Presynaptic control of striatal glutamatergic neurotransmission by adenosine A1-A2A receptor heteromers

Abstract

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R-A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R-A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R-A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Figure 1.

A₁R–A_2AR heteromerization in HEK cells. a, Coimmunoprecipitation of A₁Rs and A_2ARs. HEK-293 cells transiently expressing HA-A_2ARs alone (lane 1), HA-A_2ARs plus Flag-A₁Rs (lane 2), or Flag-A₁Rs (lane 3) were washed, solubilized, and processed for immunoprecipitation using anti-Flag monoclonal antibody (2 μg/ml; IP:Flag) or anti-HA monoclonal antibody (2 μg/ml; IP:HA). Solubilized membranes (Crude) and immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotted using rabbit anti-A_2AR polyclonal antibody (1:2000) or rabbit anti-A₁R polyclonal antibody (1:1000) and HRP-conjugated goat anti-rabbit IgG as a secondary antibody. These blots are representative of four different experiments with similar qualitative results. IB, Immunoblot. b, BRET saturation curve. BRET was measured in HEK-293 cells coexpressing A_2AR-Rluc and A₁R-YFP (▪) or A_2AR-Rluc and GABA_BR2-YFP (▴) constructs. Cotransfections were performed with increasing amounts of plasmid DNA for the A₁R-YFP construct, whereas the DNA for the A_2AR-Rluc construct was maintained constant. Both fluorescence and luminescence of each sample were measured before every experiment to confirm equal expression of A_2AR-Rluc while monitoring the increase of YFP expression. The saturation curve include results obtained from five independent experiments; mBU is the BRET ratio × 1000 (see Materials and Methods); error bars indicate SD of mean specific BRET ratio (mBU) values of five individual experiments grouped as a function of the amount of fluorescence of the acceptor. AU, Arbitrary unit. c, Cell surface colocalization of A₁Rs and A_2ARs in HEK cells. HEK cells were transfected with A_2AR-HA, A₁R-Flag, or both receptors simultaneously (coexpression). Nonpermeabilized cotransfected cells were immunostained with mouse anti-HA monoclonal antibody and rabbit anti-Flag polyclonal antibody. The bound primary antibodies were detected using either Alexa Fluor 488-conjugated goat anti-mouse IgG antibody or Texas Red-conjugated goat anti-rabbit. Cells were analyzed by double immunofluorescence with confocal microscopy. Superimposition of images reveals A₁R–A_2AR cell surface colocalization in yellow (merge). Scale bar, 10 μm.

Figure 2.

A_2AR-mediated modulation of A₁R function in HEK cells. a, b, Modulation of [³H]R-PIA binding to A₁R by A_2AR activation. HEK cells were transiently cotransfected with A₁R and A_2AR (a) or with A₁R alone (b). Membranes (0.19 mg/ml) of these cells were incubated with 0.8 nm A₁R agonist [³H]R-PIA in the absence or presence of increasing concentrations of the A_2AR agonist CGS21680 in 50 mm Tris-HCl containing 10 mm MgCl₂ and 2 U/ml adenosine deaminase. Binding experiments were performed as described in Materials and Methods. The data were adjusted to one single binding site (dashed line) or to two binding sites (solid line). c, Modulation by A_2ARs of A₁R-induced Ca²⁺ mobilization. Transiently transfected HEK-293 cells were loaded with fura-2 AM, and the intracellular Ca²⁺ mobilization was determined after stimulation with R-PIA (50 nm) or CGS21680 (50 nm). The presence of CGS21680 decreases R-PIA-induced Ca²⁺ mobilization in the doubly transfected cells. These recordings are representative of 10 different experiments with similar qualitative results.

Figure 3.

