Carme Lluís | Universidad de Barcelona (original) (raw)

Papers by Carme Lluís

Research paper thumbnail of Metabotropic glutamate 1α and adenosine A1 receptors assemble into functionally interacting complexes

Journal of Biological …, Jan 1, 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.

Research paper thumbnail of Dopamine D4 receptor, but not the ADHD-associated D4.7 variant, forms functional heteromers with the dopamine D2S receptor in the brain

Molecular Psychiatry, 2012

Polymorphic variants of the dopamine D 4 receptor have been consistently associated with attentio... more Polymorphic variants of the dopamine D 4 receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However, the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here, we show that whereas the most frequent 4-repeat (D 4.4 ) and the 2-repeat (D 4.2 ) variants form functional heteromers with the short isoform of the dopamine D 2 receptor (D 2S ), the 7-repeat risk allele (D 4.7 ) does not. D 2 receptor activation in the D 2S -D 4 receptor heteromer potentiates D 4 receptormediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D 4.7 or in the striatum of knockin mutant mice carrying the 7 repeats of the human D 4.7 in the third intracellular loop of the D 4 receptor. In the striatum, D 4 receptors are localized in corticostriatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D 2S receptors. This interaction shows the same qualitative characteristics than the D 2S -D 4 receptor heteromer-mediated mitogen-activated protein kinase (MAPK) signaling and D 2S receptor activation potentiates D 4 receptor-mediated inhibition of striatal glutamate release. It is therefore postulated that dysfunctional D 2S -D 4.7 heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD.

Research paper thumbnail of Cocaine Disrupts Histamine H3 Receptor Modulation of Dopamine D1 Receptor Signaling:  1-D1-H3 Receptor Complexes as Key Targets for Reducing Cocaine's Effects

Journal of Neuroscience, 2014

The general effects of cocaine are not well understood at the molecular level. What is known is t... more The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits β-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine.

Research paper thumbnail of Ligand-induced caveolae-mediated internalization of A1 adenosine receptors: morphological evidence of endosomal sorting and receptor recycling

Experimental Cell Research, 2003

The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the re... more The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the receptor sorting and recycling was studied in the smooth muscle cell line DDT(1)MF-2, by binding assays, by confocal microscopy, and at the structural level. The use of cholera toxin-binding subunit adsorbed to gold as a specific probe for labeling the ganglioside GM(1) and immunoelectron microscopy techniques showed that agonist stimulation produced a clustering and sequestration of adenosine receptors in caveolae. Furthermore, pull-down experiments showed there to be a direct interaction between the C-terminal domain of A(1)R and caveolin-1. Addition of exogenous adenosine deaminase (ADA), a protein that binds to A(1)R and acts as a receptor activity modifying protein (RAMP) stimulated R-PIA-induced A(1) receptor internalization. Finally, the sorting and recycling of A(1)R/ADA complexes was analyzed. Detailed electron microscopy revealed that A(1)R/ADA complexes internalize together through caveolae, are differentially sorted in endosomes, and are recycled back to the cell surface by different groups of recycling endosomes. These results give insight into the spatiotemporal regulation and traffic of A(1)R and RAMPs.

Research paper thumbnail of Interactions between Intracellular Domains as Key Determinants of the Quaternary Structure and Function of Receptor Heteromers

Journal of Biological Chemistry, 2010

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional ... more G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

Research paper thumbnail of On the Nested Hierarchical Organization of CNS: Basic Characteristics of Neuronal Molecular Networks

Lecture Notes in Computer Science, 2004

It has been suggested that the Central Nervous System (CNS) is built as a “nested system of netwo... more It has been suggested that the Central Nervous System (CNS) is built as a “nested system of networks of networks” structured according to hierarchical principles. Thus, it is possible to recognize networks at multiple scales (miniaturisation principle) moving top-down from ...

