Modulation of dopamine D1 receptors via histamine H3 receptors is a novel therapeutic target for Huntington's disease.

Early Huntington's disease (HD) include over-activation of dopamine D1 receptors (D1R), producing an imbalance in dopaminergic neurotransmission and cell death. To reduce D1R over-activation, we present a strategy based on targeting complexes of D1R and histamine H3 receptors (H3R). Using an HD mouse striatal cell model and HD mouse organotypic brain slices we found that D1R-induced cell death signaling and neuronal degeneration, are mitigated by an H3R antagonist. We demonstrate that the D1R-H3R heteromer is expressed in HD mice at early but not late stages of HD, correlating with HD progression. In accordance, we found this target expressed in human control subjects and low-grade HD patients. Finally, treatment of HD mice with an H3R antagonist prevented cognitive and motor learning deficits and the loss of heteromer expression. Taken together, our results indicate that D1R - H3R heteromers play a pivotal role in dopamine signaling and represent novel targets for treating HD.

Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum.

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington's disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.

Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors.

Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.

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

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.

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

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.

NCS-1 associates with adenosine A(2A) receptors and modulates receptor function.

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.

Cannabinoid receptors CB1 and CB2 form functional heteromers in brain.

Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.

A new D2 dopamine receptor agonist allosterically modulates A(2A) adenosine receptor signalling by interacting with the A(2A)/D2 receptor heteromer.

The structural and functional interaction between D2 dopamine receptor (DR) and A(2A) adenosine receptor (AR) has suggested these two receptors as a pharmacological target in pathologies associated with dopamine dysfunction, such as Parkinson's disease. In transfected cell lines it has been demonstrated the activation of D2DR induces a significant negative regulation of A(2A)AR-mediated responses, whereas few data are at now available about the regulation of A(2A)AR by D2DR agonists at receptor recognition site. In this work we confirmed that in A(2A)AR/D2DR co-transfected cells, these receptors exist as homo- and hetero-dimers. The classical D2DR agonists were able to negatively modulate both A(2A)AR affinity and functionality. These effects occurred even if any significant changes in A(2A)AR/D2DR energy transfer interaction could be detected in BRET experiments. Since the development of new molecules able to target A(2A)/D2 dimers may represent an attractive tool for innovative pharmacological therapy, we also identified a new small molecule, 3-(3,4-dimethylphenyl)-1-(2-piperidin-1-yl)ethyl)piperidine (compound 1), full agonist of D2DR and modulator of A(2A)-D2 receptor dimer. This compound was able to negatively modulate A(2A)AR binding properties and functional responsiveness in a manner comparable to classical D2R agonists. In contrast to classical agonists, compound 1 led to conformational changes in the quaternary structure in D2DR homomers and heteromers and induced A(2A)AR/D2DR co-internalization. These results suggest that compound 1 exerts a high control of the function of heteromers and could represent a starting point for the development of new drugs targeting A(2A)AR/D2 DR heteromers.

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

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.

Expression of the mRNA coding the cannabinoid receptor 2 in the pallidal complex of Macaca fascicularis.

The putative presence of the cannabinoid receptor type 2 (CB(2)-R) in the central nervous system is still a matter of debate. Although first described in peripheral and immune tissues, evidence suggesting the existence of CB(2)-Rs in glial cells and even neurons has been made available more recently. By taking advantage of newly designed CB(2)-R mRNA riboprobes, we have demonstrated by in situ hybridization and PCR the existence of CB2-R transcripts in a variety of brain areas of the primate Macaca fascicularis, including the cerebral cortex and the hippocampus, as well as in the external and internal divisions of the globus pallidus, both pallidal segments showing the highest abundance of CB(2)-R transcripts. In this regard, the presence of the messenger coding CB(2)-Rs within the pallidal complex highlights their consideration as potential targets for the treatment of movement disorders of basal ganglia origin.

Direct involvement of sigma-1 receptors in the dopamine D1 receptor-mediated effects of cocaine.

It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D(1) receptors (D(1)Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D(1)R and σ(1)R were found to heteromerize in transfected cells, where cocaine robustly potentiated D(1)R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D(1)R stimulation in a dopamine transporter-independent and σ(1)R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ(1)R KO mice, providing evidence for the existence of σ(1)R-D(1)R heteromers in the brain. Therefore, these results provide a molecular explanation for which D(1)R plays a more significant role in the behavioral effects of cocaine, through σ(1)R-D(1)R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction.

Prime time for G-protein-coupled receptor heteromers as therapeutic targets for CNS disorders: the dopamine D1-D3 receptor heteromer.

