DOPAMINE D₂ RECEPTOR-MEDIATED MODULATION OF ADENOSINE A_2A RECEPTOR AGONIST BINDING WITHIN THE A_2AR/D₂R OLIGOMER FRAMEWORK

Abstract

The molecular interaction between adenosine A_2A and dopamine D₂ receptors (A_2ARs and D₂Rs, respectively) within an oligomeric complex has been postulated to play a pivotal role in the adenosine-dopamine interplay in the central nervous system, in both normal and pathological conditions (e.g. Parkinson’s disease). While the effects of A_2AR challenge on D₂R functioning have been largely studied, the reverse condition is still unexplored, a fact that might have impact in therapeutics. Here, we aimed to examine in a real-time mode the D₂R-mediated allosteric modulation of A_2AR binding when an A_2AR/D₂R oligomer is established. Thus, we synthesized fluorescent A_2AR agonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A_2AR binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential D₂R-mediated negative allosteric modulation on A_2AR agonist binding that was oligomer-formation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A_2AR agonist binding. Overall, the here-developed methods were found valid to prospect the ability of drugs acting on D₂Rs to modulate A_2AR binding, thus featuring as possible helpful tools for the preliminary selection of D₂R-like candidate drugs in the management of Parkinson’s disease.

1. Introduction

Parkinson’s disease (PD) is a progressive systemic neurodegenerative disorder associated with, but not exclusively, the loss of pigmented dopaminergic neurons located at the substantia nigra pars compacta (Lang and Lozano, 1998). Interestingly, the control of this circuitry both in normal and pathological conditions is not only mediated by dopamine receptors, but also by other G protein-coupled receptors (GPCRs), for instance adenosine receptors, which may functionally and molecularly interact with the dopaminergic system (Ferre et al., 2004). Indeed, an interaction between adenosine A_2A and dopamine D₂ receptors (A_2ARs and D₂Rs, respectively) has been demonstrated in heterologous expression systems and there is accumulated evidence for the coexistence of reciprocal antagonistic interactions between A_2ARs and D₂Rs in the same neurons, the GABAergic enkephalinergic neurons (Ferre et al., 2008). Thus, since drugs acting on A_2ARs would modulate dopaminergic neurotransmission they have been proposed as therapeutic tools in PD, and in fact, A_2AR antagonists have been recently introduced in the symptomatic treatment of the former pathology (for review see (Xu et al., 2005,Morelli et al., 2009,Schwarzschild et al., 2006)). Hence, it has been suggested that the modulation of a feasible A_2AR/D₂R complex by means of simultaneous D₂R activation and A_2AR inhibition would represent a valuable target for PD pharmacotherapy. Accordingly, in the present work we aimed to analyze the reciprocal A_2AR-D₂R antagonistic interaction, concretely the possible D₂R-mediated allosteric modulation of A_2AR agonist binding. Thus, by means of a real-time fluorescence resonance energy transfer (FRET)- and a flow cytometry-assays, we evaluated the effects of different antiparkinsonian drugs (i.e. pramipexole, rotigotine and apomorphine), in order to provide further information regarding D₂R-based drugs before being selected for PD treatment.

2. Materials and Methods

2.1. Materials

The primary antibodies used were: rabbit anti-A_2AR polyclonal antibody (Ciruela et al., 2004), rabbit anti-D₂R polyclonal antibody (Millipore, Temecula, CA, USA), and mouse anti-A_2AR monoclonal antibody (clone 7F6-G5-A2; Millipore). The secondary antibodies were: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL, USA) and Cy3-conjugated donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The ligands used were: adenosine, pramipexole, apomorphine from Sigma-Aldrich (St. Louis, MO, USA); CGS21680, ZM241,385, rotigotine from Tocris Bioscience (Ellisville, MI, USA). Adenosine deaminase was purchased from Roche Diagnostics (GmbH, Mannheim, Germany) and zardaverine from Calbiochem (San Diego, CA, USA).

2.2. Synthesis of the A_2AR fluorescent agonists

The APEC^Alexa488 (MRS5206) and the APEC^Alexa532 (MRS5424) are functional agonists at the adenosine A_2AR, in which the fluorescent dyes Alexa488 and 532, respectively, are covalently attached to the A_2AR agonist 2-[[2-[4-[2-(2-aminoethyl)-aminocarbonyl] ethyl]phenyl]ethylamino]-5′-N-ethyl-carboxamidoadenosine (APEC). MRS5424, and similarly MRS5206, were synthesized as previously described (Fernandez-Duenas et al., 2012).

