Francisco Ciruela - Academia.edu (original) (raw)

Papers by Francisco Ciruela

Cardiovascular Research, Nov 1, 2006

Background: Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor ... more Background: Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor (RyR2) have been proposed to account for increased spontaneous calcium release from the sarcoplasmic reticulum (SR) in patients with heart failure, ventricular tachyarrhythmias and atrial fibrillation. While the adenosine A 2A receptor (A 2A R) is known to regulate cyclic AMP levels, expression and function of this receptor in human cardiac myocytes has not been investigated. Methods: PCR, western blotting and immunofluorescence were used to identify the A 2A R, and functional effects of A 2A R stimulation were measured with confocal calcium imaging and patch-clamp technique. Results: The A 2A R is expressed in the human right atrium and distributed in a banded pattern along the Z-lines, overlapping with the ryanodine receptor. A 2A R stimulation caused a protein kinase A dependent increase in spontaneous SR calcium release in isolated human atrial myocytes. The A 2A R agonist CGS21680 increased the frequency of calcium sparks from 0.12 ± 0.03 to 0.31 ± 0.08 sparks•μm min − 1 (p b 0.05) and calcium waves from 0.65 ± 0.31 to 5.11 ± 1.84 waves•min − 1 (p b 0.03). Moreover, spontaneous Na-Ca exchange currents (I NCX) increased from 1.19 ± 0.17 to 2.50 ± 0.42 min − 1 (p b 0.001). In contrast, CGS21680 did not alter caffeine inducible calcium release (6.98 ± 0.52 vs. 6.82 ± 0.57 amol pF − 1 , p = 0.6) or the spontaneous I NCX amplitude (0.32 ± 0.05 vs. 0.29 ± 0.04 pA pF − 1 , p = 0.2). Currentvoltage relationship and amplitude of the L-type calcium current (1.62 ± 0.18 vs. 1.80 ± 0.18 pA pF − 1) were not altered, but calcium release dependent inactivation was faster with CGS21680 (13.4 ± 0.7 vs. 15.8 ± 1.0 ms, p b 0.001). Conclusions: Adenosine A 2A receptors are expressed in the human atrial myocardium and modulate the frequency of spontaneous calcium release from the SR.

BMC Biology, 2020

Background It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabi... more Background It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. Results Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure...

Scientific reports, May 12, 2017

Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment ... more Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D2 receptors (D2Rs). Striatal D2R functioning may be finely regulated by either adenosine A2A receptor (A2AR) or angiotensin receptor type 1 (AT1R) through putative receptor heteromers. Here, we examined whether A2AR and AT1R may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical AT1R-A2AR interaction in cultured cells. Interestingly, by protein-protein docking and molecular dynamics simulations, we described that a stable heterotetrameric interaction may exist between AT1R and A2AR bound to antagonists (i.e. losartan and istradefylline, respectively). Accordingly, we subsequently ascertained the existence of AT1R/A2AR heteromers in the striatum by proximity ligation in situ assay. Finally, we took advantage of a TD animal...

Molecular systems biology, Mar 15, 2017

G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with ke... more G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable re...

Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, Jan 19, 2017

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cogn... more The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically-modified animal models, together with biochemical and pharmacological approaches, we ...

The Journal of Neuroscience, 2014

Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitab... more Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca2+wave-dependent activation of small-conductance Ca2+-activated K+(SK) channels. Here, we show that mGlu5receptors and SK2 channels coassemble in heterologous coexpression systems and in rat brain. Further, in cotransfected cells or rat primary hippocampal neurons, mGlu5receptor stimulation activated apamin-sensitive SK2-mediated K+currents. In addition, coexpression of mGlu5receptors and SK2 channels promoted plasma membrane targeting of both proteins and correlated with increased mGlu5receptor function that was unexpectedly blocked by apamin. These results demonstrate a reciprocal functional interaction between mGlu5receptors and SK2 channels that reflects their molecular coassembly.

Disease Models & Mechanisms, 2014

Parkinson’s disease (PD) is a dopaminergic-related pathology in which basal ganglia functioning a... more Parkinson’s disease (PD) is a dopaminergic-related pathology in which basal ganglia functioning are altered. It has been postulated that a direct receptor-receptor – i.e. dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AR) – interaction may be finely regulating this brain area. Accordingly, elucidating whether the pathology prompts changes on these structures could grant valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning D2R-A2AR assembly in native tissue. Thus, by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R/A2AR oligomers in rat striatum. Subsequently, we determined that under pathological conditions (i.e. in a rat PD model) D2R-A2AR interaction was impaired. Collectively, these results provide definitive evidence for a native D2R/A2AR oligomer alteration in experimental parkinsonism, t...

