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G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β 2 adrenergic receptor (β 2 AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the G s heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β 2 AR–G s complexes are formed and the efficiency of nucleotide exchange leading to G s activation. These data also reveal transient nucleotide-bound β 2 AR–G s species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism. Single-molecule FRET imaging provides insights into the allosteric link between the ligand-binding and G-protein nucleotide-binding pockets of the β 2 adrenergic receptor (β 2 AR) and improved understanding of the G-protein activation mechanism. Monitoring G-protein activation by a GPCR G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease, and understanding the molecular basis of ligand efficacy downstream of receptor activation is important for therapeutic development. For the GPCR β 2 adrenergic receptor (β 2 AR), receptor activation and coupling to the G protein G s involve outward movements of the receptor transmembrane helix 6 (TM6). Here, Scott Blanchard and colleagues apply single-molecule fluorescence resonance energy transfer (smFRET) imaging methods to directly monitor movements of TM6 in β 2 AR bound to a range of ligands with distinct efficacy profiles. They find that partial and full agonists affect TM6 motions in an efficacy-dependent manner. These motions differentially regulate the rate at which β 2 AR couples with GDP-bound G s and the efficiency of nucleotide exchange leading to G s activation. The work provides single-molecule insight into the allosteric link between the ligand- and G-protein-nucleotide-binding pockets of the receptor and improved understanding of the G-protein activation mechanism.

Biased agonism describes the ability of ligands to stabilize different conformations of a GPCR linked to distinct functional outcomes and offers the prospect of designing pathway-specific drugs that avoid on-target side effects. This mechanism is usually inferred from pharmacological data with the assumption that the confounding influences of observational (that is, assay dependent) and system (that is, cell background dependent) bias are excluded by experimental design and analysis. Here we reveal that ‘kinetic context’, as determined by ligand-binding kinetics and the temporal pattern of receptor-signalling processes, can have a profound influence on the apparent bias of a series of agonists for the dopamine D 2 receptor and can even lead to reversals in the direction of bias. We propose that kinetic context must be acknowledged in the design and interpretation of studies of biased agonism. Biased agonists act at a receptor to preferentially induce distinct intracellular signalling responses over others. Here the authors show how kinetics of ligand binding and signaling responses greatly influence observed bias profiles, and hence must be considered when studying biased agonists.

Dopamine D2 receptors (D2Rs) in the nucleus accumbens (NAc) regulate motivated behavior, but the underlying neurobiological mechanisms remain unresolved. Here, we show that selective upregulation of D2Rs in the indirect pathway of the adult NAc enhances the willingness to work for food. Mechanistic studies in brain slices reveal that D2R upregulation attenuates inhibitory transmission at two main output projections of the indirect pathway, the classical long-range projections to the ventral pallidum (VP), as well as local collaterals to direct pathway medium spiny neurons. In vivo physiology confirms the reduction in indirect pathway inhibitory transmission to the VP, and inhibition of indirect pathway terminals to VP is sufficient to enhance motivation. In contrast, D2R upregulation in the indirect pathway does not disinhibit neuronal activity of the direct pathway in vivo. These data suggest that D2Rs in ventral striatal projection neurons promote motivation by weakening the canonical output to the ventral pallidum. Dopamine D2 receptor activity in the nucleus accumbens is associated with regulation of motivated responding. Here the authors show that overexpression of D2 receptors specifically in ventral striatal projection neurons leads to an increase in the willingness to work by reducing inhibitory transmission to ventral pallidal neurons.

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.

Metabotropic glutamate (mGlu) receptor protomers can heterodimerize, leading to different pharmacology compared to their homodimeric counterparts. Here, we use complemented donor-acceptor resonance energy transfer (CODA-RET) technology that distinguishes signaling from defined mGlu heterodimers or homodimers, together with targeted mutagenesis of receptor protomers and computational docking, to elucidate the mechanism of activation and differential pharmacology in mGlu 2/4 heteromers. We demonstrate that positive allosteric modulators (PAMs) that bind an upper allosteric pocket in the mGlu 4 transmembrane domain are active at both mGlu 4/4 homomers and mGlu 2/4 heteromers, while those that bind a lower allosteric pocket within the same domain are efficacious in homomers but not heteromers. We further demonstrate that both protomers of mGlu 2/4 heteromers are cis-activated by their orthosteric agonists, signaling independently with no trans-activation detected. Intriguingly, however, upper pocket mGlu 4 PAMs enable trans-activation in mGlu 2/4 heteromers from mGlu 4 to the mGlu 2 protomer and also enhance cis-activation of the mGlu 2 protomer. While mGlu 2  PAMs enhanced mGlu 2 cis-activation in the heterodimer, we were unable to detect trans-activation in the opposite direction from mGlu 2 to the mGlu 4 protomer, suggesting an asymmetry of signaling. These insights into the molecular logic of this receptor heteromer are critical to building toward precision targeted therapies for multiple neuropsychiatric disorders. Metabotropic glutamate (mGlu) receptors form both homo- and heterodimers that respond to ligands differently. Here, the authors elucidate the mechanism of activation and differential pharmacology in mGlu 2/4 heteromers, which may guide targeted therapy.

