Vicent Casadó

Presynaptic Control of Striatal Glutamatergic Neurotransmission by Adenosine A1–A2A Receptor Heteromers

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.

Synergistic interaction between adenosine A2A and glutamate mGlu5 receptors: Implications for striatal neuronal function

The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic glutamate receptors in γ- aminobutyric acid (GABA)ergic striatal neurons is intriguing. Here we provide in vitro and in vivo evidence for a synergism between adenosine and glutamate based on subtype 5 metabotropic glutamate (mGluR5) and adenosine A2A (A2AR) receptor/receptor interactions. Colocalization of A2AR and mGluR5 at the membrane level was demonstrated in nonpermeabilized human embryonic kidney (HEK)-293 cells transiently cotransfected with both receptors by confocal laser microscopy. Complexes containing A2AR and mGluR5 were demonstrated by Western blotting of immunoprecipitates of either Flag-A2AR or hemagglutinin-mGluR5 in membrane preparations from cotransfected HEK-293 cells and of native A2AR and mGluR5 in rat striatal membrane preparations. In cotransfected HEK-293 cells a synergistic effect on extracellular signal-regulated kinase 1/2 phosphorylation and c-fos expression was demonstrated upon A2AR/mGluR5 costimulation. No synergistic effect was observed at the second messenger level (cAMP accumulation and intracellular calcium mobilization). Accordingly, a synergistic effect on c-fos expression in striatal sections and on counteracting phencyclidine-induced motor activation was also demonstrated after the central coadministration of A2AR and mGluR5 agonists to rats with intact dopaminergic innervation. The results suggest that a functional mGluR5/A2AR interaction is required to overcome the well-known strong tonic inhibitory effect of dopamine on striatal adenosine A2AR function.

G Protein–Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives

Most evidence indicates that, as for family C G protein–coupled receptors (GPCRs), family A GPCRs form homo- and heteromers. Homodimers seem to be a predominant species, with potential dynamic formation of higher-order oligomers, particularly tetramers. Although monomeric GPCRs can activate G proteins, the pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may provide a main functional unit, and oligomeric entities can be viewed as multiples of dimers. It still needs to be resolved if GPCR heteromers are preferentially heterodimers or if they are mostly constituted by heteromers of homodimers. Allosteric mechanisms determine a multiplicity of possible unique pharmacological properties of GPCR homomers and heteromers. Some general mechanisms seem to apply, particularly at the level of ligand-binding properties. In the frame of the dimer-cooperativity model, the two-state dimer model provides the most practical method to analyze ligand–GPCR interactions when considering receptor homomers. In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to different intrinsic efficacy of ligands for different signaling pathways (functional selectivity). This gives a rationale for the use of GPCR oligomers, and particularly heteromers, as novel targets for drug development. Herein, we review the functional and pharmacological properties of GPCR oligomers and provide some guidelines for the application of discrete direct screening and high-throughput screening approaches to the discovery of receptor-heteromer selective compounds.

Cell surface adenosine deaminase: Much more than an ectoenzyme

During the last 10 years, adenosine deaminase (ADA), an enzyme considered to be cytosolic, has been found on the cell surface of many cells, therefore it can be considered an ectoenzyme. EctoADA, which seems to be identical to intracellular ADA and has a globular structure, does not interact with membranes but with membrane proteins. Two of these cell surface receptors for ectoADA have been identified: CD26 and A1 adenosine receptors (A1R). Apart from degradation of extracellular adenosine another functional role of ectoADA has been assigned. EctoADA is able to transmit signals when interacting with either CD26 or A1R. In this way, it acts as a co-stimulatory molecule which facilitates a variety of specific signalling events in different cell types. The heterogeneous distribution of the enzyme in the nervous system indicates that ectoADA may be a neuroregulatory molecule. On the other hand, ectoADA might act as a bridge between two different cells thus raising the possibility that it may be important for the development of the nervous system.

Striatal adenosine A2A and cannabinoid CB1 receptors form functional heteromeric complexes that mediate the motor effects of cannabinoids

The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB1 receptors, is still a matter of debate. In the present study, we report that CB1 and adenosine A2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB1 receptor signaling was found to be completely dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A2A and CB1 receptors.

Identification of Dopamine D1–D3 Receptor Heteromers: INDICATIONS FOR A ROLE OF SYNERGISTIC D1–D3 RECEPTOR INTERACTIONS IN THE STRIATUM*

The function of dopamine D3 receptors present in the striatum has remained elusive. In the present study evidence is provided for the existence of dopamine D1–D3 receptor heteromers and for an intramembrane D1–D3 receptor cross-talk in living cells and in the striatum. The formation of D1–D3 receptor heteromers was demonstrated by fluorescence resonance energy transfer and bioluminescence resonance energy transfer techniques in transfected mammalian cells. In membrane preparations from these cells, a synergistic D1–D3 intramembrane receptor-receptor interaction was observed, by which D3 receptor stimulation enhances D1 receptor agonist affinity, indicating that the D1–D3 intramembrane receptor-receptor interaction is a biochemical characteristic of the D1–D3 receptor heteromer. The same biochemical characteristic was also observed in membrane preparations from brain striatum, demonstrating the striatal co-localization and heteromerization of D1 and D3 receptors. According to the synergistic D1–D3 intramembrane receptor-receptor interaction, experiments in reserpinized mice showed that D3 receptor stimulation potentiates D1 receptor-mediated behavioral effects by a different mechanism than D2 receptor stimulation. The present study shows that a main functional significance of the D3 receptor is to obtain a stronger dopaminergic response in the striatal neurons that co-express the two receptors.