öThe role of adenosine deaminase in the interactions between adenosine A 1 and dopamine D 1 recep... more öThe role of adenosine deaminase in the interactions between adenosine A 1 and dopamine D 1 receptors was studied in a mouse ¢broblast cell line stably cotransfected with human D 1 receptor and A 1 receptor cDNAs (A 1 D 1 cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A 1 D 1 cells but not of the D 1 cells (only cotransfected with human D 1 receptor cDNAs). In double immunolabelling experiments in A 1 D 1 cells and cortical neurons a marked overlap in the distribution of the A 1 receptor and adenosine deaminase immunoreactivities and of the D 1 receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A 1 D 1 cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A 1 and D 1 receptor immunoreactivity, respectively. The A 1 receptor agonist caused in A 1 D 1 cells and in cortical neurons coaggregation of A 1 receptors and adenosine deaminase, and of D 1 receptors and adenosine deaminase. The A 1 receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D 1 receptor antagonist [ 3 H]SCH-23390 showed that the D 1 receptors had a better ¢t for two binding sites for dopamine, and treatment with the A 1 receptor agonist produced a disappearance of the high-a⁄nity site for dopamine at the D 1 receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A 1 receptors, and which is crucial for the high-a⁄nity state of the A 1 receptor, also blocked the A 1 receptor agonist-induced loss of high-a⁄nity D 1 receptor binding. The conclusion of the present studies is that the high-a⁄nity state of the A 1 receptor is essential for the A 1 receptormediated antagonistic modulation of D 1 receptors and for the A 1 receptor-induced coaggregates of A 1 and adenosine deaminase, and of D 1 and adenosine deaminase. Thus, the confocal experiments indicate that both A 1 and D 1 receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A 1 receptors is important for the A 1^D1 receptor^receptor interaction at the level of the D 1 receptor recognition.

Current Medicinal Chemistry-Central Nervous System Agents, 2003

TheScientificWorldJournal, Jan 11, 2008

Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) r... more Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) receptors, between CB1R and adenosine A2A (A2AR) receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells.

Proceedings of the National Academy of Sciences, 2002

The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic... more 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 expressio...

Neuropsychopharmacology, 2006

In the striatum, dopamine and acetylcholine (ACh) modulate dopamine release by acting, respective... more In the striatum, dopamine and acetylcholine (ACh) modulate dopamine release by acting, respectively, on dopamine D 2 autoreceptors and nicotinic ACh (nACh) heteroreceptors localized on dopaminergic nerve terminals. The possibility that functional interactions exist between striatal D 2 autoreceptors and nACh receptors was studied with in vivo microdialysis in freely moving rats. Local perfusion of nicotine in the ventral striatum (shell of the nucleus accumbens) produced a marked increase in the extracellular levels of dopamine, which was completely counteracted by co-perfusion with either the non-a 7 nACh receptor antagonist dihydro-b-erythroidine or the D 2À3 receptor agonist quinpirole. Local perfusion of the D 2À3 receptor antagonist raclopride produced an increase in the extracellular levels of dopamine, which was partially, but significantly, counteracted by coperfusion with dihydro-b-erythroidine. These findings demonstrate a potent crosstalk between G protein-coupled receptors and ligand-gated ion channels in dopaminergic nerve terminals, with the D 2 autoreceptor modulating the efficacy of non-a 7 nACh receptor-mediated modulation of dopamine release. We further demonstrate physical interactions between b 2 subunits of non-a 7 nicotinic acetylcholine receptors and D 2 autoreceptors in coimmunoprecipitation experiments with membrane preparations from co-transfected mammalian cells and rat striatum. These results reveal that striatal non-a 7 nicotinic acetylcholine receptors form part of heteromeric dopamine autoreceptor complexes that modulate dopamine release.

Neuropsychopharmacology, 2007

The mechanism of action responsible for the motor depressant effects of cannabinoids, which opera... more The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB 1 receptors, is still a matter of debate. In the present study, we report that CB 1 and adenosine A 2A 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, CB 1 receptor signaling was found to be completely dependent on A 2A receptor activation. Accordingly, blockade of A 2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB 1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A 2A and CB 1 receptors.

