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The article focuses on deciphering downstream receptor signaling, particularly the molecular mechanisms governing ligand efficacy and potency in G protein–coupled receptors (GPCRs). Topics discussed include exploring how individual amino acids in the b2-adrenergic receptor interpret information encoded in the atoms of its endogenous agonist, adrenaline, to drive efficacy and potency and the potential use of identified residues for guiding ligand design in drug discovery.

Maintaining the integrity of cellular membranes is critical to protecting metabolic activities and genetic information from the environment. Regulation of transport across membranes of essential chemicals, including water, nutrients, hormones and many drugs, is therefore key to cellular homeostasis and physiological processes. The two main transporter superfamilies are ATP-binding cassette (ABC) transporters that primarily function as efflux transporters, and the solute carrier (SLC) transporters. SLC transporters encompass 52 gene families with almost 400 different human transporter genes. Although long under-explored, SLC transporters are an emerging drug target class and the molecular target of several approved inhibitor drugs, such as selective serotonin reuptake inhibitors (SSRIs) for depression and sodium/glucose co-transporter (SGLT2) inhibitors for diabetes. Interestingly though, although loss-of-function mutations in numerous human SLC transporters are linked to Mendelian diseases, few reports of SLC transporter activators have appeared, and only inhibitors have been advanced to clinical studies. In this commentary, we discuss several strategies for potentiating SLC transporter function, from direct acting potentiators to modulators of transcription, translation or trafficking. We review the progress made in recent years toward the understanding of the structural and molecular basis of SLC transporter function and the pathways and mechanisms that regulate SLC expression, and describe the opportunities these new insights present for discovery of SLC transporter potentiators. Finally, we highlight the challenges associated with the various approaches and provide some thoughts on future directions that might facilitate the search for SLC potentiators with therapeutic potential. [ABSTRACT FROM AUTHOR]

Background: The dopamine transporter (DAT), a member of the neurotransmitter:Na+ symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end. Methodology/Principal Findings: Key findings are (1) a second substrate binding site in the extracellular vestibule, and (2) models of the conformational states identified as occluded, doubly occupied, and inward-facing. The transition between these states involve a spatially ordered sequence of interactions between the two substrate-binding sites, followed by rearrangements in structural elements located between the primary binding site and the cytoplasmic end. These rearrangements are facilitated by identified conserved hinge regions and a reorganization of interaction networks that had been identified as gates. Conclusions/Significance: Computational simulations supported by information available from experiments in DAT and other NSS transporters have produced a detailed mechanistic proposal for the dynamic changes associated with substrate transport in DAT. This allosteric mechanism is triggered by the binding of substrate in the S2 site in the presence of the substrate in the S1 site. Specific structural elements involved in this mechanism, and their roles in the conformational transitions illuminated here describe, a specific substrate-driven allosteric mechanism that is directly amenable to experiment as shown previously for LeuT. [ABSTRACT FROM AUTHOR]

The author reflects on the pharmacology of schizophrenia. The author claims that practitioners in the field of psychiatry have been dedicated to searching new drugs that offer novel treatments for schizophrenia. Meanwhile, a descriptive chart is provided by the author in order to understand the acetylcholine actions necessary to cholinergic therapies in schizophrenia.

Transport proteins constitute ≈10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment. [ABSTRACT FROM AUTHOR]

Dopaminergic neurotransmission is fine-tuned by the rate of removal of dopamine (DA) from the extracellular space via the Na+/Cl--dependent DA transporter (DAT). DAT is a target of psychostimulants such as amphetamine (AMPH) and cocaine. Previously, we reported that AMPH redistributes the human DAT away from the cell surface. This process was associated with a reduction in transport capacity. This loss of transport capacity may result either from a modification of the function of DAT that is independent of its cell surface redistribution and/or from a reduction in the number of active transporters at the plasma membrane that results from DAT trafficking. To discriminate between these possibilities, we stably transfected HEK-293 cells with a yellow fluorescent protein (YFP)-tagged human DAT (hDAT cells). In hDAT cells, acute exposure to AMPH induced a time-dependent loss of hDAT activity. By coupling confocal imaging with patch-clamp whole-cell recordings, we have demonstrated for the first time that the loss of AMPH-induced hDAT activity temporally parallels the accumulation of intracellular hDAT. In addition, presteady-state current analysis revealed a cocaine-sensitive, voltage-dependent capacitance current that correlated with the level of transporter membrane expression and in turn served to monitor the AMPH-induced trafficking of hDAT. We found that the decrease in hDAT cell surface expression induced by AMPH was not paralleled by changes in the ability of the single transporter to carry charges. Quasi-stationary noise analysis of the AMPHinduced hDAT currents revealed that the unitary transporter current remained unaltered during the loss of hDAT membrane expression. Taken together, these data strongly suggest that the AMPH-induced reduction of hDAT transport capacity results from the removal of active hDAT from the plasma membrane. [ABSTRACT FROM AUTHOR]