A₁R–A_2AR colocalization in rat striatum. a–c, Immunogold particles for the A_2ARs were observed at the presynaptic level along the extrasynaptic plasma membrane (arrows) of axon terminal (B), as well as in the presynaptic active zone (crossed arrows), facing dendritic shafts (D) and spines. A_2ARs were also observed at the postsynaptic level along the extrasynaptic and perisynaptic plasma membrane of dendritic shafts (D) and spines (arrowheads), establishing asymmetrical synapses with axon terminals. d, e, Similarly to A_2ARs, immunogold particles for the A₁R were localized along the presynaptic active zone of axon terminals (B; crossed arrows) and along the extrasynaptic plasma membrane of dendritic shafts (D) and spines (arrowheads) establishing asymmetrical synapses with axon terminals. f, g, Presynaptic colocalization of A₁Rs and A_2ARs. Peroxidase reaction product (immunoreactivity for A₁Rs) filled axon terminals (B) establishing asymmetrical synapses with dendritic shafts (D) or spines (S), in which immunoparticles (immunoreactivity for A_2ARs) were localized along the extrasynaptic plasma membrane (arrows). h, Postsynaptic colocalization of A₁Rs and A_2ARs. Peroxidase reaction product (immunoreactivity for A₁Rs) filled dendritic shafts (D) establishing asymmetrical synapses with axon terminals (B), in which immunoparticles (immunoreactivity for A_2ARs) were localized along the synaptic plasma membrane (arrowhead). Scale bar, 0.2 μm.

Figure 4.

Intramembrane A₁R–A_2AR interaction in rat striatum. Effect of chronic caffeine treatment. Modulation of [³H]R-PIA binding to A₁Rs by A_2AR activation. Striatal membranes (0.25 mg/ml) of control (a) or caffeine-treated rats (b) were incubated with 0.9 nm [³H]R-PIA in the absence or presence of increasing concentrations of CGS21680 in 50 mm Tris-HCl containing 10 mm MgCl2 and 2 U/ml adenosine deaminase. Binding experiments were performed as described in Materials and Methods. The data were adjusted to one single binding site (dashed line) or to two binding sites (solid line).

Figure 5.

Adenosine receptors in glutamatergic striatal nerve terminals. a, Immunocytochemical identification of A_2AR (green) and A₁R (blue) in the glutamatergic population of rat striatal nerve terminals (identified as vGluT-1 and vGluT-2 immunoreactive; red). In the superimposed picture of this triple-immunocytochemical labeling (merge) of this representative field, the arrows indicate the A₁R/A_2AR/vGluT-containing nerve terminals. b, The quantification of images of three different fields per coverslip from four experiments using different synaptosomal preparations from different animals confirmed the predominant colocalization of A₁Rs and A_2ARs in rat striatal glutamatergic terminals (results are means ± SEM). c, Coimmunoprecipitation of A₁R and A_2AR from rat striatal synaptosomes. Solubilized synaptosomes were immunoprecipitated using rabbit anti-A₁R polyclonal antibody (5 μg) (lane 1), irrelevant rabbit polyclonal antibody (5 μg) (lane 2), and mouse anti-A_2AR monoclonal antibody (2 μg) (lane 3). Solubilized membranes (Crude) and immunoprecipitates (lanes 1–3) were analyzed by SDS-PAGE and immunoblotted using rabbit rabbit anti-A₁R antibody (1:1000) and HRP-conjugated swine anti-rabbit IgG as a secondary antibody. IB, Immunoblot.

Figure 6.

Effect of A₁R and A_2AR activation on evoked release of glutamate from striatal nerve terminals. Superfused synaptosomes, previously loaded with [³H]glutamate, were chemically (20 mm K⁺ for 30 s) stimulated twice (S1 and S2). a, CPA (100 nm) and CGS21680 (10 nm), present in S2, inhibited and facilitated, respectively, the evoked release of glutamate in a manner prevented by the A₁R and A_2AR antagonists DPCPX (50 nm) and SCH58261 (50 nm), respectively (present in S1 and S2). The inhibitory effect of CPA (100 nm) was also abolished when CGS21680 (10 nm) was present during S1 and S2, whereas the facilitatory effect of CGS21680 was not modified when CPA (100 nm) was present in S1 and S2. Coapplication of CPA (100 nm) and CGS (10 nm) in S2 facilitated the evoked release of glutamate. The dashed bar illustrates the arithmetic sum of the effects of 10 nm CGS21680 and 100 nm CPA, if these effects were purely additive. The results are means ± SEM of three to seven experiments. *p < 0.05 versus 0%; **p < 0.05 between bars. b, Increasing concentrations of adenosine (0.1–100 μm) produced a biphasic effect, with with low and high concentrations inhibiting and stimulating the evoked release of glutamate, respectively. The results are means ± SEM of six to eight experiments. *p < 0.05 versus 0%.