Abstract
The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.
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Introduction
The first pieces of evidence for antagonistic glutamate receptor with dopamine D2 receptor (D2R) interactions were found in 1983–1984 through the ability of glutamate to reduce the affinity of the high-affinity D2R agonist binding sites in striatal membrane preparations. Subsequently, it was observed that mGluR5 agonists alone or combined with an A2AR agonist (CGS-21680) can reduce the affinity of the high-affinity state of D2R for agonist binding sites in the rat striatum [1]. Co-immunoprecipitation experiments also indicated the existence of A2AR-mGluR5 heteroreceptor complexes in HEK293 cells and rat striatal membrane preparations [2]. The colocation of the receptors in striatal neurons was demonstrated [3, 4] as well as their synergistic interactions as studied with in vivo microdialysis and intracellular signalling in striatal preparations [2, 5, 6].
In 1974, the discovery that the methylxanthines caffeine and theophylline could enhance the contralateral turning behaviour induced by levodopa and dopamine receptor agonists in the hemi-Parkinsonian rat model was one early finding leading to the hypothesis that antagonistic adenosine-dopamine interactions existed [7, 8]. Today, a considerable amount of molecular and functional experimental data supports the view that A2AR and D2R form heteroreceptor complexes with antagonistic receptor-receptor interactions on the plasma membrane [9,10,11,12,13,14,15,16].
The existence of A2AR-D2R-mGluR5 higher-order oligomers was postulated, and it was proposed that the receptor-receptor interactions within this high-order complex are important to modulate the dorsal and ventral striatal-pallidal GABA neurons [2, 3, 8]. Years later, it was proposed that combined treatment with A2AR and mGluR5 agonists targeting A2AR-D2R-mGluR5 heteroreceptor complexes in the ventral striatal-pallidal GABA pathway can represent a new strategy for the treatment of schizophrenia [17]. Also, the combine treatment with selective A2A and mGluR5 receptor antagonists represents an alternative therapeutic approach to Parkinson’s disease [18,19,20].
A combination of bimolecular fluorescence complementation assays and bioluminescence resonance energy transfer assays as well as the sequential resonance energy transfer technique was used to show that A2AR-D2R-mGluR5 heteroreceptor complexes exist in living cells [21]. In addition, high-resolution immunoelectron microscopy was also used to further demonstrate their existence in striatal glutamate synapses [21]. An integrative role of these receptor complexes in adenosine, dopamine and glutamate transmission was also proposed [8, 22, 23]. Recently, A2AR, D2R and mGluR5 receptor-receptor interactions were also found to modulate the activity of the striatal-pallidal GABA neurons based on in vivo dual-probe microdialysis [24].
Herein, new findings that further expand the understanding of A2AR-D2R-mGluR5 heteroreceptor complexes are presented. Results in cellular models first demonstrated that A2AR promotes the D2R and mGluR5 receptor-receptor interactions, and its participation increases the density of the D2R-mGluR5 heterocomplexes. Binding and functional experiments indicated that A2AR and mGluR5 upon agonist activation play a significant role in modulating the composition, density and signalling of A2AR-D2R-mGluR5 heteroreceptor complexes. This was also observed in A2AR or D2R knockout mice when studying the effects of the mGluR5 negative allosteric modulator raseglurant on locomotor activity.
Methods
Plasmid Constructs
The cDNA encoding the rat mGluR5 was cloned (without stop codon) in pGFP2-N1 vector (PerkinElmer, Waltham, MA, USA) using standard molecular biology techniques. The D2RRluc construct used has been described previously in Borroto-Escuela et al. 2010 [25].
Drugs and Chemicals
The A2AR agonist 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS-21680), the selective A2AR antagonist 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385), the mGluR5 agonist (RS)-2-Chloro-5-hydroxyphenylglycine sodium salt (CHPG), the mGluR5 antagonist 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and the D2R antagonist 4-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone hydrochloride (haloperidol) were purchased from Tocris Bioscience (UK), and the mGluR5 negative allosteric modulator 2-[(3-Fluorophenyl)ethynyl]-4,6-dimethyl-3-pyridinamine hydrochloride (raseglurant) was purchased from Hello Bio (Republic of Ireland). The concentrations of CGS-21680 (100 nM) and ZM-241385 (1 μM) were chosen in agreement with our previous studies [26, 27]. The concentrations of CHPG (500 nM) and MPEP (300 nM) have been selected on the basis of previous studies suggesting that, in this concentration range, the compounds selectively act as agonist or antagonist of mGluR5, respectively [18, 24, 28, 29]. Finally, the dose of haloperidol (1 mg/kg) and raseglurant (1 mg/kg) used in mouse behavioural experiments was previously described [30, 31]. Also, isobutyl-1-methylxanthine (IBMX) and 4-(3-butoxy-4-methoxybenzyl) imidazolidone (Ro 20-1724) were purchased from Tocris Bioscience (Bristol, UK).
