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Dopamine (DA) release in striatal circuits, including the nucleus accumbens medial shell (mNAcSh), tracks separable features of reward like motivation and reinforcement. However, the cellular and circuit mechanisms by which DA receptors transform DA release into distinct constructs of reward remain unclear. Here we show that DA D3 receptor (D3R) signaling in the mNAcSh drives motivated behavior in mice by regulating local microcircuits. Furthermore, D3Rs coexpress with DA D1 receptors, which regulate reinforcement, but not motivation. Paralleling dissociable roles in reward function, we report nonoverlapping physiological actions of D3R and DA D1 receptor signaling in mNAcSh neurons. Our results establish a fundamental framework wherein DA signaling within the same nucleus accumbens cell type is physiologically compartmentalized via actions on distinct DA receptors. This structural and functional organization provides neurons in a limbic circuit with the unique ability to orchestrate dissociable aspects of reward-related behaviors relevant to the etiology of neuropsychiatric disorders., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Aberrant dopamine (DA) signaling is implicated in schizophrenia, bipolar disorder (BPD), autism spectrum disorder (ASD), substance use disorder, and attention-deficit/hyperactivity disorder (ADHD). Treatment of these disorders remains inadequate, as exemplified by the therapeutic use of d-amphetamine and methylphenidate for the treatment of ADHD, agents with high abuse liability. In search for an improved and non-addictive therapeutic approach for the treatment of DA-linked disorders, we utilized a preclinical mouse model expressing the human DA transporter (DAT) coding variant DAT Val559, previously identified in individuals with ADHD, ASD, or BPD. DAT Val559, like several other disease-associated variants of DAT, exhibits anomalous DA efflux (ADE) that can be blocked by d-amphetamine and methylphenidate. Kappa opioid receptors (KORs) are expressed by DA neurons and modulate DA release and clearance, suggesting that targeting KORs might also provide an alternative approach to normalizing DA-signaling disrupted by perturbed DAT function. Here we demonstrate that KOR stimulation leads to enhanced surface trafficking and phosphorylation of Thr53 in wildtype DAT, effects achieved constitutively by the Val559 mutant. Moreover, these effects can be rescued by KOR antagonism of DAT Val559 in ex vivo preparations. Importantly, KOR antagonism also corrected in vivo DA release as well as sex-dependent behavioral abnormalities observed in DAT Val559 mice. Given their low abuse liability, our studies with a construct valid model of human DA associated disorders reinforce considerations of KOR antagonism as a pharmacological strategy to treat DA associated brain disorders., Competing Interests: Conflicts of Interest All authors declare no conflicts of interest

The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for β-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for β-arrestin recruitment to the D2R, and the role of GRKs in D2R-β-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which β-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated β-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased β-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R-β-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance β-arrestin recruitment and activation independently of receptor phosphorylation.

Rational drug design for G protein-coupled receptors (GPCRs) remains a challenging area. A new study from the Xu, Roth, and Zhang groups provides a complete set of active structures for the entire dopamine receptor family bound with rotigotine that will aid in designing selective agonists for these important therapeutic targets., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Ltd.)

The D 1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein β-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting β-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using β-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive β-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for β-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in β-arrestin binding and activation.

The D3 dopamine receptor (D3R) has been suggested as a drug target for the treatment of a number of neuropsychiatric disorders, including substance use disorders (SUD). Many D3R-selective antagonists are bivalent in nature in that they engage two distinct sites on the receptor-a primary pharmacophore binds to the orthosteric site, where dopamine binds, whereas a secondary pharmacophore interacts with a unique secondary binding pocket (SBP). When engagement of the secondary pocket exerts allosteric activity, the compound is said to be bitopic. We recently reported the synthesis and characterization of two bitopic antagonists of the D3R, (±)-VK04-87 and (±)-VK05-95, which incorporated a racemic trans -cyclopropylmethyl linking chain. To gain a better understanding of the role of chirality in determining the pharmacology of such compounds, we resolved the enantiomers of (±)-VK04-87. We found that the (+)-isomer displays higher affinity for the D3R and exhibits greater selectivity versus the D2R than the (-)-isomer. Strikingly, using functional assays, we found that (+)-VK04-87 inhibits the D3R in a noncompetitive manner, while (-)-VK04-87 behaves as a purely competitive antagonist, indicating that the apparent allosteric activity of the racemate is due to the (+)-isomer. Molecular dynamic simulations of (+)-VK04-87 and (-)-VK04-87 binding to the D3R suggest that the (+)-isomer is able to interact with the SBP of the receptor whereas the (-)-isomer bends away from this pocket, thus potentially explaining their differing pharmacology. These results emphasize the importance of the linker, and its isomeric conformations, within extended-length molecules for their positioning and engagement within GPCR binding pockets.

To identify novel D 3 dopamine receptor (D3R) agonists, we conducted a high-throughput screen using a β-arrestin recruitment assay. Counterscreening of the hit compounds provided an assessment of their selectivity, efficacy, and potency. The most promising scaffold was optimized through medicinal chemistry resulting in enhanced potency and selectivity. The optimized compound, ML417 ( 20 ), potently promotes D3R-mediated β-arrestin translocation, G protein activation, and ERK1/2 phosphorylation (pERK) while lacking activity at other dopamine receptors. Screening of ML417 against multiple G protein-coupled receptors revealed exceptional global selectivity. Molecular modeling suggests that ML417 interacts with the D3R in a unique manner, possibly explaining its remarkable selectivity. ML417 was also found to protect against neurodegeneration of dopaminergic neurons derived from iPSCs. Together with promising pharmacokinetics and toxicology profiles, these results suggest that ML417 is a novel and uniquely selective D3R agonist that may serve as both a research tool and a therapeutic lead for the treatment of neuropsychiatric disorders.

