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An increasing number of G protein-coupled receptors (GPCRs) have been reported to be expressed in the plasma membrane as dimers. Since most ligand binding data are currently fitted by classical equations developed only for monomeric receptors, the interpretation of data could be misleading in the presence of GPCR dimers. On the other hand, the equations developed from dimer receptor models assuming the existence of two orthosteric binding sites within the dimeric molecule offer the possibility to directly calculate macroscopic equilibrium dissociation constants for the two sites, an index of cooperativity (D(c)) that reflects the molecular communication within the dimer and, importantly, a constant of radioligand-competitor allosteric interaction (K(DAB)) in competitive assays. Here, we provide a practical way to fit competitive binding data that allows the interpretation of apparently anomalous results, such as competition curves that could be either bell-shaped, monophasic or biphasic depending on the assay conditions. The consideration of a radioligand-competitorǀ allosteric interaction allows fitting these curve patterns both under simulation conditions and in real radioligand binding experiments, obtaining competitor affinity parameters closer to the actual values. Our approach is the first that, assuming the formation of receptor homodimers, is able to explain several experimental results previously considered erroneous due to their impossibility to be fitted. We also deduce the radioligand concentration responsible for the conversion of biphasic to monophasic or to bell-shaped curves in competitive radioligand binding assays. In conclusion, bell-shaped curves in competitive binding experiments constitute evidence for GPCR homodimerization.

Polymorphic alleles of the human dopamine D(4) receptor gene (DRD4) have been consistently associated with individual differences in personality traits and neuropsychiatric disorders, particularly between the gene encoding dopamine D(4.7) receptor variant and attention deficit hyperactivity disorder (ADHD). The α(2A) adrenoceptor gene has also been associated with ADHD. In fact, drugs targeting the α(2A) adrenoceptor (α(2A)R), such as guanfacine, are commonly used in ADHD treatment. In view of the involvement of dopamine D(4) receptor (D(4)R) and α(2A)R in ADHD and impulsivity, their concurrent localization in cortical pyramidal neurons and the demonstrated ability of D(4)R to form functional heteromers with other G protein-coupled receptors, in this study we evaluate whether the α(2A)R forms functional heteromers with D(4)R and weather these heteromers show different properties depending on the D(4)R variant involved. Using cortical brain slices from hD(4.7)R knock-in and wild-type mice, here, we demonstrate that α(2A)R and D(4)R heteromerize and constitute a significant functional population of cortical α(2A)R and D(4)R. Moreover, in cortical slices from wild-type mice and in cells transfected with α(2A)R and D(4.4)R, we detect a negative crosstalk within the heteromer. This negative crosstalk is lost in cortex from hD(4.7)R knock-in mice and in cells expressing the D(4.7)R polymorphic variant. We also show a lack of efficacy of D(4)R ligands to promote G protein activation and signaling only within the α(2A)R-D(4.7)R heteromer. Taken together, our results suggest that α(2A)R-D(4)R heteromers play a pivotal role in catecholaminergic signaling in the brain cortex and are likely targets for ADHD pharmacotherapy.

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 striatal cell model and HD 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 animal models 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, as well as 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.Impact StatementProgression of Huntington’s disease can be slowed by altering dopamine signalling through the Dopamine 1 receptor - Histamine 3 receptor heteromer.

Bivalent ligands have emerged as chemical tools to study G protein-coupled receptor dimers. Using a combination of computational, chemical, and biochemical tools, here we describe the design of bivalent ligand 13 with high affinity (K(DB1)=21 pM) for the dopamine D(2) receptor (D(2)R) homodimer. Bivalent ligand 13 enhances the binding affinity relative to monovalent compound 15 by 37-fold, indicating simultaneous binding at both protomers. Using synthetic peptides with amino acid sequences of transmembrane (TM) domains of D(2)R, we provide evidence that TM6 forms the interface of the homodimer. Notably, the disturber peptide TAT-TM6 decreased the binding of bivalent ligand 13 by 52-fold and had no effect on monovalent compound 15, confirming the D(2)R homodimer through TM6 ex vivo. In conclusion, using a versatile multivalent chemical platform, we have developed a precise strategy to generate a true bivalent ligand that simultaneously targets both orthosteric sites of the D(2)R homodimer.

The poor norepinephrine innervation and high density of Gi/o-coupled α2A- and α2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D2-like receptor ligands, such as the D3 receptor agonist 7-OH-PIPAT and the D4 receptor agonist RO-105824, to α2-adrenoceptors in cortical and striatal tissue, which express α2A-adrenoceptors and both α2A- and α2C-adrenoceptors, respectively. The affinity of dopamine for α2-adrenoceptors was found to be similar to that for D1-like and D2-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α2A- and α2C-adrenoceptors. Their ability to activate Gi/o proteins through α2A- and α2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α2-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α2A- and α2C-adrenoceptors was nearly identical to its binding to the crystallized D3 receptor. Therefore, we provide conclusive evidence that α2A- and α2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D2-like receptor ligands, which calls for revisiting previous studies with those ligands.

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., It is unclear whether GPCRs, G proteins and adenylyl cyclase (AC) associate through random collisions or defined pre-coupling mechanisms. Using a peptide-based approach, the authors show that heteromers of adenosine A2A and dopamine D2 receptors form pre-coupled complexes with their cognate G proteins and AC5.

