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1.
Eur J Med Chem ; 2622023 Dec 15.
Article in English | MEDLINE | ID: mdl-38523699

ABSTRACT

The EphA4 receptor tyrosine kinase plays a role in neurodegenerative diseases, inhibition of nerve regeneration, cancer progression and other diseases. Therefore, EphA4 inhibition has potential therapeutic value. Selective EphA4 kinase inhibitors are not available, but we identified peptide antagonists that inhibit ephrin ligand binding to EphA4 with high specificity. One of these peptides is the cyclic APY-d3 (ßAPYCVYRßASWSC-NH2), which inhibits ephrin-A5 ligand binding to EphA4 with low nanomolar binding affinity and is highly protease resistant. Here we describe modifications of APY-d3 that yield two different key derivatives with greatly increased half-lives in the mouse circulation, the lipidated APY-d3-laur8 and the PEGylated APY-d3-PEG4. These two derivatives inhibit ligand induced EphA4 activation in cells with sub-micromolar potency. Since they retain high potency and specificity for EphA4, lipidated and PEGylated APY-d3 derivatives represent new tools for discriminating EphA4 activities in vivo and for preclinical testing of EphA4 inhibition in animal disease models.


Subject(s)
Ephrin-A5 , Receptor, EphA4 , Mice , Animals , Receptor, EphA4/metabolism , Ligands , Half-Life , Ephrin-A5/metabolism , Polyethylene Glycols
2.
iScience ; 25(3): 103870, 2022 Mar 18.
Article in English | MEDLINE | ID: mdl-35243233

ABSTRACT

The EphA2 receptor tyrosine kinase activates signaling pathways with different, and sometimes opposite, effects in cancer and other pathologies. Thus, highly specific and potent biased ligands that differentially control EphA2 signaling responses could be therapeutically valuable. Here, we use EphA2-specific monomeric peptides to engineer dimeric ligands with three different geometric configurations to combine a potential ability to differentially modulate EphA2 signaling responses with the high potency and prolonged receptor residence time characteristic of dimeric ligands. The different dimeric peptides readily induce EphA2 clustering, autophosphorylation and signaling, the best with sub-nanomolar potency. Yet, there are differences in two EphA2 signaling responses induced by peptides with different configurations, which exhibit distinct potency and efficacy. The peptides bias signaling when compared with the ephrinA1-Fc ligand and do so via different mechanisms. These findings provide insights into Eph receptor signaling, and proof-of-principle that different Eph signaling responses can be distinctly modulated.

3.
J Biol Chem ; 297(1): 100876, 2021 07.
Article in English | MEDLINE | ID: mdl-34139238

ABSTRACT

The Eph receptor tyrosine kinases and their ephrin ligands regulate many physiological and pathological processes. EphA4 plays important roles in nervous system development and adult homeostasis, while aberrant EphA4 signaling has been implicated in neurodegeneration. EphA4 may also affect cancer malignancy, but the regulation and effects of EphA4 signaling in cancer are poorly understood. A correlation between decreased patient survival and high EphA4 mRNA expression in melanoma tumors that also highly express ephrinA ligands suggests that enhanced EphA4 signaling may contribute to melanoma progression. A search for EphA4 gain-of-function mutations in melanoma uncovered a mutation of the highly conserved leucine 920 in the EphA4 sterile alpha motif (SAM) domain. We found that mutation of L920 to phenylalanine (L920F) potentiates EphA4 autophosphorylation and signaling, making it the first documented EphA4 cancer mutation that increases kinase activity. Quantitative Föster resonance energy transfer and fluorescence intensity fluctuation (FIF) analyses revealed that the L920F mutation induces a switch in EphA4 oligomer size, from a dimer to a trimer. We propose this switch in oligomer size as a novel mechanism underlying EphA4-linked tumorigenesis. Molecular dynamics simulations suggest that the L920F mutation alters EphA4 SAM domain conformation, leading to the formation of EphA4 trimers that assemble through two aberrant SAM domain interfaces. Accordingly, EphA4 wild-type and the L920F mutant are affected differently by the SAM domain and are differentially regulated by ephrin ligand stimulation. The increased EphA4 activation induced by the L920F mutation, through the novel mechanism we uncovered, supports a functional role for EphA4 in promoting pathogenesis.


