Antonio Cortés

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.

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.

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.

The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding

mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding. The hydrophilic and stable cMDH is bound by cpn60 but released by MG‐ATP alone, while the hydrophobic and unstable mMDH requires both Mg‐ATP and cpn 10. Citrate equalises the stability of the native state of the two proteins but has no effect on the co‐chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor. The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg‐ATP. In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin‐assisted folding is 3–4‐times faster.

Adenosine A2A Receptors and A2A Receptor Heteromers as Key Players in Striatal Function

A very significant density of adenosine A2A receptors (A2ARs) is present in the striatum, where they are preferentially localized postsynaptically in striatopallidal medium spiny neurons (MSNs). In this localization A2ARs establish reciprocal antagonistic interactions with dopamine D2 receptors (D2Rs). In one type of interaction, A2AR and D2R are forming heteromers and, by means of an allosteric interaction, A2AR counteracts D2R-mediated inhibitory modulation of the effects of NMDA receptor stimulation in the striatopallidal neuron. This interaction is probably mostly responsible for the locomotor depressant and activating effects of A2AR agonist and antagonists, respectively. The second type of interaction involves A2AR and D2R that do not form heteromers and takes place at the level of adenylyl cyclase (AC). Due to a strong tonic effect of endogenous dopamine on striatal D2R, this interaction keeps A2AR from signaling through AC. However, under conditions of dopamine depletion or with blockade of D2R, A2AR-mediated AC activation is unleashed with an increased gene expression and activity of the striatopallidal neuron and with a consequent motor depression. This interaction is probably the main mechanism responsible for the locomotor depression induced by D2R antagonists. Finally, striatal A2ARs are also localized presynaptically, in cortico-striatal glutamatergic terminals that contact the striato-nigral MSN. These presynaptic A2ARs heteromerize with A1 receptors (A1Rs) and their activation facilitates glutamate release. These three different types of A2ARs can be pharmacologically dissected by their ability to bind ligands with different affinity and can therefore provide selective targets for drug development in different basal ganglia disorders.

Metabolic control analysis aimed at the ribose synthesis pathways of tumor cells: a new strategy for antitumor drug development.

Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with lower concentrations of drug, if an enzyme with a high control coefficient on tumor growth is being inhibited. Here we measure glucose-6-phosphate dehydrogenase (G6PDH) control coefficient on in vivo tumor growth using mice bearing Ehrlich ascites tumor cells. We used dehydroepiandrosterone-sulphate (DHEA-S), an uncompetitive inhibitor of this enzyme and the in situ cytochemical method to measure the enzyme activity changes that accompany changes on tumor cell growth. This method ensures that the enzyme activity determined is the one existing in the in situ conditions and enables computing a control coefficient in in situ conditions. From the data obtained on tumor cell number and the in situ enzyme activities in absence and presence of DHEA-S, a control coefficient of 0.41 for G6PDH on tumor cell growth was computed. This value is approximately the half of the transketolase control coefficient value of 0.9 previously reported. Moreover, the use of in situ methods to assess enzyme activities, applied for first time for the calculation of control coefficients in this study, opens new avenues to the use of uncompetitive inhibitors for the measurement of in situ control coefficients.

Novel pharmacological targets based on receptor heteromers

Studies performed in the last 10 years have provided solid evidence indicating that G-protein-coupled receptors are expressed on the plasma membrane as homo and heterodimers. The first consequence of this fact is that homo and heterodimers are the true targets of natural (hormones, neurotransmitters) and synthetic drugs. Furthermore a given receptor in a heteromer may display a different functional and/or pharmacological profile than the same receptor characterized as monomer or as homodimer. Recent evidence indicates that receptor heteromers are sensors that lead to a fine-tuning in neurotransmission or hormone regulation; mainly this is achieved by a modification of the signaling pathways activated via a given receptor when it is forming a given heteromer. Quite often antagonists display variable affinities when a given receptor is expressed with different heteromeric partners. This fact should be taken into account in the development of new drugs. Finally it should be pointed out that radioligand binding data has to be analyzed by a model that considers receptors as dimers and not as monomers. This model provides a novel approach to characterize drugs interacting with the orthosteric center (agonists/antagonists) or with allosteric centers (allosteric regulators).

Orexin–Corticotropin-Releasing Factor Receptor Heteromers in the Ventral Tegmental Area as Targets for Cocaine

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking.

Heptaspanning membrane receptors and cytoskeletal/scaffolding proteins: Focus on adenosine, dopamine, and metabotropic glutamate receptor function

Most cellular functions are mediated by multiprotein complexes. In neurons, these complexes are directly involved in the proper neuronal transmission, which is responsible for phenomena like learning, memory, and development. In recent years studies based on two-hybrid screens and proteomic, biochemical, and cell biology approaches have shown that intracellular domains of G protein-coupled receptors (GPCRs) or heptaspanning membrane receptors (HSMRs) interact with intracellular proteins. These interactions are the basis of a protein network associated with these receptors, which includes scaffolding proteins containing one or several PDZ (post-synaptic-density-95/discs-large/zona occludens-1) domains, signaling proteins, and proteins of the cytoskeleton. The present article is focused on the emerging evidence for interactions of adenosine, dopamine, and metabotropic glutamate receptors, with scaffolding and cytoskeletal proteins that play a role in the targeting and anchoring of these receptors to the plasma membrane, thus contributing to neuronal development and plasticity. Finally, given the complexity of neurological disorders such as ischemic stroke, Alzheimer’s disease, and epilepsy, exploitation of these HSMR-associated interactions might prove to be efficient in the treatment of such disorders.

Partners for adenosine A1 receptors.

G protein-coupled receptors (GPCRs) are targets for therapy in a variety of neurological diseases. Using adenosine A1 receptors (A1Rs) as paradigm of GPCRs, this review focuses on how protein-protein interactions, from monomers to heteromers, can contribute to hormone/neurotransmitter/neuromodulator regulation. The interaction of A1Rs with other membrane receptors, enzymes, and adaptor and scaffolding proteins is relevant for receptor traffic, internalization, and desensitization, and A1Rs are extremely important in driving signaling through different intracellular pathways. There is even the possibility of linking together GPCR heteromeric complexes with ion channel receptors in a receptor mosaic that might have special integrative value and might constitute the molecular basis for learning and memory.