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BACKGROUND AND PURPOSE The cannabinoid CB2 receptor (CB2 ) is a promising therapeutic target for modulating inflammation. However, little is known surrounding the mechanisms underpinning CB2 desensitisation and regulation, particularly the role of G protein-coupled receptor kinases (GRKs). Here, we evaluated the role of six GRK isoforms in β-arrestin recruitment to CB2 . Mutagenesis of several distal C-terminal aspartic acid residues was also performed in an attempt to delineate additional structural elements involved in the regulation of CB2 . EXPERIMENTAL APPROACH In CB2 -expressing HEK 293 cells, β-arrestin translocation was measured using real-time BRET assays. G protein dissociation BRET assays were performed to assess the activation and desensitisation of CB2 in the presence of β-arrestin 2. KEY RESULTS Overexpression of GRK isoforms 1-6 failed to considerably improve translocation of either β-arrestin 1 or β-arrestin 2 to CB2 . Consistent with this, inhibition of endogenous GRK2/3 did not substantially reduce β-arrestin 2 translocation. Mutagenesis of C-terminal aspartic acid residues resulted in attenuation of β-arrestin 2 translocation, which translated to a reduction in desensitisation of G protein activation. CONCLUSION AND IMPLICATIONS Our findings suggest that CB2 does not adhere to the classical GPCR regulatory paradigm, entailing GRK- and β-arrestin-mediated desensitisation. Instead, C-terminal aspartic acid residues may act as phospho-mimics to induce β-arrestin activation. This study provides novel insights into the regulatory mechanisms of CB2 , which may aid in our understanding of drug tolerance and dependence.

Cholinergic interneurons (CINs) in the striatum respond to salient stimuli with a multiphasic response, including a pause, in neuronal activity. Slice-physiology experiments have shown the importance of dopamine D2 receptors (D2Rs) in regulating CIN pausing, yet the behavioral significance of the CIN pause and its regulation by dopamine in vivo is still unclear. Here, we show that D2R upregulation in CINs of the nucleus accumbens (NAc) lengthens the pause in CIN activity ex vivo and enlarges a stimulus-evoked decrease in acetylcholine (ACh) levels during behavior. This enhanced dip in ACh levels is associated with a selective deficit in the learning to inhibit responding in a Go/No-Go task. Our data demonstrate, therefore, the importance of CIN D2Rs in modulating the CIN response induced by salient stimuli and point to a role of this response in inhibitory learning. This work has important implications for brain disorders with altered striatal dopamine and ACh function, including schizophrenia and attention-deficit hyperactivity disorder (ADHD).

The formation of neural circuits requires extensive interactions of cell-surface proteins to guide axons to their correct target neurons. Trans-cellular interactions of the adhesion G protein-coupled receptor latrophilin-2 (Lphn2) with its partner teneurin-3 instruct the precise assembly of hippocampal networks by reciprocal repulsion. Lphn2 acts as a repulsive receptor in distal CA1 neurons to direct their axons to the proximal subiculum, and as a repulsive ligand in the proximal subiculum to direct proximal CA1 axons to the distal subiculum. It remains unclear if Lphn2-mediated intracellular signaling is required for its role in either context. Here, we show that Lphn2 couples to Gα12/13 in heterologous cells; this coupling is increased by constitutive exposure of the tethered agonist. Specific mutations of Lphn2’s tethered agonist region disrupt its G protein coupling and autoproteolytic cleavage, whereas mutating the autoproteolytic cleavage site alone prevents cleavage but preserves a functional tethered agonist. Using an in vivo misexpression assay, we demonstrate that wild-type Lphn2 misdirects proximal CA1 axons to the proximal subiculum and that Lphn2 tethered agonist activity is required for its role as a repulsive receptor in axons. By contrast, neither tethered agonist activity nor autoproteolysis were necessary for Lphn2’s role as a repulsive ligand in the subiculum target neurons. Thus, tethered agonist activity is required for Lphn2-mediated neural circuit assembly in a context-dependent manner.