The binding site of the dopamine D[SUB2] receptor (D2R), like those of homologous rhodopsin-like G protein-coupled receptors (GPCRs) that bind small molecules, is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). The high- resolution structure of bovine rhodopsin, however, revealed that the second extracellular loop (E2), which connects TM4 and TM5, folds down into the transmembrane domain and forms part of the ligand-binding surface for retinal. Whether E2 plays a related role in other rhodopsin-like GPCRs is unclear. To address this issue, we have now mutated to cysteine, one at a time, 10 consecutive residues in E2 of D2R. The reaction of five of these mutants with sulfhydryl reagents inhibited antagonist binding, and bound antagonist protected two, N184C and N186C, from reaction. The pattern of accessibility in E2 is consistent with a structure similar to that of bovine rhodopsin, in which the region C-terminal to the conserved disulfide bond is deeper in the binding-site crevice than is the N-terminal part of E2. Thus, E2 likely contributes to the binding site in the D2R and probably in other aminergic GPCRs as well. Knowledge of its detailed positioning and interactions with ligand would benefit GPCR molecular modeling and facilitate the design of novel drugs. [ABSTRACT FROM AUTHOR]

Examines the approaches to the identification of the residues forming the binding sites for agonists, antagonists, and allosteric modulators in the family of aminergic G protein-coupled receptors (GPCR). Factors that altered the pharmacology of GPCR; Strategy for comparing the aligned residues in different GPCRs; Methods to identify binding site residues.

Examines the fourth transmembrane segment (TMS) of the dopamine D2 receptor. Use of substituted cysteine accessibility method for residue mapping of binding site-crevices; Account on the residues-to-cysteine mutations in the fourth TMS; Implication of homologous G-protein-coupled receptors sequence variation on domain morphology.

Examines the electrostatic and aromatic microdomains within the binding-site crevice of dopamine D2 receptor. Binding-site location; Residue mapping in the third, fifth, sixth and seventh membrane-spanning segments; Reaction of cysteine mutants with charged, hydrophilic, lipophobic and sulfhydryl-specific reagents; Pattern of accessibility.

Reports on the application of substituted-cysteine accessibility method in identifying residues in the sixth membrane-spanning segment of the dopamine D2 receptor which contribute to the binding-site crevice. Methodology used in the study; Effects of cysteine substitution on antagonist binding; Results of the study; Discussion on the results of the study.

Presents a study on the identity of the cysteine residues, the modification of which affects [3H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl) tropane (CFT) binding, and the determination of the topology of reactive cysteines. Mutation of the cysteines; What was observed about CFT binding in X5C; Impact of the binding of cocaine on the reaction of MTS ethylammonium (MTSEA).

Presents a study that identifies the residues that form the surface of the binding-site crevice in the human dopamine D2 receptor. Substituted-cysteine accessibility method (SCAM); Mapping of the residues in the third membrane-spanning segment that are exposed in the binding site crevice; Formation of a fixed alpha-helix exposed in the binding-site crevice.

The article looks at issues concerning class A G-protein-coupled receptors (GPCR) specifically its structural properties and the relevance of oligomerization to receptor function. Topics covered include biophysical methods used to demonstrate the existence of GPCR oligomers in cells, main weakness of resonance energy transfer (RET) methods, and observation on the ternary structure of dimer or oligomer. Also cited are the core functions of GPCR oligomers.

Background and Purpose: Mitragynine, the major alkaloid in Mitragyna speciosa (kratom), is a partial agonist at the μ opioid receptor. CYP3A‐dependent oxidation of mitragynine yields the metabolite 7‐OH mitragynine, a more efficacious μ receptor agonist. While both mitragynine and 7‐OH mitragynine can induce anti‐nociception in mice, recent evidence suggests that 7‐OH mitragynine formed as a metabolite is sufficient to explain the anti‐nociceptive effects of mitragynine. However, the ability of 7‐OH mitragynine to induce μ receptor‐dependent respiratory depression has not yet been studied. Experimental Approach: Respiration was measured in awake, freely moving, male CD‐1 mice, using whole body plethysmography. Anti‐nociception was measured using the hot plate assay. Morphine, mitragynine, 7‐OH mitragynine and the CYP3A inhibitor ketoconazole were administered orally. Key Results: The respiratory depressant effects of mitragynine showed a ceiling effect, whereby doses higher than 10 mg·kg−1 produced the same level of effect. In contrast, 7‐OH mitragynine induced a dose‐dependent effect on mouse respiration. At equi‐depressant doses, both mitragynine and 7‐OH mitragynine induced prolonged anti‐nociception. Inhibition of CYP3A reduced mitragynine‐induced respiratory depression and anti‐nociception without affecting the effects of 7‐OH mitragynine. Conclusions and Implications: Both the anti‐nociceptive effects and the respiratory depressant effects of mitragynine are partly due to its metabolic conversion to 7‐OH mitragynine. The limiting rate of conversion of mitragynine into its active metabolite results in a built‐in ceiling effect of the mitragynine‐induced respiratory depression. These data suggest that such 'metabolic saturation' at high doses may underlie the improved safety profile of mitragynine as an opioid analgesic. [ABSTRACT FROM AUTHOR]

Focuses on probes that reversibly recognize oligohistidine sequences engineered into proteins, which can provide insights into the structure and location of proteins in living cells. Introduction of a modular labeling method to study protein structure and localization in living cells; Use of substituted cysteine accessibility method (SCAM) for gaining information about structure, function and topology of membrane proteins in living cells; Description of the mechanism of fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) methods; Development of membrane-permeant, red biarsenical dye (ReAsH)-based chromophore-assisted light inactivation (CALI) for targeted proteins.

Dopamine (DA) receptors play critical roles in a wide range of behaviours, including sensory processing, motor function, reward and arousal. As such, aberrant DA signalling is associated with numerous neurological and psychiatric disorders. Therefore, understanding the mechanisms by which DA neurotransmission drives intracellular signalling pathways that modulate behaviour can provide critical insights to guide the development of targeted therapeutics. Drosophila melanogaster has emerged as a powerful model with unique advantages to study the mechanisms underlying DA neurotransmission and associated behaviours in a controlled and systematic manner. Many regions in the fly brain innervated by dopaminergic neurons have been mapped and linked to specific behaviours, including associative learning and arousal. Here, we provide an overview of the homology between human and Drosophila dopaminergic systems and review the current literature on the pharmacology, molecular signalling mechanisms and behavioural outcome of DA receptor activation in the Drosophila brain. [ABSTRACT FROM AUTHOR]

Background and Purpose: Opioid-induced respiratory depression limits the use of μ- opioid receptor agonists in clinical settings and is the main cause of opioid overdose fatalities. The relative potential of different opioid agonists to induce respiratory depression at doses exceeding those producing analgesia is understudied despite its relevance to assessments of opioid safety. Here we evaluated the respiratory depressant and anti-nociceptive effects of three novel opioids and relate these measurements to their in vitro efficacy. Experimental Approach: Respiration was measured in awake, freely moving male CD-1 mice using whole body plethysmography. Anti-nociception was measured using the hot plate test. Morphine, oliceridine and tianeptine were administered intraperitoneally, whereas methadone, oxycodone and SR-17018 were administered orally. Receptor activation and arrestin-3 recruitment were measured in HEK293 cells using BRET assays. Key Results: Across the dose ranges examined, all opioids studied depressed respiration in a dose-dependent manner, with similar effects at the highest doses, and with tianeptine and oliceridine showing reduced duration of effect, when compared with morphine, oxycodone, methadone and SR-17018. When administered at doses that induced similar respiratory depression, all opioids induced similar anti-nociception, with tianeptine and oliceridine again showing reduced duration of effect. These data were consistent with the in vitro agonist activity of the tested compounds. Conclusion and Implications: In addition to providing effective anti-nociception, the novel opioids, oliceridine, tianeptine and SR-17018 depress respiration in male mice. However, the different potencies and kinetics of effect between these novel opioids may be relevant to their therapeutic application in different clinical settings. [ABSTRACT FROM AUTHOR]

The authors discuss the detection of conformational changes in metabotropic glutamate receptors (mGluRs), with particular focus on G protein-coupled receptors. They explore the activation mechanisms of mGluRs and its role in new drug development. They reference the study by Doumazane and colleagues which developed the fluorescence resonance energy transfer (FRET)-based approach to monitor the conformational changes on ligand binding.

In this article, the author talks about the genetic convergence of neurodevelopmental disorders modeled in drosophila. Topics include Neurodevelopmental disorders comprising diverse and complex conditions characterized by impairments in cognitive function, adaptive behavior, or psychomotor skills; and how the Attention deficit hyperactivity disorder (ADHD) risk alleles has been challenging, mainly because of the polygenic nature of the disorder.