Cell Culture and Transfection
Human embryonic kidney 293T (HEK293T cells (American Type Culture Collection, Manassas, VA, USA) cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin/streptomycin and 10% (v/v) foetal bovine serum at 37 °C in an atmosphere of 5% CO2. Cells were plated in 6-well plates (1 × 106cells/well), 96-well plates (1 × 104cells/well) or in 75 cm2 flasks and cultured overnight prior to transfection or experimental procedures. Cells were transiently transfected using linear polyethyleneimines (Polysciences Inc., Warrington, PA, USA) according to the manufacturer’s instructions.
Animals
A2AR−/− and D2R−/− mice generated on a CD-1 genetic background [30, 32] and the corresponding wild-type littermates weighing 20–25 g were used. The animal protocol (no. 7085) was approved by the University of Barcelona Committee on Animal Use and Care. Animals were housed and tested in compliance with the guidelines provided by the Guide for the Care and Use of Laboratory Animals [33] and following the European Union directives (2010/63/EU), the ARRIVE guidelines [34]. Mice were housed in groups of five in standard cages with access to food and water ad libitum while maintained under a 12-h dark/light cycle (starting at 7:30 AM), 22 °C temperature and 66% humidity (standard conditions). All animal experimentation was carried out in a period comprehended between 9:00 AM and 6:00 PM by a researcher blind to drug treatments.
Locomotor Activity Tests
Mice spontaneous or drug-induced locomotor activity was assessed by the open field test. In brief, animals were administered intraperitoneal (i.p.) with raseglurant (1 mg/kg) or vehicle-saline with 5% DMSO and 5% Tween 20 30 min before the testing session. Non-habituated mice were placed in the centre of an activity field arena (30 × 30 cm, surrounded by four 50-cm-high black-painted walls) equipped with a camera above to record activity and connected to the light source. The total distance travelled was analysed using SPOT tracker function from ImageJ (NIH, Bethesda, MD, USA), as previously described [30].
Catalepsy Test
Mouse catalepsy was induced by the administration (i.p.) of haloperidol (1 mg/kg) [30]. After 1 h, haloperidol-induced catalepsy was measured as the duration in seconds of an abnormal upright posture in which the forepaws of the mouse were placed on a horizontal wooden bar (0.6 cm of diameter) that was located 4.5 cm above the floor. Subsequently, mice were administered (i.p.) with either vehicle (i.e. saline with 5% DMSO and 5% Tween) or raseglurant (1 mg/kg). After 20 min, a second haloperidol-induced catalepsy measurement was performed.
The rationale for the use of raseglurant (a mGluR5-negative allosteric modulator) instead of a full antagonist was based on the theoretical advantages that allosteric modulators offer compared with their competitive counterparts. mGluR5 allosteric modulators (negative allosteric modulators (NAM) and positive allosteric modulators (PAM)) have the potential for greater subtype selectivity when compared to orthosteric ligands. Also, mGluR5 NAM and PAM do not possess intrinsic activity and are assumed to be quiescent in the absence of an endogenous agonist and only modulate receptor function when the endogenous agonist is present. In this manner, NAM and PAM have the potential to retain spatial and temporal aspects of endogenous receptor signalling. This is of particular interest for CNS targets where optimal neurotransmission is likely to have an improved therapeutic outcome as opposed to sustained receptor blockade or activation.
Haloperidol-Induced Catalepsy
Mice (n = 10) were randomly assigned to treatment groups, and behavioural testing was performed blind to treatment. The dopamine D2 receptor (D2R) antagonist, haloperidol (1 mg/kg, s.c.), was administered to induce catalepsy. Thirty minutes after the haloperidol administration, mice experienced a full cataleptic response. At this time point, for each mouse, the state of catalepsy was tested by gently placing their front limbs over an 8-cm-high horizontal bar. The intensity of catalepsy was assessed by measuring the time the mice remain in this position being completely immobile for a maximum of 120 s. Only mice that remained cataleptic for the entire 120 s were used for subsequent drug testing. After 30 min of the baseline measurement vehicle (0.5% methylcellulose and 2% DMSO), PBF509 was administered orally via gavage (3, 10 or 30 mg/kg, p.o.), and the catalepsy was then determined at 15, 30 and 60 min PBF509 administration. For each time point, the number of responding mice and the total cataleptic time for each animal were determined.
Membrane Preparation
HEK293T cells or mouse striata were homogenized in ice-cold 10 mM Tris HCl, pH 7.4, 1 mM EDTA and 300 mM KCl buffer containing a protease inhibitor cocktail (Roche, Penzberg, Germany) using a Polytron for three periods of 10 s each. The homogenate was centrifuged for 10 min at 1000 × g. The resulting supernatant was centrifuged for 30 min at 12,000 × g. The membranes were dispersed in 50 mM Tris HCl (pH 7.4) and 10 mM MgCl2, washed and resuspended in the same medium. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA).
