We study the functional and pharmacological properties of G protein-coupled receptor (GPCR) heteromers localized in brain circuits that are dysfunctional in Substance Use Disorders (SUD) and other neuropsychiatric disorders, such as L-DOPA-induced dyskinesia (LID), attention deficit hyperactivity disorder (ADHD) and Restless Legs Syndrome (RLS), and their possible role as targets for the treatment of these neuropsychiatric disorders. During the last year we have focused on: molecular mechanisms of striatal heteromers of dopamine D1 and D3 receptors (D1R-D3R heteromers) and their implications for the treatment of LID; molecular mechanisms of striatal heteromers of dopamine D2 and D4 receptors (D2R-D4R heteromers), which represent targets for the treatment of ADHD and RLS; molecular mechanisms of striatal heteromers of adenosine A1 and A2A receptors (A1R-A2AR heteromers) and their implications for the treatment of RLS; and molecular mechanisms of heteromers of mu-opioid and galanin Gal1 receptors (MOR-Gal1R heteromers) localized in the ventral tegmental area (VTA) and their implications for the treatment of opioid use disorder (OUD). Striatal D1R-D3R heteromers: We previously 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. We now demonstrated, for the first time, the presence of D1R-D3R heteromers in the mouse ventral striatum by using a synthetic peptide that selectively destabilizes D1R-D3R heteromers (1). Application of the destabilizing peptide in transfected cells and in the ventral striatum allowed demonstrating that both, in vitro and in vivo, co-activation of D1R and D3R induces a switch from a G protein-dependent to a G protein-independent D1R-mediated MAPK signaling mediated by the D1R-D3R heteromers (1). The results suggested that the locomotor and MAPK-activating synergistic effects of D1R and D3R agonists in reserpinized mice can be used as a proxy animal model of LID (1). Previous studies have shown that D3R are upregulated in patients with LID and in well-accepted rat and monkey models of LID. The effect of D1R and D3R agonists were then evaluated on their ability to produce LID-like movements in a rat model of LID. Each agonist dose-dependently induced dyskinesia, but, more importantly, when threshold doses were co-administered, rats displayed synergistic exacerbation of dyskinesia (2). In correlation with our studies in reserpinized mice, D1R-D3R co-stimulation was associated with a significant increase in striatal MAPK activation only in rats with LID (2). Our results support that D1R-D3R heteromers are specially involved in the pathogenesis of LID. Striatal D2R-D4R heteromers: The two most common polymorphisms of the human DRD4 gene encode a D4R with four or seven repeats of a proline-rich sequence of 16 amino acids (D4.4R or D4.7R). Although D4.7R has been repeatedly associated with ADHD and SUD, the differential functional properties between D4.4R and D4.7R remained enigmatic until recent studies indicated a gain of function of D4.7R. Since no clear differences in the biochemical properties of individual D4.4R and D4.7R have been reported, it was previously suggested that those differences emerge upon heteromerization with D2R, which co-localizes with D4R in the brain, in cortico-striatal glutamatergic terminals. Using a biophysical assay that allows the measurement of ligand-induced changes in the interaction between G protein-coupled receptors (GPCRs) forming homomers or heteromers with their cognate G protein (3), we found a significant increase and decrease in the constitutive activity of D2R upon heteromerization with D4.7R and D4.4R, respectively, providing the first clear mechanism for a functional difference between both products of polymorphic variants and for a gain of function of the D4.7R (3). Striatal A1R-A2AR heteromers: Adenosine also plays an important modulatory role driving the function of cortico-striatal glutamatergic terminals. This control is mediated by adenosine A1R and A2AR heteromers, which work as an adenosine concentration-dependent switch. Low concentrations of adenosine primarily activate A1R, which induces an inhibition of glutamate release. On the contrary, with high concentrations of adenosine, the simultaneous activation of A2AR leads to an allosteric modulation in the A1R-A2AR heteromer with a reduction of affinity and efficacy of adenosine for the A1R. Under these conditions the prevailing A2AR signaling imduces glutamate release. Using biophysical techniques in transfected mammalian cells and computational analysis we found results indicating that the mechanism by which the A1R-A2AR acts as an adenosine concentration-dependent switch implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR (4). Brain iron deficiency (BID) is the initial pathogenetic mechanism in RLS. We previously found evidence for an increased sensitivity of cortico-striatal terminals in an animal model of RLS, the rat with BID, that could relate to RLS sensorimotor symptoms. Also based on previous results from our research group, we postulated the adenosine hypothesis of RLS, the existence of a hypoadenosinergic state that mostly results from a BID-induced reduction in the brain levels of A1R (5). We also postulated that the mechanism responsible for the increased sensitivity of cortico-striatal terminals is related to an A1R downregulation, leading to an excess of the A2AR not forming heteromers with A1R. Our hypothesis was supported by a clinical study with the inhibitor of adenosine transport dipyridamole (see previous Annual Report) and by our more recent optogenetic-microdialysis in the rat with BID. We first demonstrated in control rats that the frequency of optogenetic stimulation that was ineffective at inducing cortico-striatal glutamate release became effective with the local perfusion of a selective A1R antagonist (6). Furthermore, in rats with and without BID, the striatal application of dipyridamole blocked the optogenetic-induced glutamate release and decreased basal levels of glutamate, which was counteracted by the A1R antagonist (6). MOR-Gal1R heteromers in the VTA: We previously demonstrated that MOR-Gal1R heteromers constitute a main population of MOR that modulate dopamine neuronal function in the VTA. We also demonstrated that Gal1R ligands counteract MOR-mediated signaling in the MOR-Gal1R heteromer. We have now demonstrated that these heteromers are the main mediators of the dopaminergic effects of opioids and are most probably involved in their euphoric effects (7). The study included preclinical and clinical data, from patients with OUD and RLS and included research groups from NIDA, the University of Maryland, Harvard Medical School and the University of Barcelona. In radioligand binding experiments, we first found a significant ability of Gal1R ligands to decrease the affinity of MOR agonist binding in cells expressing the MOR-Gal1R heteromers (7). Using several in vitro and in vivo techniques, we also found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of MOR with Gal1R, rendering a profound decrease in the potency of methadone (7). This finding was explained by the weaker proficiency of methadone in activating the dopaminergic system as compared with morphine and predicted a dissociation of the therapeutic and euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-reports of feeling high in methadone-medicated patients (7). The results suggest a lower addictive liability of some opioids, such as methadone, due to their selective low potency for the MOR-Gal1R heteromer.
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