The authors comment on an article that demonstrated how dimer-colored glasses can filter perception, impair recall and provide alternative hypotheses and contradictory findings. Topics covered include observations concerning rhodopsin dimers and the potential of heterodimers for improved pharmacotherapies. Also cited is an observation concerning rhodopsin monomers from detergent.

The dopamine transporter (DAT), which mediates the inactivation of released dopamine through its reuptake, is the primary molecular target for the actions of psychostimulants. An increasing number of studies support an essential role for phosphorylation of serines (Ser) in the distal amino (N) terminus of DAT in regulating its function. Still, the molecular details of the regulation of phosphorylation and its impact on function are not fully understood. To address this, we have developed and characterized two distinct phospho-antibodies that recognize human DAT when it is phosphorylated at Ser7 or Ser12. Our data show that treatment of cells with phorbol 12-myristate 13-acetate (PMA), amphetamine (AMPH) or okadaic acid (OA) leads to an increase in the phosphorylation of DAT at both residues and that these responses are dependent on the activity of protein kinase C. We also show that AMPH-induced and OA-induced phosphorylation of DAT are dependent on Ca 2+ /calmodulin-dependent protein kinase α. Our data further suggest that the lipid raft localization of DAT is necessary for efficient N-terminal phosphorylation and for the associated behavioral effects of AMPH, demonstrating the potential of these novel antibodies as powerful tools to study DAT regulation and function in vivo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 immuno-blotting 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. [ABSTRACT FROM AUTHOR]

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 mGlu7 negative allosteric modulator (NAM), ADX71743. Curiously, a different mGlu7 NAM, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazo nolo[4,5-c]pyridin-4(5H)-one, failed to block these responses in brain slices despite its robust activity at mGlu7 homodimers in vitro. We hypothesized that this might result from heterodimerization of mGlu7 with another mGlu receptor protomer and focused on mGlu8 as a candidate given the reported effects of mGlu8-targeted compounds in the hippocampus. Here, we used complemented donor acceptor-resonance energy transfer to study mGlu7/8 heterodimer activation in vitro and observed that ADX71743 blocked responses of both mGlu7/7 homodimers and mGlu7/8 heterodimers, whereas 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one only antagonized responses of mGlu7/7 homodimers. Taken together with our electrophysiology observations, these results suggest that a receptor with pharmacology consistent with an mGlu7/8 heterodimer modulates the activity of SC-CA1 synapses. Building on this hypothesis, we identified two additional structurally related mGlu7 NAMs that also differ in their activity at mGlu7/8 heterodimers, in a manner consistent with their ability to inhibit synaptic transmission and plasticity at SC-CA1. Thus, we propose that mGlu7/8 heterodimers are a key molecular target for modulating the activity of hippocampal SC-CA1 synapses. [ABSTRACT FROM AUTHOR]

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 GPCR 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‐mediated 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. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB5 toxin, inhibits Gαi/o G proteins and has proven useful for interrogating inhibitory G protein signalling. Pertussis toxin, however, does not inhibit one member of the inhibitory G protein family, Gαz. The role of Gαz signalling has been neglected largely due to a lack of inhibitors. Recently, the identification of another Pertussis-like AB5 toxin was described. Here we show that this toxin, that we call OZITX, specifically inhibits Gαi/o and Gαz G proteins and that expression of the catalytic S1 subunit is sufficient for this inhibition. We identify mutations that render Gα subunits insensitive to the toxin that, in combination with the toxin, can be used to interrogate the signalling of each inhibitory Gα G protein. A recently identified pertussis toxin-like AB5 toxin, OZITX, is found to inhibit Gαi/o and Gαz G proteins. In combination with directed mutations, it is a useful tool for interrogating Gαi/o/z G protein subunits individually. [ABSTRACT FROM AUTHOR]

Abstract: Positron Emission Tomography (PET) has been used for more than three decades to image and quantify dopamine D2 receptors (D2R) in vivo with antagonist radioligands but in the recent years agonist radioligands have also been employed. In vitro competition studies have demonstrated that agonists bind to both a high and a low-affinity state of the D2Rs, of which the high affinity state reflects receptors that are coupled to G-proteins and the low-affinity state reflects receptors uncoupled from G-proteins. In contrast, antagonists bind with uniform affinity to the total pool of receptors. Results of these studies led to the proposal that D2Rs exist in high and low-affinity states for agonists in vivo and sparked the development and use of agonist radioligands for PET imaging with the primary purpose of measuring the proportion of receptors in the high-affinity (activating) state. Although several lines of research support the presence of high and low-affinity states of D2Rs and their detection by in vivo imaging paradigms, a growing body of controversial data has now called this into question. These include both in vivo and ex vivo studies of anesthesia effects, rodent models with increased proportions of high-affinity state D2Rs as well as the molecular evidence for stable receptor–G-protein complexes. In this commentary we review these data and discuss the evidence for the in vivo existence of D2Rs configured in high and low-affinity states and whether or not the high-affinity state of the D2R can, in fact, be imaged in vivo with agonist radioligands. [Copyright &y& Elsevier]