Introduction: G protein-coupled receptors (GPCRs) are a superfamily of membrane proteins highly expressed in the brain that are involved in almost all functions of the CNS. During the last twenty years, a large number of GPCRs have been reported to form homodimers, heterodimers and higher order oligomers. Areas covered: This review summarizes the functional and pharmacological characteristics of the dopamine D1 receptor (D1R) interactome constituted by heteromers with GPCRs or non-GPCRs. The review also focuses on heteromer-selective ligands reported for GPCRs, including those for the receptor-based interactome of D1R. Expert opinion: Since the D1R plays a key role in basal ganglia motor control, where all the mentioned D1R heteromers are present, the study of allosteric interactions within the D1R interactome may be of high therapeutic interest for treating motor dysfunction. Moreover, several of these heteromers have also been detected in the prefrontal cortex and hippocampus, where they are involved in learning, memory and attention dysfunction. We propose that drugs targeting specific D1R heteromers in the CNS will be more effective and safer, resulting in a reduction of side effects compared with traditional drugs targeting monomeric receptors. Heteromer-selective ligands will have a big impact on drug development with many pharmacological and clinical implications.

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.

Dopaminergic and purinergic signaling play a pivotal role in neurological diseases associated with motor symptoms, including Parkinson's disease (PD), multiple sclerosis, amyotrophic lateral sclerosis, Huntington disease, Restless Legs Syndrome (RLS), spinal cord injury (SCI), and ataxias. Extracellular dopamine and adenosine exert their functions interacting with specific dopamine (DR) or adenosine (AR) receptors, respectively, expressed on the surface of target cells. These receptors are members of the family A of G protein-coupled receptors (GPCRs), which is the largest protein superfamily in mammalian genomes. GPCRs are target of about 40% of all current marketed drugs, highlighting their importance in clinical medicine. The striatum receives the densest dopamine innervations and contains the highest density of dopamine receptors. The modulatory role of adenosine on dopaminergic transmission depends largely on the existence of antagonistic interactions mediated by specific subtypes of DRs and ARs, the so-called A

Several studies found in vitro evidence for heteromerization of dopamine D(1) receptors (D1R) and D(3) receptors (D3R), and it has been postulated that functional D1R-D3R heteromers that are normally present in the ventral striatum mediate synergistic locomotor-activating effects of D1R and D3R agonists in rodents. Based also on results obtained in vitro, with mammalian transfected cells, it has been hypothesized that those behavioral effects depend on a D1R-D3R heteromer-mediated G protein-independent signaling. Here, we demonstrate the presence on D1R-D3R heteromers in the mouse ventral striatum by using a synthetic peptide that selectively destabilizes D1R-D3R heteromers. Parallel locomotor activity and ex vivo experiments in reserpinized mice and in vitro experiments in D1R-D3R mammalian transfected cells were performed to dissect the signaling mechanisms of D1R-D3R heteromers. Co-administration of D1R and D3R agonists in reserpinized mice produced synergistic locomotor activation and a selective synergistic AKT phosphorylation in the most ventromedial region of the striatum, in the shell of the nucleus accumbens. Application of the destabilizing peptide in transfected cells and in the shell of the nucleus accumbens allowed demonstrating that, both in vitro and in vivo, co-activation of D3R induces a switch from G protein-dependent to G protein-independent D1R-mediated signaling determined by D1R-D3R heteromerization. The results therefore demonstrate that a biased G protein-independent signaling of D1R-D3R heteromers localized in the shell of the nucleus accumbens mediate the locomotor synergistic effects of D1R and D3R agonists in reserpinized mice.

Dopaminergic and purinergic signaling play a pivotal role in neurological diseases associated with motor symptoms, including Parkinson's disease (PD), multiple sclerosis, amyotrophic lateral sclerosis, Huntington disease, Restless Legs Syndrome (RLS), spinal cord injury (SCI), and ataxias. Extracellular dopamine and adenosine exert their functions interacting with specific dopamine (DR) or adenosine (AR) receptors, respectively, expressed on the surface of target cells. These receptors are members of the family A of G protein-coupled receptors (GPCRs), which is the largest protein superfamily in mammalian genomes. GPCRs are target of about 40% of all current marketed drugs, highlighting their importance in clinical medicine. The striatum receives the densest dopamine innervations and contains the highest density of dopamine receptors. The modulatory role of adenosine on dopaminergic transmission depends largely on the existence of antagonistic interactions mediated by specific subtypes of DRs and ARs, the so-called A2AR–D2R and A1R–D1R interactions. Due to the dopamine/adenosine antagonism in the CNS, it was proposed that ARs and DRs could form heteromers in the neuronal cell surface. Therefore, adenosine can affect dopaminergic signaling through receptor–receptor interactions and by modulations in their shared intracellular pathways in the striatum and spinal cord. In this work we describe the allosteric modulations between GPCR protomers, focusing in those of adenosine and dopamine within the A1R–D1R heteromeric complex, which is involved in RLS. We also propose that the knowledge about the intricate allosteric interactions within the A1R–D1R heterotetramer, may facilitate the treatment of motor alterations, not only when the dopamine pathway is hyperactivated (RLS, chorea, etc.) but also when motor function is decreased (SCI, aging, PD, etc.).