Research paper thumbnail of Adenosine A2A-dopamine D2 receptor–receptor heteromers. Targets for neuro-psychiatric disorders

Emerging evidence shows that G protein-coupled receptors can form homo- and heteromers. These inc... more Emerging evidence shows that G protein-coupled receptors can form homo- and heteromers. These include adenosine A(2A) receptor-dopamine D(2) receptor heteromers, which are most probably localized in the dendritic spines of the striatopallidal GABAergic neurons, where they are in a position to modulate glutamatergic neurotransmission. The discovery of A(2A) receptor-dopamine D(2) receptor heteromers gives a frame for the well-known antagonistic interaction between both receptors, which is the bases for a new therapeutic approach for neuro-psychiatric disorders, such as Parkinson's disease and schizoprenia. The present review deals mainly with the biochemical and molecular aspects of A(2A) receptor-dopamine D(2) receptor interactions. Recent results at the molecular level show that A(2A) receptor-dopamine D(2) receptor heteromers represent the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization. Most probably A(2A) receptor-D(2) receptor heteromerization is not static, but subject to a dynamic regulation, related to the phosphorylation dependence of the A(2A) receptor epitope and to the ability of the D(2) receptor epitope to bind different partners. Finding out the mechanisms involved in this dynamic regulation can have important implications for the treatment of basal ganglia disorders, schizophrenia and drug addiction.

Research paper thumbnail of Modulation of GABA Transport by Adenosine A1R-A2AR Heteromers, Which Are Coupled to Both Gs- and Gi/o-Proteins

Journal of Neuroscience, 2011

Astrocytes play a key role in modulating synaptic transmission by controlling the available extra... more Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A(1) (A(1)R) and A(2A) (A(2A)R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A(1)R-A(2A)R that signal via two different G-proteins, G(s) and G(i/o), and either enhances (A(2A)R) or inhibits (A(1)R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

Research paper thumbnail of Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

PLoS ONE, 2013

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking c... more Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D 1 -like family of dopamine receptors and inputs of aversion coming from neurons containing the D 2 -like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D 1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D 2 receptors function, we present evidence of s 1 receptor molecular and functional interaction with dopamine D 2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D 2 receptors (the long isoform of the D 2 receptor) can complex with s 1 receptors, a result that is specific to D 2 receptors, as D 3 and D 4 receptors did not form heteromers. We demonstrate that the s 1 -D 2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to s 1 -D 2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from s 1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D 2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D 1 and D 2 receptor containing neurons in the brain.

Research paper thumbnail of Neurotransmitter receptor heteromers and their integrative role in ‘local modules’: The striatal spine module

Brain Research Reviews, 2007

Local module' is a fundamental functional unit of the central nervous system that can be defined ... more Local module' is a fundamental functional unit of the central nervous system that can be defined as the minimal portion of one or more neurons and-or one or more glial cells that operates as an independent integrative unit. This review focuses on the importance of neurotransmitter receptor heteromers for the operation of local modules. To illustrate this, we use the striatal spine module (SSM), comprised of the dendritic spine of the medium spiny neuron (MSN), its glutamatergic and dopaminergic terminals and astroglial processes. The SSM is found in the striatum, and although aspects such as neurotransmitters and receptors will be specific to the SSM, some general principles should apply to any local module in the brain. The analysis of some of the receptor heteromers in the SSM shows that receptor heteromerization is associated with particular elaborated functions in this local module. Adenosine A 2A receptor-dopamine D 2 receptor-glutamate metabotropic mGlu 5 receptor heteromers are located adjacent to the glutamatergic synapse of the dendritic spine of the enkephalin MSN, and their cross-talk within the receptor heteromers helps to modulate postsynaptic plastic changes at the glutamatergic synapse. A 1 receptor-A 2A receptor heteromers are found in the glutamatergic terminals and the molecular cross-talk between the two receptors in the heteromer helps to modulate glutamate release. Finally, dopamine D 2 receptor-non-α 7 nicotinic acetylcholine receptor heteromers, which are located in dopaminergic terminals, introduce the new concept of autoreceptor heteromer.

Research paper thumbnail of Increase in A 2A receptors in the nucleus accumbens after extended cocaine self-administration and its disappearance after cocaine withdrawal

Brain Research, 2007

Effects of extended cocaine self-administration and its withdrawal have been studied on A2A and D... more Effects of extended cocaine self-administration and its withdrawal have been studied on A2A and D2 receptor binding characteristics and expression in the nucleus accumbens and the anterior and posterior dorsal striatum of the rat (Rattus norvegicus). Biochemical binding techniques have been used with the D2-like receptor antagonist radioligand [3H]-Raclopride and the A2A receptor antagonist radioligand [3H]-ZM 241385 and immunoblots to

Research paper thumbnail of A method for binding parameters estimation of A adenosine receptor subtype: A practical approach

Analytical Biochemistry, 1990

Working with pig brain striatum in which A1 and A2 adenosine receptor subtypes coexist, we descri... more Working with pig brain striatum in which A1 and A2 adenosine receptor subtypes coexist, we describe an uncomplicated method for unequivocally obtaining the equilibrium parameters (KD and binding capacity) of A1 receptor without interference from ligand binding to A2 receptor. Also, the equilibrium parameter estimation method we propose avoids the experimental determination of nonspecific binding by the inclusion of the corresponding unknown parameter in the function. This not only saves time but also avoids the use of expensive radioligands in saturation experiments. The method is suitable for any system with two different receptor subtypes for the same physiological ligand, and good estimates of the equilibrium parameters corresponding to the subtype displaying the higher affinity for the ligand can be obtained.

