Gemma Navarro - Academia.edu (original) (raw)
Papers by Gemma Navarro
Springer eBooks, 2017
Heteromerization alters GPCR recognition, G-protein activation, receptor signaling and traffickin... more Heteromerization alters GPCR recognition, G-protein activation, receptor signaling and trafficking, thus changing receptor protomer pharmacology and function. This review deals mainly with the A2AR-D2R and A1R-D1R heteroreceptor complexes, their balance with dopamine and adenosine isoreceptor complexes and their role in Parkinson’s disease and its treatment. The major technique used for the visualization of the heteroreceptor complexes in the brain was the proximity ligation assay. A1R-D1R and putative A1R-D1R-D3R heteroreceptor complexes appear to exist in the direct pathway. Upon agonist activation the A1R protomer exerts a brake on the D1R protomer signaling of these complexes reducing the activity of the direct pathway with reduction of movement initiation. D1R-NMDAR and D1R-H3R-NMDAR heteroreceptor complexes in the striatal glutamate synapses integrate synaptic and volume transmission, where in the former complexes the D1R protomer enhances NMDAR signaling with enhancement of movements. A2AR-D2R and A2AR-D2R-mGlu5R heteroreceptor complexes with antagonistic receptor-receptor interactions exist in the dorsal striato-pallidal GABA neurons mediating motor inhibition. The A2AR and mGlu5R antagonists synergize to increase D2R protomer signaling by removing the A2AR and mGlu5R brakes on the D2R protomer signaling and heterobivalent compounds built of A2AR and mGlu5R antagonists may specifically and substantially remove these brakes reducing motor inhibition with development of antiparkinson actions.
Frontiers in Molecular Neuroscience, 2012
Modulation of G protein-coupled receptor (GPCR) signaling by local changes in intracellular calci... more Modulation of G protein-coupled receptor (GPCR) signaling by local changes in intracellular calcium concentration is an established function of Calmodulin (CaM) which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca 2+-sensor proteins that frequently associate with CaM targets with different functional outcome. In the present study we aimed to investigate if a target of CaM-the A 2A adenosine receptor is able to associate with two other neuronal calcium binding proteins (nCaBPs), namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments we show the existence of A 2A-NCS-1 complexes in living cells whereas caldendrin did not associate with A 2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A 2A receptor intracellular signaling in a Ca 2+-dependent manner. Taken together this study provides further evidence that neuronal Ca 2+-sensor proteins play an important role in modulation of GPCR signaling.
Neuropharmacology, May 1, 2016
Purinergic signaling modulates dopaminergic neurotransmission in health and disease. Classically ... more Purinergic signaling modulates dopaminergic neurotransmission in health and disease. Classically adenosine A1 and A2A receptors have been considered key for the fine tune control of dopamine actions in the striatum, the main CNS motor control center. The main adenosine signaling mechanism is via the cAMP pathway but the future will tell whether calcium signaling is relevant in adenosinergic control of striatal function. Very relevant is the recent approval in Japan of the adenosine A2A receptor antagonist, istradefylline, for use in Parkinson's disease patients. Purine nucleotides are also regulators of striatal dopamine neurotransmission via P2 purinergic receptors. In parallel to the alpha-synuclein hypothesis of Parkinson's disease etiology, purinergic P2X1 receptors have been identified as mediators of accumulation of the Lewy-body enriched protein alpha-synuclein. Of note is the expression in striatum of purinergic-receptor-containing heteromers that are potential targets of anti-Parkinson's disease therapies and should be taken into account in drug discovery programs.
Springer eBooks, 2018
While adrenergic receptors were instrumental to start to understand the role of GPCRs, other rece... more While adrenergic receptors were instrumental to start to understand the role of GPCRs, other receptors are taking the lead to understand why GPCR homo−/ heteromers are needed and to address their physiological consequences in both healthy/homeostatic conditions and disease. Adenosine and dopamine receptors in the CNS are instrumental to understand pathogenic mechanisms in Parkinson's disease and to know the role of receptor heteromers. We here provide the account of the heteroreceptor complexes formed by adenosine receptors (A 1 , A 2A , A 2B , and A 3), and their potential as therapeutic targets. Both adenosine (A 1 or A 2A)-dopamine (D 1 or D 2) and adenosine A 1 A 2A heteroreceptor complexes are therapeutic targets in Parkinson's disease and may be altered after chronic levodopa treatment. A short account on the potential of adenosine receptors as targets in schizophrenia is also provided. Apart from potential in combating symptoms, adenosine receptors have potential as targets for neuroprotection. However, the design of neuroprotective drugs requires to understand how adenosine affects microglia and which adenosinereceptor-containing heteromers may be targeted.