Neurochemistry International, 2013

The molecular interaction between adenosine A 2A and dopamine D 2 receptors (A 2A Rs and D 2 Rs, ... more The molecular interaction between adenosine A 2A and dopamine D 2 receptors (A 2A Rs and D 2 Rs, respectively) within an oligomeric complex has been postulated to play a pivotal role in the adenosine-dopamine interplay in the central nervous system, in both normal and pathological conditions (e.g. Parkinson's disease). While the effects of A 2A R challenge on D 2 R functioning have been largely studied, the reverse condition is still unexplored, a fact that might have impact in therapeutics. Here, we aimed to examine in a real-time mode the D 2 R-mediated allosteric modulation of A 2A R binding when an A 2A R/D 2 R oligomer is established. Thus, we synthesized fluorescent A 2A R agonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A 2A R binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential D 2 R-mediated negative allosteric modulation on A 2A R agonist binding that was oligomerformation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A 2A R agonist binding. Overall, the here-developed methods were found valid to prospect the ability of drugs acting on D 2 Rs to modulate A 2A R binding, thus featuring as possible helpful tools for the preliminary selection of D 2 R-like candidate drugs in the management of Parkinson's disease.

Journal of Neurochemistry, 2009

G protein‐coupled receptors are known to form homo‐ and heteromers at the plasma membrane, but th... more G protein‐coupled receptors are known to form homo‐ and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu5 receptors (mGlu5R) and dopamine D2 receptors (D2R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu5R, D2R and adenosine A2A receptor (A2AR). Interestingly, by using high‐resolution immunoelectron microscopy we could confirm that the three receptors co‐distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co‐immunoprecipitatio...

Journal of Neurochemistry, 2013

Journal of Neurochemistry, 2012

In the CNS, an antagonistic interaction has been shown between adenosine A2A and dopamine D2 rece... more In the CNS, an antagonistic interaction has been shown between adenosine A2A and dopamine D2 receptors (A2ARs and D2Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor–receptor interaction have recently been explored, as the fine tuning of this target (namely the A2AR/D2R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer‐based approach to examine the allosteric modulation of the D2R within the A2AR/D2R oligomer and the dependence of this receptor–receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D2R. Interestingly, we observed a negative allosteric effect of the D2R agonist quinpirole on A2AR ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217–222 and 267–269) on intracellular loop 3 of...

Journal of Biological Chemistry, 2003

The abbreviations used are: HSMR, heptaspanning membrane receptor; GPCR, G protein-coupled recept... more The abbreviations used are: HSMR, heptaspanning membrane receptor; GPCR, G protein-coupled receptor; YFP, yellow fluorescent protein; BRET, bioluminescence resonance energy transfer; FRET, fluorescence resonance energy transfer; E n , extracellular loop n; I n , intracellular loop n; Mes, 4-morpholineethanesulfonic acid; ANOVA, analysis of variance; Rluc, Renilla luciferase; EYFP enhanced yellow fluorescent protein; CHO, Chinese hamster ovary; GFP, green fluorescent protein; PDB, Protein Data Bank; GABA, ␥-aminobutyric acid; PBS, phosphate-buffered saline; CH, channel; A 2A R, adenosine A 2A receptor; D n R, dopamine D n receptor.

Journal of Biological Chemistry, 2001

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be presen... more Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo-and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G proteincoupled receptors, namely the metabotropic glutamate type 1␣ (mGlu 1␣ receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu 1␣ and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu 1␣ and A1 receptors in receptorevoked [Ca 2؉ ] i signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu 1␣ and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.

Journal of Biological Chemistry, 2003

Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be ... more Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast twohybrid screen, we identified ␣-actinin, a major F-actincross-linking protein, as a binding partner for the Cterminal domain of the adenosine A 2A receptor (A 2A R). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A 2A R and ␣-actinin in transfected HEK-293 cells and also in rat striatal tissue. A 2A R activation by agonist induced the internalization of the receptor by a process that involved rapid ␤-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A 2A ⌬CTR, a mutant version of A 2A R that lacks the Cterminal domain and does not interact with ␣-actinin, was not able to internalize when activated by agonist. Interestingly, A 2A ⌬CTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an ␣-actinin-dependent association between the actin cytoskeleton and A 2A R trafficking.