Research paper thumbnail of On the Molecular Basis of the Receptor Mosaic Hypothesis of the Engram

Cellular and Molecular Neurobiology, 2004

1. This paper revisits the so-called “receptor mosaic hypothesis” for memory trace formation in t... more 1. This paper revisits the so-called “receptor mosaic hypothesis” for memory trace formation in the light of recent findings in “functional (or interaction) proteomics.” The receptor mosaic hypothesis maintains that receptors may form molecular aggregates at the plasma membrane level representing part of the computational molecular networks.

Research paper thumbnail of Role of Histidine Residues in Agonist and Antagonist Binding Sites of A 1 Adenosine Receptor

Journal of Neurochemistry, 1993

The influence of pH on the equilibrium dissociation constant and on kinetic association and disso... more The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-@- [~denine-2,8-~H, ethyl-

Research paper thumbnail of Allosteric interactions between agonists and antagonists within the adenosine A 2A receptor-dopamine D 2 receptor heterotetramer

Proceedings of the National Academy of Sciences, 2015

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striata... more Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

Research paper thumbnail of Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer

Neuropharmacology, 2015

The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provid... more The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provides a main functional unit and oligomeric entities can be viewed as multiples of dimers. For GPCR heteromers, experimental evidence supports a tetrameric structure, comprised of two different homodimers, each able to signal with its preferred G protein. GPCR homomers and heteromers can act as the conduit of allosteric interactions between orthosteric ligands. The well-known agonist/agonist allosteric interaction in the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer, by which A2AR agonists decrease the affinity of D2R agonists, gave the first rationale for the use of A2AR antagonists in Parkinson's disease. We review new pharmacological findings that can be explained in the frame of a tetrameric structure of the A2AR-D2R heteromer: first, ligand-independent allosteric modulations by the D2R that result in changes of the binding properties of A2AR ligands; second, differential modulation of the intrinsic efficacy of D2R ligands for G protein-dependent and independent signaling; third, the canonical antagonistic Gs-Gi interaction within the frame of the heteromer; and fourth, the ability of A2AR antagonists, including caffeine, to also exert the same allosteric modulations of D2R ligands than A2AR agonists, while A2AR agonists and antagonists counteract each other's effects. These findings can have important clinical implications when evaluating the use of A2AR antagonists. They also call for the need of monitoring caffeine intake when evaluating the effect of D2R ligands, when used as therapeutic agents in neuropsychiatric disorders or as probes in imaging studies. This article is part of a Special Issue entitled 'Purines-Neurodegeneration'.

Research paper thumbnail of A Solid-Phase Combinatorial Approach for Indoloquinolizidine-Peptides with High Affinity at D1 and D2 Dopamine Receptors

European Journal of Medicinal Chemistry, 2015

Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number o... more Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D 1 R and D 2 R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D 2 R, a receptor that is crucial to re-establish activity on dopaminedepleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D 2 R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D 1 and D 2 dopamine receptors.

Research paper thumbnail of Functional selectivity of allosteric interactions within G protein-coupled receptor oligomers: the dopamine D1-D3 receptor heterotetramer

Molecular pharmacology, 2014

The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential thera... more The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R a...

Research paper thumbnail of Reply: Does the adenosine A2A receptor stimulate the ryanodine receptor?

In our paper on the expression and function of the adenosine A2A receptor (A2AR) in human atrial ... more In our paper on the expression and function of the adenosine A2A receptor (A2AR) in human atrial myocytes (1), we show that agonist stimulation of this receptor induces a protein kinase A (PKA)-dependent increase in the frequency of calcium sparks and spontaneous calcium waves. We therefore investigated whether A2AR stimulation affected the L-type calcium current, sarcoplasmic reticulum (SR) calcium uptake,

Research paper thumbnail of G protein-coupled receptor heteromers as new targets for drug development

Progress in Molecular Biology and Translational Science, 2010

Research paper thumbnail of Metabotropic glutamate 1α and adenosine A1 receptors assemble into functionally interacting complexes

Journal of Biological …, Jan 1, 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.