Receptor-Receptor Interactions in the Central Nervous System, 2018
The Journal of Neuroscience, 2021
Ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R1a), is coexpressed wi... more Ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R1a), is coexpressed with its truncated isoform GHS-R1b, which does not bind ghrelin or signal, but oligomerizes with GHS-R1a, exerting a complex modulatory role that depends on its relative expression. D1dopamine receptor (D1R) and D5R constitute the two D1-like receptor subtypes. Previous studies showed that GHS-R1b also facilitates oligomerization of GHS-R1a with D1R, conferring GHS-R1a distinctive pharmacological properties. Those include a switch in the preferred coupling of GHS-R1a from Gq to Gs and the ability of D1R/D5R agonists and antagonists to counteract GHS-R1a signaling. Activation of ghrelin receptors localized in the ventral tegmental area (VTA) seems to play a significant role in the contribution of ghrelin to motivated behavior. In view of the evidence indicating that dopaminergic cells of the VTA express ghrelin receptors and D5R, but not D1R, we investigated the possible existence of functi...
Possible interfaces in A2AR homodimers in complex with Gs. In A–E, the A2AR homodimer was modeled... more Possible interfaces in A2AR homodimers in complex with Gs. In A–E, the A2AR homodimer was modeled through TM4 using the H1-receptor structure as template (A), through TM5 using the structure of squid rhodopsin (B), through TM4/5 using the β1-receptor structure (C), and via TM5/6 (D) and TM1 (E) using the μ-OR structure. TM helices 1, 4, and 5 involved in receptor dimerization are highlighted in dark blue, light blue, and gray, respectively. A2AR protomers bound to Gs (in gray) are shown in light green, whereas Gs-unbound A2AR protomers are shown in dark green. Rluc (blue) is attached to the N-terminal αN helix of Gs, and YFP (yellow) is attached to the C-terminal domain of the Gs-unbound A2AR protomer (light green). It is important to note that the position of YFP is highly dependent on the orientation of the long and highly flexible C-tail of A2AR (102 amino acids, Gln311–Ser412), which was modeled as described for the OXER [32] (see Additional file 9: Figure S9 for details). Despi...
Graphical description of the stoichiometry of A1R-GFP, A2AR-mCherry or both A1-GFP and A2A-mCherr... more Graphical description of the stoichiometry of A1R-GFP, A2AR-mCherry or both A1-GFP and A2A-mCherry. The fluorescence intensity signal distribution (gray area) detected for more than 7000 independent observations is given for HEK-293T cells expressing A1-GFP (A), A2A-mCherry (D), or both A1-GFP and A2A-mCherry (B, E). The stoichiometry analysis was performed for A1-GFP (A, B) and A2A-mCherry (D, E). Curves approximately delineating the amount of monomers, dimers, trimers, and tetramers are displayed in green for A1-GFP (A, B) and in red for A2A-mCherry (D-E). The occurrence on the cell surface of monomers, dimers, trimers, and tetramers for A1-GFP (C) expressed alone (black bars) or in the presence of A2A-mCherry (blue bars) and for A2A-mCherry (F) expressed alone (black bars) or in the presence of A1-GFP (blue bars) was calculated by stoichiometry analysis from results shown in A, B, D, and E. (TIF 455 kb)
Molecular dynamics (MD) simulation of the adenosine A1R-A2AR heterotetramer in complex with Gi an... more Molecular dynamics (MD) simulation of the adenosine A1R-A2AR heterotetramer in complex with Gi and Gs. (A) Root-mean-square deviations (rmsd) on protein α-carbons of the whole system (black solid line), of the two A1Rs (orange and red solid lines), of the two A2ARs (light and dark green solid lines), of Gi (gray solid line), and of Gs (gray dotted line) throughout the MD simulation. This color scheme matches with the color of the different proteins depicted in the two adjacent schematic representations. (B) Intermolecular distances between the N-terminal helices of the γ-subunit of Gi and Gs (magenta line), the N-terminal helices of the α-subunit of Gi and Gs (gray line), the N-terminal helix of the α-subunit of Gi and the C-terminal helix (Hx8) of inactive A1R (orange line), the N-terminal helix of the α-subunit of Gs and the C-terminal Hx8 of inactive A2AR (green line), the C-terminal Hx8 of A1R and A2AR (blue lines). These computed intermolecular distances are depicted as double ...
