The heterotetrameric structure of the adenosine A1-dopamine D1 receptor complex: Pharmacological implication for restless legs syndrome.

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.).

Biased G Protein-Independent Signaling of Dopamine D1-D3 Receptor Heteromers in the Nucleus Accumbens.

Several studies found in vitro evidence for heteromerization of dopamine D1 receptors (D1R) and D3 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.

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

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.

Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

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.

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

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 (ß-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 ß-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.

Functional µ-Opioid-Galanin Receptor Heteromers in the Ventral Tegmental Area.

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 identified in situ in slices of rat ventral tegmental area (VTA) with MAPK activation and two additional cell signaling pathways, AKT and CREB phosphorylation. Furthermore, in vivo microdialysis 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 STATEMENT: The µ-opioid receptor (MOR) localized in the ventral tegmental area (VTA) plays a key role in the reinforcing and addictive properties of opioids. With parallel in vitro experiments in mammalian transfected cells and in situ and in vivo experiments 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.

Evidence for the heterotetrameric structure of the adenosine A2A-dopamine D2 receptor complex.

Heteromers of G-protein-coupled receptors (GPCRs) have emerged as potential novel targets for drug development. Accumulating evidence indicates that GPCRs can form homodimers and heteromers, with homodimers being the predominant species and oligomeric receptors being formed as multiples of dimers. Recently, heterotetrameric structures have been proposed for dopamine D1receptor (D1R)-dopamine D3receptor (D3R) and adenosine A2Areceptor (A2AR)-dopamine D2receptor (D2R) heteromers. The structural model proposed for these complexes is a heteromer constituted by two receptor homodimers. The existence of GPCR homodimers and heteromers provides a structural basis for inter-protomer allosteric mechanisms that might account for a multiplicity of unique pharmacological properties. In this review, we focus on the A2AR-D2R heterotetramer as an example of an oligomeric structure that is key in the modulation of striatal neuronal function. We also review the interfaces involved in this and other recently reported heteromers of GPCRs. Furthermore, we discuss several published studies showing theex vivoexpression of A2AR-D2R heteromers. The ability of A2AR agonists to decrease the affinity of D2R agonists has been reported and, on the basis of this interaction, A2AR antagonists have been proposed as potential drugs for the treatment of Parkinson's disease. The heterotetrameric structure of the A2AR-D2R complex offers a novel model that can provide new clues about how to adjust the drug dosage to the expected levels of endogenous adenosine.

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

BACKGROUND: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. RESULTS: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. CONCLUSIONS: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

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

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

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.

Moonlighting adenosine deaminase: a target protein for drug development.

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.

Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson's disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

Cocaine disrupts histamine H3 receptor modulation of dopamine D1 receptor signaling: σ1-D1-H3 receptor complexes as key targets for reducing cocaine's effects.

The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits ß-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine.

L-DOPA disrupts adenosine A(2A)-cannabinoid CB(1)-dopamine D(2) receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: biochemical and behavioral studies.

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 naïve 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 naïve 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.

L-DOPA-treatment in primates disrupts the expression of A(2A) adenosine-CB(1) cannabinoid-D(2) dopamine receptor heteromers in the caudate nucleus.

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 naïve 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.

Homodimerization of adenosine A1 receptors in brain cortex explains the biphasic effects of caffeine.

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.

Detection of receptor heteromers involving dopamine receptors by the sequential BRET-FRET technology.

Until very recently, dopamine receptors, like other G-protein-coupled receptors, were believed to function as individual units on the cell surface. Now it has been described by several groups including ours that dopamine receptors not only function as homomers but also form heteromers with other receptors at the membrane level. Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) based techniques have been very useful to determine the interaction between two receptors, but to demonstrate the existence of higher-order complexes involving more than two molecules requires more sophisticated techniques. Combining BRET and FRET in the Sequential BRET-FRET (SRET) technique permits heteromers formed by three different proteins to be identified. In SRET experiments, the oxidation of a Renilla Luciferase substrate triggers acceptor excitation by BRET and subsequent energy transfer to a FRET acceptor. Using this methodology here we describe the heteromerization between adenosine A(2A), dopamine D(2), and cannabinoids CB(1) receptors in living cells.

The catalytic site structural gate of adenosine deaminase allosterically modulates ligand binding to adenosine receptors.

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.

Psychostimulant pharmacological profile of paraxanthine, the main metabolite of caffeine in humans.

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 A(1) than an adenosine A(2A) receptor agonist. In drug discrimination experiments in rats trained to discriminate a maximal locomotor activating dose of caffeine, paraxanthine, unlike theophylline, 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 paraxanthine, 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 paraxanthine, 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.

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.