Subject(s)
Mutation, Missense , Neoplasms/genetics , Receptor, EphA4/chemistry , Signal Transduction , Sterile Alpha Motif , HEK293 Cells , Humans , Protein Multimerization , Receptor, EphA4/genetics , Receptor, EphA4/metabolism
4.
Hum Mol Genet ; 29(4): 605-617, 2020 03 13.
Article in English | MEDLINE | ID: mdl-31814004

ABSTRACT

Worldwide, stroke is the main cause of long-term adult disability. After the initial insult, most patients undergo a subacute period with intense plasticity and rapid functional improvements. This period is followed by a chronic phase where recovery reaches a plateau that is only partially modifiable by rehabilitation. After experimental stroke, various subacute rehabilitation paradigms improve recovery. However, in order to reach the best possible outcome, a combination of plasticity-promoting strategies and rehabilitation might be necessary. EphA4 is a negative axonal guidance regulator during development. After experimental stroke, reduced EphA4 levels improve functional outcome with similar beneficial effects upon the inhibition of EphA4 downstream targets. In this study, we assessed the effectiveness of a basic enriched environment in the chronic phase after photothrombotic stroke in mice as well as the therapeutic potential of EphA4 targeted therapy followed by rehabilitation. Our findings show that environmental enrichment in the chronic phase improves functional outcome up to 2 months post-stroke. Although EphA4 levels increase after experimental stroke, subacute EphA4 inhibition followed by environmental enrichment does not further increase recovery. In conclusion, we show that environmental enrichment during the chronic phase of stroke improves functional outcome in mice with no synergistic effects of the used EphA4 targeted therapy.


Subject(s)
Disease Models, Animal , Peptide Fragments/pharmacology , Receptor, EphA4/antagonists & inhibitors , Recovery of Function , Stroke Rehabilitation/methods , Stroke/drug therapy , Animals , Male , Mice , Mice, Inbred C57BL , Molecular Targeted Therapy , Phosphorylation , Stroke/etiology , Stroke/pathology
5.
Int J Mol Sci ; 20(14)2019 Jul 23.
Article in English | MEDLINE | ID: mdl-31340557

ABSTRACT

Background: Several biophysical techniques have been successfully implemented to detect G protein-coupled receptors (GPCRs) heteromerization. Although these approaches have made it possible to ascertain the presence of GPCR heteromers in animal models of disease, no success has been accomplished in pathological human post-mortem brains. The AlphaScreen technology has been consistently used to quantify small analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range and sensitivity exhibited by this technology support that it may be suitable to detect GPCR heteromers even under non-optimal conditions. Methods: Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. Results: Adenosine A2A-dopamine D2 receptor (A2AR/D2R) heteromer formation was monitored in caudate from healthy and Parkinson's disease (PD) subjects. The approach was first validated using striatal membranes from wild type and A2AR deficient mice. Secondly, we took advantage of the 6-hydroxydopamine hemiparkinsonian rat model to validate previous results. In addition, finally, A2AR/D2R heteromer formation was assessed in caudate membranes from human post-mortem brains. Importantly, our preliminary results revealed an increase in A2AR/D2R heteromer formation in PD brains. Conclusions: The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity.


