Significant Functional Differences Between Dopamine D4 Receptor Polymorphic Variants Upon Heteromerization with α1A Adrenoreceptors.

The functional role of the dopamine D4 receptor (D4R) and its main polymorphic variants has become more evident with the demonstration of heteromers of D4R that control the function of frontal cortico-striatal neurons. Those include heteromers with the α2A adrenoceptor (α2AR) and with the D2R, localized in their cortical somato-dendritic region and striatal nerve terminals, respectively. By using biophysical and cell-signaling methods and heteromer-disrupting peptides in mammalian transfected cells and rat brain slice preparations, here we provide evidence for a new functionally relevant D4R heteromer, the α1AR-D4R heteromer, which is also preferentially localized in cortico-striatal glutamatergic terminals. Significant differences in allosteric modulations between heteromers of α1AR with the D4.4R and D4.7R polymorphic variants could be evidenced with the analysis of G protein-dependent and independent signaling. Similar negative allosteric modulations between α1AR and D4R ligands could be demonstrated for both α1AR-D4.4R and α1AR-D4.7R heteromers on G protein-independent signaling, but only for α1AR-D4.4R on G protein-dependent signaling. From these functional differences, it is proposed that the D4.4R variant provides a gain of function of the α1AR-mediated noradrenergic stimulatory control of cortico-striatal glutamatergic neurotransmission, which could result in a decrease in the vulnerability for impulse control-related neuropsychiatric disorders and increase in the vulnerability for posttraumatic stress disorder.

Functional and pharmacological role of the dopamine D4 receptor and its polymorphic variants.

The functional and pharmacological significance of the dopamine D4 receptor (D4R) has remained the least well understood of all the dopamine receptor subtypes. Even more enigmatic has been the role of the very prevalent human DRD4 gene polymorphisms in the region that encodes the third intracellular loop of the receptor. The most common polymorphisms encode a D4R with 4 or 7 repeats of a proline-rich sequence of 16 amino acids (D4.4R and D4.7R). DRD4 polymorphisms have been associated with individual differences linked to impulse control-related neuropsychiatric disorders, with the most consistent associations established between the gene encoding D4.7R and attention-deficit hyperactivity disorder (ADHD) and substance use disorders. The function of D4R and its polymorphic variants is being revealed by addressing the role of receptor heteromerization and the relatively avidity of norepinephrine for D4R. We review the evidence conveying a significant and differential role of D4.4R and D4.7R in the dopaminergic and noradrenergic modulation of the frontal cortico-striatal pyramidal neuron, with implications for the moderation of constructs of impulsivity as personality traits. This differential role depends on their ability to confer different properties to adrenergic α2A receptor (α2AR)-D4R heteromers and dopamine D2 receptor (D2R)-D4R heteromers, preferentially localized in the perisomatic region of the frontal cortical pyramidal neuron and its striatal terminals, respectively. We also review the evidence to support the D4R as a therapeutic target for ADHD and other impulse-control disorders, as well as for restless legs syndrome.

Pharmacological targeting of G protein-coupled receptor heteromers.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Preferential Gs protein coupling of the galanin Gal1 receptor in the µ-opioid-Gal1 receptor heterotetramer.

Recent studies have proposed that heteromers of µ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance.

Heteromerization between α2A adrenoceptors and different polymorphic variants of the dopamine D4 receptor determines pharmacological and functional differences. Implications for impulsive-control disorders.

Polymorphic alleles of the human dopamine D4 receptor gene (DRD4) have been consistently associated with individual differences in personality traits and neuropsychiatric disorders, particularly between the gene encoding dopamine D4.7 receptor variant and attention deficit hyperactivity disorder (ADHD). The α2A adrenoceptor gene has also been associated with ADHD. In fact, drugs targeting the α2A adrenoceptor (α2AR), such as guanfacine, are commonly used in ADHD treatment. In view of the involvement of dopamine D4 receptor (D4R) and α2AR in ADHD and impulsivity, their concurrent localization in cortical pyramidal neurons and the demonstrated ability of D4R to form functional heteromers with other G protein-coupled receptors, in this study we evaluate whether the α2AR forms functional heteromers with D4R and weather these heteromers show different properties depending on the D4R variant involved. Using cortical brain slices from hD4.7R knock-in and wild-type mice, here, we demonstrate that α2AR and D4R heteromerize and constitute a significant functional population of cortical α2AR and D4R. Moreover, in cortical slices from wild-type mice and in cells transfected with α2AR and D4.4R, we detect a negative crosstalk within the heteromer. This negative crosstalk is lost in cortex from hD4.7R knock-in mice and in cells expressing the D4.7R polymorphic variant. We also show a lack of efficacy of D4R ligands to promote G protein activation and signaling only within the α2AR-D4.7R heteromer. Taken together, our results suggest that α2AR-D4R heteromers play a pivotal role in catecholaminergic signaling in the brain cortex and are likely targets for ADHD pharmacotherapy.

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BACKGROUND: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. RESULTS: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. CONCLUSIONS: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

Opioid-galanin receptor heteromers mediate the dopaminergic effects of opioids.