Research paper thumbnail of Dopamine D4 receptor, but not the ADHD-associated D4.7 variant, forms functional heteromers with the dopamine D2S receptor in the brain

Molecular Psychiatry, 2012

Polymorphic variants of the dopamine D 4 receptor have been consistently associated with attentio... more Polymorphic variants of the dopamine D 4 receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However, the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here, we show that whereas the most frequent 4-repeat (D 4.4 ) and the 2-repeat (D 4.2 ) variants form functional heteromers with the short isoform of the dopamine D 2 receptor (D 2S ), the 7-repeat risk allele (D 4.7 ) does not. D 2 receptor activation in the D 2S -D 4 receptor heteromer potentiates D 4 receptormediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D 4.7 or in the striatum of knockin mutant mice carrying the 7 repeats of the human D 4.7 in the third intracellular loop of the D 4 receptor. In the striatum, D 4 receptors are localized in corticostriatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D 2S receptors. This interaction shows the same qualitative characteristics than the D 2S -D 4 receptor heteromer-mediated mitogen-activated protein kinase (MAPK) signaling and D 2S receptor activation potentiates D 4 receptor-mediated inhibition of striatal glutamate release. It is therefore postulated that dysfunctional D 2S -D 4.7 heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD.

Research paper thumbnail of Cocaine Disrupts Histamine H3 Receptor Modulation of Dopamine D1 Receptor Signaling:  1-D1-H3 Receptor Complexes as Key Targets for Reducing Cocaine's Effects

Journal of Neuroscience, 2014

The general effects of cocaine are not well understood at the molecular level. What is known is t... more The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits β-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine.

Research paper thumbnail of Ligand-induced caveolae-mediated internalization of A1 adenosine receptors: morphological evidence of endosomal sorting and receptor recycling

Experimental Cell Research, 2003

The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the re... more The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the receptor sorting and recycling was studied in the smooth muscle cell line DDT(1)MF-2, by binding assays, by confocal microscopy, and at the structural level. The use of cholera toxin-binding subunit adsorbed to gold as a specific probe for labeling the ganglioside GM(1) and immunoelectron microscopy techniques showed that agonist stimulation produced a clustering and sequestration of adenosine receptors in caveolae. Furthermore, pull-down experiments showed there to be a direct interaction between the C-terminal domain of A(1)R and caveolin-1. Addition of exogenous adenosine deaminase (ADA), a protein that binds to A(1)R and acts as a receptor activity modifying protein (RAMP) stimulated R-PIA-induced A(1) receptor internalization. Finally, the sorting and recycling of A(1)R/ADA complexes was analyzed. Detailed electron microscopy revealed that A(1)R/ADA complexes internalize together through caveolae, are differentially sorted in endosomes, and are recycled back to the cell surface by different groups of recycling endosomes. These results give insight into the spatiotemporal regulation and traffic of A(1)R and RAMPs.

Research paper thumbnail of Interactions between Intracellular Domains as Key Determinants of the Quaternary Structure and Function of Receptor Heteromers

Journal of Biological Chemistry, 2010

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional ... more G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

Research paper thumbnail of On the Nested Hierarchical Organization of CNS: Basic Characteristics of Neuronal Molecular Networks

Lecture Notes in Computer Science, 2004

It has been suggested that the Central Nervous System (CNS) is built as a “nested system of netwo... more It has been suggested that the Central Nervous System (CNS) is built as a “nested system of networks of networks” structured according to hierarchical principles. Thus, it is possible to recognize networks at multiple scales (miniaturisation principle) moving top-down from ...

