell differentiation⁴. The activation of D₂R induces ERK phosphorylation²⁰. Thus, the roles of A_2aR/D₂R heterodimerization in D₂R-induced ERK activation were also investigated.

Western blot analysis showed that the activation of A_2aR did not affect D₂R-induced ERK phosphorylation (Figure 6A), whereas ZM241385 reduced D₂R-induced ERK phosphorylation in a dose-dependent manner (Figure 6B). These results were confirmed in HEK293 cells (Figure 6D).

Figure 6

Effect of ZM241385 on quinpirole-induced ERK1/2 activation. (A–C) Neurons were treated with the A_2aR agonist CGS21680, the D₂R agonist quinpirole (100 nmol/L), and the A_2aR antagonist ZM241385 at the indicated concentrations alone or in combination for 10 min. (D–E) HEK293 cells were co-transfected with D₂Rmyc and HAA_2aR. At 48 h after transfection, the cells were treated with ZM241385 (100 nmol/L) or the D₂R agonist quinpirole (1 μmol/L) alone or in combination for 5 min. The cells were lysed, and the cell lysates were analyzed by SDS-PAGE and immunoblotting for ERK1/2 phosphorylation using a phospho-specific (Thr202/Tyr204) antibody. The data from B and D were quantitated, and the means±SEM are shown in C and E, respectively (n=3). ^bP<0.05 compared with the untreated control. ^eP<0.05 compared with the quinpirole-treated group.

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Src kinase and β-arrestin participated in D₂R-induced ERK phosphorylation when D₂R was co-expressed with A_2aR

D₂R induces ERK activation in a G protein-dependent manner, and Src kinase participates in this activation^2,4. However, the mechanism of ERK activation might be different under different conditions. The formation of heterodimers might affect receptor-induced ERK activation²¹. In some cases, signal proteins, such as β-arrestin, not only participate in receptor internalization but also are involved in ERK activation^22,23.

Striatal neurons were treated with the Src kinase-specific inhibitor PP2, followed by treatment with the D₂R agonist quinpirole. PP2 inhibited quinpirole-induced ERK phosphorylation (Figure 7), suggesting that Src kinase is involved in D₂R-induced ERK phosphorylation, consistent with previously reported data².

Figure 7

Effects of Src kinase on ERK phosphorylation induced by D₂R activation. The neurons were treated with PP2 (5 μmol/L) for 30 min before stimulation with the D₂R agonist quinpirole (1 μmol/L) for 10 min (A). The cell lysates were analyzed by SDS-PAGE and immunoblotting for ERK1/2 phosphorylation using a phospho-specific (Thr202/Tyr204) antibody. The data from B were quantitated, and the mean±SEM are shown in the lower panel (n=5). ^cP<0.01 compared with the untreated control. ^fP<0.01 compared with the quinpirole-treated group.

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Some studies have implicated β-arrestin in mechanisms of the GPCR downstream signaling pathway, such as ERK phosphorylation^21,22,24. The mutant β-arrestin (319–418) was used to investigate effects of β-arrestins in D₂ receptor-induced ERK activation. The data showed that in HEK 293 cells expressing D₂R alone, the expression of β-arrestin (319–418) did not affect quinpirole-induced ERK phosphorylation (Figure 8A), consistent with previous reports²⁰. Interestingly, in HEK293 cells co-expressing D₂R and A_2aR, the expression of β-arrestin (319–418) attenuated quinpirole-induced ERK phosphorylation (Figure 8B), whereas β-arrestin (319–418) expression in HEK293 cells co-expressing D₂R and A₁R did not affect quinpirole-induced ERK phosphorylation (Figure 8C). These results suggested that D₂R/A_2aR heterodimerization changed D₂R-induced ERK phosphorylation.

Figure 8

Effects of β-arrestins on D₂R-induced ERK phosphorylation. HEK293 cells were transfected with D₂Rmyc (A), D₂Rmyc and HAA_2aR (B), D₂Rmyc or A₁R (C), alone or in combination with β-arrestin (319–418). At 48 h after transfection, the cells were treated with quinpirole (1 μmol/L) for 5 min, followed by lysis and analysis by SDS-PAGE and immunoblotting for ERK1/2 phosphorylation using a phospho-specific (Thr202/Tyr204) antibody. The data were quantitated, and the means±SEM are shown (n=3). NT represents cells not transfected with β-arrestin (319–418). DN represents cells transfected with β-arrestin (319–418). ^cP<0.01 compared with the quinpirole-treated non-transfected β-arrestin (319–418) cells in each group.