BRET assays in cells expressing fusion proteins containing hemi-Rluc8 and hemi-Venus moieties fus... more BRET assays in cells expressing fusion proteins containing hemi-Rluc8 and hemi-Venus moieties fused to adenosine receptors or containing the ghrelin GHS1a receptor instead of one of the adenosine receptors. (A) Saturation BRET curve in HEK-293T co-transfected with 1.5 μg of the two cDNAs corresponding to A1R-cRLuc8 and A2AR-nRLuc8 and with increasing amounts of cDNAs corresponding to A1R-nVenus and A2AR-cVenus (equal amounts of the two cDNAs). BRETmax was 35 ± 2 mBU and BRET50 was 16 ± 3 mBU. BRET in cells expressing cRluc8 instead of A1R-cRluc8 gave a linear, non-saturable signal. (B) Comparison of BRET responses using complementary and non-complementary pairs, or replacing one adenosine receptor with the ghrelin GHS1a (gn) receptor. Data are mean ± SD of three different experiments grouped as a function of the amount of BRET acceptor. ***p
Receptor-Receptor Interactions in the Central Nervous System, 2018
International Journal of Molecular Sciences, 2021
Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect... more Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A2A receptor (A2AR). First, we noticed that methamphetamine does not affect A2A functionality if the receptor is expressed in a heterologous system. However, A2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB1 receptor (CB1R) and the sigma 1 receptor (σ1R). Signaling via both adenosine A2AR and cannabinoid CB1R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A2AR–CB1R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A2AR- and the CB1R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusi...
Glia, 2019
Neuroprotective M2‐skewed microglia appear as promising to alter the course of neurodegenerative ... more Neuroprotective M2‐skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein‐coupled receptors (GPCRs) are potential targets to achieve such microglial polarization. A common feature of adenosine A2A (A2AR) and cannabinoid CB2 (CB2R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB2R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A2AR antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A2A‐CB2 receptor heteromer (A2A‐CB2Het). Particularly relevant is the upregulation of A2A‐CB2Het expression in samples from the APPSw,Ind AD transgenic mice model. T...
Often, in vitro or in vivo enzyme-mediated catalytic events occur far from equilibrium and, there... more Often, in vitro or in vivo enzyme-mediated catalytic events occur far from equilibrium and, therefore, substrate affinity measured as the inverse of ES ⇄ E+S dissociation equilibrium constant (Kd) has a doubtful physiological meaning; in practice it is almost impossible to determine Kd (except using stopped-flow or other sophisticated methodologies). The Michaelis-Menten constant (Km), the concentration of substrate ([S]) providing half of enzyme maximal activity, is not the (Kd). In the simple E+S ⇄ ES → E+P or in more complex models describing S conversion into P, Km must be considered the constant defining the steady state at any substrate concentration. Enzyme kinetics is based on initial rate determination, i.e. in the linear part of the S to P conversion when the concentration of [ES] remains constant while steady state occurs. We also show that Systems Biology issues such as the time required to respond to a system perturbation, is more dependent on k1, the kinetic constant d...
Nature Communications, 2018
G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the mos... more G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A2A receptor and dopamine D2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.
Frontiers in molecular neuroscience, 2018
N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions ... more N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK-and Ca 2+-mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APP Sw,Ind transgenic Alzheimer's disease model. Interestingly, a very marked increase in NMDAR-NCS1 complexes was identified in neurons and a marked increase of both NMDAR-NCS1 and NMDAR-CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the
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](Frontiers in pharmacology, 2018
The central adenosine system and adenosine receptors play a fundamental role in the modulation of... more The central adenosine system and adenosine receptors play a fundamental role in the modulation of dopaminergic neurotransmission. This is mostly achieved by the strategic co-localization of different adenosine and dopamine receptor subtypes in the two populations of striatal efferent neurons, striatonigral and striatopallidal, that give rise to the direct and indirect striatal efferent pathways, respectively. With optogenetic techniques it has been possible to dissect a differential role of the direct and indirect pathways in mediating "Go" responses upon exposure to reward-related stimuli and "NoGo" responses upon exposure to non-rewarded or aversive-related stimuli, respectively, which depends on their different connecting output structures and their differential expression of dopamine and adenosine receptor subtypes. The striatopallidal neuron selectively expresses dopamine D receptors (D2R) and adenosine A receptors (A2AR), and numerous experiments using mult...