Cardiovascular Research, Nov 1, 2006

Background: Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor ... more Background: Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor (RyR2) have been proposed to account for increased spontaneous calcium release from the sarcoplasmic reticulum (SR) in patients with heart failure, ventricular tachyarrhythmias and atrial fibrillation. While the adenosine A 2A receptor (A 2A R) is known to regulate cyclic AMP levels, expression and function of this receptor in human cardiac myocytes has not been investigated. Methods: PCR, western blotting and immunofluorescence were used to identify the A 2A R, and functional effects of A 2A R stimulation were measured with confocal calcium imaging and patch-clamp technique. Results: The A 2A R is expressed in the human right atrium and distributed in a banded pattern along the Z-lines, overlapping with the ryanodine receptor. A 2A R stimulation caused a protein kinase A dependent increase in spontaneous SR calcium release in isolated human atrial myocytes. The A 2A R agonist CGS21680 increased the frequency of calcium sparks from 0.12 ± 0.03 to 0.31 ± 0.08 sparks•μm min − 1 (p b 0.05) and calcium waves from 0.65 ± 0.31 to 5.11 ± 1.84 waves•min − 1 (p b 0.03). Moreover, spontaneous Na-Ca exchange currents (I NCX) increased from 1.19 ± 0.17 to 2.50 ± 0.42 min − 1 (p b 0.001). In contrast, CGS21680 did not alter caffeine inducible calcium release (6.98 ± 0.52 vs. 6.82 ± 0.57 amol pF − 1 , p = 0.6) or the spontaneous I NCX amplitude (0.32 ± 0.05 vs. 0.29 ± 0.04 pA pF − 1 , p = 0.2). Currentvoltage relationship and amplitude of the L-type calcium current (1.62 ± 0.18 vs. 1.80 ± 0.18 pA pF − 1) were not altered, but calcium release dependent inactivation was faster with CGS21680 (13.4 ± 0.7 vs. 15.8 ± 1.0 ms, p b 0.001). Conclusions: Adenosine A 2A receptors are expressed in the human atrial myocardium and modulate the frequency of spontaneous calcium release from the SR.

BMC Biology, 2020

Background It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabi... more Background It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. Results Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure...

Scientific reports, May 12, 2017

Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment ... more Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D2 receptors (D2Rs). Striatal D2R functioning may be finely regulated by either adenosine A2A receptor (A2AR) or angiotensin receptor type 1 (AT1R) through putative receptor heteromers. Here, we examined whether A2AR and AT1R may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical AT1R-A2AR interaction in cultured cells. Interestingly, by protein-protein docking and molecular dynamics simulations, we described that a stable heterotetrameric interaction may exist between AT1R and A2AR bound to antagonists (i.e. losartan and istradefylline, respectively). Accordingly, we subsequently ascertained the existence of AT1R/A2AR heteromers in the striatum by proximity ligation in situ assay. Finally, we took advantage of a TD animal...

Molecular systems biology, Mar 15, 2017

G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with ke... more G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable re...

Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, Jan 19, 2017

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cogn... more The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically-modified animal models, together with biochemical and pharmacological approaches, we ...

The Journal of Neuroscience, 2014

Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitab... more Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca2+wave-dependent activation of small-conductance Ca2+-activated K+(SK) channels. Here, we show that mGlu5receptors and SK2 channels coassemble in heterologous coexpression systems and in rat brain. Further, in cotransfected cells or rat primary hippocampal neurons, mGlu5receptor stimulation activated apamin-sensitive SK2-mediated K+currents. In addition, coexpression of mGlu5receptors and SK2 channels promoted plasma membrane targeting of both proteins and correlated with increased mGlu5receptor function that was unexpectedly blocked by apamin. These results demonstrate a reciprocal functional interaction between mGlu5receptors and SK2 channels that reflects their molecular coassembly.

Disease Models & Mechanisms, 2014

Parkinson’s disease (PD) is a dopaminergic-related pathology in which basal ganglia functioning a... more Parkinson’s disease (PD) is a dopaminergic-related pathology in which basal ganglia functioning are altered. It has been postulated that a direct receptor-receptor – i.e. dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AR) – interaction may be finely regulating this brain area. Accordingly, elucidating whether the pathology prompts changes on these structures could grant valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning D2R-A2AR assembly in native tissue. Thus, by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R/A2AR oligomers in rat striatum. Subsequently, we determined that under pathological conditions (i.e. in a rat PD model) D2R-A2AR interaction was impaired. Collectively, these results provide definitive evidence for a native D2R/A2AR oligomer alteration in experimental parkinsonism, t...