2.3. Plasmid constructs

To perform FRET experiments with the MRS5424 fluorescent agonist, a CFP-tagged A_2AR (A_2AR^CFP) and an AGT-tagged D₂R (D₂R^AGT) were cloned, as previously described (Fernandez-Duenas et al., 2012).

2.4. Cell culture, transfection and membrane preparation

Human embryonic kidney (HEK)-293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL streptomycin, 100 mg/mL penicillin and 5% (v / v) fetal bovine serum at 37° C and in an atmosphere of 5% CO₂. HEK-293 cells growing in 25 cm² flasks or six-well plates containing 18 mm coverslips were used for western blot and fluorescence imaging, respectively, were transiently transfected with the cDNA encoding the specified proteins using TransFectin™ Lipid Reagent (Bio-Rad Laboratories, Hercules, CA, USA). Membrane suspensions from transfected HEK cells were obtained as described previously (Fernandez-Duenas et al., 2012).

2.5. Gel electrophoresis, immunoblotting and immunocytochemistry

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 7.5 % polyacrylamide gels. Proteins were transferred to PVDF membranes using a semi-dry transfer system, immunoblotted with the indicated antibodies and then bands detected using a chemiluminescent detection kit (Pierce). Immunocytochemistry experiments were performed as previously described (Ciruela et al., 2006).

2.6. [³H]-cAMP assay

The accumulation of cAMP was measured with a [³H]-cAMP assay system (GE Healthcare, Piscataway, NJ, USA). In brief, transiently transfected HEK-293 cells were grown O/N in serum-free DMEM containing adenosine deaminase (0.5 U/mL), preincubated with 50 µM of the phosphodiesterase inhibitor zardaverine for 10 min and then stimulated with adenosine (10 µM) for 15 min at 37° C. Then, we performed the assay protocol, adding the labelled [³H]-cAMP, as described in the manufacturer's manual. The radioactivity was determined in scintillation vials using a Packard 1600 TRI-CARB scintillation counter (PerkinElmer, Waltham, MA, USA).

2.7. Microscopic FRET measurements

Single-cell real-time FRET experiments with the MRS5424 ligand, in cells expressing the A_2AR^CFP construct and the D₂R^AGT stained by means of the SNAP-Surface 647 ligand (New England BioLabs, Ipswich, MA, USA), were performed as previously described (Fernandez-Duenas et al., 2012).

2.8. Flow cytometry analysis

Transiently transfected HEK-293 cells were detached with accutase (Sigma-Aldrich) and resuspended in Hank’s balanced salt solution containing 1 g/l glucose (HBSS). Then, cells were incubated with the proper ligands in silicon-coated reaction vessels (3×10⁵ cells/50 µl) during 60 minutes at room temperature and used directly for flow cytometric measurements. For each binding condition tested 10⁴ cells were analyzed using a FACSCalibur™ flow cytometer (Becton Dickinson, Heidelberg, Germany) with excitation at 488 nm and emission at 510 nm. Samples were maintained in the dark during the analysis to avoid photobleaching. Fluorescence intensities were obtained in the FL-1 channel in log mode. Data were collected using Cell Quest Pro software (BD, Franklin Lakes, NJ) and analyzed by GraphPad Prism (GraphPad Software, La Jolla, CA, USA).

2.9. Statistics

The number of samples (n) in each experimental condition is indicated in figure legends. Statistical analysis was performed by one-way ANOVA followed by Dunnett`s multiple comparison post-hoc test. Statistical significance is indicated for each experiment.

3. Results and discussion

In the next years, the search for new drugs specifically targeting the A_2AR/D₂R complex may represent a novel and therapeutically superior approach for the management of PD. In such way, drugs intended to overcome the tonic A_2AR-mediated inhibition of D₂R signalling promise to be of great value. Accordingly, in the present work we aimed to study the D₂R-mediated allosteric modulation of A_2AR agonist binding within the A_2AR/D₂R oligomer framework. Hence, we developed a FRET- and a flow cytometry-based agonist binding assays devoted to study the adenosine-dopamine receptor-receptor interaction.