Subject(s)
Autopsy/methods , Corpus Striatum/metabolism , High-Throughput Screening Assays/instrumentation , Parkinson Disease, Secondary/genetics , Parkinson Disease/genetics , Receptor, Adenosine A2A/genetics , Aged , Aged, 80 and over , Animals , Autopsy/instrumentation , Case-Control Studies , Corpus Striatum/pathology , Disease Models, Animal , Female , Gene Expression , Humans , Male , Mice , Mice, Knockout , Oxidopamine/administration & dosage , Parkinson Disease/diagnosis , Parkinson Disease/pathology , Parkinson Disease, Secondary/chemically induced , Parkinson Disease, Secondary/pathology , Protein Multimerization , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A2A/chemistry , Receptor, Adenosine A2A/metabolism
6.
J Biol Chem ; 294(22): 8791-8805, 2019 05 31.
Article in English | MEDLINE | ID: mdl-31015204

ABSTRACT

The EPH receptor A2 (EphA2) tyrosine kinase plays an important role in a plethora of biological and disease processes, ranging from angiogenesis and cancer to inflammation and parasitic infections. EphA2 is therefore considered an important drug target. Two short peptides previously identified by phage display, named YSA and SWL, are widely used as EphA2-targeting agents owing to their high specificity for this receptor. However, these peptides have only modest (micromolar) potency. Lack of structural information on the binding interactions of YSA and SWL with the extracellular EphA2 ligand-binding domain (LBD) has for many years precluded structure-guided improvements. We now report the high-resolution (1.53-2.20 Å) crystal structures of the YSA peptide and several of its improved derivatives in complex with the EphA2 LBD, disclosing that YSA targets the ephrin-binding pocket of EphA2 and mimics binding features of the ephrin-A ligands. The structural information obtained enabled iterative peptide modifications conferring low nanomolar potency. Furthermore, contacts observed in the crystal structures shed light on how C-terminal features can convert YSA derivatives from antagonists to agonists that likely bivalently interact with two EphA2 molecules to promote receptor oligomerization, autophosphorylation, and downstream signaling. Consistent with this model, quantitative FRET measurements in live cells revealed that the peptide agonists promote the formation of EphA2 oligomeric assemblies. Our findings now enable rational strategies to differentially modify EphA2 signaling toward desired outcomes by using appropriately engineered peptides. Such peptides could be used as research tools to interrogate EphA2 function and to develop pharmacological leads.


Subject(s)
Peptides/metabolism , Receptor, EphA2/metabolism , Signal Transduction , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Ephrin-A1/chemistry , Ephrin-A1/metabolism , Humans , Ligands , Molecular Dynamics Simulation , Peptides/chemistry , Peptides/pharmacology , Phosphorylation , Protein Binding , Protein Engineering , Protein Multimerization , Protein Structure, Tertiary , Receptor, EphA2/agonists , Receptor, EphA2/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Signal Transduction/drug effects
7.
Front Neurosci ; 12: 604, 2018.
Article in English | MEDLINE | ID: mdl-30233293

ABSTRACT

Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. The main treatment of PD consists of medication with dopamine-based drugs, which palliate the symptoms but may produce adverse effects after chronic administration. Accordingly, there is a need to develop novel neuroprotective therapies. Several studies suggest that omega-3 polyunsaturated fatty acids (n-3 PUFA) might provide protection against brain damage. Here, we studied several experimental models of PD, using striatal neuronal cultures, striatal slices, and mice, to assess the neuroprotective effects of docosahexaenoic acid (DHA), the main n-3 PUFA in the brain, administered in its triglyceride form (TG-DHA). Hence, we determined the beneficial effects of TG-DHA on neural viability following 6-hydroxydopamine (6-OHDA)-induced neurotoxicity, a well-established PD model. We also implemented a novel mouse behavioral test, the beam walking test, to finely assess mouse motor skills following dopaminergic denervation. This test showed potential as a useful behavioral tool to assess novel PD treatments. Our results indicated that TG-DHA-mediated neuroprotection was independent of the net incorporation of PUFA into the striatum, thus suggesting a tight control of brain lipid homeostasis both in normal and pathological conditions.