A number of G-protein-coupled receptors (GPCRs) are currently under consideration as potential therapeutic targets for drugs acting in the central nervous system (CNS). Attempts to discover new medications have operated under the assumption that GPCRs are monomers and that a specific drug activates one single receptor coupled to one single signal transduction mechanism. In the neuronal membrane, GPCRs are now known to be arranged into homo- and hetero-oligomers; drugs acting on a single receptor within a specific heteromer context are thought to induce a particular downstream signaling. However, there is recent evidence showing that heteromer-tailored drugs can be designed that display different affinities for a given receptor depending on the receptor partners contained within the heteromer. It can therefore be predicted that customized drugs targeting a specific receptor heteromer in the CNS might improve safety and efficacy for their therapeutic targets. Finally, it will be important to identify receptor heteromers that are involved in the pathogenesis of diseases, such as the recently discovered dopamine D1-D3 receptor heteromer, which might play a key role in L-DOPA-induced dyskinesia in Parkinson's disease.

Gi protein coupling to adenosine A1-A2A receptor heteromers in human brain caudate nucleus.

Pharmacological characterization of adenosine A(1) and A(2A) receptors in human brain caudate nucleus membranes led to non-cooperative binding of radiolabelled ligands. In human caudate nucleus but not in cortex, the agonist binding to A(1) receptors was modulated by the agonist binding to A(2A) receptors indicating a functional negative cross-talk. Accordingly, the A(1) receptor-activation-mediated G(i)-dependent guanosine 5'-o-(3-[(35)S]thio-triphosphate) binding was modulated by agonist binding to A(2A) receptors. A(2A) receptors occupation led to a decrease in the potency of A(1) receptor agonists. These results indicate that A(1) but not A(2A) receptors activation, likely occurring at low adenosine concentrations, engages a G(i)-mediated signaling; however, when both receptors are occupied by adenosine, there is an A(2A) receptor-mediated impairment of G(i)-operated transducing units. These findings are relevant to get insight into the complex relationships derived from co-expression of multiple neurotransmitter/neuromodulator receptors subtypes that individually are coupled to different G proteins. A further finding was the demonstration that the A(2A) receptor agonist, CGS 21680, at high concentrations able to significantly bind to the A(1) receptor, behaved as a partial agonist of the later receptor. This fact might be taken into account when characterizing CGS 21680 actions in human cells expressing A(1) receptors when the compound is used at micromolar concentrations.

A hybrid indoloquinolizidine peptide as allosteric modulator of dopamine D1 receptors.

The indoloquinolizidine-peptide 28 [(3S,12bR)-N-((S)-1-((S)-1-((S)-2-carbamoylpyrrolidin-1-yl)-3-(4-fluorophenyl)-1-oxopropan-2-ylamino)-4-cyclohexyl-1-oxobutan-2-yl)-1,2,3,4,6,7,12, 12b-octahydroindolo[2,3-a]quinolizine-3-carboxamide], a trans-indoloquinolizidine-peptide hybrid obtained by a combinatorial approach, behaved as an orthosteric ligand of all dopamine D(2)-like receptors (D(2), D(3), and D(4)) and dopamine D(5) receptors, but as a negative allosteric modulator of agonist and antagonist binding to striatal dopamine D(1) receptors. Indoloquinolizidine-peptide 28 induced a concentration-dependent hyperbolic increase in the antagonist apparent equilibrium dissociation constant values and altered the dissociation kinetics of dopamine D(1) receptor antagonists. The negative allosteric modulation was also found when agonist binding to D(1) receptors was assayed. Indoloquinolizidine-peptide 28 was a weak ago-allosteric modulator but markedly led to a decreased potency without decreasing the maximum partial/full agonist-mediated effect on cAMP levels. Compounds able to decrease the potency while preserving the efficacy of D(1) receptor agonists are promising for exploration in psychotic pathologies.

GPCR homomers and heteromers: a better choice as targets for drug development than GPCR monomers?

G protein-coupled receptors (GPCR) are targeted by many therapeutic drugs marketed to fight against a variety of diseases. Selection of novel lead compounds are based on pharmacological parameters obtained assuming that GPCR are monomers. However, many GPCR are expressed as dimers/oligomers. Therefore, drug development may consider GPCR as homo- and hetero-oligomers. A two-state dimer receptor model is now available to understand GPCR operation and to interpret data obtained from drugs interacting with dimers, and even from mixtures of monomers and dimers. Heteromers are distinct entities and therefore a given drug is expected to have different affinities and different efficacies depending on the heteromer. All these concepts would lead to broaden the therapeutic potential of drugs targeting GPCRs, including receptor heteromer-selective drugs with a lower incidence of side effects, or to identify novel pharmacological profiles using cell models expressing receptor heteromers.