Background G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. Results We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. Conclusions We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR.

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

The poor norepinephrine innervation and high density of Gi/o-coupled α(2A) and α(2C) adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine and several compounds reported as selective dopamine D(2)-like receptor ligands, such as the D(3) receptor agonist 7-OH-PIPAT and the D(4) receptor agonist RO-105824, to α(2) adrenoceptors in cortical and striatal tissue, which express α(2A) adrenoceptors and both α(2A) and α(2C) adrenoceptors, respectively. The affinity of dopamine for α(2) adrenoceptors was found to be similar to that for D(1)-like and D(2)-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α(2A) and α(2C) adrenoceptors. Their ability to activate Gi/o proteins through α(2A) and α(2C) adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α(2) adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α(2A) and α(2C) adrenoceptors was nearly identical to its binding to the crystallized D(3) receptor. Therefore, we provide conclusive evidence that α(2A) and α(2C) adrenoceptors are functional receptors for norepinephrine, dopamine and other previously assumed selective D(2)-like receptor ligands, which calls for revisiting previous studies with those ligands.

Background G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. Results We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. Conclusions We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR. Electronic supplementary material The online version of this article (10.1186/s12915-018-0491-x) contains supplementary material, which is available to authorized users.

The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists.

Interest in adenosine deaminase (ADA) in the context of medicine has mainly focused on its enzymatic activity. This is justified by the importance of the reaction catalyzed by ADA not only for the intracellular purine metabolism, but also for the extracellular purine metabolism as well, because of its capacity as a regulator of the concentration of extracellular adenosine that is able to activate adenosine receptors (ARs). In recent years, other important roles have been described for ADA. One of these, with special relevance in immunology, is the capacity of ADA to act as a costimulator, promoting T-cell proliferation and differentiation mainly by interacting with the differentiation cluster CD26. Another role is the ability of ADA to act as an allosteric modulator of ARs. These receptors have very general physiological implications, particularly in the neurological system where they play an important role. Thus, ADA, being a single chain protein, performs more than one function, consistent with the definition of a moonlighting protein. Although ADA has never been associated with moonlighting proteins, here we consider ADA as an example of this family of multifunctional proteins. In this review, we discuss the different roles of ADA and their pathological implications. We propose a mechanism by which some of their moonlighting functions can be coordinated. We also suggest that drugs modulating ADA properties may act as modulators of the moonlighting functions of ADA, giving them additional potential medical interest.

Using bioluminescence resonance energy transfer and proximity ligation assays, we obtained the first direct evidence that adenosine A₁ receptors (A₁Rs) form homomers not only in cell cultures but also in brain cortex. By radioligand binding experiments in the absence or in the presence of the A₁Rs allosteric modulator, adenosine deaminase, and by using the two-state dimer receptor model to fit binding data, we demonstrated that the protomer-protomer interactions in the A₁R homomers account for some of the pharmacological characteristics of agonist and antagonist binding to A₁Rs. These pharmacological properties include the appearance of cooperativity in agonist binding, the change from a biphasic saturation curve to a monophasic curve in self-competition experiments and the molecular cross-talk detected when two different specific molecules bind to the receptor. In this last case, we discovered that caffeine binding to one protomer increases the agonist affinity for the other protomer in the A₁R homomer, a pharmacological characteristic that correlates with the low caffeine concentrations-induced activation of agonist-promoted A₁R signaling. This pharmacological property can explain the biphasic effects reported at low and high concentration of caffeine on locomotor activity.

Caffeine induces locomotor activation by its ability to block adenosine receptors. Caffeine is metabolized to several methylxanthines, with paraxanthine being the main metabolite in humans. In this study we show that in rats paraxanthine has a stronger locomotor activating effect than caffeine or the two other main metabolites of caffeine, theophylline and theobromine. As previously described for caffeine, the locomotor activating doses of paraxanthine more efficiently counteract the locomotor depressant effects of an adenosine A1 than an adenosine A2A receptor agonist. In drug discrimination experiments in rats trained to discriminate a maximal locomotor activating dose of caffeine, paraxanthine, unlike theoph- ylline, generalized poorly to caffeine suggesting the existence of additional mechanisms other than adenosine antagonism in the behavioral effects of paraxanthine. Pretreatment with the nitric oxide inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) reduced the locomotor activating effects of par- axanthine, but not caffeine. On the other hand, pretreatment with the selective cGMP-preferring phosphodiesterase PDE9 inhibitor BAY 73-6691, increased locomotor activity induced by caffeine, but not paraxanthine. Ex vivo experiments demonstrated that paraxanthine, but not caffeine, can induce cGMP accumulation in the rat striatum. Finally, in vivo microdialysis experiments showed that para- xanthine, but not caffeine, significantly increases extracellular levels of dopamine in the dorsolateral striatum, which was blocked by L-NAME. These findings indicate that inhibition of cGMP-preferring PDE is involved in the locomotor activating effects of the acute administration of paraxanthine. The present results demonstrate a unique psychostimulant profile of paraxanthine, which might contribute to the reinforcing effects of caffeine in humans. Published by Elsevier Ltd.