8.
Mol Neurobiol ; 55(6): 4952-4958, 2018 Jun.
Article in English | MEDLINE | ID: mdl-28779351

ABSTRACT

Dopamine D2 receptor (D2R) activation triggers both G protein- and ß-arrestin-dependent signaling. Biased D2R ligands activating ß-arrestin pathway have been proposed as potential antipsychotics. The ability of D2R to heteromerize with adenosine A2A receptor (A2AR) has been associated to D2R agonist-induced ß-arrestin recruitment. Accordingly, here we aimed to demonstrate the A2AR dependence of D2R/ß-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between ß-arrestin-2 tagged with yellow fluorescent protein and bimolecular luminescence complementation (BiLC) of D2R/A2AR homomers and heteromers, we demonstrated that the D2R agonists quinpirole and UNC9994 could promote ß-arrestin-2 recruitment only when A2AR/D2R heteromers were expressed. Subsequently, the role of A2AR in the antipsychotic-like activity of UNC9994 was assessed in wild-type and A2AR-/- mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in A2AR-/- mice, it failed to reduce PCP-induced hyperlocomotion or produced only a moderate reduction of AMPH-mediated hyperlocomotion. Overall, the results presented here reinforce the notion that D2R/A2AR heteromerization facilitates D2R ß-arrestin recruitment, and furthermore, reveal a pivotal role for A2AR in the antipsychotic-like activity of the ß-arrestin-biased D2R ligand, UNC9994.


Subject(s)
Antipsychotic Agents/pharmacology , Motor Activity/drug effects , Receptor, Adenosine A2A/metabolism , Receptors, Dopamine D2/agonists , Signal Transduction/drug effects , Adenosine/analogs & derivatives , Adenosine/pharmacology , Amphetamine/pharmacology , Animals , Dimerization , Dopamine Agents/pharmacology , Dopamine Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Mice , Mice, Knockout , Phencyclidine/pharmacology , Phenethylamines/pharmacology , Quinpirole/pharmacology , Receptor, Adenosine A2A/genetics
9.
Sci Rep ; 6: 19839, 2016 Jan 22.
Article in English | MEDLINE | ID: mdl-26796668

ABSTRACT

Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson's disease.


Subject(s)
Cell Membrane/metabolism , Fatty Acids, Omega-3/pharmacology , Protein Multimerization/drug effects , Receptor, Adenosine A2A/metabolism , Receptors, Dopamine D2/metabolism , Cell Membrane/drug effects , Cell Survival/drug effects , Diffusion , Docosahexaenoic Acids/pharmacology , Fatty Acids/analysis , HEK293 Cells , Humans , Kinetics , Molecular Dynamics Simulation , Phospholipids/analysis , Probability , Protein Aggregates/drug effects , Time Factors
10.
Behav Brain Res ; 282: 103-10, 2015 Apr 01.
Article in English | MEDLINE | ID: mdl-25557798

ABSTRACT

N-methyl-d-aspartate (NMDA) preconditioning is induced by subtoxic doses of NMDA and it promotes a transient state of resistance against subsequent lethal insults. Interestingly, this mechanism of neuroprotection depends on adenosine A1 receptors (A1R), since blockade of A1R precludes this phenomenon. In this study we evaluated the consequences of NMDA preconditioning on the hippocampal A1R biology (i.e. expression, binding properties and functionality). Accordingly, we measured A1R expression in NMDA preconditioned mice (75mg/kg, i.p.; 24h) and showed that neither the total amount of receptor, nor the A1R levels in the synaptic fraction was altered. In addition, the A1R binding affinity to the antagonist [(3)H] DPCPX was slightly increased in total membrane extracts of hippocampus from preconditioned mice. Next, we evaluated the impact of NMDA preconditioning on A1R functioning by measuring the A1R-mediated regulation of glutamate uptake into hippocampal slices and on behavioral responses in the open field and hot plate tests. NMDA preconditioning increased glutamate uptake into hippocampal slices without altering the expression of glutamate transporter GLT-1. Interestingly, NMDA preconditioning also induced antinociception in the hot plate test and both effects were reversed by post-activation of A1R with the agonist CCPA (0.2mg/kg, i.p.). NMDA preconditioning or A1R modulation did not alter locomotor activity in the open field. Overall, the results described herein provide new evidence that post-activation of A1R modulates NMDA preconditioning-mediated responses, pointing to the importance of the cross-talk between glutamatergic and adenosinergic systems to neuroprotection.