öThe role of adenosine deaminase in the interactions between adenosine A 1 and dopamine D 1 recep... more öThe role of adenosine deaminase in the interactions between adenosine A 1 and dopamine D 1 receptors was studied in a mouse ¢broblast cell line stably cotransfected with human D 1 receptor and A 1 receptor cDNAs (A 1 D 1 cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A 1 D 1 cells but not of the D 1 cells (only cotransfected with human D 1 receptor cDNAs). In double immunolabelling experiments in A 1 D 1 cells and cortical neurons a marked overlap in the distribution of the A 1 receptor and adenosine deaminase immunoreactivities and of the D 1 receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A 1 D 1 cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A 1 and D 1 receptor immunoreactivity, respectively. The A 1 receptor agonist caused in A 1 D 1 cells and in cortical neurons coaggregation of A 1 receptors and adenosine deaminase, and of D 1 receptors and adenosine deaminase. The A 1 receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D 1 receptor antagonist [ 3 H]SCH-23390 showed that the D 1 receptors had a better ¢t for two binding sites for dopamine, and treatment with the A 1 receptor agonist produced a disappearance of the high-a⁄nity site for dopamine at the D 1 receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A 1 receptors, and which is crucial for the high-a⁄nity state of the A 1 receptor, also blocked the A 1 receptor agonist-induced loss of high-a⁄nity D 1 receptor binding. The conclusion of the present studies is that the high-a⁄nity state of the A 1 receptor is essential for the A 1 receptormediated antagonistic modulation of D 1 receptors and for the A 1 receptor-induced coaggregates of A 1 and adenosine deaminase, and of D 1 and adenosine deaminase. Thus, the confocal experiments indicate that both A 1 and D 1 receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A 1 receptors is important for the A 1^D1 receptor^receptor interaction at the level of the D 1 receptor recognition.

Current Medicinal Chemistry-Central Nervous System Agents, 2003

TheScientificWorldJournal, Jan 11, 2008

Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) r... more Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) receptors, between CB1R and adenosine A2A (A2AR) receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells.

Proceedings of the National Academy of Sciences, 2002

The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic... more 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 expressio...

Neuropsychopharmacology, 2006

In the striatum, dopamine and acetylcholine (ACh) modulate dopamine release by acting, respective... more In the striatum, dopamine and acetylcholine (ACh) modulate dopamine release by acting, respectively, on dopamine D 2 autoreceptors and nicotinic ACh (nACh) heteroreceptors localized on dopaminergic nerve terminals. The possibility that functional interactions exist between striatal D 2 autoreceptors and nACh receptors was studied with in vivo microdialysis in freely moving rats. Local perfusion of nicotine in the ventral striatum (shell of the nucleus accumbens) produced a marked increase in the extracellular levels of dopamine, which was completely counteracted by co-perfusion with either the non-a 7 nACh receptor antagonist dihydro-b-erythroidine or the D 2À3 receptor agonist quinpirole. Local perfusion of the D 2À3 receptor antagonist raclopride produced an increase in the extracellular levels of dopamine, which was partially, but significantly, counteracted by coperfusion with dihydro-b-erythroidine. These findings demonstrate a potent crosstalk between G protein-coupled receptors and ligand-gated ion channels in dopaminergic nerve terminals, with the D 2 autoreceptor modulating the efficacy of non-a 7 nACh receptor-mediated modulation of dopamine release. We further demonstrate physical interactions between b 2 subunits of non-a 7 nicotinic acetylcholine receptors and D 2 autoreceptors in coimmunoprecipitation experiments with membrane preparations from co-transfected mammalian cells and rat striatum. These results reveal that striatal non-a 7 nicotinic acetylcholine receptors form part of heteromeric dopamine autoreceptor complexes that modulate dopamine release.

Neuropsychopharmacology, 2007

The mechanism of action responsible for the motor depressant effects of cannabinoids, which opera... more The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB 1 receptors, is still a matter of debate. In the present study, we report that CB 1 and adenosine A 2A 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, CB 1 receptor signaling was found to be completely dependent on A 2A receptor activation. Accordingly, blockade of A 2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB 1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A 2A and CB 1 receptors.