DAR-0100A, the active enantiomer of dihydrexidine, is a potent dopamine D1 agonist under investigation for treatment of cognitive impairment and negative symptoms of schizophrenia. We measured the dose-occupancy relationship for DAR-0100A at D1 receptors using positron emission tomography (PET) imaging in baboons with [11C] NNC112 and its binding to D2 with [11C] raclopride. Two baboons were scanned with [11C] NNC112 at baseline and after three different doses of DAR-0100A. Two baboons were scanned with [11C] raclopride at baseline and after one dose of DAR-0100A. Occupancy (ΔBPND) was computed in the striatum and cortex. A clear relationship was observed between plasma concentration of DAR-0100A and ΔBPND. ΔBPND was larger in the striatum than in the cortex, consistent with reports showing that 25% of [11C] NNC112 BPND in the cortex is attributed to 5-HT2A. Plasma EC50 estimates ranged from 150 to 550 ng/mL according to the constraints on the model. There was no detectable effect of DAR-0100A on [11C] raclopride BPND. These data suggest that at doses likely to be administered to patients, occupancy will not be detectable with [11C] NNC112 PET and binding of DAR-0100A to D2 will be negligible. This is the first demonstration with PET of a significant occupancy by a full D1 agonist in vivo. [ABSTRACT FROM AUTHOR]

Dopamine D2 receptor antagonism is a unifying property of all antipsychotic drugs in clinical use. Remarkably, the effector molecules through which these medications exert their actions remain poorly characterized. Increasing attention is being focused on Akt/glycogen synthase kinase-3 (GSK-3) and wingless (Wnt) signaling pathways, which have been associated with schizophrenia in a number of genetic and postmortem studies. Antipsychotic medications may treat symptoms of psychosis, at least in part, through modulation of levels and activity of Akt, GSK-3, and Wnt-related intracellular signaling. The authors review evidence that Akt/GSK-3 and Wnt-related pathways are involved in the pathogenesis of schizophrenia as well as details of intracellular events related to these molecules mediated by both typical and atypical antipsychotic medications. Further study of Akt/ GSK-3 and Wnt signaling may ultimately lead to alternative therapeutics of schizophrenia- related disorders. [ABSTRACT FROM AUTHOR]

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serlne/threonine residues within the third cytoplasmic loop of the receptor that are phuspborylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using biolumineseence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phusphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the 2 DARs to the cell surface, subsequent to agonist-induced endocytusis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphoryiated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. [ABSTRACT FROM AUTHOR]

Amphetamine (AMPH) is a widely abused psychostimulant that acts as a substrate for the human dopamine transporter (hDAT). Using a piezoelectric rapid application system, we measured AMPH-induced currents mediated by hDAT. Whole-cell patch-clamp recordings in a heterologous expression system reveal that AMPH induces a rapidly activating and subsequently decaying inward current mediated by hDAT. We hypothesize that this transient inward current reflects a conformational change associated with substrate translocation. The AMPH-induced current strictly depends on extracellular Na+. Elevated intracellular Na_ has no effect on the peak AMPH-induced current amplitude but inhibits the steady-state current. In addition, elevated intracellular Na_ causes an overshoot outward current upon washout of AMPH that reflects hDAT locked in a Na+-exchange mode. Furthermore, elevated intracellular Na_ dramatically accelerates the recovery time from desensitization of the AMPH-induced current, revealing a new role for intracellular Na+in promoting the transition to the hDAT "outward-facing" conformation. Ion substitution suggests that both extracellular and intracellular Cl- facilitate transporter turnover in contrast to the classical model of Cl-as a cotransported ion. We present an alternating-access model of hDAT function that accurately fits the main features of the experimental data. The model predicts that translocation of substrate occurs within milliseconds of substrate binding but that slow reorientation of the empty transporter is the rate-limiting factor for turnover. The model provides a framework for interpreting perturbations of hDAT activity. [ABSTRACT FROM AUTHOR]