It was first posited, more than five decades ago, that the etiology of schizophrenia involves overstimulation of dopamine receptors. Since then, advanced clinical research methods, including brain imaging, have refined our understanding of the relationship between striatal dopamine and clinical phenotypes as well as disease trajectory. These studies point to striatal dopamine D2 receptors, the main target for all current antipsychotic medications, as being involved in both positive and negative symptoms. Simultaneously, animal models have been central to investigating causal relationships between striatal dopamine D2 receptors and behavioral phenotypes relevant to schizophrenia. We begin this article by reviewing the circuit, cell-type and subcellular locations of dopamine D2 receptors and their downstream signaling pathways. We then summarize results from several mouse models in which D2 receptor levels were altered in various brain regions, cell-types and developmental periods. Behavioral, electrophysiological and anatomical consequences of these D2 receptor perturbations are reviewed with a selective focus on striatal circuit function and alterations in motivated behavior, a core negative symptom of schizophrenia. These studies show that D2 receptors serve distinct physiological roles in different cell types and at different developmental time points, regulating motivated behaviors in sometimes opposing ways. We conclude by considering the clinical implications of this complex regulation of striatal circuit function by D2 receptors.

Adhesion G protein-coupled receptor latrophilin 3 (ADGRL3), a cell adhesion molecule highly expressed in the central nervous system, acts in synapse formation through trans interactions with its ligands. It is largely unknown if these interactions serve a purely adhesive function or can modulate G protein signaling. To assess how different structural elements of ADGRL3 (e.g., the adhesive domains, autoproteolytic cleavage site, or tethered agonist (TA)) impact receptor function, we require constructs that disrupt specific receptor features without impacting others. While we showed previously that mutating conserved Phe and Met residues in the TA of ADGRL3-C-terminal fragment (CTF), a CTF truncated to the G protein-coupled receptor proteolysis site, abolishes receptor-mediated G protein activation, we now find that autoproteolytic cleavage is disrupted in the full-length version of this construct. To identify a construct that disrupts TA-dependent activity without impacting proteolysis, we explored other mutations in the TA. We found that mutating the sixth and seventh residues of the TA, Leu and Met, to Ala impaired activity in a serum response element activity assay for both full-length and CTF constructs. We confirmed this activity loss results from impaired G protein coupling using an assay that acutely exposes the TA through controlled proteolysis. The ADGRL3 mutant expresses normally at the cell surface, and immunoblotting shows that it undergoes normal autoproteolysis. Thus, we found a construct that disrupts tethered agonism while retaining autoproteolytic cleavage, providing a tool to disentangle these functions in vivo. Our approach and specific findings are likely to be broadly applicable to other adhesion receptors.

Glutamate acts at eight metabotropic glutamate (mGlu) receptor subtypes expressed in a partially overlapping fashion in distinct brain circuits. Recent evidence indicates that specific mGlu receptor protomers can heterodimerize and that these heterodimers can exhibit different pharmacology when compared to their homodimeric counterparts. Group III mGlu agonist-induced suppression of evoked excitatory potentials and induction of long-term potentiation at Schaffer collateral-CA1 (SC-CA1) synapses in the rodent hippocampus can be blocked by the selective mGlu

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

The dopamine D2 receptor (D2R) is the target of drugs used to treat the symptoms of Parkinson’s disease and schizophrenia. The D2R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D2R arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of D2R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D2R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD2R and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the D2R. We compared measurements of D2R phosphorylation with other measurements of D2R signalling to profile selected D2R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D2R regulation and signalling.

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of β-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

Abuse of psychostimulants, including amphetamines (AMPHs), is a major public health problem with profound psychiatric, medical, and psychosocial complications. The actions of these drugs at the dopamine transporter (DAT) play a critical role in their therapeutic efficacy as well as their liability for abuse and dependence. To date, however, the mechanisms that mediate these actions are not well-understood, and therapeutic interventions for AMPH abuse have been limited. Drug exposure can induce broad changes in gene expression that can contribute to neuroplasticity and effect long-lasting changes in neuronal function. Identifying genes and gene pathways perturbed by drug exposure is essential to our understanding of the molecular basis of drug addiction. In this study, we used

The four vertebrate arrestins play a key role in the desensitization and internalization of G protein-coupled receptors (GPCRs) and also mediate receptor-dependent signaling. Recent work has shown that bias for arrestin vs G protein signaling could offer certain therapeutic advantages (or disadvantages) in different systems, making assays that measure arrestin binding to receptors important for drug discovery efforts. Herein, we briefly review several commonly used techniques for measuring arrestin binding to receptors, as well as provide an in-depth and methodologically focused review of two methods that do not require receptor modification. The first approach measures direct binding between purified arrestin and rhodopsin, and the second measures the recruitment of arrestin to receptors in living cells.