Research paper thumbnail of Adenosine A2A-dopamine D2 receptor–receptor heteromers. Targets for neuro-psychiatric disorders

Emerging evidence shows that G protein-coupled receptors can form homo- and heteromers. These inc... more Emerging evidence shows that G protein-coupled receptors can form homo- and heteromers. These include adenosine A(2A) receptor-dopamine D(2) receptor heteromers, which are most probably localized in the dendritic spines of the striatopallidal GABAergic neurons, where they are in a position to modulate glutamatergic neurotransmission. The discovery of A(2A) receptor-dopamine D(2) receptor heteromers gives a frame for the well-known antagonistic interaction between both receptors, which is the bases for a new therapeutic approach for neuro-psychiatric disorders, such as Parkinson's disease and schizoprenia. The present review deals mainly with the biochemical and molecular aspects of A(2A) receptor-dopamine D(2) receptor interactions. Recent results at the molecular level show that A(2A) receptor-dopamine D(2) receptor heteromers represent the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization. Most probably A(2A) receptor-D(2) receptor heteromerization is not static, but subject to a dynamic regulation, related to the phosphorylation dependence of the A(2A) receptor epitope and to the ability of the D(2) receptor epitope to bind different partners. Finding out the mechanisms involved in this dynamic regulation can have important implications for the treatment of basal ganglia disorders, schizophrenia and drug addiction.

Research paper thumbnail of Modulation of GABA Transport by Adenosine A1R-A2AR Heteromers, Which Are Coupled to Both Gs- and Gi/o-Proteins

Journal of Neuroscience, 2011

Astrocytes play a key role in modulating synaptic transmission by controlling the available extra... more Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A(1) (A(1)R) and A(2A) (A(2A)R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A(1)R-A(2A)R that signal via two different G-proteins, G(s) and G(i/o), and either enhances (A(2A)R) or inhibits (A(1)R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

Research paper thumbnail of Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

PLoS ONE, 2013

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking c... more Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D 1 -like family of dopamine receptors and inputs of aversion coming from neurons containing the D 2 -like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D 1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D 2 receptors function, we present evidence of s 1 receptor molecular and functional interaction with dopamine D 2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D 2 receptors (the long isoform of the D 2 receptor) can complex with s 1 receptors, a result that is specific to D 2 receptors, as D 3 and D 4 receptors did not form heteromers. We demonstrate that the s 1 -D 2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to s 1 -D 2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from s 1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D 2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D 1 and D 2 receptor containing neurons in the brain.

Research paper thumbnail of Neurotransmitter receptor heteromers and their integrative role in ‘local modules’: The striatal spine module

Brain Research Reviews, 2007

Local module' is a fundamental functional unit of the central nervous system that can be defined ... more Local module' is a fundamental functional unit of the central nervous system that can be defined as the minimal portion of one or more neurons and-or one or more glial cells that operates as an independent integrative unit. This review focuses on the importance of neurotransmitter receptor heteromers for the operation of local modules. To illustrate this, we use the striatal spine module (SSM), comprised of the dendritic spine of the medium spiny neuron (MSN), its glutamatergic and dopaminergic terminals and astroglial processes. The SSM is found in the striatum, and although aspects such as neurotransmitters and receptors will be specific to the SSM, some general principles should apply to any local module in the brain. The analysis of some of the receptor heteromers in the SSM shows that receptor heteromerization is associated with particular elaborated functions in this local module. Adenosine A 2A receptor-dopamine D 2 receptor-glutamate metabotropic mGlu 5 receptor heteromers are located adjacent to the glutamatergic synapse of the dendritic spine of the enkephalin MSN, and their cross-talk within the receptor heteromers helps to modulate postsynaptic plastic changes at the glutamatergic synapse. A 1 receptor-A 2A receptor heteromers are found in the glutamatergic terminals and the molecular cross-talk between the two receptors in the heteromer helps to modulate glutamate release. Finally, dopamine D 2 receptor-non-α 7 nicotinic acetylcholine receptor heteromers, which are located in dopaminergic terminals, introduce the new concept of autoreceptor heteromer.

Research paper thumbnail of Increase in A 2A receptors in the nucleus accumbens after extended cocaine self-administration and its disappearance after cocaine withdrawal

Brain Research, 2007

Effects of extended cocaine self-administration and its withdrawal have been studied on A2A and D... more Effects of extended cocaine self-administration and its withdrawal have been studied on A2A and D2 receptor binding characteristics and expression in the nucleus accumbens and the anterior and posterior dorsal striatum of the rat (Rattus norvegicus). Biochemical binding techniques have been used with the D2-like receptor antagonist radioligand [3H]-Raclopride and the A2A receptor antagonist radioligand [3H]-ZM 241385 and immunoblots to

Research paper thumbnail of A method for binding parameters estimation of A adenosine receptor subtype: A practical approach

Analytical Biochemistry, 1990

Working with pig brain striatum in which A1 and A2 adenosine receptor subtypes coexist, we descri... more Working with pig brain striatum in which A1 and A2 adenosine receptor subtypes coexist, we describe an uncomplicated method for unequivocally obtaining the equilibrium parameters (KD and binding capacity) of A1 receptor without interference from ligand binding to A2 receptor. Also, the equilibrium parameter estimation method we propose avoids the experimental determination of nonspecific binding by the inclusion of the corresponding unknown parameter in the function. This not only saves time but also avoids the use of expensive radioligands in saturation experiments. The method is suitable for any system with two different receptor subtypes for the same physiological ligand, and good estimates of the equilibrium parameters corresponding to the subtype displaying the higher affinity for the ligand can be obtained.