Bioluminescence Resonance Energy Transfer Saturation Assay
BRET2 saturation curves have been particularly used with the aim to establish the oligomeric order of receptor complexes, as well as the proportion of receptors engaged in dimers or oligomers (BRETmax). In the current work, bioluminescence resonance energy transfer (BRET2) saturation assays were carried out using plasmids encoding for D2RRluc and mGluR5GFP2 according to previously published methods [9, 26, 35, 36]. The netBRET2 ratio was defined as the BRET ratio for co-expressed Rluc and GFP2 constructs normalized against the BRET ratio for the Rluc expression construct alone: netBRET2 ratio = [(GFP2 emission at 515 ± 30 nm)/(Rluc emission 410 ± 80 nm)]-cf. The correction factor, cf, corresponds to (emission at 515 ± 30 nm)/(emission at 410 ± 80 nm) found with the receptor-Rluc construct expressed alone in the same experiment. The maximal value of BRET (netBRET2max) corresponds to the situation when all available donor molecules are paired up with acceptor molecules [8]. Also, saturation assay was used to compare the relative affinity of receptors for each other and their probability to form a complex, the so-called BRET50, which represents the acceptor/donor ratio giving 50% of the maximal signal. The ratio is calculated from fluorescence and bioluminescence values expressed as arbitrary units. BRET50 values should not be regarded as a common or classical value to expressed affinities as Molar units. Pairs with low BRET50 value thought to form oligomers or an increased tendency to dimerize, while high BRET50 values indicate weak interaction or the absence of interaction between the investigated receptors. The specificity of D2RRluc-mGluR5GFP2 interactions was assessed by comparison with co-expression of A1RGFP2 and D2RRluc.
In Situ PLA in Cultured Cells
In situ proximity ligation assay (PLA) in cultured cells was performed using the Duolink in situ PLA detection kit (Sigma-Aldrich, St. Louis, MO, USA), following the protocol described previously [11, 37, 38] using mouse monoclonal anti-D2R (2 μg/ml, MABN53; Millipore, Billerica, MA, USA) and rabbit polyclonal anti-mGluR5 (2 μg/ml, AB5675; Millipore) primary antibodies. PLA control experiments employed only one primary antibody. The PLA signal was visualized and quantified by using a TCS-SL confocal microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) and the Duolink ImageTool software. High magnifications of the microphotograph were taken and visualized using multiple z-scan projections.
The background signal was estimated from both PLA control experiments and from PLA experiments performed on non-transfected HEK293T cells (HEK293T cell line expresses endogenously small amount of D2R, A2AR and mGluR5). In general, the positive PLA values obtained in these experiments were residuals. The assay cut-off value was set to two standard deviations over the background signal. Therefore, samples with values below this cut-off were negative for the interaction of interest, while samples with values higher than the threshold were positive.
Immunohistofluorescence and In Situ PLA in Mouse Brain
Mice were anaesthetized and intracardially perfused with 50–200 ml of ice-cold 4% formaldehyde solution (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS; 1.47 mM KH2PO4, 8.07 mM Na2HPO4, 137 mM NaCl, 0.27 mM KCl with pH 7.2). The brains were post-fixed overnight in the same 4% formaldehyde solution at 4 °C. The vibratome (Leica Lasertechnik GmbH, Heidelberg, Germany) was used to make coronal section (50 μm). Slices were collected and kept in Walter’s antifreezing solution (30% glycerol, 30% ethylene glycol in PBS with pH 7.2) at −20 °C until further processing [39].
For immunohistofluorescence (IHF), experiments coronal brain slices were washed three times with PBS for 10 min at 22 C, then permeabilized with 0.3% Triton X-100 in PBS (2 h at 22 °C) and rinsed (3×) with washing solution (PBS containing 0.05% Triton X-100, 10 min, at 22 °C). Blocking of the slices was performed with washing solution containing 10% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 2 h at 22 °C. To avoid unspecific binding, the slices were incubated with secondary anti-mouse IgG (no. 715-005-150; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) in washing solution (2 h at 22 °C). Then, the slices were incubated with mouse anti-mGluR5 monoclonal (20 μg/ml, MABN540; Millipore) and rabbit anti-D2R polyclonal (1 μg/ml, D2R-Rb-Af960; Frontier Institute Co. Ltd, Shinko-nishi, Ishikari, Hokkaido, Japan) in washing solution with 5% NDS overnight at 4 °C. Subsequently, the slices were washed twice with a washing solution containing 1% NDS (10 min at 22 °C). Next, the slices were incubated with anti-Cy2 donkey anti-rabbit (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and anti-Cy3 donkey anti-mouse (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in washing solution with 1% NDS for 2 h at 22 °C. Finally, slices were washed two times with washing solution containing 1% NDS (10 min at 22 °C), two times with PBS (10 min at 22 °C) and then mounted with Duolink® in situ mounting medium with DAPI (Sigma-Aldrich). The Leica TCS 4D confocal scanning laser microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) was used to capture the fluorescence striatal images.