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.

Arrestin translocation and signaling have come to the fore of the G protein-coupled receptor molecular pharmacology field. Some receptor-arrestin interactions are relatively well understood and considered responsible for specific therapeutic or adverse outcomes. Coupling of arrestins with cannabinoid receptors 1 (CB ) and 2 (CB ) has been reported, though the majority of studies have not systematically characterized the differential ligand dependence of this activity. In addition, many prior studies have utilized bovine (rather than human) arrestins, and the most widely applied assays require reporter-tagged receptors, which prevent meaningful comparison between receptor types. We have employed a bioluminescence resonance energy transfer (BRET) method that does not require the use of tagged receptors and thereby allows comparisons of arrestin translocation between receptor types, as well as with cells lacking the receptor of interest - an important control. The ability of a selection of CB and CB agonists to stimulate cell surface translocation of human and bovine β-arrestin-1 and -2 was assessed. We find that some CB ligands induce moderate β-arrestin-2 translocation in comparison with vasopressin V receptor (a robust arrestin recruiter); however, CB coupling with β-arrestin-1 and CB with either arrestin elicited low relative efficacies. A range of efficacies between ligands was evident for both receptors and arrestins. Endocannabinoid 2-arachidonoylglycerol stood out as a high efficacy ligand for translocation of β-arrestin-2 via CB . Δ -tetrahydrocannabinol was generally unable to elicit translocation of either arrestin subtype via CB or CB ; however, control experiments revealed translocation in cells not expressing CB /CB , which may assist in explaining some discrepancy with the literature. Overexpression of GRK2 had modest influence on CB /CB -induced arrestin translocation. Results with bovine and human arrestins were largely analogous, but a few instances of inconsistent rank order potencies/efficacies between bovine and human arrestins raise the possibility that subtle differences in receptor conformation stabilized by these ligands manifest in disparate affinities for the two arrestin species, with important potential consequences for interpretation in ligand bias studies. As well as contributing important information regarding CB /CB ligand-dependent arrestin coupling, our study raises a number of points for consideration in the design and interpretation of arrestin recruitment assays.

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. Gα 12/13 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. Among the adhesion receptor class of GPCRs, which are understudied, the adhesion receptor ADGRL3 can be activated by its own tethered agonist and couples to G protein G12/13 and somewhat to Gq.

Neurotransmitter:Na + symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na + gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport. Conformational states of a membrane transporter The neurotransmitter:Na + symporter (NSS) family stops cellular signalling by recapturing released neurotransmitters from the synapse in a reuptake process driven by the Na + gradient. Drugs that interfere with this mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. The conformational changes associated with substrate binding have been studied in the prokaryotic NSS homologue LeuT using single-molecule fluorescence imaging experiments and molecular dynamics simulations. The observed single-molecule transitions reflect the structural dynamics of the intracellular region of the transporter, and can be interpreted in the context of an allosteric mechanism coupling ion and substrate binding to transport. Neurotransmitter:Na + symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na + gradient. Here, single-molecule fluorescence imaging assays have been combined with molecular dynamics simulations to probe the conformational changes that are associated with substrate binding and transport by a prokaryotic NSS homologue, LeuT. The findings are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.

LeuT is a Na + -coupled amino acid transporter that is similar in sequence and function to eukaryotic neurotransmitter/sodium symporters, which are active in reuptake of neurotransmitters from the synapse. Distance measurements between spin-label pairs are used to identify ligand-dependent structural transitions in LeuT. The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated, and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we used distance measurements between spin-label pairs to elucidate Na + - and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.