The cannabinoid CBIn CBOverexpression of GRK isoforms 1-6 failed to considerably improve translocation of either β-arrestin 1 or β-arrestin 2 to CBOur findings suggest that CB

Description, D2R-NMDAR receptor heteromers are potential therapeutic targets in drug addiction., Addictive drugs increase dopamine in the nucleus accumbens (NAc), where it persistently shapes excitatory glutamate transmission and hijacks natural reward processing. Here, we provide evidence, from mice to humans, that an underlying mechanism relies on drug-evoked heteromerization of glutamate N-methyl-d-aspartate receptors (NMDAR) with dopamine receptor 1 (D1R) or 2 (D2R). Using temporally controlled inhibition of D1R-NMDAR heteromerization, we unraveled their selective implication in early phases of cocaine-mediated synaptic, morphological, and behavioral responses. In contrast, preventing D2R-NMDAR heteromerization blocked the persistence of these adaptations. Interfering with these heteromers spared natural reward processing. Notably, we established that D2R-NMDAR complexes exist in human samples and showed that, despite a decreased D2R protein expression in the NAc, individuals with psychostimulant use disorder display a higher proportion of D2R forming heteromers with NMDAR. These findings contribute to a better understanding of molecular mechanisms underlying addiction and uncover D2R-NMDAR heteromers as targets with potential therapeutic value.

Tianeptine is an atypical antidepressant used in Europe to treat patients who respond poorly to selective serotonin reuptake inhibitors (SSRIs). The recent discovery that tianeptine is a mu opioid receptor (MOR) agonist has provided a potential avenue for expanding our understanding of antidepressant treatment beyond the monoamine hypothesis. Thus, our studies aim to understand the neural circuits underlying tianeptine's antidepressant effects. We show that tianeptine induces rapid antidepressant-like effects in mice after as little as one week of treatment. Critically, we also demonstrate that tianeptine's mechanism of action is distinct from fluoxetine in two important aspects: (1) tianeptine requires MORs for its chronic antidepressant-like effect, while fluoxetine does not, and (2) unlike fluoxetine, tianeptine does not promote hippocampal neurogenesis. Using cell-type specific MOR knockouts we further show that MOR expression on GABAergic cells-specifically somatostatin-positive neurons-is necessary for the acute and chronic antidepressant-like responses to tianeptine. Using central infusion of tianeptine, we also implicate the ventral hippocampus as a potential site of antidepressant action. Moreover, we show a dissociation between the antidepressant-like phenotype and other opioid-like phenotypes resulting from acute tianeptine administration such as analgesia, conditioned place preference, and hyperlocomotion. Taken together, these results suggest a novel entry point for understanding what circuit dysregulations may occur in depression, as well as possible targets for the development of new classes of antidepressant drugs.

Mutations in the GNB1 gene, encoding the G beta(1) subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three G beta(1) mutations (K78R, I80N and I80T) using computational and functional approaches. In heterologous expression models, these mutations did not alter the coupling between G protein-coupled receptors to G(i/o), or the G beta gamma regulation of the neuronal voltage-gated Ca2+ channel Ca(V)2.2. However, the mutations profoundly affected the G beta gamma regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in G beta(1) protein expression levels, G beta gamma binding to cytosolic segments of GIRK subunits, and in G beta gamma function, and included gain-of-function for K78R or loss-of-function for I80T/N, which were GIRK subunit-specific. Our findings offer new insights into subunit-dependent gating of GIRKs by G beta gamma, and indicate diverse etiology of GNB1 Encephalopathy cases, bearing a potential for personalized treatment.

