The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their functional significance is being revealed. 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. The occurrence of receptor heteromers with different pharmacological and signaling properties opens a complete new field to search for novel drug targets useful against a variety of neuropsychiatric disorders with potentially less side effects. Our program can be divided into two main lines of research: First (A), the discovery of receptor heteromers with functional or pathological significance in the CNS;and second (B), discovery of drugs that target selectively those receptor heteromers. A). The identification of functionally significant receptor heteromers implies, first, the study of heteromerization of transfected receptors in mammalian cell lines using Bioluminescence Resonance Energy Transfer (BRET) and the identification of their unique biochemical properties. This can be achieved by identifying the molecular determinants that determine heteromerization or the right quaternary structure of the receptor heteromer. Modification or occupation of those determinants (by using artificial or natural mutants or receptor variants or blocking peptides) should lead to disruption of heteromerization or to changes in the quaternary structure of the receptor heteromer and its function (allosteric interaction in the receptor heteromer or specific signaling). Once a biochemical property of the heteromer is determined, it can be used as a biochemical fingerprint of the receptor heteromer in native tissues. Nevertheless, a real in situ demonstration can be achieved by using the same strategies than those used in transfected cells (transgenic animals or peptide delivery or expression). Finally, the use of electrophysiological techniques or the study of neurotransmitter release with in vitro or in vivo approaches (also using transgenic animals or peptide delivery or expression) can allow establishing the functional significance of the receptor heteromer. By using this approach we discovered the existence of receptor heteromers between the short isoform of the dopamine D2 receptor (D2S) and the D4 receptor that modulate striatal glutamatergic neurotransmission (1). Importantly, from the three main human variants of D4 receptors (D4.2, D4.4 and D4.7), the ADHD-associated D4.7 variant does not heteromerize with D2S. This allowed the identification of a biochemical property of the D2S-D4 receptor heteromer, which is the ability of D2S receptor stimulation to potentiate D4 receptor-mediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D4.7 or in the striatum of knockin mutant mice carrying the 7 repeats of the human D4.7 in the third intracellular loop of the D4 receptor (1). In the striatum, D4 receptors were found in glutamatergic terminals, where they selectively modulated glutamatergic neurotransmission by interacting with D2S receptors. This interaction showed the same qualitative characteristics than the D2S-D4 receptor heteromer-mediated MAPK signaling, and D2S receptor activation potentiated D4 receptor-mediated inibition of striatal glutamate release (1). It was therefore postulated that dysfunctional D2S-D4.7 heteromers impair presynaptic dopaminergic control of cortico-striatal glutamatergic neurotransmission and explain functional deficits associated with ADHD. The same approach was also used to demonstrate the existence of heteromers between dopamine D1-like and galanin 1 (Gal1) receptors that modulate hippocampal cholinergic neurotransmission (2). Previous studies had shown that dopamine and galanin modulate cholinergic transmission in the hippocampus, but little was known about the mechanisms involved and their possible interactions. By using BRET in transfected mammalian cells, we demonstrated the existence of heteromers between dopamine D1-like (D1 and D5) and galanin 1 (Gal1) receptors, but not with the homologous Gal2 receptor. Within the D1-Gal1 and D5-Gal1 receptor heteromers, dopamine receptor activation potentiated and dopamine receptor blockade counteracted MAPK activation induced by stimulation of Gal1 receptors. The ability of a D1-like receptor antagonist to block galanin-induced MAPK activation (cross-antagonism) was used as a biochemical fingerprint of D1-like-Gal1 receptor heteromers, allowing their identification in the rat ventral hippocampus (2). The functional role of D1-like-Gal1 receptor heteromers was demonstrated in synaptosomes from rat ventral hippocampus, where galanin facilitated acetylcholine release, but only with co-stimulation of D1-like receptors (2). Furthermore, electrophysiological experiments in rat ventral hippocampal slices showed that these receptor interactions modulate hippocampal synaptic transmission (2). D1-like-Gal1 receptor heteromers could constitute another target for drugs active in drug addiction since the ventral hippocampus is an important source of glutamatergic afferents to the ventral striatum. Furthermore, D1-like-Gal1 receptor heteromers could also be found in the VTA and substantia nigra, where D1, D5 and Gal1 receptors are also co-localized. It is well known that blockade of the dopamine transporter is a main mechanism of action of cocaine. But this mechanism should lead to a general increase in dopaminergic neurotransmission, and yet D1 receptors 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. We found that D1 and σ1 receptors heteromerize in transfected cells, where cocaine robustly potentiated D1 receptor-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK signaling induced by D1 receptor activation in a dopamine transporter-independent and σ1 receptor-dependent manner (3). Some of these effects were also demonstrated in murine striatal slices and were absent in σ1 receptor KO mice, providing evidence for the existence of σ1-D1 receptor heteromers in the brain (3). Therefore, our results provide a molecular explanation for which D1 receptor plays a more significant role in the behavioral effects of cocaine and identify σ1-D1 receptor heteromers as possible new targets for cocaine addiction. Finally, during the last year we also found evidence for the existence of functionally significant heteromers between D1 and histamine H3 receptors in the brain, in the striatum (4). Previously, we identified receptor heteromers between those receptors in transfected cells and demonstrated some of their biochemical characteristics. We have now extended this work to show that the D1-H3 receptor heteromer provides a novel link between the MAPK pathway and the medium spiny neurons (MSNs) of the direct striatal efferent pathway. Thus, the H3 receptor agonist-induced MAPK activation in striatal slices is mediated by D1-H3 receptor heteromers (4). In view of the possible role of the direct striatal efferent pathway in the mediation of L-dopa-induced dyskinesia in patients with Parkinsons disease and in relapse to drugs of abuse, the D1-H3 receptor heteromer could be considered as a possible target for drugs with a potential use in these disorders. B). Receptor heteromer-selective drugs could in principle be ligands that bind with more affinity to any of the two protomers of the heteromer than to the same receptor units when not forming heteromers. The idea is that receptor heteromerization can change the tertiary structure of a receptor and consequently its ligand binding propertie
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