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The functional and pharmacological significance of the dopamine D

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.

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.

The two highly homologous subtypes of stimulatory G proteins Gαs (Gs) and Gαolf (Golf) display contrasting expression patterns in the brain. Golf is predominant in the striatum, while Gs is predominant in the cortex. Yet, little is known about their functional distinctions. The dopamine D1 receptor (D1R) couples to Gs/olf and is highly expressed in cortical and striatal areas, making it an important therapeutic target for neuropsychiatric disorders. Using novel drug screening methods that allow analysis of specific G-protein subtype coupling, we found that, relative to dopamine, dihydrexidine and N-propyl-apomorphine behave as full D1R agonists when coupled to Gs, but as partial D1R agonists when coupled to Golf. The Gs/Golf-dependent biased agonism by dihydrexidine was consistently observed at the levels of cellular signaling, neuronal function, and behavior. Our findings of Gs/Golf-dependent functional selectivity in D1R ligands open a new avenue for the treatment of cortex-specific or striatum-specific neuropsychiatric dysfunction., D1-like dopamine receptors are coupled to Golf proteins in the dorsal striatum but Gs in cortical and other areas. Here, the authors demonstrate selective agonism of Gs-coupled versus Golf-coupled D1 receptors.

Gαs (Gs) and Gαolf (Golf) are highly homologous G-protein α subunits that activate adenylate cyclase, thereby serving as crucial mediators of intracellular signaling. Because of their dramatically different brain expression patterns, we studied similarities and differences between their activation processes with the aim of comparing their receptor coupling mechanisms. We engineered novel luciferase- and Venus-fused Gα constructs that can be used in bioluminescence resonance energy transfer assays. In conjunction with molecular simulations, these novel biosensors were used to determine receptor activation–induced changes in conformation. Relative movements in Gs were consistent with the crystal structure of β2 adrenergic receptor in complex with Gs. Conformational changes in Golf activation are shown to be similar to those in Gs. Overall the current study reveals general similarities between Gs and Golf activation at the molecular level and provides a novel set of tools to search for Gs- and Golf-specific receptor pharmacology. In view of the wide functional and pharmacological roles of Gs- and Golf-coupled dopamine D1 receptor and adenosine A2A receptor in the brain and other organs, elucidating their differential structure–function relationships with Gs and Golf might provide new approaches for the treatment of a variety of neuropsychiatric disorders. In particular, these novel biosensors can be used to reveal potentially therapeutic dopamine D1 receptor and adenosine A2A receptor ligands with functionally selective properties between Gs and Golf signaling.

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.

The two most common polymorphisms of the human DRD4 gene encode a dopamine D(4) receptor (D4R) with four or seven repeats of a proline-rich sequence of 16 amino acids (D4.4R or D4.7R). Although the seven-repeat polymorphism has been repeatedly associated with attention deficit hyperactivity disorder and substance use disorders, the differential functional properties between D4.4R and D4.7R remained enigmatic until recent electrophysiological and optogenetic-microdialysis experiments 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 dopamine D(2) receptor (D2R), which co-localizes with D4R in the brain. However, contrary to a gain of function, experiments in mammalian transfected cells suggested that heteromerization with D2R results in lower MAPK signaling by D4.7R as compared to D4.4R. In the present study, we readdressed the question of functional differences of D4.4R and D4.7R forming homomers or heteromers with the short isoform of D2R (D2SR), using a functional bioluminescence resonance energy transfer (BRET) 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. Significant functional and pharmacological differences between D4.4R and D4.7R were only evident upon heteromerization with the short isoform of D2R (D2SR). The most dramatic finding was a significant increase and decrease in the constitutive activity of D2S 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.

Protein complementation assays (PCA) are used as pharmacological tools, enabling a wide array of applications, ranging from studies of protein–protein interactions to second messenger effects. Methods to detect activities of G protein-coupled receptors (GPCRs) have particular relevance for drug screening. Recent development of an engineered luciferase NanoLuc created the possibility of generating a novel PCA, which in turn could open a new avenue for developing drug screening assays. Here we identified a novel split position for NanoLuc and demonstrated its use in a series of fusion constructs to detect the activity of GPCRs. The split construct can be applied to a variety of pharmacological screening systems.

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1receptor (CRF1R) and orexin OX1receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking.

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.

The development of bivalent ligands has attracted interest as a way to potentially improve the selectivity and/or affinity for a specific receptor subtype. The ability to bind two distinct receptor binding sites simultaneously can allow the selective activation of specific G-protein dependent or β-arrestin-mediated cascade pathways. Herein, we developed an extended SAR study using sumanirole (1) as the primary pharmacophore. We found that substitutions in the N-1- and/or N-5-positions, physiochemical properties of those substituents, and secondary aromatic pharmacophores can enhance agonist efficacy for the cAMP inhibition mediated by G(i/o)-proteins, while reducing or suppressing potency and efficacy toward β-arrestin recruitment. Compound 19 was identified as a new lead for its selective D(2) G-protein biased agonism with an EC(50) in the subnanomolar range. Structure—activity correlations were observed between substitutions in positions N-1 and/or N-5 of 1 and the capacity of the new bivalent compounds to selectively activate G-proteins versus β-arrestin recruitment in D(2)R-BRET functional assays.