Plasma membrane neurotransmitter transporters for monoamines, GABA, glycine and excitatory amino acids are homologous to two sizable families of bacterial amino acid transporters. Recently, a high resolution structure was determined for a thermophilic glutamate transporter. Also, a bacterial tryptophan transporter related to the family of biogenic amine neurotransmitter transporters was functionally expressed. Structural insights from these and other bacterial transporters will help to rationalize the mechanisms for the increasingly complex functions that have been described for mammalian transporters, in addition to their modes of regulation. We touch on recent insights into the functions of neurotransmitter transporters in their physiological contexts. [Copyright &y& Elsevier]

The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake, and thereby plays an important homeostatic role in dopaminergic neurotransmission. Amphetamines exert their stimulant effects by targeting DAT and inducing the reverse transport of DA, leading to a dramatic increase of extracellular DA. Animal models have proven critical to investigating the molecular and cellular mechanisms underlying transporter function and its modulation by psychostimulants such as amphetamine. Here we establish a behavioral model for amphetamine action using adult Drosophila melanogaster. We use it to characterize the effects of amphetamine on sleep and sleep architecture. Our data show that amphetamine induces hyperactivity and disrupts sleep in a DA-dependent manner. Flies that do not express a functional DAT (dDAT null mutants) have been shown to be hyperactive and to exhibit significantly reduced sleep at baseline. Our data show that, in contrast to its action in control flies, amphetamine decreases the locomotor activity of dDAT null mutants and restores their sleep by modulating distinct aspects of sleep structure. To begin to explore the circuitry involved in the actions of amphetamine on sleep, we also describe the localization of dDAT throughout the fly brain, particularly in neuropils known to regulate sleep. Together, our data establish Drosophila as a robust model for studying the regulatory mechanisms that govern DAT function and psychostimulant action. [ABSTRACT FROM AUTHOR]

Recently we showed evidence that mutation of Tyr335 to Ala (Y335A) in the human dopamine transporter (hDAT) alters the conformational equilibrium of the transport cycle. Here, by substituting, one at a time, 16 different bulky or charged intracellular residues, we identify three residues, Lys-264, Asp-345, and Asp-436, the mutation of which to alanine produces a phenotype similar to that of Y335A. Like Y335A, the mutants (K264A, D345A, and D436A) were characterized by low uptake capacity that was potentiated by Zn[sup 2+]. Moreover, the mutants displayed lower affinity for cocaine and other inhibitors, suggesting a role for these residues in maintaining the structural integrity of the inhibitor binding crevice. The conformational state of K264A, Y335A, and D345A was investigated by assessing the accessibility to MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate) of a cysteine engineered into position 159 (I159C) in transmembrane segment 3 of the MTSET-insensitive "E2C" background (C90A/C306A). Unlike its effect at the corresponding position in the homologous norepinephrine transporter (NET I155C), MTSET did not inhibit uptake mediated by E2C I159C. Furthermore, no inhibition was observed upon treatment with MTSET in the presence of dopamine, cocaine, or Zn[sup 2+]. Without Zn[sup 2+], E2C I159C/K264A, E2C I159C] Y335A, and E2C I159C/D345A were also not inactivated by MTSET. In the presence of Zn[sup 2+] (10 µM), however, MTSET (0.5 mM) caused up to ∼60% inactivation. As in NET I155C, this inactivation was protected by dopamine and enhanced by cocaine. These data are consistent with a Zn[sup 2+]-dependent partial reversal of a constitutively altered conformational equilibrium in the mutant transporters. They also suggest that the conformational equilibrium produced by the mutations resembles that of the NET more than that of the DAT. Moreover, the data provide evidence that the cocaine-bound state of both DAT mutants and of the NET is structurally distinct from the cocaine-bound state of the DAT. [ABSTRACT FROM AUTHOR]