Subject(s)
Behavior, Animal/drug effects , Excitatory Amino Acid Agonists/pharmacology , Hippocampus/drug effects , N-Methylaspartate/pharmacology , Receptor, Adenosine A1/metabolism , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adenosine A1 Receptor Antagonists/pharmacology , Animals , Excitatory Amino Acid Transporter 2/metabolism , Glutamic Acid/metabolism , Hippocampus/metabolism , Locomotion/drug effects , Male , Mice , Mice, Inbred Strains , Motor Activity/drug effects , Purinergic P1 Receptor Agonists/pharmacology , Xanthines/pharmacology
11.
Dis Model Mech ; 8(1): 57-63, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25398851

ABSTRACT

Parkinson's disease (PD) is a dopaminergic-related pathology in which functioning of the basal ganglia is altered. It has been postulated that a direct receptor-receptor interaction - i.e. of dopamine D2 receptor (D2R) with adenosine A2A receptor (A2AR) (forming D2R-A2AR oligomers) - finely regulates this brain area. Accordingly, elucidating whether the pathology prompts changes to these complexes could provide valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning whether D2R-A2AR assembly occurs in native tissue: by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R-A2AR oligomers in rat striatum. Subsequently, we determined that, under pathological conditions (i.e. in a rat PD model), D2R-A2AR interaction was impaired. Collectively, these results provide definitive evidence for alteration of native D2R-A2AR oligomers in experimental parkinsonism, thus conferring the rationale for appropriate oligomer-based PD treatments.


Subject(s)
Dopamine/chemistry , Parkinsonian Disorders/metabolism , Receptors, Dopamine/chemistry , Receptors, Purinergic P1/chemistry , Animals , Brain/pathology , Cell Membrane/metabolism , Corpus Striatum/metabolism , Disease Models, Animal , Fluorescence Resonance Energy Transfer , Humans , Immunohistochemistry , Ligands , Mice , Mice, Knockout , Microscopy, Immunoelectron , Oxidopamine/chemistry , Parkinsonian Disorders/drug therapy , Plasmids/metabolism , Rats , Rats, Sprague-Dawley
12.
J Neurosci ; 34(44): 14793-802, 2014 Oct 29.
Article in English | MEDLINE | ID: mdl-25355231

ABSTRACT

Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca(2+) wave-dependent activation of small-conductance Ca(2+)-activated K(+) (SK) channels. Here, we show that mGlu5 receptors and SK2 channels coassemble in heterologous coexpression systems and in rat brain. Further, in cotransfected cells or rat primary hippocampal neurons, mGlu5 receptor stimulation activated apamin-sensitive SK2-mediated K(+) currents. In addition, coexpression of mGlu5 receptors and SK2 channels promoted plasma membrane targeting of both proteins and correlated with increased mGlu5 receptor function that was unexpectedly blocked by apamin. These results demonstrate a reciprocal functional interaction between mGlu5 receptors and SK2 channels that reflects their molecular coassembly.


Subject(s)
Hippocampus/metabolism , Neurons/metabolism , Receptor, Metabotropic Glutamate 5/metabolism , Small-Conductance Calcium-Activated Potassium Channels/metabolism , Animals , Apamin/pharmacology , Calcium/metabolism , HEK293 Cells , Hippocampus/drug effects , Hippocampus/ultrastructure , Humans , Mice , Neurons/drug effects , Neurons/ultrastructure , Rats
13.
ACS Chem Biol ; 9(11): 2496-501, 2014 Nov 21.
Article in English | MEDLINE | ID: mdl-25268872

ABSTRACT

Caffeine, the most consumed psychoactive substance worldwide, may have beneficial effects on Parkinson's disease (PD) therapy. The mechanism by which caffeine contributes to its antiparkinsonian effects by acting as either an adenosine A2A receptor (A2AR) neutral antagonist or an inverse agonist is unresolved. Here we show that caffeine is an A2AR inverse agonist in cell-based functional studies and in experimental parkinsonism. Thus, we observed that caffeine triggers a distinct mode, opposite to A2AR agonist, of the receptor's activation switch leading to suppression of its spontaneous activity. These inverse agonist-related effects were also determined in the striatum of a mouse model of PD, correlating well with increased caffeine-mediated motor effects. Overall, caffeine A2AR inverse agonism may be behind some of the well-known physiological effects of this substance both in health and disease. This information might have a critical mechanistic impact for PD pharmacotherapeutic design.


Subject(s)
Caffeine/pharmacology , Receptor, Adenosine A2A/drug effects , Animals , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Mice , Parkinsonian Disorders
14.
Curr Pharm Biotechnol ; 15(10): 962-70, 2014.
Article in English | MEDLINE | ID: mdl-25213309

ABSTRACT

G-protein-coupled receptors (GPCRs) represent the main family of cell surface receptors and are virtually expressed in all eukaryotic cells. Interestingly, a large number of clinically used drugs exert their pharmacological effect via a GPCR, thus it seems crucial to deeply understand the biology of these receptors. The study of GPCR activation and signaling has been classically performed by physiological, biochemical and pharmacological approaches using radioactivity-based tools. However, apart from the potential hazards of radioisotope handling and environmental burden, these approaches have some technical limitations. Therefore, the development of fluorescence-based techniques in general and fluorescence and bioluminescence resonance energy transfer (FRET and BRET) in particular have revolutionized the way to study GPCR functioning both in vitro and in vivo. Indeed, these techniques allow the characterization and visualization of all the individual GPCR signaling steps (i.e. ligand binding, receptor activation, G-protein coupling, G-protein activation, GPCR desensitization) with high temporal and spatial resolution. Here, we review the use and impact of fluorescent-based methodologies on the deciphering of GPCR biology.


Subject(s)
Receptors, G-Protein-Coupled/metabolism , Energy Transfer , Fluorescence , Humans , Ligands
15.
J Med Chem ; 57(1): 238-42, 2014 Jan 09.
Article in English | MEDLINE | ID: mdl-24354313

ABSTRACT

We have developed a novel methodology for monitoring the σ1 receptor activation switch in living cells. Our assay uncovered the intrinsic nature of σ1 receptor ligands by recording the ligand-mediated conformational changes of this chaperone protein. The change triggered by each ligand correlated well with its ability to attenuate formalin induced nociception in an animal model of pain. This tool may assist in predicting the antinociceptive efficacy of σ1 receptor ligands.


Subject(s)
Analgesics/pharmacology , Biosensing Techniques/methods , Fluorescence Resonance Energy Transfer/methods , Receptors, sigma/drug effects , Animals , Ligands , Mice , Receptors, sigma/chemistry , Sigma-1 Receptor
16.
Neurochem Int ; 63(1): 42-6, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23619397

ABSTRACT

The molecular interaction between adenosine A2A and dopamine D2 receptors (A2ARs and D2Rs, respectively) within an oligomeric complex has been postulated to play a pivotal role in the adenosine-dopamine interplay in the central nervous system, in both normal and pathological conditions (e.g. Parkinson's disease). While the effects of A2AR challenge on D2R functioning have been largely studied, the reverse condition is still unexplored, a fact that might have impact in therapeutics. Here, we aimed to examine in a real-time mode the D2R-mediated allosteric modulation of A2AR binding when an A2AR/D2R oligomer is established. Thus, we synthesized fluorescent A2AR agonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A2AR binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential D2R-mediated negative allosteric modulation on A2AR agonist binding that was oligomer-formation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A2AR agonist binding. Overall, the here-developed methods were found valid to explore the ability of drugs acting on D2Rs to modulate A2AR binding, thus serving to facilitate the preliminary selection of D2R-like candidate drugs in the management of Parkinson's disease.