For in situ PLA in mouse brain, the Duolink in situ PLA detection kit (Sigma-Aldrich) was used as previously described [37, 39, 40]. Thus, the experimental procedure until the secondary antibody incubation step was the same as the IHF (see above). Subsequently, the following steps were performed according to the manufacturer’s protocol. Images were acquired and analysed as previously described [39]. The background signal was estimated from PLA control experiments, and the assay cut-off value was performed as described above.
Radioligand Competition Binding Experiments
For the binding experiments, membrane preparations (60 μg protein/ml) were obtained from HEK293T cells expressing either D2R and mGluR5 or A2AR, D2R and mGluR5, and [3H]-raclopride (Novandi Chemistry AB, Södertälje, Sweden) competition assays with minor modifications were performed according to previously published methods [26, 27, 41]. [3H]-raclopride (75 Ci/mmol), a D2-like receptor antagonist competing [42] with quinpirole for binding to D2-like receptors in HEK293T membrane preparations, was used to determine the D2R high-affinity (Ki, High) and D2R low-affinity (Ki, Low) values. (+)-Butaclamol, a selective D2R antagonist (100 μM, Sigma-Aldrich), was used to determine the non-specific binding. The amount of bound [3H]-raclopride was determined by liquid scintillation spectrometry.
cAMP Functional Assay
Intracellular cAMP levels were determined using a cAMP-Glo™ assay detection kit (Promega, Madison, WI, USA). HEK293T cells expressing either D2R and mGluR5 or A2AR, D2R and mGluR5 were plated at a density of 10,000 cells/well in 96-well microtiter plates coated with poly-L-lysine (Sigma-Aldrich) and incubated overnight. Culture medium was then removed; cells were washed with 1 × PBS before the induction buffer (red phenol/serum-free DMEM containing 500 μM IBMX and 100 μM Ro 20-1724) was added. The cells were incubated for 1 h prior to drug incubation. To examine the Gi protein-mediated inhibition of adenylyl cyclase, the levels of cAMP were first raised with 5 µM forskolin for 10 min. Drug dilutions were prepared in the induction buffer, and the temperature- and carbon dioxide-equilibrated drug dilutions (37 °C cell culture incubator for 30 min) were added as indicated, and cells were then incubated at 37 °C for 30 min. The assay was performed accordingly to the manufacturer’s specifications (Promega, Sweden). Readings of luminescence intensity were performed using the POLARstar Optima plate reader (BMG Lab Technologies, Offenburg, Germany). cAMP levels in non-transfected, non-treated cells and non-transfected cells treated only with forskolin were defined as basal and control, respectively.
Gel Electrophoresis and Immunoblotting
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 7% polyacrylamide gels. Proteins were transferred to Hybond-LFP polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA) using the Trans-Blot Turbo™ transfer system (Bio-Rad, Hercules, CA, USA) at 200 mA/membrane for 30 min. PVDF membranes were blocked with 5% (wt/vol) dry non-fat milk in phosphate-buffered saline (PBS; 8.07 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 0.27 mM KCl, pH 7.2) containing 0.05% Tween-20 (PBS-T) during 1 h at 20 °C before being immunoblotted with the indicated antibody in blocking solution overnight at 4 °C. PVDF membranes were washed with PBS-T three times (5 min each) before incubation with either a HRP-conjugated rabbit anti-mouse IgG (1/10,000) or HRP-conjugated goat anti-rabbit IgG (1/30,000) in blocking solution at 20 °C during 2 h. After washing the PVDF membranes with PBS-T three times (5 min each), the immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific) and detected with an Amersham Imager 600 (GE Healthcare Europe, Barcelona, Spain).
Statistical Analysis
The number of independent experiments (n) in each group is indicated in figure legends. Data are represented as mean ± standard error of mean (SEM). Outliers were assessed by the ROUT method [43]; thus, subjects were excluded assuming a Q-value of 1% in GraphPad Prism 9 (San Diego, CA, USA). Data normality was assessed by the Shapiro-Wilk normality test (p < 0.05). When two groups were evaluated, unpaired Student’s t-test or Mann-Whitney U-test was used. Comparisons among more than two experimental groups were performed by one-, two- or three-way factor analysis of variance (ANOVA) followed by either Dunnett’s, Šídák’s or Tukey post hoc test using GraphPad Prism 9, as indicated in the figure legends. A p-value ≤ 0.05 was considered significant.
Results
BRET.2 Experiments : Transient Co-expression of A 2A R with D 2 R and mGluR 5 Had a Significant Impact on D 2 R-mGluR 5 Heteroreceptor Complex Formation
HEK293T cells were transiently transfected with constant amounts of D2RRluc and increasing amounts of plasmids encoding for mGluR5GFP2 with/without transient co-expression of A2AR. The transient co-expression of A2AR with D2RRluc and mGluR5GFP2 had a significant impact on D2RRluc-mGluR5GFP2 heteroreceptor complex formation (Fig. 1A). Transient co-expression of A2AR promoted a significant increase of netBRET2max ratio value (0.084 ± 0.003 AU) compared to that found in cells without transient co-expression of A2AR (0.043 ± 0.002 AU) (Fig. 1B). When the A2AR was coexpressed with D2RRluc and mGluR5GFP2, these receptors hence showed an increased ability to heteromerize.