Using cysteine cross-linking, we demonstrated previously that the dopamine transporter (DAT) is at least a homodimer, with the extracellular end of transmembrahe segment (TM) 6 at a symmetrical dimer interface. We have now explored the possibility that DAT exists as a higher order oligomer in the plasma membrane. Cysteine cross-linking of wild type DAT resulted in bands on SDS-PAGE consistent with dimer, trimer, and tetramer, suggesting that DAT forms a tetramer in the plasma membrane. A cysteine-depleted DAT (CD-DAT) into which only Cys[sup 243] or Cys[sup 306] was reintroduced was cross-linked to dimer, suggesting that these endogenous cysteines in TM4 and TM6, respectively, were crosslinked at a symmetrical dimer interface. Reintroduction of both Cys[sup 243] and Cys[sup 306] into CD-DAT led to a pattern of cross-linking indistinguishable from that of wild type, with dimer, trimer, and tetramer bands. This indicated that the TM4 interface and the TM6 interface are distinct and further suggested that DAT may exist in the plasma membrane as a dimer of dimers, with two symmetrical homodimer interfaces. The cocaine analog MFZ 2-12 and other DAT inhibitors, including benztropine and mazindol, protected Cys[sup 243] against cross-linking. In contrast, two substrates of DAT, dopamine and tyramine, did not significantly impact cross-linking. We propose that the impairment of cross-linking produced by the inhibitors results from a conformational change at the TM4 interface, further demonstrating that these compounds are not neutral blockers but by themselves have effects on the structure of the transporter. [ABSTRACT FROM AUTHOR]

Peroxynitrite and nitrogen dioxide (NO[sub 2]) are reactive nitrogen species that have been implicated as causal factors in neurodegenerative conditions. Peroxynitriteinduced nitration of tyrosine residues in tyrosine hydroxylase (TH) may even be one of the earliest biochemical events associated with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced damage to dopamine neurons. Exposure of TH to peroxynitrite or NO[sub 2] results in nitration of tyrosine residues and modification of cysteines in the enzyme as well as inactivation of catalytic activity. Dopamine (DA), its precursor 3,4-dihydroxyphenylalanine, and metabolite 3,4-dihydroxyphenylacetic acid completely block the nitrating effects of peroxynitrite and NO[sub 2] on TH but do not relieve the enzyme from inhibition. o-Quinones formed in the reaction of catechols with either peroxynitrite or NO[sub 2] react with cysteine residues in TH and inhibit catalytic function. Using direct, real-time evaluation of tyrosine nitration with a green fluorescent protein-TH fusion protein stably expressed in intact cells (also stably expressing the human DA transporter), DA was also found to prevent NO[sub 2]-induced nitration while leaving TH activity inhibited. These results show that peroxynitrite and NO[sub 2] react with DA to form quinones at the expense of tyrosine nitration. Endogenous DA may therefore play an important role in determining how DA neurons are affected by reactive nitrogen species by shifting the balance of their effects away from tyrosine nitration and toward o-quinone formation. [ABSTRACT FROM AUTHOR]

Identifies the fourth transmembrane segment as a symmetrical dimer interface in the dopamine D2 receptor. Effects of cross-linking on receptor activation; Accessibility of adjacent residues in the segment; Functional significance of the interface.

Examines the symmetrical dimer of the human dopamine transporter (DAT). Expression of DAT in human embryonic kidney 293 cells; Effect of DAT cross-linking on tyramines uptake; Replacement of non-disulfide-bonded cysteines in the extracellular and membrane domains.

Depression and anxiety, two of the most common mental health disorders, share common symptoms and treatments. Most pharmacological agents available to treat these disorders target monoamine systems. Currently, finding the most effective treatment for an individual is a process of trial and error. To better understand how disease etiology may predict treatment response, we studied mice exposed developmentally to the selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX). These mice show the murine equivalent of anxiety- and depression-like symptoms in adulthood and here we report that these mice are also behaviorally resistant to the antidepressant-like effects of adult SSRI administration. We investigated whether tianeptine (TIA), which exerts its therapeutic effects through agonism of the mu-opioid receptor instead of targeting monoaminergic systems, would be more effective in this model. We found that C57BL/6J pups exposed to FLX from postnatal day 2 to 11 (PNFLX, the mouse equivalent in terms of brain development to the human third trimester) showed increased avoidant behaviors as adults that failed to improve, or were even exacerbated, by chronic SSRI treatment. By contrast, avoidant behaviors in these same mice were drastically improved following chronic treatment with TIA. Overall, this demonstrates that TIA may be a promising alternative treatment for patients that fail to respond to typical antidepressants, especially in patients whose serotonergic system has been altered by in utero exposure to SSRIs. [ABSTRACT FROM AUTHOR]

The article presents a research report on disrupting D1-NMDA or D2-NMDA receptor heteromerization prevents cocaine's rewarding effects but preserves natural reward processing. Topics include addictive drugs increase dopamine in the nucleus accumbens, where it persistently shapes excitatory glutamate transmission and hijacks natural reward processing; and understanding of molecular mechanisms underlying addiction and uncover D2R-NMDAR heteromers as targets with potential therapeutic value.