Neurochemistry International, 2013

The molecular interaction between adenosine A 2A and dopamine D 2 receptors (A 2A Rs and D 2 Rs, ... more The molecular interaction between adenosine A 2A and dopamine D 2 receptors (A 2A Rs and D 2 Rs, respectively) within an oligomeric complex has been postulated to play a pivotal role in the adenosine-dopamine interplay in the central nervous system, in both normal and pathological conditions (e.g. Parkinson's disease). While the effects of A 2A R challenge on D 2 R functioning have been largely studied, the reverse condition is still unexplored, a fact that might have impact in therapeutics. Here, we aimed to examine in a real-time mode the D 2 R-mediated allosteric modulation of A 2A R binding when an A 2A R/D 2 R oligomer is established. Thus, we synthesized fluorescent A 2A R agonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A 2A R binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential D 2 R-mediated negative allosteric modulation on A 2A R agonist binding that was oligomerformation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A 2A R agonist binding. Overall, the here-developed methods were found valid to prospect the ability of drugs acting on D 2 Rs to modulate A 2A R binding, thus featuring as possible helpful tools for the preliminary selection of D 2 R-like candidate drugs in the management of Parkinson's disease.

Journal of Neurochemistry, 2009

G protein‐coupled receptors are known to form homo‐ and heteromers at the plasma membrane, but th... more G protein‐coupled receptors are known to form homo‐ and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu5 receptors (mGlu5R) and dopamine D2 receptors (D2R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu5R, D2R and adenosine A2A receptor (A2AR). Interestingly, by using high‐resolution immunoelectron microscopy we could confirm that the three receptors co‐distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co‐immunoprecipitatio...

Journal of Neurochemistry, 2013

Journal of Neurochemistry, 2012

In the CNS, an antagonistic interaction has been shown between adenosine A2A and dopamine D2 rece... more In the CNS, an antagonistic interaction has been shown between adenosine A2A and dopamine D2 receptors (A2ARs and D2Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor–receptor interaction have recently been explored, as the fine tuning of this target (namely the A2AR/D2R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer‐based approach to examine the allosteric modulation of the D2R within the A2AR/D2R oligomer and the dependence of this receptor–receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D2R. Interestingly, we observed a negative allosteric effect of the D2R agonist quinpirole on A2AR ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217–222 and 267–269) on intracellular loop 3 of...

Journal of Biological Chemistry, 2003

The abbreviations used are: HSMR, heptaspanning membrane receptor; GPCR, G protein-coupled recept... more The abbreviations used are: HSMR, heptaspanning membrane receptor; GPCR, G protein-coupled receptor; YFP, yellow fluorescent protein; BRET, bioluminescence resonance energy transfer; FRET, fluorescence resonance energy transfer; E n , extracellular loop n; I n , intracellular loop n; Mes, 4-morpholineethanesulfonic acid; ANOVA, analysis of variance; Rluc, Renilla luciferase; EYFP enhanced yellow fluorescent protein; CHO, Chinese hamster ovary; GFP, green fluorescent protein; PDB, Protein Data Bank; GABA, ␥-aminobutyric acid; PBS, phosphate-buffered saline; CH, channel; A 2A R, adenosine A 2A receptor; D n R, dopamine D n receptor.

Journal of Biological Chemistry, 2001

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be presen... more Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo-and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G proteincoupled receptors, namely the metabotropic glutamate type 1␣ (mGlu 1␣ receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu 1␣ and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu 1␣ and A1 receptors in receptorevoked [Ca 2؉ ] i signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu 1␣ and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.

Journal of Biological Chemistry, 2003

Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be ... more Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast twohybrid screen, we identified ␣-actinin, a major F-actincross-linking protein, as a binding partner for the Cterminal domain of the adenosine A 2A receptor (A 2A R). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A 2A R and ␣-actinin in transfected HEK-293 cells and also in rat striatal tissue. A 2A R activation by agonist induced the internalization of the receptor by a process that involved rapid ␤-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A 2A ⌬CTR, a mutant version of A 2A R that lacks the Cterminal domain and does not interact with ␣-actinin, was not able to internalize when activated by agonist. Interestingly, A 2A ⌬CTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an ␣-actinin-dependent association between the actin cytoskeleton and A 2A R trafficking.