Receptor heteromer is defined as a macromolecular complex composed of at least two (functional) receptor units with biochemical properties that are demonstrably different from those of its individual components. We have two main lines of research in relation to receptor heteromers: First, the discovery of receptor heteromers with functional or pathological significance in the CNS, particularly those localized in brain circuits involved in drug addiction;second, preclinical and clinical pharmacological targeting of those receptor heteromers. The identification of functionally significant receptor heteromers implies, first, the study of heteromerization of transfected receptors in mammalian cell lines using resonance energy transfer techniques, such as bioluminescence and fluorescence resonance energy transfer;BRET and FRET, respectively) and the identification of their unique biochemical properties. Once a biochemical property of the heteromer is determined, it can be used as a biochemical fingerprint of the receptor heteromer in native tissues. By using this approach we have previously discovered a number of dopamine and adenosine receptor heteromers. We have also contributed to the field with the development of new biophysical techniques, such as sequential BRET-FRET (SRET), which allows the detection of heteromerization of more than two proteins in living cells (1). With this technique and BRET with luminescence/fluorescence complementation approaches we demonstrated that sigma-1 (σ1) receptors form higher heteromers with dopamine D2 receptors, with the minimal structural unit being σ1-1-D2-D2 receptor heterotetramer (2). In the D2 receptor family, this is specific of the D2 receptor subtype (long isoform), as D3 and D4 receptor did not form heteromers with σ1 receptors. We also demonstrated that the σ1-D2 receptor heteromers are found in mouse striatum and that cocaine, by binding to σ1-D2 receptor heteromers, inhibits D2 receptor-mediated signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes were not found and this inhibition was not observed (2). Together with previously published studies from our group on σ1-D1 heteromers, our results provide a mechanism of action that explains the predominant D1-receptor mediated effects of cocaine upon acute administration. Adenosine receptor heteromers have been a major focus of our preclinical and clinical studies, among other reasons, because they are main targets for the central effects of caffeine. Both striatal A1 and A2A receptors are involved in the locomotor-activating and probably reinforcing effects of caffeine, although they play a different role under conditions of acute or chronic caffeine administration. Our studies have previously showed that adenosine acts both pre- and post-synaptically through multiple mechanisms, which depend on heteromerization of A1 and A2A receptors among themselves and with D1 and D2 receptors, respectively. A critical aspect of the mechanisms of the psychostimulant effects of caffeine is its ability to release the pre- and post-synaptic brakes that adenosine imposes on dopaminergic neurotransmission by acting on different adenosine receptor heteromers localized in different striatal neuronal elements. There has been a long debate about role of pre-synaptic mechanisms in the psychostimulant effects of caffeine, if caffeine can produce significant dopamine release in the striatum. Our new study demonstrates that paraxanthine, the main metabolite of caffeine in humans does in fact have a powerful dopamine-releasing effect. We first showed that, in rats, paraxanthine has a stronger locomotor activating effect than caffeine or the two other main metabolites of caffeine, theophylline and theobromine (3). As we 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 theophylline, generalized poorly to caffeine suggesting the existence of additional mechanisms other than adenosine antagonism in the behavioral effects of paraxanthine (3). Pretreatment with the nitric oxide inhibitor 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 (3). Ex vivo experiments demonstrated that paraxanthine, but not caffeine, can induce cGMP accumulation in the rat striatum (3). 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 (3). 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. Based on this psychostimulant pharmacological profile of paraxanthine, we are going to initiate clinical studies to evaluate if this methylxanthine could be used as a substitutive for classical psychostimulants, such as cocaine and amphetamine. Adenosine receptors also interact with cannabinoid receptors. In previous studies we demonstrated the existence of functional A2A-CB1 receptor heteromers in the striatum, involved in the motor effects of cannabinoids. Cannabis and caffeine are two of the most widely used psychoactive substances. Δ(9)-Tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis, induces deficits in short-term memory. Caffeine attenuates some memory deficits, but there have been few studies addressing the effects of caffeine and THC in combination. By evaluating the effects of these drugs using a rodent model of working memory, we found out that caffeine did not counteract memory deficits induced by THC but actually exacerbated them (4). Our results are consistent with recent demonstrations of antagonistic interactions between adenosine A1 receptors and cannabinoid CB1 receptors in the hippocampus, and they indicate that the combined administration of THC and caffeine can have a deleterious effect on cognitive function.

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Ferré, Sergi; Earley, Christopher; Gulyani, Seema et al. (2017) In search of alternatives to dopaminergic ligands for the treatment of restless legs syndrome: iron, glutamate, and adenosine. Sleep Med 31:86-92
Earley, Christopher J; Uhl, George R; Clemens, Stefan et al. (2017) Connectome and molecular pharmacological differences in the dopaminergic system in restless legs syndrome (RLS): plastic changes and neuroadaptations that may contribute to augmentation. Sleep Med 31:71-77
Guitart, Xavier; Chern, Yijuang; Ferré, Sergi (2017) Targeting the equilibrative nucleoside transporter ENT1 in Huntington disease. Oncotarget 8:12550-12551
Moreno, Estefanía; Quiroz, César; Rea, William et al. (2017) Functional ?-Opioid-Galanin Receptor Heteromers in the Ventral Tegmental Area. J Neurosci 37:1176-1186
Bonaventura, Jordi; Quiroz, César; Cai, Ning-Sheng et al. (2017) Key role of the dopamine D4 receptor in the modulation of corticostriatal glutamatergic neurotransmission. Sci Adv 3:e1601631
Ferré, Sergi (2017) Hormones and Neuropeptide Receptor Heteromers in the Ventral Tegmental Area. Targets for the Treatment of Loss of Control of Food Intake and Substance Use Disorders. Curr Treat Options Psychiatry 4:167-183
Quiroz, César; Orrú, Marco; Rea, William et al. (2016) Local Control of Extracellular Dopamine Levels in the Medial Nucleus Accumbens by a Glutamatergic Projection from the Infralimbic Cortex. J Neurosci 36:851-9
Casadó-Anguera, Verònica; Bonaventura, Jordi; Moreno, Estefanía et al. (2016) Evidence for the heterotetrameric structure of the adenosine A2A-dopamine D2 receptor complex. Biochem Soc Trans 44:595-600
Guitart, Xavier; Bonaventura, Jordi; Rea, William et al. (2016) Equilibrative nucleoside transporter ENT1 as a biomarker of Huntington disease. Neurobiol Dis 96:47-53
Ferré, Sergi; Sebastião, Ana Maria (2016) Dissecting striatal adenosine-cannabinoid receptor interactions. New clues from rats over-expressing adenosine A2A receptors. J Neurochem 136:897-9

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