The neuropeptide galanin has been shown to interact with the opioid system. More specifically, galanin counteracts the behavioral effects of the systemic administration of μ-opioid receptor (MOR) agonists. Yet the mechanism responsible for this galanin–opioid interaction has remained elusive. Using biophysical techniques in mammalian transfected cells, we found evidence for selective heteromerization of MOR and the galanin receptor subtype Gal1 (Gal1R). Also in transfected cells, a synthetic peptide selectively disrupted MOR–Gal1R heteromerization as well as specific interactions between MOR and Gal1R ligands: a negative cross talk, by which galanin counteracted MAPK activation induced by the endogenous MOR agonist endomorphin-1, and a cross-antagonism, by which a MOR antagonist counteracted MAPK activation induced by galanin. These specific interactions, which represented biochemical properties of the MOR-Gal1R heteromer, could then be identifiedin situin slices of rat ventral tegmental area (VTA) with MAPK activation and two additional cell signaling pathways, AKT and CREB phosphorylation. Furthermore,in vivomicrodialysis experiments showed that the disruptive peptide selectively counteracted the ability of galanin to block the dendritic dopamine release in the rat VTA induced by local infusion of endomorphin-1, demonstrating a key role of MOR-Gal1R heteromers localized in the VTA in the direct control of dopamine cell function and their ability to mediate antagonistic interactions between MOR and Gal1R ligands. The results also indicate that MOR-Gal1R heteromers should be viewed as targets for the treatment of opioid use disorders.SIGNIFICANCE STATEMENTThe μ-opioid receptor (MOR) localized in the ventral tegmental area (VTA) plays a key role in the reinforcing and addictive properties of opioids. With parallelin vitroexperiments in mammalian transfected cells andin situandin vivoexperiments in rat VTA, we demonstrate that a significant population of these MORs form functional heteromers with the galanin receptor subtype Gal1 (Gal1R), which modulate the activity of the VTA dopaminergic neurons. The MOR-Gal1R heteromer can explain previous results showing antagonistic galanin–opioid interactions and offers a new therapeutic target for the treatment of opioid use disorder.

Dopamine is a neurotransmitter widely distributed in both the periphery and the central nervous system (CNS). Its physiological effects are mediated by five closely related G protein-coupled receptors (GPCRs) that are divided into two major subclasses: the D1-like (D1, D5) and the D2-like (D2, D3, D4) receptors. D3 receptors (D3Rs) have the highest density in the limbic areas of the brain, which are associated with cognitive and emotional functions. These receptors are therefore attractive targets for therapeutic management.This review summarizes the functional and pharmacological characteristics of D3Rs, including the design and clinical relevance of full agonists, partial agonists and antagonists, as well as the capacity of these receptors to form active homodimers, heterodimers or higher order receptor complexes as pharmacological targets in several neurological and neurodegenerative disorders.The high sequence homology between D3R and the D2-type challenges the development of D3R-selective compounds. The design of new D3R-preferential ligands with improved physicochemical properties should provide a better pharmacokinetic/bioavailability profile and lesser toxicity than is found with existing D3R ligands. It is also essential to optimize D3R affinity and, especially, D3R vs. D2-type binding and functional selectivity ratios. Developing allosteric and bitopic ligands should help to improve the D3R selectivity of these drugs. As most evidence points to the ability of GPCRs to form homomers and heteromers, the most promising therapeutic strategy in the future is likely to involve the application of heteromer-selective drugs. These selective ligands would display different affinities for a given receptor depending on the receptor partners within the heteromer. Therefore, designing novel compounds that specifically target and modulate D1R-D3R heteromers would be an interesting approach for the treatment of levodopa (L-DOPA)-induced dyskinesias.

Sensory information in the mammalian brain is encoded in the cortex. The brain structure is considered to store, at least partially, long-term memories. This area modulates planning, cognitive flexibility, abstract thinking, rule acquisition, implementation, behavioral inhibition, and the selection of relevant sensory information, playing a key role in almost all aspects of cognition. One factor affecting cognitive functions is psychostimulant usage. Psychostimulants are a broad class of sympathomimetic drugs that, at low doses, can increase arousal, vigilance, vigor and attention, and cognitive enhancement; however, at high doses, their ability to induce cognitive deficits as well as addiction has been described. Caffeine is one of the most important naturally occurring methylated xanthine alkaloids and it is the most widely used psychoactive drug in the world. The biological effects of caffeine in the brain are developed throughout a wide range of molecular targets. The most important of them are adenosine receptors, where xanthinic compounds act as antagonists with a low micromolar range of affinities. Among these, adenosine A 1 and A 2A receptors are the preferential targets for caffeine in the brain, since physiological extracellular levels of adenosine are sufficient to occupy and stimulate these receptors. However, the amount of A 2A subtype in the brain cortex is negligible in comparison with the levels of A 1 . For this reason, the cortical effects of caffeine are mainly by A 1 antagonism. Using bioluminescence resonance energy transfer and proximity ligation assays, the first direct evidence that adenosine A 1 receptors form homomers in the brain cortex has been obtained. Moreover, by radioligand binding experiments and fitting the data obtained using the two-state dimer receptor model, it has been demonstrated that the protomer–protomer interactions within A 1 receptor homomers account for some unexpected pharmacological characteristics that can explain some biphasic effects reported at both low and high concentrations of caffeine.