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous protein-truncating mutation in CNTNAP2, to study its effects on embryonic cortical development. Patients with this mutation present with clinical characteristics of brain overgrowth. Patient-derived forebrain organoids displayed an increase in volume and total cell number that is driven by increased neural progenitor proliferation. Single-cell RNA sequencing revealed PFC-excitatory neurons to be the key cell types expressing CNTNAP2. Gene ontology analysis of differentially expressed genes (DEgenes) corroborates aberrant cellular proliferation. Moreover, the DEgenes are enriched for ASD-associated genes. The cell-type-specific signature genes of the CNTNAP2-expressing neurons are associated with clinical phenotypes previously described in patients. The organoid overgrowth phenotypes were largely rescued after correction of the mutation using CRISPR-Cas9. This CNTNAP2-organoid model provides opportunity for further mechanistic inquiry and development of new therapeutic strategies for ASD., Mutations in CNTNAP2 have been associated with a syndromic form of Autism Spectrum Disorder. Here the authors show that forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of ASD with a homozygous truncating mutation in CNTNAP2 displayed an increase in volume and total cell number, which is driven by abnormal cellular proliferation and neurogenesis.

Decades of research have highlighted the importance of optimal stimulation of cortical dopaminergic receptors, particularly the D1R receptor (D1R), for prefrontal-mediated cognition. This mechanism is particularly relevant to the cognitive deficits in schizophrenia, given the abnormalities in cortical dopamine (DA) neurotransmission and in the expression of D1R. Despite the critical need for D1R-based therapeutics, many factors have complicated their development and prevented this important therapeutic target from being adequately interrogated. Challenges include determination of the optimal level of D1R stimulation needed to improve cognitive performance, especially when D1R expression levels, affinity states, DA levels, and the resulting D1R occupancy by DA, are not clearly known in schizophrenia, and may display great interindividual and intraindividual variability related to cognitive states and other physiological variables. These directly affect the selection of the level of stimulation necessary to correct the underlying neurobiology. The optimal mechanism for stimulation is also unknown and could include partial or full agonism, biased agonism, or positive allosteric modulation. Furthermore, the development of D1R targeting drugs has been complicated by complexities in extrapolating from in vitro affinity determinations to in vivo use. Prior D1R-targeted drugs have been unsuccessful due to poor bioavailability, pharmacokinetics, and insufficient target engagement at tolerable doses. Newer drugs have recently become available, and these must be tested in the context of carefully designed paradigms that address methodological challenges. In this paper, we discuss how a better understanding of these challenges has shaped our proposed experimental design for testing a new D1R/D5R partial agonist, PF-06412562, renamed CVL-562.

Substance use and related mental health epidemics are causing increasing suffering and death in diverse communities.1,2Despite extensive efforts focused on developing pharmacotherapies for treating substance use disorders, there is an urgent need for radically different therapeutic approaches.3,4Ibogaine provides an important drug prototype in this direction, as a psychoactive iboga alkaloid suggested to have the ability to interrupt opioid use in drug-dependent humans.5However, ibogaine and its major metabolite noribogaine present considerable safety risk associated with cardiac arrhythmias.6We introduce a new class of iboga alkaloids - “oxa-iboga” - defined as benzofuran-containing iboga analogs and created via structural editing of the iboga skeleton. The oxa-iboga compounds act as potent kappa opioid receptor agonistsin vitroandin vivo, but exhibit atypical behavioral features compared to standard kappa psychedelics. We show that oxa-noribogaine has greater therapeutic efficacy in rat models of opioid use, and no cardiac pro-arrhythmic potential, in contrast to noribogaine. Oxa-noribogaine induces long-lasting suppression of morphine and fentanyl intake after a single dose, persistent reduction of morphine intake and reinforcing efficacy after a short treatment regimen, and suppression of morphine and fentanyl drug seeking in relapse models. Oxa-noribogaine also induces a lasting elevation of neurotrophin proteins in the ventral tegmental area and medial prefrontal cortex, consistent with targeted neuroplasticity induction and alteration of addiction-like states. As such, oxa-iboga compounds represent candidates for a novel type of pharmacotherapy for treatment of opioid use disorder.