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Taken together, these results showed that Src kinase (Figure 7) and β-arrestin (Figure 8) are involved in D₂R-induced ERK activation and that ZM241385 reduces D₂R-induced Src kinase phosphorylation and β-arrestin recruitment (Figure 5), thereby reducing D₂R-induced ERK activation through ZM241385 (Figure 6).

Discussion

D₂R and A_2aR form heterodimers, and the activation of either D₂R or A_2aR results in the co-internalization of D₂R/A_2aR heterodimers⁹. The results of the present study confirmed these observations in striatal neurons and HEK293 cells co-expressing D₂R/A_2aR. The activation of A_2aR induced the co-internalization of D₂R/A_2aR heterodimers, whereas ZM241385 reduced D₂R internalization, indicating that D₂R/A_2aR receptor heterodimerization affects D₂R internalization.

The present study also demonstrated that after activation, D₂R is internalized through a β-arrestin/clathrin-dependent pathway involving Src kinase. Western blot analysis revealed that ZM241385 reduces D₂R-induced Src kinase phosphorylation, and co-immunoprecipitation showed that ZM241385 reduces the D₂R-mediated recruitment of β-arrestin2. These results indicated that ZM241385 reduces D₂R-induced Src kinase phosphorylation and the recruitment of β-arrestin2, thereby reducing D₂R internalization. Fuxe et al demonstrated that A_2aR activation enhances D₂R binding with β-arrestin2 and that D₂R, A_2aR, and β-arrestin2 might form a complex²⁵. In the present study, we showed that ZM241385 reduces the D₂R-mediated recruitment of β-arrestin2. Thus, it is possible that A_2aR is involved in the interaction of β-arrestin2 and D₂R/A_2aR heterodimers. ZM241385 maintains A_2aR in an inactive status^26,27 and affects the interaction between D₂R/A_2aR heterodimer and β-arrestin2, thereby reducing D₂R internalization. Clinical studies have shown that the A_2aR antagonist prolongs the effective time and shortens the “off time” of L-dopa^11,28, potentially reflecting D₂R internalization.

In this study, we showed that the A_2aR antagonist reduces D₂R-induced Src kinase phosphorylation and β-arrestin2 recruitment; thus, the A_2aR antagonist might not only reduce D₂R internalization but also modulate D₂R downstream signaling. The results in the present study also showed that D₂R-induced ERK activation is Src kinase dependent. Moreover, the expression of the mutant β-arrestin (319–418) also reduced D₂R-induced ERK activation when D₂R was co-expressed with A_2aR, suggesting that both Src kinase and β-arrestin participate in D₂R-induced ERK activation. The A_2aR antagonist reduces D₂R-induced Src kinase phosphorylation and β-arrestin2 recruitment; thus, the A_2aR antagonist might also reduce D₂R-induced ERK activation.

Interestingly, β-arrestin was also involved in D₂R-induced ERK phosphorylation when D₂R was co-expressed with A_2aR, but not when D₂R was expressed alone. ERK phosphorylation might be influenced by many factors, and receptor dimerization might also influence ERK activation²¹. To our knowledge, most studies on D₂R-induced ERK activation have utilized cells expressing D₂R alone, showing that the expression of β-arrestin (319–418) had no effects on D₂R-induced ERK activation²⁰; these results were also confirmed in the present study. However, when D₂R and A_2aR were co-expressed, β-arrestin participated in D₂R-induced ERK activation. In vivo, particularly in the striatum, D₂R exists not only as a monomer or homodimer but also as a D₂R/A_2aR heterodimer⁹. Thus, the results of the present study enhance the current understanding of the mechanism underlying D₂R-induced ERK activation under physiological conditions. Different mechanisms of ERK activation might exert different downstream effects. Distinguishing D₂R monomer-induced ERK phosphorylation from D₂R/A_2a R heterodimer-induced ERK activation is difficult, as the development of a system containing only D₂R/A_2aR receptor heterodimers and no D₂R monomers is not easy. However, this study showed the importance of heterodimers in receptor downstream signaling pathway.

Thus, this study showed that D₂R and A_2aR co-internalize. The A_2aR antagonist reduced D₂R-induced Src kinase phosphorylation and β-arrestin2 recruitment, thereby reducing D₂R internalization and ERK phosphorylation. These observations showed that D₂R/A_2aR heterodimers play an important role in D₂R signaling pathway and trafficking.

Author contribution

Lin-yin FENG and Li HUANG designed the research; Li HUANG, Dong-dong WU, and Lei ZHANG performed the research; Li HUANG analyzed the data; and Lin-yin FENG and Li HUANG wrote the paper.