Evidence for Adenosine/Dopamine Receptor Interactions: Indications for Heteromerization

Evidence has been obtained for adenosine/dopamine interactions in the central nervous system. There exists an anatomical basis for the existence of functional interactions between adenosine A1R and dopamine D1R and between adenosine A2A and dopamine D2 receptors in the same neurons. Selective A1R agonists affect negatively the high affinity binding of D1 receptors. Activation of A2A receptors leads to a decrease in receptor affinity for dopamine agonists acting on D2 receptors, specially of the high-affinity state. These interactions have been reproduced in cell lines and found to be of functional significance. Adenosine/dopamine interactions at the behavioral level probably reflect those found at the level of dopamine receptor binding and transduction. All these findings suggest receptor subtype-specific interactions between adenosine and dopamine receptors that may be achieved by molecular interactions (e.g., receptor heterodimerization). At the molecular level adenosine receptors can serve as a model for homomeric and heteromeric protein–protein interactions. A1R forms homodimers in membranes and also form high-order molecular structures containing also heterotrimeric G-proteins and adenosine deaminase. The occurrence of clustering also clearly suggests that G-protein- coupled receptors form high-order molecular structures, in which multimers of the receptors and probably other interacting proteins form functional complexes. In view of the occurrence of homodimers of adenosine and of dopamine receptors it is speculated that heterodimers between these receptors belonging to two different families of G-protein-coupled receceptors can be formed. Evidence that A1/D1 can form heterodimers in cotransfected cells and in primary cultures of neurons has in fact been obtained. In the central nervous system direct and indirect receptor–receptor interactions via adaptor proteins participate in neurotransmission and neuromodulation and, for example, in the establishment of high neural functions such as learning and memory.

Interactions between histamine H3 and dopamine D2 receptors and the implications for striatal function

The striatum contains a high density of histamine H(3) receptors, but their role in striatal function is poorly understood. Previous studies have demonstrated antagonistic interactions between striatal H(3) and dopamine D(1) receptors at the biochemical level, while contradictory results have been reported about interactions between striatal H(3) and dopamine D(2) receptors. In this study, by using reserpinized mice, we demonstrate the existence of behaviorally significant antagonistic postsynaptic interactions between H(3) and D(1) and also between H(3) and dopamine D(2) receptors. The selective H(3) receptor agonist imetit inhibited, while the H(3) receptor antagonist thioperamide potentiated locomotor activation induced by either the D(1) receptor agonist SKF 38393 or the D(2) receptor agonist quinpirole. High scores of locomotor activity were obtained with H(3) receptor blockade plus D(1) and D(2) receptor co-activation, i.e., when thioperamide was co-administered with both SKF 38393 and quinpirole. Radioligand binding experiments in striatal membrane preparations showed the existence of a strong and selective H(3)-D(2) receptor interaction at the membrane level. In agonist/antagonist competition experiments, stimulation of H(3) receptors with several H(3) receptor agonists significantly decreased the affinity of D(2) receptors for the agonist. This kind of intramembrane receptor-receptor interactions are a common biochemical property of receptor heteromers. In fact, by using Bioluminescence Resonance Energy Transfer techniques in co-transfected HEK-293 cells, H(3) (but not H(4)) receptors were found to form heteromers with D(2) receptors. This study demonstrates an important role of postsynaptic H(3) receptors in the modulation of dopaminergic transmission by means of a negative modulation of D(2) receptor function.

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide‐induced NO release and NO synthase‐II expression in a time‐ and concentration‐dependent manner. CGS21680 potentiation of lipopolysaccharide‐induced NO release was suppressed by the A2A receptor antagonist ZM‐241385 and did not occur on mixed glial cultures from A2A receptor‐deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase‐II inhibitor 1400W abolished NO production, and NO synthase‐II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase‐II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.

A1 Adenosine Receptors Accumulate in Neurodegenerative Structures in Alzheimer's Disease and Mediate Both Amyloid Precursor Protein Processing and Tau Phosphorylation and Translocation

Immunostaining of adenosine receptors in the hippocampus and cerebral cortex from necropsies of Alzheimers disease (AD) patients shows that there is a change in the pattern of expression and a redistribution of receptors in these brain areas when compared with samples from controls. Adenosine A1 receptor (A1R) immunoreactivity was found in degenerating neurons with neurofibrillary tangles and in dystrophic neurites of senile plaques. A high degree of colocalization for A1R and pA4 amyloid in senile plaques and for A1R and tau in neurons with tau deposition, but without tangles, was seen. Additionally, adenosine A2A receptors, located mainly in striatal neurons in controls, appeared in glial cells in the hippocampus and cerebral cortex of patients. On comparing similar samples from controls and patients, no significant change was evident for metabotropic glutamate receptors. In the human neuroblastoma SH‐SY5Y cell line, agonists for A1R led to a dose‐dependent increase in the production of soluble forms of amyloid precursor protein in a process mediated by PKC. A1R agonist induced p21 Ras activation and ERK1/2 phosphorylation. Furthermore, activation of A1R led to and ERK‐dependent increase of tau phosphorylation and translocation towards the cytoskeleton. These results indicate that adenosine receptors are potential targets for AD.