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. [ABSTRACT FROM AUTHOR]

Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here, we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex with MP1104, to rationally design novel mixed mu (MOR) and kappa (KOR) opioid receptor agonists with reduced arrestin signaling. Analysis of structure-activity relationships for new MP1104 analogs points to a region between transmembrane 5 (TM5) and extracellular loop (ECL2) as key for modulation of arrestin recruitment to both MOR and KOR. The lead compounds, MP1207 and MP1208, displayed MOR/KOR Gi-partial agonism with diminished arrestin signaling, showed efficient analgesia with attenuated liabilities, including respiratory depression and conditioned place preference and aversion in mice. The findings validate a novel structure-inspired paradigm for achieving beneficial in vivo profiles for analgesia through different mechanisms that include bias, partial agonism, and dual MOR/KOR agonism. [ABSTRACT FROM AUTHOR]

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+‐dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate‐bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly‐Met‐Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity. Synopsis: Bacillus halodurans MhsT transporter is the homologue of human SLC6 transporters and mediates uptake of a broad range of hydrophobic amino acids. Here, structural and functional data provide insight into the substrate recognition mechanism of MhsT with ramifications for SLC6 transporters and the larger family of LeuT‐fold proteins. Crystal structures of six MhsT/substrate complexes in the inward‐facing conformation reveal the role of the conserved unwound region of transmembrane segment (TM) 6 in substrate recognition, promiscuity and transport.The conserved (G/A/C)ΦG motif within the unwound part of TM6 is crucial for tailoring the volume of the central substrate site, thereby differentiating between branched aliphatic and larger aromatic amino acid substrates.The interaction between Glu66 located in TM2 and the unwound part of TM6 is conserved in all known conformational states and appears as a crucial mechanistic element of SLC6 transporters. [ABSTRACT FROM AUTHOR]

The neurotransmitter:sodium symporter (NSS) homolog LeuT from Aquifex aeolicus has proven to be a valuable model for studying the transport mechanism of the NSS family. Crystal structures have captured LeuT in key conformations visited during the transport cycle, allowing for the construction of a nearly complete model of transport, with much of the conformational dynamics studied by computational simulations. Here, we report crystal structures of LeuT representing new intermediate conformations between the outward-facing open and occluded states. These structures, combined with binding and accessibility studies, reveal details of conformational dynamics that can follow substrate binding at the central substrate binding site (S1) of LeuT in outward-facing states, suggesting a potential competition for direction between the outward-open and outward-occluded states at this stage during substrate transport. Our structures further support an intimate interplay between the protonation state of Glu290 and binding of Na1 that may ultimately regulate the outward-open-to-occluded transition. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by ß-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or ß-arrestinsignaling pathways. However, nearly all screening techniques for measuring ß-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure ß-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to ß-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, ß-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of ß-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring ß-arrestin recruitment to diverse GPCR types in heterologous or native cells. [ABSTRACT FROM AUTHOR]

Several letters to the editor are presented in response to the article "Cholinergic agonists as novel treatments for schizophrenia: the promise of rational drug development for psychiatry," by J.A. Lieberman and colleagues in the August 2008 issue.

The metabotropic glutamate receptor 5 (mGluR5) is a promising treatment target for psychiatric disorders due to its modulatory effects on glutamate transmission. Using [11C]ABP688, we previously showed that the rapidly acting antidepressant ketamine decreases mGluR5 availability. The mGluR5 radioligand [18F]FPEB offers key advantages over [11C]ABP688; however, its suitability for drug challenge studies is unknown. We evaluated whether [18F]FPEB can be used to capture ketamine-induced effects on mGluR5. Seven healthy subjects participated in three [18F]FPEB scans: a baseline, a same-day post-ketamine, and a 24-h post-ketamine scan. The outcome measure was V T/ f P, obtained using a two-tissue compartment model and a metabolite-corrected arterial input function. Dissociative symptoms, heart rate and blood pressure increased following ketamine infusion. [18F]FPEB V T/ f P decreased by 9% across the cortex after ketamine infusion, with minimal difference between baseline and 24-h scans. Compared to our previous work using [11C]ABP688, the magnitude of the ketamine-induced change in mGluR5 was smaller using [18F]FPEB; however, effect sizes were similar for the same-day post-ketamine vs. baseline scan (Cohen's d = 0.75 for [18F]FPEB and 0.88 for [11C]ABP688). [18F]FPEB is therefore able to capture some of the effects of ketamine on mGluR5, but [11C]ABP688 appears to be more suitable in drug challenge paradigms designed to probe glutamate transmission. [ABSTRACT FROM AUTHOR]

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging.“Self-healing”dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen.We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design. [ABSTRACT FROM AUTHOR]