Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes

Abstract

The receptor-receptor interaction (RRI) of G protein-coupled receptors (GPCRs) leads to new functional entities that are conceptually distinct from the simple addition of signals mediated by the activation of the receptors that form the heteromers. Focusing on astrocytes, there is evidence for the existence of inhibitory and facilitatory RRIs, including the heteromers formed by the adenosine A2A and the dopamine D2 receptors, by A2A and the oxytocin receptor (OTR), and the D2-OTR heteromers. The possible involvement of these receptors in mosaicism has never been investigated in striatal astrocytes. By biophysical and functional approaches, we focused our attention on the existence of an A2A-D2-OTR high-order receptor complex and its role in modulating cytosolic calcium levels and endogenous glutamate release, when striatal astrocyte processes were stimulated with 4-aminopyridine. Functional data indicate a permissive role of OTR on dopamine signaling in the regulation of the glutamatergic transmission, and an inhibitory control mediated by A2A on both the D2-mediated signaling and on the OTR-facilitating effect on D2. Imaging biochemical and bioinformatic evidence confirmed the existence of the A2A-D2-OTR complex and its ternary structure in the membrane. In conclusion, the D2 receptor appears to be a hotspot in the control of the glutamate release from the astrocytic processes and may contribute to the regulation and integration of different neurotransmitter-mediated signaling in the striatum by the A2A-D2-OTR heterotrimers. Considering the possible selectivity of allosteric interventions on GPCRs organized as receptor mosaics, A2A-D2-OTR heterotrimers may offer selective pharmacological targets in neuropsychiatric disorders and neurodegenerative diseases.

Keywords: adenosine receptor; astrocyte process; dopamine receptor; glutamate; heteromers; high-order receptor complex; intracellular calcium; neuroglia; oxytocin; receptor mosaic.

Figure 1

Striatal astrocytes express the A2A, D2, and OT receptors. Representative confocal images showing the presence of the A2A, D2, and OTR in rat striatum and their co-localization with GFAP. Immunofluorescence analysis was conducted in rat hemibrain slices (see Section 4 for details). Maximum intensity projections and their merges for representative fields (with a dimension of 60 × 60 µm; at least 11 z-stacks) are shown; GFAP (green, (A,C,E,G,H,J)), A2A (red, (B,C)), D2 (red, (F,G)), and OTR (red, (I,J)). Astrocytes were positively stained with mouse anti-GFAP (red (K,M)) and goat anti-GFAP (blue (L,M)) primary antibodies. The scale bars are indicated in the merged images. (D) Cresyl violet staining of a close-up section used in Figure 2. GFAP, glial fibrillary acidic protein; A2A, adenosine receptor; D2, dopamine receptor; OTR, oxytocin receptor.

Figure 2

Astrocytes express the A2A receptors and D2-OT heteromers. Representative confocal images showing the presence of the D2-OTR heteromers in rat striatum and their co-localization with A2A receptors. The immunofluorescence analysis was conducted by combing the in situ PLA with anti-D2 receptor and anti-OTR primary antibodies, and the classical immunofluorescence technique using anti-A2A receptor, anti-GFAP, and anti-ezrin primary antibodies in rat hemibrain slices (for further details, see Materials and Methods). (A–E) Maximum intensity projections for single channels and the merged images of a representative field (29.13 × 29.13 µm; 11 z stacks) are shown; GFAP + ezrin (blue, (A,E)), PLA D2-OTR (green, (B,D,E)), and A2A (red, (C–E)). (F–J) Enlarged images for each channel and the merged confocal images of a single z-stack is shown; this is the same field of the maximum intensity projections shown in (A–E); GFAP + ezrin (blue, (F,J)), PLA D2-OTR (green, (G,I,J)), and A2A (red, (H–J)). The yellow line in (J) was used to create the profile plot shown in (K). The colours of the different lines (blue for the GFAP + ezrin signal, green for the PLAD2-OTR signal, red for the A2A signal) correspond to those used in image (J). (L) A representative merge control image obtained without the primary antibody for OTR or D2 during the PLA protocol. GFAP, glial fibrillary acidic protein; A2A, adenosine receptor; D2, dopamine receptor; OTR, oxytocin receptor; PLA, proximity ligation assay.