The enzyme adenosine deaminase (ADA) is a multifunctional protein that can both degrade adenosine and bind extracellularly to adenosine receptors, acting as an allosteric modulator regulating the hormonal effects of adenosine. The molecular regions of ADA responsible for the latter are unknown. In this work, alanine scanning mutagenesis of various ADA amino acid stretches, selected through in silico docking experiments, allowed us to identify regions of the enzyme responsible for modulating both its catalytic activity and its ability to modulate agonist binding to A and A adenosine receptors (AR and AR). The combination of computational and in vitro experiments show that the structural gate to the catalytic site; i.e., the α-1 helix containing residues L58-I72 and the loop containing residues A184-I188 of ADA, were important to maintain both the catalytic efficiency of the enzyme and its action as an allosteric modulator of the adenosine receptors. These data are consistent with a predicted supramolecular assembly, in which ADA bridges AR and CD26 and are in line with the notion that the interaction of ADA with adenosine receptors has an important role in the immunosynapse. We propose that it is the ADA open form, but not the closed one, that is responsible for the functional interaction with A₁R and A₂AR.

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.

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.

Background and purpose: Functional interactions between the G protein-coupled dopamine D1 and histamine H3 receptors have been described in the brain. In the present study we investigated the existence of D1–H3 receptor heteromers and their biochemical characteristics. Experimental approach: D1–H3 receptor heteromerization was studied in mammalian transfected cells with Bioluminescence Resonance Energy Transfer and binding assays. Furthermore, signalling through mitogen-activated protein kinase (MAPK) and adenylyl cyclase pathways was studied in co-transfected cells and compared with cells transfected with either D1 or H3 receptors. Key results: Bioluminescence Resonance Energy Transfer and binding assays confirmed that D1 and H3 receptors can heteromerize. Activation of histamine H3 receptors did not lead to signalling towards the MAPK pathway unless dopamine D1 receptors were co-expressed. Also, dopamine D1 receptors, usually coupled to Gs proteins and leading to increases in cAMP, did not couple to Gs but to Gi in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor. Conclusions and implications: D1–H3 receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a Gs-independent and Gi-dependent manner. An antagonist of one of the receptor units in the D1–H3 receptor heteromer can induce conformational changes in the other receptor unit and block specific signals originating in the heteromer. This gives rise to unsuspected therapeutic potentials for G protein-coupled receptor antagonists.

It has been shown that adenosine deaminase (ADA; EC 3.5.4.4) behaves as an ecto-enzyme anchored to membrane proteins, among them A1 adenosine receptors (A1Rs). Bovine ADA interacts with A1Rs from many species and regulates agonists binding to receptors in an activity-independent form. However, it was not known whether human ADA exerted any effect on the agonist binding to human A1Rs, because of both technical difficulties in obtaining pure human ADA and tissues containing human A1Rs. In this study, human ADA was purified to homogeneity. Taking in consideration that A1Rs form homodimers and taking advantage of a new procedure to fit binding data to receptors dimers, which allows to calculate ligand dissociation constants and the degree of cooperativity between the two subunits in the dimer, here it is demonstrated that human ADA markedly enhances the agonist and antagonist affinity and abolishes the negative cooperativity on agonist binding to human striatal A1Rs. ADA also increases the ability of the agonist to decrease the forskolin-induced cAMP levels. The results show that human ADA, apart from reducing the adenosine concentration and thus preventing A1R desensitization, binds to A1R behaving as an allosteric effector that markedly enhances agonist affinity and increases receptor functionality. The physiological role of the interaction is to make receptors more sensitive to adenosine. This powerful regulation has important implications for the physiology and pharmacology of neuronal A1Rs.

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.

Almost all existing models for G-protein-coupled receptors (GPCRs) are based on the occurrence of monomers. Recent studies show that many GPCRs are dimers. Therefore for some receptors dimers and not monomers are the main species interacting with hormones/neurotransmitters/drugs. There are reasons for equivocal interpretations of the data fitting to receptor dimers assuming they are monomers. Fitting data using a dimer-based model gives not only the equilibrium dissociation constants for high and low affinity binding to receptor dimers but also a ‘cooperativity index’ that reflects the molecular communication between monomers within the dimer. The dimer cooperativity index (DC) is a valuable tool that enables to interpret and quantify, for instance, the effect of allosteric regulators. For different receptors heteromerization confers a specific functional property for the receptor heteromer that can be considered as a ‘dimer fingerprint’. The occurrence of heteromers with different pharmacological and signalling properties opens a complete new field to search for novel drug targets useful to combat a variety of diseases and potentially with fewer side effects. Antagonists, which are quite common marketed drugs targeting GPCRs, display variable affinities when a given receptor is expressed with different heteromeric partners. This fact should be taken into account in the development of new drugs. British Journal of Pharmacology (2008) 153, S90–S98; doi:10.1038/sj.bjp.0707571; published online 26 November 2007

Almost all existing models that explain heptahelical G-protein-coupled receptor (GPCR) operation are based on the occurrence of monomeric receptor species. However, an increasing number of studies show that many G-protein-coupled heptahelical membrane receptors (HMR) are expressed in the plasma membrane as dimers. We here review the approaches for fitting ligand binding data that are based on the existence of receptor monomers and also the new ones based on the existence of receptor dimers. The reasons for equivocal interpretations of the fitting of data to receptor dimers, assuming they are monomers, are also discussed. A recently devised model for receptor dimers provides a new approach for fitting data that eventually gives more accurate and physiological relevant parameters. Fitting data using the new procedure gives not only the equilibrium dissociation constants for high- and low-affinity binding to receptor dimers but also a "cooperativity index" that reflects the molecular communication within the dimer. A comprehensive way to fit binding data from saturation isotherms and from competition assays to a dimer receptor model is reported and compared with the traditional way of fitting data. The new procedure can be applied to any receptor forming dimers; from receptor tyrosine kinases to intracellular receptors (e.g., estrogen receptor) and in general for ligand binding to proteins forming dimers.