Identifying non-addictive opioid medications is a high priority in medical sciences, but µ-opioid receptors mediate both the analgesic and addictive effects of opioids. We found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of µ-opioid receptors with galanin Gal1 receptors, rendering a profound decrease in the potency of methadone. This was explained by methadone's weaker proficiency to activate the dopaminergic system as compared to morphine and predicted a dissociation of therapeutic versus euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-report of "high" in methadone-maintained patients. These results suggest that µ-opioid-Gal1 receptor heteromers mediate the dopaminergic effects of opioids that may lead to a lower addictive liability of opioids with selective low potency for the µ-opioid-Gal1 receptor heteromer, exemplified by methadone.

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.

Revisiting the Functional Role of Dopamine D4 Receptor Gene Polymorphisms: Heteromerization-Dependent Gain of Function of the D4.7 Receptor Variant.

The two most common polymorphisms of the human DRD4 gene encode a dopamine D4 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 D2 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.

Luciferase complementation based-detection of G-protein-coupled receptor activity.

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.

Essential Control of the Function of the Striatopallidal Neuron by Pre-coupled Complexes of Adenosine A2A-Dopamine D2 Receptor Heterotetramers and Adenylyl Cyclase.

The central adenosine system and adenosine receptors play a fundamental role in the modulation of dopaminergic neurotransmission. This is mostly achieved by the strategic co-localization of different adenosine and dopamine receptor subtypes in the two populations of striatal efferent neurons, striatonigral and striatopallidal, that give rise to the direct and indirect striatal efferent pathways, respectively. With optogenetic techniques it has been possible to dissect a differential role of the direct and indirect pathways in mediating "Go" responses upon exposure to reward-related stimuli and "NoGo" responses upon exposure to non-rewarded or aversive-related stimuli, respectively, which depends on their different connecting output structures and their differential expression of dopamine and adenosine receptor subtypes. The striatopallidal neuron selectively expresses dopamine D2 receptors (D2R) and adenosine A2A receptors (A2AR), and numerous experiments using multiple genetic and pharmacological in vitro, in situ and in vivo approaches, demonstrate they can form A2AR-D2R heteromers. It was initially assumed that different pharmacological interactions between dopamine and adenosine receptor ligands indicated the existence of different subpopulations of A2AR and D2R in the striatopallidal neuron. However, as elaborated in the present essay, most evidence now indicates that all interactions can be explained with a predominant population of striatal A2AR-D2R heteromers forming complexes with adenylyl cyclase subtype 5 (AC5). The A2AR-D2R heteromer has a tetrameric structure, with two homodimers, which allows not only multiple allosteric interactions between different orthosteric ligands, agonists, and antagonists, but also the canonical Gs-Gi antagonistic interaction at the level of AC5. We present a model of the function of the A2AR-D2R heterotetramer-AC5 complex, which acts as an integrative device of adenosine and dopamine signals that determine the excitability and gene expression of the striatopallidal neurons. The model can explain most behavioral effects of A2AR and D2R ligands, including the psychostimulant effects of caffeine. The model is also discussed in the context of different functional striatal compartments, mainly the dorsal and the ventral striatum. The current accumulated knowledge of the biochemical properties of the A2AR-D2R heterotetramer-AC5 complex offers new therapeutic possibilities for Parkinson's disease, schizophrenia, SUD and other neuropsychiatric disorders with dysfunction of dorsal or ventral striatopallidal neurons.

α2A- and α2C-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands.

The poor norepinephrine innervation and high density of Gi/o-coupled α2A- and α2C-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 D2-like receptor ligands, such as the D3 receptor agonist 7-OH-PIPAT and the D4 receptor agonist RO-105824, to α2-adrenoceptors in cortical and striatal tissue, which express α2A-adrenoceptors and both α2A- and α2C-adrenoceptors, respectively. The affinity of dopamine for α2-adrenoceptors was found to be similar to that for D1-like and D2-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α2A- and α2C-adrenoceptors. Their ability to activate Gi/o proteins through α2A- and α2C-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 α2-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 α2A- and α2C-adrenoceptors was nearly identical to its binding to the crystallized D3 receptor. Therefore, we provide conclusive evidence that α2A- and α2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D2-like receptor ligands, which calls for revisiting previous studies with those ligands.

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.

Gs- versus Golf-dependent functional selectivity mediated by the dopamine D1 receptor.

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.

Development of novel biosensors to study receptor-mediated activation of the G-protein α subunits Gs and Golf.

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 ß2 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.

Novel Bivalent Ligands Based on the Sumanirole Pharmacophore Reveal Dopamine D2 Receptor (D2R) Biased Agonism.

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 ß-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 Gi/o-proteins, while reducing or suppressing potency and efficacy toward ß-arrestin recruitment. Compound 19 was identified as a new lead for its selective D2 G-protein biased agonism with an EC50 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 ß-arrestin recruitment in D2R-BRET functional assays.

Key role of the dopamine D4 receptor in the modulation of corticostriatal glutamatergic neurotransmission.

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.

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 Noncanonical Neurotransmitter Activation: Norepinephrine as a Dopamine D2-Like Receptor Agonist.

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 ß 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.

Orexin-corticotropin-releasing factor receptor heteromers in the ventral tegmental area as targets for cocaine.

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 CRF1 receptor (CRF1R) and orexin OX1 receptors (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 σ1 receptor (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.