Immunodensity and mRNA expression of A2A adenosine, D2 dopamine, and CB1 cannabinoid receptors in postmortem frontal cortex of subjects with schizophrenia: effect of antipsychotic treatment.

RATIONALE: Dopamine D2 receptors are the main target of antipsychotic drugs. In the brain, D2 receptors coexpress with adenosine A2A and CB1 cannabinoid receptors, leading to functional interactions. OBJECTIVES: The protein and messenger RNA (mRNA) contents of A2A, D2, and CB1 receptors were quantified in postmortem prefrontal cortex of subjects with schizophrenia. MATERIALS AND METHODS: The study was performed in subjects suffering schizophrenia (n=31) who mainly died by suicide, matched with non-schizophrenia suicide victims (n=13) and non-suicide controls (n=33). The density of receptor proteins was evaluated by immunodetection techniques, and their relative mRNA expression was quantified by quantitative real-time polymerase chain reaction. RESULTS: In schizophrenia, the densities of A2A (90+/-6%, n=24) and D2-like receptors (95+/-5%, n=22) did not differ from those in controls (100%). Antipsychotic treatment did not induce changes in the protein expression. In contrast, the immunodensity of CB1 receptors was significantly decreased (71+/-7%, n=11; p<0.05) in antipsychotic-treated subjects with schizophrenia but not in drug-free subjects (104+/-13%, n=11). The relative mRNA amounts encoding for A2A, D2, and CB1 receptors were similar in brains of drug-free, antipsychotic-treated subjects with schizophrenia and controls. CONCLUSIONS: The findings suggest that antipsychotics induce down-regulation of CB1 receptors in brain. Since A2A, D2, and CB1 receptors coexpress on brain GABAergic neurons and reductions in markers of GABA neurotransmission have been identified in schizophrenia, a lower density of CB1 receptor induced by antipsychotics could represent an adaptative mechanism that reduces the endocannabinoid-mediated suppression of GABA release, contributing to the normalization of cognitive functions in the disorder.

Useful pharmacological parameters for G-protein-coupled receptor homodimers obtained from competition experiments. Agonist-antagonist binding modulation.

Many G-protein-coupled receptors (GPCRs) are expressed on the plasma membrane as dimers. Since drug binding data are currently fitted using equations developed for monomeric receptors, the interpretation of the pharmacological data are equivocal in many cases. As reported here, GPCR dimer models account for changes in competition curve shape as a function of the radioligand concentration used, something that cannot be explained by monomeric receptor models. Macroscopic equilibrium dissociation constants for the agonist and homotropic cooperativity index reflecting the intramolecular communication within the dopamine D1 or adenosine A2A receptor homodimer as well as hybrid equilibrium dissociation constant, which reflects the antagonist/agonist modulation may be calculated by fitting binding data from antagonist/agonist competition experiments to equations developed from dimer receptor models. Comparing fitting the data by assuming a classical monomeric receptor model or a dimer model, it is shown that dimer receptor models provide more clues useful in drug discovery than monomer-based models.

Adenosine A2A receptor-antagonist/dopamine D2 receptor-agonist bivalent ligands as pharmacological tools to detect A2A-D2 receptor heteromers.

Adenosine A(2A) (A(2A)R) and dopamine D(2) (D(2)R) receptors mediate the antagonism between adenosinergic and dopaminergic transmission in striatopallidal GABAergic neurons and are pharmacological targets for the treatment of Parkinson's disease. Here, a family of heterobivalent ligands containing a D(2)R agonist and an A(2A)R antagonist linked through a spacer of variable size was designed and synthesized to study A(2A)R-D(2)R heteromers. Bivalent ligands with shorter linkers bound to D(2)R or A(2A)R with higher affinity than the corresponding monovalent controls in membranes from brain striatum and from cells coexpressing both receptors. In contrast, no differences in affinity of bivalent versus monovalent ligands were detected in experiments using membranes from cells expressing only one receptor. These findings indicate the existence of A(2A)R-D(2)R heteromers and of a simultaneous interaction of heterobivalent ligands with both receptors. The cooperative effect derived from the simultaneous interaction suggests the occurrence of A(2A)R-D(2)R heteromers in cotransfected cells and in brain striatum. The dopamine/adenosine bivalent action could constitute a novel concept in Parkinson's disease pharmacotherapy.

Interactions between calmodulin, adenosine A2A, and dopamine D2 receptors.

The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents.