Publications by authors named "Marc Brugarolas"

10 Publications

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Computational modeling of the olfactory receptor Olfr73 suggests a molecular basis for low potency of olfactory receptor-activating compounds.

Commun Biol 2019 23;2:141. Epub 2019 Apr 23.

2Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

The mammalian olfactory system uses hundreds of specialized G-protein-coupled olfactory receptors (ORs) to discriminate a nearly unlimited number of odorants. Cognate agonists of most ORs have not yet been identified and potential non-olfactory processes mediated by ORs are unknown. Here, we used molecular modeling, fingerprint interaction analysis and molecular dynamics simulations to show that the binding pocket of the prototypical olfactory receptor Olfr73 is smaller, but more flexible, than binding pockets of typical non-olfactory G-protein-coupled receptors. We extended our modeling to virtual screening of a library of 1.6 million compounds against Olfr73. Our screen predicted 25 Olfr73 agonists beyond traditional odorants, of which 17 compounds, some with therapeutic potential, were validated in cell-based assays. Our modeling suggests a molecular basis for reduced interaction contacts between an odorant and its OR and thus the typical low potency of OR-activating compounds. These results provide a proof-of-principle for identifying novel therapeutic OR agonists.
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http://dx.doi.org/10.1038/s42003-019-0384-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478719PMC
April 2020

Cross-communication between G and G in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain.

BMC Biol 2018 02 28;16(1):24. Epub 2018 Feb 28.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, University of Barcelona, 08028, Barcelona, Spain.

Background: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (AR or AR) can form AR-AR heteromers (A-AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for AR and AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream G- and G-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of AR impedes signaling via AR.

Results: We examined the mechanism by which A-AHet integrates G- and G-dependent signals. AR blockade by AR in the A-AHet is not observed in the absence of AR activation by agonists, in the absence of the C-terminal domain of AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A-AHet, indicating that signaling mediated by AR and AR is controlled by both G and G proteins.

Conclusions: We identified a new mechanism of signal transduction that implies a cross-communication between G and G proteins guided by the C-terminal tail of the AR. This mechanism provides the molecular basis for the operation of the A-AHet as an adenosine concentration-sensing device that modulates the signals originating at both AR and AR.
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http://dx.doi.org/10.1186/s12915-018-0491-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389107PMC
February 2018

Equilibrative nucleoside transporter ENT1 as a biomarker of Huntington disease.

Neurobiol Dis 2016 Dec 24;96:47-53. Epub 2016 Aug 24.

Integrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD 21224, United States. Electronic address:

The initial goal of this study was to investigate alterations in adenosine A receptor (AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease.
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http://dx.doi.org/10.1016/j.nbd.2016.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102769PMC
December 2016

Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

BMC Biol 2016 Apr 5;14:26. Epub 2016 Apr 5.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.

Background: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown.

Results: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins.

Conclusions: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.
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http://dx.doi.org/10.1186/s12915-016-0247-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822319PMC
April 2016

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

Proc Natl Acad Sci U S A 2015 Jul 22;112(27):E3609-18. Epub 2015 Jun 22.

Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, and Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas and Institute of Biomedicine of the University of Barcelona, 08028 Barcelona, Spain;

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.
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http://dx.doi.org/10.1073/pnas.1507704112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500251PMC
July 2015

G-protein-coupled receptor heteromers as key players in the molecular architecture of the central nervous system.

CNS Neurosci Ther 2014 Aug 9;20(8):703-9. Epub 2014 May 9.

Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona (UB), Barcelona, Spain; Centro investigación biomédica en red enfermedades neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain.

The overall architecture of the nervous system, especially the CNS, is remarkable. The anatomy of the nervous system is constituted not only by macroscopic and microscopy identifiable regions and neuronal cell types, but also by protein complexes whose identification and localization require sophisticated techniques. G-protein-coupled receptors (GPCRs) constitute an example of proteins that are the key factors in the framework needed to sustain brain and nerve structure and function. The versatility underlying nervous system anatomy takes advantage of a recently discovered feature of GPCRs, the possibility to form heteromers that, placed at specific neuronal subsets and at specific locations (pre-, post-, or peri-synaptic), contribute to attain unique neural functions.
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http://dx.doi.org/10.1111/cns.12277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493065PMC
August 2014

A1R-A2AR heteromers coupled to Gs and G i/0 proteins modulate GABA transport into astrocytes.

Purinergic Signal 2013 Sep 10;9(3):433-49. Epub 2013 May 10.

Institute of Pharmacology and Neurosciences, Faculty of Medicine, University of Lisbon, Av. Professor Egas Moniz, Edificio Egas Moniz, 1649-028, Lisbon, Portugal.

Astrocytes play a key role in modulating synaptic transmission by controlling extracellular gamma-aminobutyric acid (GABA) levels via GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that a further level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through A1R-A2AR heteromers that signal via two different G proteins, Gs and Gi/0, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where 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.
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http://dx.doi.org/10.1007/s11302-013-9364-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757138PMC
September 2013

Cocaine inhibits dopamine D2 receptor signaling via sigma-1-D2 receptor heteromers.

PLoS One 2013 18;8(4):e61245. Epub 2013 Apr 18.

Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) and Institute of Biomedicine of the University of Barcelona (IBUB) and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain.

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 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 D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 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 D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061245PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630156PMC
December 2013

Modulation of GABA transport by adenosine A1R-A2AR heteromers, which are coupled to both Gs- and G(i/o)-proteins.

J Neurosci 2011 Nov;31(44):15629-39

Institute of Pharmacology and Neurosciences, Faculty of Medicine, Unit of Neurosciences, Institute of Molecular Medicine, University of Lisbon, 1649-028 Lisbon, Portugal.

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.
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http://dx.doi.org/10.1523/JNEUROSCI.2526-11.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6623011PMC
November 2011

Striatal pre- and postsynaptic profile of adenosine A(2A) receptor antagonists.

PLoS One 2011 Jan 11;6(1):e16088. Epub 2011 Jan 11.

National Institute on Drug Abuse, IRP, NIH, DHHS, Baltimore, Maryland, United States of America.

Striatal adenosine A(2A) receptors (A(2A)Rs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D(2) receptors (D(2)Rs). A(2A)Rs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A(1) receptors (A(1)Rs). It has been hypothesized that postsynaptic A(2A)R antagonists should be useful in Parkinson's disease, while presynaptic A(2A)R antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A(2A)R antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A(2A)R-D(2)R and A(1)R-A(2A)R heteromers to determine possible differences in the affinity of these compounds for different A(2A)R heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A(2A)R when co-expressed with D(2)R than with A(1)R. KW-6002 showed the best relative affinity for A(2A)R co-expressed with D(2)R than co-expressed with A(1)R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential pre- versus postsynaptic actions, SCH-442416 and KW-6002 may be used as lead compounds to obtain more effective antidyskinetic and antiparkinsonian compounds, respectively.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016088PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019225PMC
January 2011