Subject(s)
Adenosine A2 Receptor Agonists/metabolism , Biopolymers/metabolism , Receptor, Adenosine A2A/metabolism , Receptors, Dopamine D2/physiology , Flow Cytometry , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Protein Binding , Receptors, Dopamine D2/metabolism
17.
J Neurochem ; 123(3): 373-84, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22924752

ABSTRACT

In the CNS, an antagonistic interaction has been shown between adenosine A(2A) and dopamine D(2) receptors (A(2A)Rs and D(2)Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor-receptor interaction have recently been explored, as the fine tuning of this target (namely the A(2A)R/D(2)R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer-based approach to examine the allosteric modulation of the D(2)R within the A(2A)R/D(2)R oligomer and the dependence of this receptor-receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D(2)R. Interestingly, we observed a negative allosteric effect of the D(2)R agonist quinpirole on A(2A)R ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217-222 and 267-269) on intracellular loop 3 of the D(2)R, thus demonstrating a major role of these positively charged residues in mediating the observed receptor-receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A(2A)R/D(2)R oligomer in living cells.


Subject(s)
Receptor, Adenosine A2A/chemistry , Receptor, Adenosine A2A/genetics , Receptors, Dopamine D2/chemistry , Receptors, Dopamine D2/physiology , Adenosine A2 Receptor Agonists/pharmacology , Adenosine A2 Receptor Antagonists/pharmacology , Allosteric Regulation/drug effects , Allosteric Regulation/genetics , Arginine/genetics , Dopamine Agonists/pharmacology , HEK293 Cells , Humans , Intracellular Fluid/chemistry , Intracellular Fluid/drug effects , Intracellular Fluid/physiology , Mutagenesis, Site-Directed , Protein Binding/drug effects , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Quinpirole/pharmacology , Receptor, Adenosine A2A/metabolism , Receptors, Dopamine D2/genetics
18.
Neuropharmacology ; 61(5-6): 937-49, 2011.
Article in English | MEDLINE | ID: mdl-21752340

ABSTRACT

Voltage sensitivity has been demonstrated for some GPCRs. At the dopamine D(2S) receptor, this voltage sensitivity is agonist-specific; some agonists, including dopamine, exhibit decreased potency at depolarized potentials, whereas others are not significantly affected. In the present study, we examined some of the receptor-agonist interactions contributing to these differences, and investigated how dopamine D(2S) receptor voltage sensitivity affects clinically used dopamine agonists. GIRK channel activation in voltage-clamped Xenopus oocytes was used as readout of receptor activation. Structurally distinct agonists and complementary site-directed mutagenesis of the receptor's binding site were used to investigate the role of agonist-receptor interactions. We also confirmed that the depolarization-induced decrease of dopamine potency in GIRK activation is correlated by decreased binding of radiolabeled dopamine, and by decreased potency in G protein activation. In the mutagenesis experiments, a conserved serine residue as well as the conserved aspartate in the receptor's binding site were found to be important for voltage sensitive potency of dopamine. Furthermore, the voltage sensitivity of the receptor had distinct effects on different therapeutic D(2) agonists. Depolarization decreased the potency of several compounds, whereas for others, efficacy was reduced. For some agonists, both potency and efficacy were diminished, whereas for others still, neither parameter was significantly altered. The present work identifies some of the ligand-receptor interactions which determine agonist-specific effects of voltage at the dopamine D(2S) receptor. The observed differences between therapeutic agonists might be clinically relevant, and make them potential tools for investigating the roles of dopamine D(2) receptor voltage sensitivity in native tissue.