Dopamine receptors in striatum are important for healthy brain functioning and are the target of levodopa-based therapy in Parkinson's disease. Lateralization of dopaminergic neurotransmission in striata from different hemispheres occurs in patients, but also in healthy individuals. Our data show that the affinity of dopamine binding to dopamine D1 receptors is significantly higher in left than in right striatum. Analysis of data from radioligand binding to striatal samples from naive, 6-hydroxydopamine lesioned, levodopa-treated and levodopa-induced dyskinetic rats shows differential receptor structure and gives hints on the causes of right/left lateralization of dopamine binding to striatal D1 receptors. Moreover, binding data showed loss of lateralization in levodopa (L-DOPA)-induced dyskinetic rats.

Significance G protein-coupled receptors (GPCRs) constitute the largest plasma membrane protein family involved in cell signaling. GPCR homodimers are predominant species, and GPCR heteromers likely are constituted by heteromers of homodimers. The adenosine A 2A receptor (A 2A R)-dopamine D 2 receptor (D 2 R) heteromer is a target for the nonselective adenosine receptor antagonist caffeine. This study uncovers allosteric modulations of A 2A R antagonists that mimic those of A 2A R agonists, challenging the traditional view of antagonists as inactive ligands. These allosteric modulations disappear when agonist and antagonist are coadministered, however. A model is proposed that considers A 2A R-D 2 R heteromers as heterotetramers, constituted by A 2A R and D 2 R homodimers. The model predicted that high concentrations of A 2A R antagonists would behave as A 2A R agonists and decrease D 2 R function in the brain.

Since 1990 it has been known that dimers are the basic functional form of nearly all G-protein-coupled receptors (GPCRs) and that homo- and heterodimerization may play a key role in correct receptor maturation and trafficking to the plasma membrane. Nevertheless, homo- and heterodimerization of GPCR has become a matter of debate especially in the search for the precise physiological meaning of this phenomenon. This article focuses on how heterodimerization of adenosine A1 and A2A receptors, which are coupled to apparently opposite signalling pathways, allows adenosine to exert a fine-tuning modulation of striatal glutamatergic neurotransmission, providing a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release.

Background: After pancreas transplantation (PTx) some patients report occasional symptoms of hypoglycaemia and at times, serious hypoglycaemia. Continuous blood glucose monitoring (CBGM) allows determination of the daily glucose profile and detection of unrecognized hypoglycaemia. The aims of our study were to determine the incidence of hypoglycaemia in PTx and evaluate whether the use of CBGM helps to detect unrecognized nocturnal hypoglycaemia. Patients and methods: We studied 12 patients (six males) with normal functioning PTx and kidney transplantation for more than 3 yr, with systemic drainage of endocrine secretion and stable immunosuppression. A 24-h CBGM using a microdialysis technique (GlucoDay, A. Menarini Diagnostics, Florence, Italy) was performed in all the patients. Results: Three patients had asymptomatic recorded glucose levels below 3.3 nmol/L during the nocturnal period (01:00–07:00 hours) with the glucose levels during these episodes being 2.6, 2.5 and 2.5 nmol/L, and the duration of nocturnal hypoglycaemia being 27, 62 and 93 min, respectively, rising spontaneously without intervention. Patients with hypoglycaemia presented lower glycosylated haemoglobin levels when compared with those not presenting hypoglycaemic episodes, although basal glucose and insulin levels and insulin antibody titres were similar. In one of the three patients presenting hypoglycaemia CBGM was re-evaluated after including an extra snack at bedtime, with subsequent normalization of the blood glucose profile being observed. Conclusion: Unrecognized nocturnal hypoglycaemia is relatively frequent in patients with PTx and 24-h CBMG may be useful to detect these episodes.

Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors.

Interest in adenosine deaminase (ADA) in the context of medicine has mainly focused on its enzymatic activity. This is justified by the importance of the reaction catalyzed by ADA not only for the intracellular purine metabolism, but also for the extracellular purine metabolism as well, because of its capacity as a regulator of the concentration of extracellular adenosine that is able to activate adenosine receptors (ARs). In recent years, other important roles have been described for ADA. One of these, with special relevance in immunology, is the capacity of ADA to act as a costimulator, promoting T-cell proliferation and differentiation mainly by interacting with the differentiation cluster CD26. Another role is the ability of ADA to act as an allosteric modulator of ARs. These receptors have very general physiological implications, particularly in the neurological system where they play an important role. Thus, ADA, being a single chain protein, performs more than one function, consistent with the definition of a moonlighting protein. Although ADA has never been associated with moonlighting proteins, here we consider ADA as an example of this family of multifunctional proteins. In this review, we discuss the different roles of ADA and their pathological implications. We propose a mechanism by which some of their moonlighting functions can be coordinated. We also suggest that drugs modulating ADA properties may act as modulators of the moonlighting functions of ADA, giving them additional potential medical interest.