The function of the dopamine D4 receptor and the product of a clinically significant gene polymorphic variant is revealed., Polymorphic variants of the dopamine D4 receptor gene (DRD4) have been repeatedly associated with numerous neuropsychiatric disorders. Yet, the functional role of the D4 receptor and the functional differences of the products of DRD4 polymorphic variants remained enigmatic. Immunohistochemical and optogenetic-microdialysis experiments were performed in knock-in mice expressing a D4 receptor with the long intracellular domain of a human DRD4 polymorphic variant associated with attention deficit hyperactivity disorder (ADHD). When compared with the wild-type mouse D4 receptor, the expanded intracellular domain of the humanized D4 receptor conferred a gain of function, blunting methamphetamine-induced cortical activation and optogenetic and methamphetamine-induced corticostriatal glutamate release. The results demonstrate a key role of the D4 receptor in the modulation of corticostriatal glutamatergic neurotransmission. Furthermore, these data imply that enhanced D4 receptor–mediated dopaminergic control of corticostriatal transmission constitutes a vulnerability factor of ADHD and other neuropsychiatric disorders.

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.

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.

The Gαi/o-coupled dopamine D2-like receptor family comprises three subtypes: the D2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D3 receptor (D3R), and D4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D2-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and β arrestin recruitment. Norepinephrine acted as a potent agonist for all D2-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR >> D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays.

Previous studies have shown that dopamine and galanin modulate cholinergic transmission in the hippocampus, but little is known about the mechanisms involved and their possible interactions. By using resonance energy transfer techniques in transfected mammalian cells, we demonstrated the existence of heteromers between the dopamine D1-like receptors (D1and D5) and galanin Gal1, but not Gal2receptors. Within the D1–Gal1and D5–Gal1receptor heteromers, dopamine receptor activation potentiated and dopamine receptor blockade counteracted MAPK activation induced by stimulation of Gal1receptors, whereas Gal1receptor activation or blockade did not modify D1-like receptor-mediated MAPK activation. Ability of a D1-like receptor antagonist to block galanin-induced MAPK activation (cross-antagonism) was used as a “biochemical fingerprint” of D1-like–Gal1receptor heteromers, allowing their identification in the rat ventral hippocampus. The functional role of D1-like–Gal receptor heteromers was demonstrated in synaptosomes from rat ventral hippocampus, where galanin facilitated acetylcholine release, but only with costimulation of D1-like receptors. Electrophysiological experiments in rat ventral hippocampal slices showed that these receptor interactions modulate hippocampal synaptic transmission. Thus, a D1-like receptor agonist that was ineffective when administered alone turned an inhibitory effect of galanin into an excitatory effect, an interaction that required cholinergic neurotransmission. Altogether, our results strongly suggest that D1-like–Gal1receptor heteromers act as processors that integrate signals of two different neurotransmitters, dopamine and galanin, to modulate hippocampal cholinergic neurotransmission.

Dopamine (DA), the most abundant catecholamine in the basal ganglia, participates in the regulation of motor functions and of cognitive processes such as learning and memory. Abnormalities in dopaminergic systems are thought to be the bases for some neuropsychiatric disorders including addiction, Parkinson's disease, and Schizophrenia. DA exerts its arrays of functions via stimulation of D1-like (D1 and D5) and D2-like (D2, D3, and D4) DA receptors which are located in various regions of the brain. The DA D1 and D2 receptors are very abundant in the basal ganglia where they exert their functions within separate neuronal cell types. The present paper focuses on a review of the effects of stimulation of DA D1 receptors on diverse signal transduction pathways and gene expression patterns in the brain. We also discuss the possible involvement of the DA D1 receptors in DA-mediated toxic effects observed both in vitro and in vivo. Future studies using more selective agonist and antagonist agents and the use of genetically modified animals should help to further clarify the role of these receptors in the normal physiology and in pathological events that involve DA.

Human immunodeficiency virus (HIV)-infected methamphetamine (METH) abusers show greater neuropathological abnormalities than HIV-negative METH abusers or HIV-positive non-METH users. Herein, we investigated the effects of METH and of the HIV protein, Tat, using the human neuroblastoma cell line, SHY5Y. METH and Tat alone caused no toxic effects. In contrast, the combination of METH and Tat caused significant increases in the percentage of dead cells observed after 48 h of exposure. These results suggest the possibility of using this model to elucidate cellular and molecular mechanisms involved in the potentiation of clinical effects observed in patient populations of HIV-positive METH-abusing patients.