Also, saturation assay was used to compare the relative affinity of receptors for each other and their probability to form a complex, the so-called BRET50, which represents the acceptor/donor ratio giving 50% of the maximal signal. The netBRET250 ratio value for D2RRluc-mGluR5GFP2 heteromerization was significantly reduced by transient co-expression of A2AR from (1.58 ± 0.09 AU) to (0.94 ± 0.11 AU) (Fig. 1C) indicating increased affinity of the two receptor protomers for each other. Pairs with low BRET50 value thought to form oligomers or an increased tendency to dimerize, while high BRET50 values indicate weak interaction or the absence of interaction between the investigated receptors.
Proximity Ligation Assay Experiments : Transient Co-expression of A 2A R Promoted the Formation D 2 R-mGluR 5 Heteroreceptor Complexes in HEK Cells
The role of A2AR in the dynamics of the D2R-mGluR5 heteromers was also evaluated by in situ proximity ligation assays (PLA) in transiently co-transfected HEK293T cells. The PLA results were in line with the results from the BRET2 assays. The in situ PLA demonstrated the existence of D2R-mGluR5 heteroreceptor complexes in cells to a low degree without transient co-expression of A2AR (Fig. 2A). Furthermore, the transient co-expression of A2AR highly significantly promoted the formation D2R-mGluR5 heteroreceptor complexes as shown by the marked increase in the number of PLA-positive D2R-mGluR5 complexes, while this was significantly reduced in HEK293T cells without co-expressing A2AR (Fig. 2 B and D). Few and weak PLA clusters were detected in the PLA-negative controls (lack of D2R antibodies) representing background labelling (Fig. 2C).
The specificity of the PLA-positive D2R-mGluR5 complexes, shown as red blobs in the mouse dorsal striatum (Fig. 3A), was demonstrated using D2R−/− mice (Fig. 3C). In the sections from the mouse striatum, the appearance of the red PLA-positive D2R-GluR5 complexes, shown as mean number of red blobs/Nucleus, was markedly and highly significantly reduced (Fig. 3D). Furthermore, the loss of the red D2R-mGluR5 blobs to the same high degree in the A2AR−/− mice (Fig. 3 B, D) likely reflects the requirement of D2R-mGluR5 heterocomplexes to be part of an A2AR-D2R-mGluR5 to be expressed in the mouse striatum, probably by dorsal striatal-pallidal GABAergic neurons. In this way, it forms D2R-mGluR5 complexes that are close enough to be visualized by PLA.
[.3 H]-Raclopride/Quinpirole Competition Experiments: the A 2A R and mGluR 5 Protomers Interact and Modulate D 2 R Protomer Recognition
In HEK293T cells expressing D2R and mGluR5, the mGluR5 agonist CHPG (500 nM) reduced the affinity of the high-affinity state (Ki, High) of the D2R for the agonist quinpirole with no effects on its low-affinity state (Ki, Low). Co-treatment with A2AR agonist CGS-21680 (100 nM) did not significantly alter the D2R Ki, High and Ki, Low values obtained when the cells were treated only with CHPG (500 nM) (Fig. 4A and Table 1). In HEK293T cells expressing A2AR, D2R and mGluR5, mGluR5 agonist stimulation also reduced the affinity of the high-affinity state (Ki, High) of the D2R for the agonist quinpirole with no statistically significant effects on its low-affinity state (Ki, Low) (Fig. 4A and Table 2). However, the transient co-expression of A2AR by itself (without agonist stimulation) potentiates mGluR5 agonist effects on the high-affinity D2R agonist binding sites (Fig. 4B, Tables 1 and 2). Finally, the co-stimulation of A2AR and mGluR5 synergistically increased in the Ki, High values of the D2R protomer upon co-expression of the A2AR (Table 2). Nevertheless, in cells expressing A2AR, D2R and mGluR5, further analysis should be performed to test the effect of combine treatment of A2AR (ZM-241385) and mGluR5 (CHPG) to figure out if the expression of A2AR, without agonist stimulation and its corresponding constitutive activity, is responsible for increased in the Ki, High values of the D2R protomer upon co-expression of the A2AR.