Research paper thumbnail of On the Molecular Basis of the Receptor Mosaic Hypothesis of the Engram

Cellular and Molecular Neurobiology, 2004

1. This paper revisits the so-called “receptor mosaic hypothesis” for memory trace formation in t... more 1. This paper revisits the so-called “receptor mosaic hypothesis” for memory trace formation in the light of recent findings in “functional (or interaction) proteomics.” The receptor mosaic hypothesis maintains that receptors may form molecular aggregates at the plasma membrane level representing part of the computational molecular networks.

Research paper thumbnail of Role of Histidine Residues in Agonist and Antagonist Binding Sites of A 1 Adenosine Receptor

Journal of Neurochemistry, 1993

The influence of pH on the equilibrium dissociation constant and on kinetic association and disso... more The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-@- [~denine-2,8-~H, ethyl-

Research paper thumbnail of Allosteric interactions between agonists and antagonists within the adenosine A 2A receptor-dopamine D 2 receptor heterotetramer

Proceedings of the National Academy of Sciences, 2015

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striata... more Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

Research paper thumbnail of Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer

Neuropharmacology, 2015

The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provid... more The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provides a main functional unit and oligomeric entities can be viewed as multiples of dimers. For GPCR heteromers, experimental evidence supports a tetrameric structure, comprised of two different homodimers, each able to signal with its preferred G protein. GPCR homomers and heteromers can act as the conduit of allosteric interactions between orthosteric ligands. The well-known agonist/agonist allosteric interaction in the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer, by which A2AR agonists decrease the affinity of D2R agonists, gave the first rationale for the use of A2AR antagonists in Parkinson's disease. We review new pharmacological findings that can be explained in the frame of a tetrameric structure of the A2AR-D2R heteromer: first, ligand-independent allosteric modulations by the D2R that result in changes of the binding properties of A2AR ligands; second, differential modulation of the intrinsic efficacy of D2R ligands for G protein-dependent and independent signaling; third, the canonical antagonistic Gs-Gi interaction within the frame of the heteromer; and fourth, the ability of A2AR antagonists, including caffeine, to also exert the same allosteric modulations of D2R ligands than A2AR agonists, while A2AR agonists and antagonists counteract each other's effects. These findings can have important clinical implications when evaluating the use of A2AR antagonists. They also call for the need of monitoring caffeine intake when evaluating the effect of D2R ligands, when used as therapeutic agents in neuropsychiatric disorders or as probes in imaging studies. This article is part of a Special Issue entitled 'Purines-Neurodegeneration'.

Research paper thumbnail of A Solid-Phase Combinatorial Approach for Indoloquinolizidine-Peptides with High Affinity at D1 and D2 Dopamine Receptors

European Journal of Medicinal Chemistry, 2015

Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number o... more Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D 1 R and D 2 R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D 2 R, a receptor that is crucial to re-establish activity on dopaminedepleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D 2 R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D 1 and D 2 dopamine receptors.

Research paper thumbnail of Functional selectivity of allosteric interactions within G protein-coupled receptor oligomers: the dopamine D1-D3 receptor heterotetramer

Molecular pharmacology, 2014

The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential thera... more The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R a...

Research paper thumbnail of Reply: Does the adenosine A2A receptor stimulate the ryanodine receptor?

In our paper on the expression and function of the adenosine A2A receptor (A2AR) in human atrial ... more In our paper on the expression and function of the adenosine A2A receptor (A2AR) in human atrial myocytes (1), we show that agonist stimulation of this receptor induces a protein kinase A (PKA)-dependent increase in the frequency of calcium sparks and spontaneous calcium waves. We therefore investigated whether A2AR stimulation affected the L-type calcium current, sarcoplasmic reticulum (SR) calcium uptake,

Research paper thumbnail of G protein-coupled receptor heteromers as new targets for drug development

Progress in Molecular Biology and Translational Science, 2010