Subject(s)
Dopamine Agonists/pharmacology , Receptors, Dopamine D2/metabolism , Animals , Apomorphine/chemistry , Apomorphine/metabolism , Apomorphine/pharmacology , Benzothiazoles/chemistry , Benzothiazoles/pharmacology , Cell Culture Techniques , Dopamine/genetics , Dopamine/metabolism , Dopamine/pharmacology , Dopamine Agonists/chemistry , Dopamine Agonists/metabolism , Dose-Response Relationship, Drug , Female , Fluorescence Resonance Energy Transfer , Indoles/chemistry , Indoles/pharmacology , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Oocytes , Patch-Clamp Techniques , Piribedil/chemistry , Piribedil/pharmacology , Pramipexole , Protein Binding , Radioligand Assay , Receptors, Dopamine D2/agonists , Sensitivity and Specificity , Serine/genetics , Serine/metabolism , Tetrahydronaphthalenes/chemistry , Tetrahydronaphthalenes/pharmacology , Thiophenes/chemistry , Thiophenes/pharmacology , Transfection , Xenopus
19.
Biochim Biophys Acta ; 1808(5): 1245-55, 2011 May.
Article in English | MEDLINE | ID: mdl-21316336

ABSTRACT

While the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last fifteen years, the existence of a multi-receptor complex involving direct receptor-receptor interactions, called receptor oligomers, begins to be widely accepted. Eventually, it has been postulated that oligomers constitute a distinct functional form of the GPCRs with essential receptorial features. Also, it has been proven, under certain circumstances, that the GPCR oligomerization phenomenon is crucial for the receptor biosynthesis, maturation, trafficking, plasma membrane diffusion, and pharmacology and signalling. Adenosine receptors are GPCRs that mediate the physiological functions of adenosine and indeed these receptors do also oligomerize. Accordingly, adenosine receptor oligomers may improve the molecular mechanism by which extracellular adenosine signals are transferred to the G proteins in the process of receptor transduction. Importantly, these adenosine receptor-containing oligomers may allow not only the control of the adenosinergic function but also the fine-tuning modulation of other neurotransmitter systems (i.e. dopaminergic and glutamatergic transmission). Overall, we underscore here recent significant developments based on adenosine receptor oligomerization that are essential for acquiring a better understanding of neurotransmission in the central nervous system under normal and pathological conditions.


Subject(s)
Brain/metabolism , Dopamine/metabolism , Glutamic Acid/metabolism , Protein Multimerization , Receptors, Purinergic P1/metabolism , Synaptic Transmission/physiology , Animals , Humans , Signal Transduction/drug effects
20.
Biochem Biophys Res Commun ; 402(4): 801-7, 2010 Nov 26.
Article in English | MEDLINE | ID: mdl-21040702

ABSTRACT

A single serine point mutation (S374A) in the adenosine A(2A) receptor (A(2A)R) C-terminal tail reduces A(2A)R-D(2)R heteromerization and prevents its allosteric modulation of the dopamine D(2) receptor (D(2)R). By means of site directed mutagenesis of the A(2A)R and synthetic transmembrane (TM) α-helix peptides of the D(2)R we further explored the role of electrostatic interactions and TM helix interactions of the A(2A)R-D(2)R heteromer interface. We found evidence that the TM domains IV and V of the D(2)R play a major role in the A(2A)R-D(2)R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A(2A)R and D(2)R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A(2A)R-D(2)R heteromers. The mutation of two negatively charged aspartates in the A(2A)R C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A(2A)R-D(2)R interaction and lost the ability of antagonistic allosteric modulation over the A(2A)R-D(2)R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A(2A)R and the intracellular loop 3 (IL3) of D(2)R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A(2A)R-D(2L)R heteromer interface together with D(2L)R TM segments IV/V interacting with A(2A)R TM-IV/V or TM-I/VII.


Subject(s)
Receptor, Adenosine A2A/chemistry , Receptors, Dopamine D2/chemistry , Allosteric Regulation , Amino Acid Sequence , Aspartic Acid/chemistry , Aspartic Acid/genetics , Cell Line , Humans , Molecular Sequence Data , Mutation , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Receptor, Adenosine A2A/genetics , Receptors, Dopamine D2/genetics , Serine/chemistry , Serine/genetics
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