In both HEK293T cells expressing D2R and mGluR5 or A2AR, D2R and mGluR5, the incubation with A2AR antagonist ZM-241385 (1 μM) and mGluR5 antagonist MPEP (300 μM) alone or in combination resulted in an almost complete blockade of the mGluR5 increase of the D2R Ki, High values and A2AR agonist-induced increase of mGluR5 agonist effects on the high-affinity D2R agonist binding sites (Tables 1 and 2).
cAMP Functional Experiments : the A 2A R and mGluR 5 Protomers Interact and Modulate D 2 R Protomer Signalling
In cells expressing D2R and mGluR5 forming D2R-mGluR5 heterocomplexes (Fig. 2), the D2R agonist activation with quinpirole (100 nM) induced a Gi protein-mediated inhibition of adenylyl cyclase that first was raised with 5 µM forskolin (Fig. 5A). This effect was highly significantly blocked by the D2R antagonist raclopride (1 μM). In these cells, the mGluR5 agonist CHPG stimulation significantly counteracted the D2R agonist-induced reduction of cAMP accumulation (Fig. 5A). The significant effect of CHPG (500 nM) was significantly reduced by the mGluR5 antagonist MPEP (300 μM). The co-treatment with the A2AR agonist did not enhance the counteraction of the inhibitory D2R signalling by CHPG (Fig. 5A).
Likewise, quinpirole significantly reduced the cAMP level in cells expressing A2AR, D2R and mGluR5 (Fig. 5B). The mGluR5 agonist CHPG had an improved ability to counteract the adenylyl cyclase inhibition produced by the D2R agonist in these cells, yielding cAMP levels similar to those obtained after blocking D2R signalling with raclopride (Fig. 5B). Upon A2AR and mGluR5 agonist co-activation, a larger counteraction of the D2R agonist action was found compared to that obtained with such a co-treatment performed in cells expressing only D2R and mGluR5. These results suggest a synergistic and significant counteraction by A2AR and mGluR5 agonists of the D2R agonist-induced decrease of cAMP accumulation (Fig. 5B). Such effects of the combined agonist treatment were only weakly reduced by the A2AR antagonist (ZM-241385). In cells not expressing the A2AR, the A2AR antagonist failed to produce any changes in the cAMP accumulation under such co-agonist treatments.
It should be noted that CHPG agonist produces similar increases in cAMP levels as found after the A2AR agonist GGS in HEK293T cells co-expressing D2R, A2AR and mGluR5 (Fig. 5C). Therefore, we should consider also that mGluR5 might simply activate Gs, inducing cAMP accumulation, independently of D2-Gi-induced inhibition of adenylate cyclase (Fig. 5 A−C).
Experiments on Haloperidol-Induced Catalepsy
Catalepsy is a nervous condition characterized by loss of muscle control and fixity of posture. It is considered a symptom of certain nervous disorders such as Parkinson’s diseases and epilepsy [44]. It is also a characteristic symptom of cocaine withdrawal, as well as one of the features of catatonia. The catalepsy is mainly produced by haloperidol induced blockade of D2R complexes in the dorsal striatal-pallidal GABA neurons within the dorsal striatum [44,45,46]. These GABA neurons mediate motor inhibition, counteracted by D2R agonist-induced activation of the D2R homo- and heterocomplexes like the D2R-A2AR or the D2R-mGluR5 heterocomplexes [24, 47,48,49,50]. The D2R activation of the dorsal striatal-pallidal GABA neurons is also essential for maintenance of normal locomotor activity.
The catalepsy induced by the D2R antagonist haloperidol was evaluated in 10-min time intervals from 60 to 90 min after the injection of haloperidol (Fig. 6). In wild-type mice, the mGluR5-negative allosteric modulator raseglurant produced in this time period a significant reduction of the catalepsy time which was in the order of 25% (Fig. 6). In contrast, such a reduction of catalepsy was not observed by raseglurant treatment of A2AR−/− mice. Furthermore, in vehicle-treated A2AR−/− animals, the haloperidol-induced catalepsy was markedly reduced compared to that obtained in vehicle treated wild-type mice (Fig. 6).
Discussion
The field of dopamine D2Rs changed markedly with the discovery of many types of D2R homo- and heteroreceptor complexes in subcortical limbic areas as well as the dorsal striatum [4, 16, 40]. The results indicate that the D2R is a hub receptor [51] which interacts not only with many other GPCRs including dopamine isoreceptors but also with ion-channel receptors, receptor tyrosine kinases, scaffolding proteins and dopamine transporters [24, 52, 53]. Disturbances in several of these D2R heteroreceptor complexes may contribute to the development of brain disorders through changes in the balance of diverse D2R homo- and heteroreceptor complexes mediating the dopamine signal, especially to the ventral striato-pallidal GABA pathway [37, 52, 54]. Of high relevance was the discovery of A2AR-D2R and A2AR-mGluR5 heteroreceptor complexes in native tissue [4, 16, 40, 55, 56]. Furthermore, the existence of the D2R-mGluR5 heterodimers in the biomembranes of living cells was demonstrated by bimolecular fluorescence complementation experiments in cellular models [21]. Although when tested by FRET microscopy in tsA 201 cells, D2R did not associate with mGluR5 [57]. Nevertheless, by combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, the occurrence of an A2AR-D2R-mGluR5 heteroreceptor complexes was observed in living cells. Furthermore, by co-immunoprecipitation, experiments validated the existence of an association of mGluR5, D2R and A2AR in rat striatum homogenates [21].