The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3receptor-mediated inhibition of D1receptor function. This blockade requires the σ1receptor and occurs upon cocaine binding to σ1-D1-H3receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1receptor that activates Gs, freely recruits β-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Usingin vitroassays with transfected cells and inex vivoexperiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1receptor, we show that blockade of σ1receptor by an antagonist restores the protective H3receptor-mediated brake on D1receptor signaling and prevents the cell death from elevated D1receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3receptor agonists could serve to reduce some effects of cocaine.

Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57-K168 and intrasubunit R149-E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149-E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15 degrees C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.

The concentration of an inhibitor that decreases the rate of an enzyme-catalysed reaction by 50%, symbolized i(0.5), is often used in pharmacological studies to characterize inhibitors. It can be estimated from the common inhibition plots used in biochemistry by means of the fact that the extrapolated inhibitor concentration at which the rate becomes infinite is equal to -i(0.5). This method is, in principle, more accurate than comparing the rates at various different inhibitor concentrations, and inferring the value of i(0.5) by interpolation. Its reciprocal, 1/i(0.5), is linearly dependent on v(0)/V, the uninhibited rate divided by the limiting rate, and the extrapolated value of v(0)/V at which 1/i(0.5) is zero allows the type of inhibition to be characterized: this value is 1 if the inhibition is strictly competitive; greater than 1 if the inhibition is mixed with a predominantly competitive component; infinite (i.e. 1/i(0.5) does not vary with v(0)/V) if the inhibition is pure non-competitive (i.e. mixed with competitive and uncompetitive components equal); negative if the inhibition is mixed with a predominantly uncompetitive component; and zero if it is strictly uncompetitive. The type of analysis proposed has been tested experimentally by examining inhibition of lactate dehydrogenase by oxalate (an uncompetitive inhibitor with respect to pyruvate) and oxamate (a competitive inhibitor with respect to pyruvate), and of cytosolic malate dehydrogenase by hydroxymalonate (a mixed inhibitor with respect to oxaloacetate). In all cases there is excellent agreement between theory and experiment.

Mitochondrial malate dehydrogenase (mMDH) shows a complex dependence upon ionic environment that includes kinetic and structural effects. We measured mMDH activity in several buffers (phosphate, MOPS, and MES) at pH 6.5 and 7.5, and in the presence of a number of anions, at highly diluted enzyme concentrations where mMDH showed significant loss of activity. Under these conditions, mMDH activity shows a non-linear dependence on enzyme concentration, in agreement with the existence of a dimer–monomer equilibrium, where only the dimeric form is active. According to this hypothesis, the dissociation constant of mMDH dimer has been determined to be 5.4 nM in the MES buffer at pH 6.5. Either the presence of a small anion like phosphate, or an increase of the pH from 6.5 to 7.5 shifts the equilibrium in favor of the dimeric form with the two effects appearing to be additive. To extend the study, we analysed the effect of a number of anions on the mMDH activity in 50 mM MOPS buffer at pH 7.5. All the anions had a dual effect: at low concentrations, they increased the activity of mMDH, while at high concentrations, they inhibited it. A more accurate analysis of the data revealed that the activation capacity of all the anions tested was similar, although they differed in their inhibitory influence. To show these differences more clearly, the experiment was repeated in 50 mM phosphate buffer at pH 7.5, under conditions where almost all activations were due to the buffer. The analysis of the results obtained under these conditions revealed the following sequence of inhibition potency: phosphate

Long-term therapy with L-3,4-dihydroxyphenylalanine (L-DOPA), still the most effective treatment in Parkinson's disease (PD), is associated with severe motor complications such as dyskinesia. Experimental and clinical data have indicated that adenosine A2A receptor antagonists can provide symptomatic improvement by potentiating L-DOPA efficacy and minimizing its side effects. It is known that the G-protein-coupled adenosine A2A, cannabinoid CB1 and dopamine D2 receptors may interact and form functional A2A-CB1-D2 receptor heteromers in co-transfected cells as well as in rat striatum. These data suggest that treatment with a combination of drugs or a single compound selectively acting on A2A-CB1-D2 heteromers may represent an alternative therapeutic treatment of PD. We investigated the expression of A2A-CB1-D2 receptor heteromers in the striatum of both naive and hemiparkinsonian rats (HPD-rats) bearing a unilateral 6-hydroxydopamine (6-OHDA) lesion, and assessed how receptor heteromer expression and biochemical properties were affected by L-DOPA treatment. Radioligand binding data showed that A2A-CB1-D2 receptor heteromers are present in the striatum of both naive and HPD-rats. However, behavioral results indicated that the combined administration of A2A (MSX-3 or SCH58261) and CB1 (rimonabant) receptor antagonists, in the presence of L-DOPA does not produce a response different from administration of the A2A receptor antagonist alone. These behavioral results prompted identification of heteromers in L-DOPA-treated animals. Interestingly, the radioligand binding results in samples from lesioned animals suggest that the heteromer is lost following acute or chronic treatment with L-DOPA.