3.1. FRET determinations of D₂R-mediated modulation of A_2AR binding

We recently described a single-cell real-time FRET-based A_2AR agonist binding procedure, in which it was possible to ascertain the negative allosteric modulatory effect of a D₂R agonist (i.e. quinpirole) on A_2AR agonist binding (Fernandez-Duenas et al., 2012). Accordingly, here we aimed to use the same experimental approach to analyze the effects of several clinically used antiparkinsonian drugs (i.e. pramipexole, rotigotine and apomorphine) on A_2AR agonist binding (Figure 1A). First, doubly A_2AR^CFP and D₂R^AGT transfected cells were visualized at 480 nm (CFP) and 688 nm (SNAP; see methods). Noteworthy, the A_2AR^CFP construct was, without losing its functionality, mostly targeted to the plasma membrane (see Supplementary Figure 1), where it co-distributed with the D₂R^AGT (Fernandez-Duenas et al., 2012). Then, we proceeded to analyze the effects of the antiparkinsonian agents on MRS5424 binding. Since it has been previously demonstrated that in real-time FRET determinations there is not receptor reserve and furthermore the measurements are done under continuous superfusion of the ligand (i.e. non-equilibrium conditions) (Ziegler et al., 2011), high saturating concentrations of the antiparkinsonian D₂R agonists (pramipexole: 120 µM, rotigotine: 1 µM and apomorphine: 6 µM) (Millan et al., 2002,Scheller et al., 2009) were used to displace the MRS5424 (2 µM) bound to A_2ARs. Interestingly, while MRS5424 binding was unaffected by any of the D₂R agonists in cells expressing only the A_2AR^CFP (data not shown), upon D₂R^AGT co-expression and challenge a significant lower FRET signal was observed (Figure 1B). A close view of the real-time FRET profiles allowed us to discern that apomorphine (58±6%, p < 0.001) was the most efficacious negative allosteric modulator (NAM) of A_2AR agonist binding, when compared to rotigotine (29±5%, p < 0.001) or pramipexole (38±5%, p < 0.001) (Figure 1C). Overall, these results indicated that all D₂R agonists tested were NAMs of A_2AR agonist binding, a phenomenon that would be dependent on A_2AR/D₂R oligomerization. Indeed, we recently demonstrated that the formation of the A_2AR/D₂R oligomer was mandatory for the observed receptor-receptor interaction (Fernandez-Duenas et al., 2012).

Figure 1. Real-time FRET determinations of antiparkinsonian D₂R agonist-mediated modulation of A_2AR agonist binding.

(A) Chemical structures of the investigated antiparkinsonian D₂R agonists (pramipexole, rotigotine and apomorphine). (B) Time-resolved changes in FRET signals engaged by MRS5424 (2 µM) in cells transfected with A_2AR^CFP and D₂R^AGT in the absence (black trace) or presence (red trace) of pramipexole (120 µM), rotigotine (1 µM) and apomorphine (6 µM) were recorded. Traces are representative of four separate experiments with similar qualitative and quantitative results. (C) The FRET increase (1/F₄₈₀) was fitted by a simple monoexponential curve and the magnitude of the FRET signal (A, see experimental procedures) calculated for each experimental condition. Data represents the average ± S.E.M. values of four independent experiments. Asterisk indicates data significantly different from the control condition (i.e. in the absence of antiparkinsonian D₂R agonist): *p < 0.01 by ANOVA with Dunnett`s multiple comparison post-hoc test.