Herein, we present new findings that further expand the understanding of A2AR-D2R-mGluR5 heteroreceptor complexes. Also, strong evidences which support that the expression of the A2AR is necessary to facilitate the association of D2R and mGluR5 in a complex.
Our new findings are that transient co-expression of A2AR in HEK293T cells together with D2RRluc and mGluR5GFP2 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heterodimer, a component of the A2AR-D2R-mGluR5 heterocomplex, based on the increase in the BRET2 max values. Such an increase could be related to the development of an increased affinity of the two D2R and mGluR5 protomers for each other due to allosteric changes related to the formation of the A2AR-D2R-mGluR5 complex. In line with this hypothesis, the BRET250 values were significantly reduced for the D2R-mGluR5 heteromeric component of this trimeric heteroreceptor complex.
These results are also supported by the demonstration with PLA that an increased density of PLA-positive D2R-mGluR5 clusters was observed when A2AR expression had been added to the cells compared to cells only expressing D2R and mGluR5. In agreement, in the mouse dorsal striatum, the D2R-mGluR5 complexes were significantly reduced in the A2AR−/− mice. Thus, it becomes clear that the expression of the A2AR in the mouse dorsal striatum is necessary to facilitate that the D2R and mGluR5 form a complex. It underlines that the multiple receptor protomers in the high-order heteroreceptor complexes are dependent on each other to improve or facilitate the formation of such complexes in the dorsal striatum.
The different results obtained on haloperidol-induced catalepsy in wild-type mice vs A2AR−/− mice are of substantial interest since they can indicate a functional role of the A2AR-D2R-mGluR5 heteroreceptor complexes in the dorsal striatum as previously discussed [8, 17]. There was a marked reduction in the haloperidol-induced catalepsy in the A2AR−/− mice compared to wild-type mice. Thus, in the absence of the A2AR, the D2R antagonist haloperidol appears to have a substantially reduced potency to block the D2R which can be caused by the loss of the antagonistic A2AR-D2R interaction [9, 58]. According to the current findings in cell lines, the D2R-mGluR5 heterocomplexes should be also formed to a much lower degree in the absence of A2AR in view of their dependency of A2AR according to the PLA experiments performed. The counteraction of the D2R-mediated inhibitory actions on cAMP signalling by CHPG, a mGluR5 agonist, was in our cell line also more effective in cells co-expressing beside D2R and mGluR5, also A2AR.
It seems likely that the formation of the A2AR-D2R-mGluR5 complex enhances the affinity of the D2R and mGluR5 protomers for each other in this complex. It is of high interest that the biochemical binding experiments reveal that the mGluR5 CHPG agonist-induced increase in D2R Ki, High values becomes significantly higher in the A2AR-D2R-mGluR5 complex compared to the D2R-mGluR5 complex despite the absence of A2AR agonist exposure. Thus, although agonist activation of the A2AR seems necessary to exert negative allosteric modulation of the D2R protomer agonist binding via heteroreceptor complexes, an increased constitutive activity of the A2AR protomer could explain the above results.
As expected, the combined incubation with CHPG and CGS-21680 led to an even stronger increase in the D2R Ki, High values of the A2AR-D2R-mGluR5 complex, demonstrating the impact of the A2AR protomer on the D2R-mGluR5 allosteric interactions, which can involve both constitutive and A2AR agonist-induced inhibition of D2R agonist binding. Our findings represent one of the first examples of integrative activity within a higher-order heteroreceptor complex and show how one receptor (A2AR) can substantially modulate the structure and recognition of a participating receptor heterodimer (D2R-mGluR5) in such a trimeric receptor complex.
The pharmacological analysis of the A2AR-D2R-mGluR5 complex and its impact on cAMP levels indicated that the A2AR can modulate the effects of the D2R-mGluR5 interactions on cAMP signalling. It was found that when the A2AR-D2R-mGluR5 complex was likely to be formed through the expression also of the A2AR, the mGluR5 agonist had an increased ability to counteract the D2 agonist-induced Gi/o-mediated inhibition of the cAMP levels in comparison with the counteraction observed in the absence of A2AR expression. The same was also true for the combined treatment with the mGluR5 agonist CHPG and the A2AR agonist CGS-21680 when the A2AR was coexpressed. A stronger counteraction of the D2R-induced inhibition of the cAMP levels was observed when A2AR expression was present.