Figure 3

Astrocytic processes express A2A, D2, and OTRs. Representative confocal images showing the presence of the studied receptors in rat striatal astrocytic processes and their co-localization with GFAP. The immunofluorescence analysis was conducted using anti-D2 receptor, anti-OTR, anti-A2A receptor, and anti-GFAP primary antibodies in gliosomes (for further details, see Materials and Methods). (A–E) A representative field is shown; A2A (green, (A,E)), D2 (red, (B,E)), OTR (blue, (C,E)), and GFAP (yellow, (D,E)). GFAP, glial fibrillary acidic protein; A2A, adenosine A2A receptor; D2, dopamine D2 receptor; OTR, oxytocin receptor.

Figure 4

Co-immunoprecipitation of the OTR, A2A, and D2 receptors in striatal astrocytic processes. The aliquots (300 µg) of Triton X-100-soluble proteins prepared from striatal fresh isolated gliosomes were immunoprecipitated with 1 µg of anti-OTR (A), or anti-A2A (B), or anti-D2 (C) antibody. The immunoprecipitated (IP) and not immunoprecipitated (O, output) materials were analyzed by Western blot (WB) for the indicated antigens. A2A, OTR, D2, and flotillin-1 (FLOT) immunoreactive bands were quantified and the data are reported in the graphs as a percentage of the total amount of the relevant protein (% of total). The values are means ± SEM (n = 5 for (A,C); n = 4 for (B)). For each protein, a representative blot is shown. The arrows indicate the expected weights of the antigens. A2A, adenosine A2A receptor; D2, dopamine D2 receptor; OTR, oxytocin receptor; FLOT, flotillin.

Figure 5

CG-loaded gliosomes were exposed to the indicated stimuli for 300 s at 37 °C. (A–C), CG-dependent fluorescence was monitored every 10 s from 0 to 300 s. [Ca²⁺]_i increase is expressed as “Delta Fluorescence”. The lines represent the mean values from 7 to 3 experiments (A) and from 5 independent experiments (C). (B–D), the Ca²⁺ influx after 300 s was estimated by calculating the areas underlying the curves (AUC) and is reported in the graphs for each experimental condition. Data are means ± SEM. ns, not statistically significant according to the Mann–Whitney test. * p < 0.01 and § p < 0.001 according to ANOVA, followed by Tukey’s post hoc test. 4-AP, 4-aminopyridine; CG, Calcium Green™-1 AM; OT, oxytocin.

Figure 6

Endogenous glutamate efflux in response to 4-AP-induced depolarization in striatal gliosomes. Modulation by the D2-OTR heteromers and A2A receptors. The inhibitory effect of OT 3 nM and quinpirole 0.1 µM on the 4-AP-evoked endogenous glutamate release; abolishment by CGS 21,680 0.01 µM and the antagonism of the A2A receptors by SCH-58261. The bars represent the overflow of the glutamate release, expressed as pmol/mg of protein, in the presence of drugs at the concentrations used. Briefly, 4-AP was added (6 min) during superfusion; OT, quinpirole, and CGS 21,680 were added together with 4-AP, while SCH-58261 was added 8 min before the agonists. Further experimental details can be found in Materials and Methods. Data are the mean ± SEM of n = 5–10 independent experiments. * p < 0.0001 compared with the effect of 4-AP; # p < 0.0001 compared with the effect of 4-AP + OT + quinpirole; $ p < 0.0001 compared with the effect of 4P + OT + quinpirole + CGS 21,680 by one-way ANOVA plus Tukey’s post hoc test. 4-AP, 4-aminopyridine; OT, oxytocin.

Figure 7

Predicted models of the A2A-D2-OTR heterotrimer. The predicted structure of the receptor complex in an “open” topological arrangement is shown in (A). It was derived by first docking A2A and D2 receptors and then associating the OTR. The left panel shows the complex in the membrane environment used for refinement by energy minimization and the resulting heterotrimer is shown in the middle panel. A solution characterized by a “closed” topological arrangement is shown in (B). It was obtained by docking the A2A receptor to the previously estimated D2-OTR heterodimer. For both cases, the obtained arrangement of the transmembrane domains (as different coloured numbers for each receptor in the illustration) is illustrated in the right panels together with the estimated change in free energy associated with complex formation. For each topology, the structure shown is the one exhibiting the best energy score according to the applied docking method.