Frontiers in synaptic neuroscience, 2018
This perspective article provides observations supporting the view that nigro-striatal dopamine n... more This perspective article provides observations supporting the view that nigro-striatal dopamine neurons and meso-limbic dopamine neurons mainly communicate through short distance volume transmission in the um range with dopamine diffusing into extrasynaptic and synaptic regions of glutamate and GABA synapses. Based on this communication it is discussed how volume transmission modulates synaptic glutamate transmission onto the D1R modulated direct and D2R modulated indirect GABA pathways of the dorsal striatum. Each nigro-striatal dopamine neuron was first calculated to form large numbers of neostriatal DA nerve terminals and then found to give rise to dense axonal arborizations spread over the neostriatum, from which dopamine is released. These neurons can through DA volume transmission directly influence not only the striatal GABA projection neurons but all the striatal cell types in parallel. It includes the GABA nerve cells forming the island-/striosome GABA pathway to the nigral...
Frontiers in neuroscience, 2018
Adenosine is a nucleoside mainly formed by degradation of ATP, located intracellularly or extrace... more Adenosine is a nucleoside mainly formed by degradation of ATP, located intracellularly or extracellularly, and acts as a neuromodulator. It operates as a volume transmission signal through diffusion and flow in the extracellular space to modulate the activity of both glial cells and neurons. The effects of adenosine are mediated via four adenosine receptor subtypes: A1R, A2AR, A2BR, A3R. The A2AR has a wide-spread distribution but it is especially enriched in the ventral and dorsal striatum where it is mainly located in the striato-pallidal GABA neurons at a synaptic and extrasynaptic location. A number of A2AR heteroreceptor complexes exist in the striatum. The existence of A2AR-D2R heteroreceptor complexes with antagonistic A2AR-D2R interactions in the striato-pallidal GABA neurons is well-known with A2AR activation inhibiting Gi/o mediated signaling of D2Rs. A2AR-mGluR5 heteroreceptor complexes were also found in with synergistic receptor-receptor interactions enhancing the inhib...
Springer eBooks, 2017
Heteromerization alters GPCR recognition, G-protein activation, receptor signaling and traffickin... more Heteromerization alters GPCR recognition, G-protein activation, receptor signaling and trafficking, thus changing receptor protomer pharmacology and function. This review deals mainly with the A2AR-D2R and A1R-D1R heteroreceptor complexes, their balance with dopamine and adenosine isoreceptor complexes and their role in Parkinson’s disease and its treatment. The major technique used for the visualization of the heteroreceptor complexes in the brain was the proximity ligation assay. A1R-D1R and putative A1R-D1R-D3R heteroreceptor complexes appear to exist in the direct pathway. Upon agonist activation the A1R protomer exerts a brake on the D1R protomer signaling of these complexes reducing the activity of the direct pathway with reduction of movement initiation. D1R-NMDAR and D1R-H3R-NMDAR heteroreceptor complexes in the striatal glutamate synapses integrate synaptic and volume transmission, where in the former complexes the D1R protomer enhances NMDAR signaling with enhancement of movements. A2AR-D2R and A2AR-D2R-mGlu5R heteroreceptor complexes with antagonistic receptor-receptor interactions exist in the dorsal striato-pallidal GABA neurons mediating motor inhibition. The A2AR and mGlu5R antagonists synergize to increase D2R protomer signaling by removing the A2AR and mGlu5R brakes on the D2R protomer signaling and heterobivalent compounds built of A2AR and mGlu5R antagonists may specifically and substantially remove these brakes reducing motor inhibition with development of antiparkinson actions.
Frontiers in Molecular Neuroscience, 2012
Modulation of G protein-coupled receptor (GPCR) signaling by local changes in intracellular calci... more Modulation of G protein-coupled receptor (GPCR) signaling by local changes in intracellular calcium concentration is an established function of Calmodulin (CaM) which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca 2+-sensor proteins that frequently associate with CaM targets with different functional outcome. In the present study we aimed to investigate if a target of CaM-the A 2A adenosine receptor is able to associate with two other neuronal calcium binding proteins (nCaBPs), namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments we show the existence of A 2A-NCS-1 complexes in living cells whereas caldendrin did not associate with A 2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A 2A receptor intracellular signaling in a Ca 2+-dependent manner. Taken together this study provides further evidence that neuronal Ca 2+-sensor proteins play an important role in modulation of GPCR signaling.