Taken together, our work on cell lines gives strong indications that, in the A2AR-D2R-mGluR5 complex, the A2AR protomer enhances the formation of the D2R-mGluR5 component of the complex with enhanced inhibition of D2R agonist binding recognition and its Gi/o-mediated cAMP signalling. The inhibitory effects by A2AR and mGluR5 on D2R recognition and signalling reveal a significant molecular integration in A2AR-D2R-mGluR5 complexes, likely formed also in the dorsal striatum. The A2AR and mGluR5 antagonists targeting the A2AR-D2R-mGluR5 complexes in dorsal striatum may reduce the haloperidol-induced catalepsy by removal of the A2AR and mGluR5 protomer-mediated allosteric inhibition of the D2R protomer. Understanding of the trimeric complexes formed by these GPCRs could provide novel strategies for development of drugs against neuropsychiatric and neurodegenerative diseases by targeting their antagonistic receptor-receptor interactions.
Data Availability
The datasets generated during and/or analysed during the current study are available (upon request) in the Fuxe Lab repository at the Department of Neuroscience, Karolinska Institutet (contact email: Kjell.Fuxe@ki.se).
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Acknowledgements
We thank Esther Castaño, Benjamín Torrejón and Álvaro Gimeno from the CCiT-Bellvitge Campus of the University of Barcelona.
Funding
Open access funding provided by Karolinska Institute. This work was supported by grants from the Swedish Research Council (2019–01022 and 62X-00715–50-3) and Parkinson Fonden to K. F, Swedish Research Council (2017–4676) to J. C. and by the Hjärnfonden (F02018-0286, F02019-0296), Karolinska Institutet Forskningsstiftelser 2020 and by project EMERGIA 2020–39318 founded by the Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020) to D. O. B-E. Furthermore, by Olle Engkvists Stiftelse (2019, 2021) to KF and D. O. B-E. Also supported by project PID2020-118511RB-I00 founded by MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa” and Generalitat de Catalunya (2017SGR1604) to FC. We thank the Centres de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya for IDIBELL institutional support.
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We confirm and declare that all authors meet the criteria for authorship according to the ICMJE, including approval of the final manuscript, and they take public responsibility for the work and have full confidence in the accuracy and integrity of the work of other group authors. They have substantially contributed to the conception or design of the work. Also, they have participated in the acquisition, analysis and interpretation of data for the current version. They have also helped revising it critically for important intellectual content and final approval of the version to be published. In addition, they have contributed to this last version of the manuscript in writing assistance, technical editing and language editing. Conceptualization, Francisco Ciruela Alferez, Kjell Fuxe and Dasiel O. Borroto-Escuela; formal analysis, Wilber Romero-Fernandez, Jaume J. Taura, René A. J Crans, Marc Lopez-Cano, Ramon Fores-Pons, Manuel Narváez, Jens Carlsson, Francisco Ciruela Alferez, Kjell Fuxe and Dasiel O. Borroto-Escuela; funding acquisition, Francisco Ciruela Alferez, Kjell Fuxe and Dasiel O. Borroto-Escuela; investigation, Wilber Romero-Fernandez, Jaume J. Taura, René A. J Crans, Marc Lopez-Cano, Ramon Fores-Pons, Manuel Narváez, Jens Carlsson, Francisco Ciruela Alferez, Kjell Fuxe and Dasiel O. Borroto-Escuela; methodology, Wilber Romero-Fernandez, Jaume J. Taura, René A. J Crans, Marc Lopez-Cano, Ramon Fores-Pons, Manuel Narváez, Jens Carlsson, Francisco Ciruela Alferez, Kjell Fuxe and Dasiel O. Borroto-Escuela; project administration, Francisco Ciruela Alferez, Kjell Fuxe and Dasiel O. Borroto-Escuela; supervision, Francisco Ciruela Alferez, Kjell Fuxe and Dasiel O. Borroto-Escuela; visualization, Wilber Romero-Fernandez, Jaume J. Taura, René A. J Crans, Marc Lopez-Cano, Ramon Fores-Pons, Manuel Narváez, Francisco Ciruela Alferez and Dasiel O. Borroto-Escuela; writing — original draft, Dasiel Oscar Borroto-Escuela and Kjell Fuxe; writing — review and editing, René A. J Crans, Jens Carlsson, Francisco Ciruela Alferez, Kjell Fuxe and Dasiel O. Borroto-Escuela. All authors read and approved the final manuscript.
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This study was performed in line with the principles of the Declaration of Helsinki. The animal protocol (no. 7085) was approved by the University of Barcelona Committee on Animal Use and Care. Animals were housed and tested in compliance with the guidelines provided by the Guide for the Care and Use of Laboratory Animals [33] and following the European Union directives (2010/63/EU), the ARRIVE guidelines [34].
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Not applicable. The current research work does not involve human subjects.
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Not applicable. This manuscript does not contain individual personal’s data in any form.
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Romero-Fernandez, W., Taura, J.J., Crans, R.A.J. et al. The mGlu5 Receptor Protomer-Mediated Dopamine D2 Receptor Trans-Inhibition Is Dependent on the Adenosine A2A Receptor Protomer: Implications for Parkinson’s Disease. Mol Neurobiol 59, 5955–5969 (2022). https://doi.org/10.1007/s12035-022-02946-9
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DOI: https://doi.org/10.1007/s12035-022-02946-9