The molecular basis of priming for L-DOPA-induced dyskinesias in Parkinson's disease (PD), which depends on the indirect pathway of motor control, is not known. In rodents, the indirect pathway contains striatopallidal GABAergic neurons that express heterotrimers composed of A(2A) adenosine, CB(1) cannabinoid and D(2) dopamine receptors that regulate dopaminergic neurotransmission. The present study was designed to investigate the expression of these heteromers in the striatum of a primate model of Parkinson's disease and to determine whether their expression and pharmacological properties are altered upon L-DOPA treatment. By using the recently developed in situ proximity ligation assay and by identification of a biochemical fingerprint, we discovered a regional distribution of A(2A)/CB(1) /D(2) receptor heteromers that predicts differential D(2)-mediated neurotransmission in the caudate-putamen of Macaca fascicularis. Whereas heteromers were abundant in the caudate nucleus of both naive and MPTP-treated monkeys, L-DOPA treatment blunted the biochemical fingerprint and led to weak heteromer expression. These findings constitute the first evidence of altered receptor heteromer expression in pathological conditions and suggest that drugs targeting A(2A)-CB(1) -D(2) receptor heteromers may be successful to either normalize basal ganglia output or prevent L-DOPA-induced side effects.

Using bioluminescence resonance energy transfer and proximity ligation assays, we obtained the first direct evidence that adenosine A₁ receptors (A₁Rs) form homomers not only in cell cultures but also in brain cortex. By radioligand binding experiments in the absence or in the presence of the A₁Rs allosteric modulator, adenosine deaminase, and by using the two-state dimer receptor model to fit binding data, we demonstrated that the protomer-protomer interactions in the A₁R homomers account for some of the pharmacological characteristics of agonist and antagonist binding to A₁Rs. These pharmacological properties include the appearance of cooperativity in agonist binding, the change from a biphasic saturation curve to a monophasic curve in self-competition experiments and the molecular cross-talk detected when two different specific molecules bind to the receptor. In this last case, we discovered that caffeine binding to one protomer increases the agonist affinity for the other protomer in the A₁R homomer, a pharmacological characteristic that correlates with the low caffeine concentrations-induced activation of agonist-promoted A₁R signaling. This pharmacological property can explain the biphasic effects reported at low and high concentration of caffeine on locomotor activity.

The role of the pineal gland is to translate the rhythmic cycles of night and day encoded by the retina into hormonal signals that are transmitted to the rest of the neuronal system in the form of serotonin and melatonin synthesis and release. Here we describe that the production of both melatonin and serotonin by the pineal gland is regulated by a circadian-related heteromerization of adrenergic and dopamine D₄ receptors. Through α(₁B)-D₄ and β₁-D₄ receptor heteromers dopamine inhibits adrenergic receptor signaling and blocks the synthesis of melatonin induced by adrenergic receptor ligands. This inhibition was not observed at hours of the day when D₄ was not expressed. These data provide a new perspective on dopamine function and constitute the first example of a circadian-controlled receptor heteromer. The unanticipated heteromerization between adrenergic and dopamine D₄ receptors provides a feedback mechanism for the neuronal hormone system in the form of dopamine to control circadian inputs.

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

The incorporation of chemical modifications into the structure of bioactive compounds is often difficult because the biological properties of the new molecules must be retained with respect to the native ligand. Ergopeptides, with their high affinities at D(1) and D(2) dopamine receptors, are particularly complex examples. Here, we report the systematic derivatization of two ergopeptides with different peptide-based spacers and their evaluation by radioligand binding assays. Selected spacer-containing ergopeptides with minimal biological alteration and a proper anchoring point were further derivatized with a biotin reporter. Detailed characterization studies identified 13 as a biotin ergopeptide maintaining high affinity and agonist behavior at dopamine receptors, being a useful tool for the study of heteromers involving D(1)R, D(2)R, or D(3)R.

Previously, using artificial cell systems, we identified receptor heteromers between the dopamine D1 or D2 receptors and the histamine H3 receptor. In addition, we demonstrated two biochemical characteristics of the dopamine D1 receptor-histamine H3 receptor heteromer. We have now extended this work to show the dopamine D 1 receptor-histamine H3 receptor heteromer exists in the brain and serves to provide a novel link between the MAPK pathway and the GABAergic neurons in the direct striatal efferent pathway. Using the biochemical characteristics identified previously, we found that the ability of H 3 receptor activation to stimulate p44 and p42 extracellular signal-regulated MAPK (ERK 1/2) phosphorylation was only observed in striatal slices of mice expressing D1 receptors but not in D1 receptor-deficient mice. On the other hand, the ability of both D1 and H3 receptor antagonists to block MAPK activation induced by either D1 or H3 receptor agonists was also found in striatal slices. Taken together, these data indicate the occurrence of D 1-H3 receptor complexes in the striatum and, more importantly, that H3 receptor agonist-induced ERK 1/2 phosphorylation in striatal slices is mediated by D1-H3 receptor heteromers. Moreover, H3 receptor-mediated phospho-ERK 1/2 labeling co-distributed with D1 receptor-containing but not with D2 receptor-containing striatal neurons. These results indicate that D 1-H3 receptor heteromers work as processors integrating dopamine- and histamine-related signals involved in controlling the function of striatal neurons of the direct striatal pathway.

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