Neuropharmacology, May 1, 2016
Purinergic signaling modulates dopaminergic neurotransmission in health and disease. Classically ... more Purinergic signaling modulates dopaminergic neurotransmission in health and disease. Classically adenosine A1 and A2A receptors have been considered key for the fine tune control of dopamine actions in the striatum, the main CNS motor control center. The main adenosine signaling mechanism is via the cAMP pathway but the future will tell whether calcium signaling is relevant in adenosinergic control of striatal function. Very relevant is the recent approval in Japan of the adenosine A2A receptor antagonist, istradefylline, for use in Parkinson's disease patients. Purine nucleotides are also regulators of striatal dopamine neurotransmission via P2 purinergic receptors. In parallel to the alpha-synuclein hypothesis of Parkinson's disease etiology, purinergic P2X1 receptors have been identified as mediators of accumulation of the Lewy-body enriched protein alpha-synuclein. Of note is the expression in striatum of purinergic-receptor-containing heteromers that are potential targets of anti-Parkinson's disease therapies and should be taken into account in drug discovery programs.
Springer eBooks, 2018
While adrenergic receptors were instrumental to start to understand the role of GPCRs, other rece... more While adrenergic receptors were instrumental to start to understand the role of GPCRs, other receptors are taking the lead to understand why GPCR homo−/ heteromers are needed and to address their physiological consequences in both healthy/homeostatic conditions and disease. Adenosine and dopamine receptors in the CNS are instrumental to understand pathogenic mechanisms in Parkinson's disease and to know the role of receptor heteromers. We here provide the account of the heteroreceptor complexes formed by adenosine receptors (A 1 , A 2A , A 2B , and A 3), and their potential as therapeutic targets. Both adenosine (A 1 or A 2A)-dopamine (D 1 or D 2) and adenosine A 1 A 2A heteroreceptor complexes are therapeutic targets in Parkinson's disease and may be altered after chronic levodopa treatment. A short account on the potential of adenosine receptors as targets in schizophrenia is also provided. Apart from potential in combating symptoms, adenosine receptors have potential as targets for neuroprotection. However, the design of neuroprotective drugs requires to understand how adenosine affects microglia and which adenosinereceptor-containing heteromers may be targeted.
Receptor-Receptor Interactions in the Central Nervous System, 2018
The Journal of Neuroscience, 2021
Ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R1a), is coexpressed wi... more Ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R1a), is coexpressed with its truncated isoform GHS-R1b, which does not bind ghrelin or signal, but oligomerizes with GHS-R1a, exerting a complex modulatory role that depends on its relative expression. D1dopamine receptor (D1R) and D5R constitute the two D1-like receptor subtypes. Previous studies showed that GHS-R1b also facilitates oligomerization of GHS-R1a with D1R, conferring GHS-R1a distinctive pharmacological properties. Those include a switch in the preferred coupling of GHS-R1a from Gq to Gs and the ability of D1R/D5R agonists and antagonists to counteract GHS-R1a signaling. Activation of ghrelin receptors localized in the ventral tegmental area (VTA) seems to play a significant role in the contribution of ghrelin to motivated behavior. In view of the evidence indicating that dopaminergic cells of the VTA express ghrelin receptors and D5R, but not D1R, we investigated the possible existence of functi...
Possible interfaces in A2AR homodimers in complex with Gs. In A–E, the A2AR homodimer was modeled... more Possible interfaces in A2AR homodimers in complex with Gs. In A–E, the A2AR homodimer was modeled through TM4 using the H1-receptor structure as template (A), through TM5 using the structure of squid rhodopsin (B), through TM4/5 using the β1-receptor structure (C), and via TM5/6 (D) and TM1 (E) using the μ-OR structure. TM helices 1, 4, and 5 involved in receptor dimerization are highlighted in dark blue, light blue, and gray, respectively. A2AR protomers bound to Gs (in gray) are shown in light green, whereas Gs-unbound A2AR protomers are shown in dark green. Rluc (blue) is attached to the N-terminal αN helix of Gs, and YFP (yellow) is attached to the C-terminal domain of the Gs-unbound A2AR protomer (light green). It is important to note that the position of YFP is highly dependent on the orientation of the long and highly flexible C-tail of A2AR (102 amino acids, Gln311–Ser412), which was modeled as described for the OXER [32] (see Additional file 9: Figure S9 for details). Despi...
Graphical description of the stoichiometry of A1R-GFP, A2AR-mCherry or both A1-GFP and A2A-mCherr... more Graphical description of the stoichiometry of A1R-GFP, A2AR-mCherry or both A1-GFP and A2A-mCherry. The fluorescence intensity signal distribution (gray area) detected for more than 7000 independent observations is given for HEK-293T cells expressing A1-GFP (A), A2A-mCherry (D), or both A1-GFP and A2A-mCherry (B, E). The stoichiometry analysis was performed for A1-GFP (A, B) and A2A-mCherry (D, E). Curves approximately delineating the amount of monomers, dimers, trimers, and tetramers are displayed in green for A1-GFP (A, B) and in red for A2A-mCherry (D-E). The occurrence on the cell surface of monomers, dimers, trimers, and tetramers for A1-GFP (C) expressed alone (black bars) or in the presence of A2A-mCherry (blue bars) and for A2A-mCherry (F) expressed alone (black bars) or in the presence of A1-GFP (blue bars) was calculated by stoichiometry analysis from results shown in A, B, D, and E. (TIF 455 kb)
Molecular dynamics (MD) simulation of the adenosine A1R-A2AR heterotetramer in complex with Gi an... more Molecular dynamics (MD) simulation of the adenosine A1R-A2AR heterotetramer in complex with Gi and Gs. (A) Root-mean-square deviations (rmsd) on protein α-carbons of the whole system (black solid line), of the two A1Rs (orange and red solid lines), of the two A2ARs (light and dark green solid lines), of Gi (gray solid line), and of Gs (gray dotted line) throughout the MD simulation. This color scheme matches with the color of the different proteins depicted in the two adjacent schematic representations. (B) Intermolecular distances between the N-terminal helices of the γ-subunit of Gi and Gs (magenta line), the N-terminal helices of the α-subunit of Gi and Gs (gray line), the N-terminal helix of the α-subunit of Gi and the C-terminal helix (Hx8) of inactive A1R (orange line), the N-terminal helix of the α-subunit of Gs and the C-terminal Hx8 of inactive A2AR (green line), the C-terminal Hx8 of A1R and A2AR (blue lines). These computed intermolecular distances are depicted as double ...
BRET assays in cells expressing fusion proteins containing hemi-Rluc8 and hemi-Venus moieties fus... more BRET assays in cells expressing fusion proteins containing hemi-Rluc8 and hemi-Venus moieties fused to adenosine receptors or containing the ghrelin GHS1a receptor instead of one of the adenosine receptors. (A) Saturation BRET curve in HEK-293T co-transfected with 1.5 μg of the two cDNAs corresponding to A1R-cRLuc8 and A2AR-nRLuc8 and with increasing amounts of cDNAs corresponding to A1R-nVenus and A2AR-cVenus (equal amounts of the two cDNAs). BRETmax was 35 ± 2 mBU and BRET50 was 16 ± 3 mBU. BRET in cells expressing cRluc8 instead of A1R-cRluc8 gave a linear, non-saturable signal. (B) Comparison of BRET responses using complementary and non-complementary pairs, or replacing one adenosine receptor with the ghrelin GHS1a (gn) receptor. Data are mean ± SD of three different experiments grouped as a function of the amount of BRET acceptor. ***p
Receptor-Receptor Interactions in the Central Nervous System, 2018
International Journal of Molecular Sciences, 2021
Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect... more Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A2A receptor (A2AR). First, we noticed that methamphetamine does not affect A2A functionality if the receptor is expressed in a heterologous system. However, A2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB1 receptor (CB1R) and the sigma 1 receptor (σ1R). Signaling via both adenosine A2AR and cannabinoid CB1R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A2AR–CB1R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A2AR- and the CB1R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusi...
Glia, 2019
Neuroprotective M2‐skewed microglia appear as promising to alter the course of neurodegenerative ... more Neuroprotective M2‐skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein‐coupled receptors (GPCRs) are potential targets to achieve such microglial polarization. A common feature of adenosine A2A (A2AR) and cannabinoid CB2 (CB2R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB2R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A2AR antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A2A‐CB2 receptor heteromer (A2A‐CB2Het). Particularly relevant is the upregulation of A2A‐CB2Het expression in samples from the APPSw,Ind AD transgenic mice model. T...
Often, in vitro or in vivo enzyme-mediated catalytic events occur far from equilibrium and, there... more Often, in vitro or in vivo enzyme-mediated catalytic events occur far from equilibrium and, therefore, substrate affinity measured as the inverse of ES ⇄ E+S dissociation equilibrium constant (Kd) has a doubtful physiological meaning; in practice it is almost impossible to determine Kd (except using stopped-flow or other sophisticated methodologies). The Michaelis-Menten constant (Km), the concentration of substrate ([S]) providing half of enzyme maximal activity, is not the (Kd). In the simple E+S ⇄ ES → E+P or in more complex models describing S conversion into P, Km must be considered the constant defining the steady state at any substrate concentration. Enzyme kinetics is based on initial rate determination, i.e. in the linear part of the S to P conversion when the concentration of [ES] remains constant while steady state occurs. We also show that Systems Biology issues such as the time required to respond to a system perturbation, is more dependent on k1, the kinetic constant d...
Nature Communications, 2018
G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the mos... more G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A2A receptor and dopamine D2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.
Frontiers in molecular neuroscience, 2018
N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions ... more N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK-and Ca 2+-mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APP Sw,Ind transgenic Alzheimer's disease model. Interestingly, a very marked increase in NMDAR-NCS1 complexes was identified in neurons and a marked increase of both NMDAR-NCS1 and NMDAR-CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the
Frontiers in pharmacology, 2018
The central adenosine system and adenosine receptors play a fundamental role in the modulation of... more The central adenosine system and adenosine receptors play a fundamental role in the modulation of dopaminergic neurotransmission. This is mostly achieved by the strategic co-localization of different adenosine and dopamine receptor subtypes in the two populations of striatal efferent neurons, striatonigral and striatopallidal, that give rise to the direct and indirect striatal efferent pathways, respectively. With optogenetic techniques it has been possible to dissect a differential role of the direct and indirect pathways in mediating "Go" responses upon exposure to reward-related stimuli and "NoGo" responses upon exposure to non-rewarded or aversive-related stimuli, respectively, which depends on their different connecting output structures and their differential expression of dopamine and adenosine receptor subtypes. The striatopallidal neuron selectively expresses dopamine D receptors (D2R) and adenosine A receptors (A2AR), and numerous experiments using mult...
Frontiers in synaptic neuroscience, 2018
This perspective article provides observations supporting the view that nigro-striatal dopamine n... more This perspective article provides observations supporting the view that nigro-striatal dopamine neurons and meso-limbic dopamine neurons mainly communicate through short distance volume transmission in the um range with dopamine diffusing into extrasynaptic and synaptic regions of glutamate and GABA synapses. Based on this communication it is discussed how volume transmission modulates synaptic glutamate transmission onto the D1R modulated direct and D2R modulated indirect GABA pathways of the dorsal striatum. Each nigro-striatal dopamine neuron was first calculated to form large numbers of neostriatal DA nerve terminals and then found to give rise to dense axonal arborizations spread over the neostriatum, from which dopamine is released. These neurons can through DA volume transmission directly influence not only the striatal GABA projection neurons but all the striatal cell types in parallel. It includes the GABA nerve cells forming the island-/striosome GABA pathway to the nigral...
Frontiers in neuroscience, 2018
Adenosine is a nucleoside mainly formed by degradation of ATP, located intracellularly or extrace... more Adenosine is a nucleoside mainly formed by degradation of ATP, located intracellularly or extracellularly, and acts as a neuromodulator. It operates as a volume transmission signal through diffusion and flow in the extracellular space to modulate the activity of both glial cells and neurons. The effects of adenosine are mediated via four adenosine receptor subtypes: A1R, A2AR, A2BR, A3R. The A2AR has a wide-spread distribution but it is especially enriched in the ventral and dorsal striatum where it is mainly located in the striato-pallidal GABA neurons at a synaptic and extrasynaptic location. A number of A2AR heteroreceptor complexes exist in the striatum. The existence of A2AR-D2R heteroreceptor complexes with antagonistic A2AR-D2R interactions in the striato-pallidal GABA neurons is well-known with A2AR activation inhibiting Gi/o mediated signaling of D2Rs. A2AR-mGluR5 heteroreceptor complexes were also found in with synergistic receptor-receptor interactions enhancing the inhib...