3.2. Flow cytometric measurements of D₂R-agonists effects on A_2AR binding

One of the main advantages of FRET-based approaches consists of providing high spatiotemporal resolution of the agonist-receptor interaction. On the contrary, this kind of tool does not permit the collection of a large number of events, therefore not suitable for high throughput screening of drugs. Accordingly, in order to validate our previous results by a different experimental approach and also to generate an easy, fast and reliable way for monitoring A_2AR-D₂R interactions we aimed to develop a flow cytometry homogeneous no-wash assay. First, a new selective A_2AR fluorescent agonist, a pure isomeric APEC^Alexa488 named MRS5206, was synthesized according to our flow cytometer needs. First, it was tested to be bound selectively to the A_2AR by live-cell detection under a fluorescence microscope (see Supplementary Figure 2). And thereafter, we attempted to quantify the occurrence of MRS5206 binding to A_2ARs by means of flow cytometry. To this end, A_2AR expressing cells were initially incubated with MRS5206 and then separated by flow cytometry with the aid of both forward and sideward scatter signals. Interestingly, while MRS5206 did not bind to mock transfected cells (data not shown), a concentration-dependent binding isotherm for A_2AR expressing cells was found when positive cells or events (Figure 2A, inset histogram) were plotted against MRS5206 concentration (Figure 2A). A close analysis of these results revealed that the maximal MRS5206 binding capacity identified ~33% of positive cells, and this was achieved at low nanomolar concentrations of MRS5206. This value may depend on the existing interrelation between cell surface receptor density and transfection efficiency achieved in our experiments. On the other hand, MRS5206 binding was completely blocked by ZM241,385 (1 µM) at any MRS5206 concentration tested, thus demonstrating the specificity of the fluorescent compound (Figure 2A). Also, we evaluated the affinity of MRS5206 by competitive ligand binding experiments. Thus, a fixed dose of MRS5206 (100 nM) was displaced with CGS21680, another A_2AR selective agonist, in a concentration-dependent manner (Figure 2B). The dissociation constant of CGS21680 (106±42 nM) was similar to that described in the literature by means of radioligand experiments (Ciruela et al., 2006). Interestingly, these results were consistent with previous observations of fluorescent ligand binding for other GPCRs using flow cytometry under equilibrium conditions (Schneider et al., 2006). Thus, we finally carried out flow cytometry binding experiments using the antiparkinsonian drugs. Importantly, while in cells transfected only with the A_2AR-containing plasmid the D₂R agonists did not alter MRS5206 binding to A_2ARs (data not shown), in A_2AR-D₂R co-expressing cells all the antiparkinsonian agents tested produced a significant reduction of MRS5206 (100 nM) binding (Figure 2C), with apomorphine again achieving the largest binding inhibition (42±7%, p < 0.01) in comparison to rotigotine (22±3%, p < 0.01) and pramipexole (29±7%, p < 0.01) (Figure 2C).

Figure 2. Flow cytometric determinations of antiparkinsonian D₂R agonist-mediated modulation of A_2AR agonist binding.

(A) The flow cytometric equilibrium binding assay. HEK-293 cells transiently transfected with A_2AR were incubated with increasing concentrations of MRS5206 and analyzed by flow cytometry. In the inset panel a representative histogram of MRS5206 (100 nM) total binding (black line) and nonspecific binding (red line, in the presence of 1 µM of ZM241,385) is shown. The specific fluorescence detection (FL-1) for each MRS5206 concentration tested was determined by M1 cursor (see histogram). Thus, specific events within the M1 region were plotted against MRS5206 concentration. (B) Competition between MRS5206 (100 nM) and increasing concentrations of CGS21680, another specific A_2AR agonist. The geometric fluorescence mean within the M1 region events was plotted against each CGS21680 concentration tested. (C) Quantification of antiparkinsonian D₂R agonists effects on MRS5206 binding. HEK-293 cells transiently transfected with A_2AR and D₂R were incubated with 100 nM of MRS5206 in the absence or presence of quinpirole (100 µM), pramipexole (120 µM), rotigotine (1 µM) and apomorphine (6 µM) and analyzed by flow cytometry. The obtained geometric fluorescence mean within the M1 region events for each equilibrium binding condition was plotted. Data represents the average ± S.E.M. values of three independent experiments. Asterisks indicate data significantly different from the control condition (i.e. in the absence of antiparkinsonian D₂R agonist): *p < 0.05 and **p < 0.01 by ANOVA with Dunnett`s multiple comparison post-hoc test.

4. Conclusion

The two different experimental approaches presented here permitted us to evaluate the degree of D₂R-mediated allosteric modulation of A_2AR agonist binding within the A_2AR/D₂R oligomer framework. Hence, it could be concluded that both our FRET- and flow cytometry-based agonist binding assays would be valuable for determining the D₂R agonist NAM activity of different antiparkinsonian agents over A_2AR functioning. Needless to say, the relevance of this information for the design of effective D₂R-based antiparkinsonian therapies is intrinsically limited, since the experiments were performed in a heterologous system. Thus, it is important to keep in mind that any new emerging D₂R-based drug with A_2AR NAM activity should be first tested in PD animal models before attempting some clinical extrapolation. And on the other hand, it would also be important to correlate the degree of the discovered properties of apomorphine, rotigotine, and pramipexole with their effectiveness as antiparkinsonian drugs. Overall, the present methods are mainly circumscribed to the mechanistic elucidation of the receptor-receptor interaction occurring between A_2ARs and D₂Rs, but they may also be viewed as possible complementary tools on the development of D₂R-like drugs targeted to the management of Parkinson’s disease.