Signaling bias is the propensity for some agonists to preferentially stimulate G protein-coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein-biased agonist of the D 2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β 2 -adrenergic receptor (β2R) to build β2R-WT and β2R-Y199 5.38 A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y199 5.38 A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y199 5.38 A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The dopamine transporter (DAT) is a plasma membrane protein that mediates the reuptake of extracellular dopamine (DA) and controls the spatiotemporal dynamics of dopaminergic neurotransmission. The transporter is subject to fine control that tailors clearance of transmitter to physiological demands, and dysregulation of reuptake induced by psychostimulant drugs, transporter polymorphisms, and signaling defects may impact transmitter tone in disease states. We previously demonstrated that DAT undergoes complex regulation by palmitoylation, with acute inhibition of the modification leading to rapid reduction of transport activity and sustained inhibition of the modification leading to transporter degradation and reduced expression. Here, to examine mechanisms and outcomes related to increased modification, we coexpressed DAT with palmitoyl acyltransferases (PATs), also known as DHHC enzymes, which catalyze palmitate addition to proteins. Of 12 PATs tested, DAT palmitoylation was stimulated by DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17, with others having no effect. Increased modification was localized to previously identified palmitoylation site Cys580 and resulted in upregulation of transport kinetics and elevated transporter expression mediated by reduced degradation. These findings confirm palmitoylation as a regulator of multiple DAT properties crucial for appropriate DA homeostasis and identify several potential PAT pathways linked to these effects. Defects in palmitoylation processes thus represent possible mechanisms of transport imbalances in DA disorders.

The D 1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D 1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D 1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and β -arrestin-mediated signaling and increase the affinity of dopamine for the D 1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both β -arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D 1 receptor PAM "Compound B," suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D 1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D 1 receptor as well as the development of optimized lead compounds for therapeutic use., (U.S. Government work not protected by U.S. copyright.)

The dopamine D2 receptor (D2R) is known to elicit effects through activating two major signaling pathways mediated by either G proteins (Gi/o) or β-arrestins. However, the specific role of each pathway in physiological or therapeutic activities is not known with certainty. One approach to the dissection of these pathways is through the use of drugs that can selectively modulate one pathway vs. the other through a mechanism known as functional selectivity or biased signaling. Our laboratory has previously described a G protein signaling-biased agonist, MLS1547, for the D2R using a variety of in vitro functional assays. To further evaluate the biased signaling activity of this compound, we investigated its ability to promote D2R internalization, a process known to be mediated by β-arrestin. Using multiple cellular systems and techniques, we found that MLS1547 promotes little D2R internalization, which is consistent with its inability to recruit β-arrestin. Importantly, we validated these results in primary striatal neurons where the D2R is most highly expressed suggesting that MLS1547 will exhibit biased signaling activity in vivo . In an effort to optimize and further explore structure-activity relationships (SAR) for this scaffold, we conducted an iterative chemistry campaign to synthesize and characterize novel analogs of MLS1547. The resulting analysis confirmed previously described SAR requirements for G protein-biased agonist activity and, importantly, elucidated new structural features that are critical for agonist efficacy and signaling bias of the MLS1547 scaffold. One of the most important determinants for G protein-biased signaling is the interaction of a hydrophobic moiety of the compound with a defined pocket formed by residues within transmembrane five and extracellular loop two of the D2R. These results shed new light on the mechanism of biased signaling of the D2R and may lead to improved functionally-selective molecules.

Compounds that target D2-like dopamine receptors (DRs) are currently used as therapeutics for several neuropsychiatric disorders including schizophrenia (antagonists) and Parkinson's disease (agonists). However, as the D 2 R and D 3 R subtypes are highly homologous, creating compounds with sufficient subtype-selectivity as well as drug-like properties for therapeutic use has proved challenging. This review summarizes the progress that has been made in developing D 2 R- or D 3 R-selective antagonists and agonists, and also describes the experimental conditions that need to be considered when determining the selectivity of a given compound, as apparent selectivity can vary widely depending on assay conditions. Future advances in this field may take advantage of currently available structural data to target alternative secondary binding sites through creating bivalent or bitopic chemical structures. Alternatively, the use of high-throughput screening techniques to identify novel scaffolds that might bind to the D 2 R or D 3 R in areas other than the highly conserved orthosteric site, such as allosteric sites, followed by iterative medicinal chemistry will likely lead to exceptionally selective compounds in the future. More selective compounds will provide a better understanding of the normal and pathological functioning of each receptor subtype, as well as offer the potential for improved therapeutics., (Published by Elsevier Inc.)

The development of bitopic ligands directed toward D 2 -like receptors has proven to be of particular interest to improve the selectivity and/or affinity of these ligands and as an approach to modulate and bias their efficacies. The structural similarities between dopamine D 3 receptor (D 3 R)-selective molecules that display bitopic or allosteric pharmacology and those that are simply competitive antagonists are subtle and intriguing. Herein we synthesized a series of molecules in which the primary and secondary pharmacophores were derived from the D 3 R-selective antagonists SB269,652 (1) and SB277011A (2) whose structural similarity and pharmacological disparity provided the perfect templates for SAR investigation. Incorporating a trans-cyclopropylmethyl linker between pharmacophores and manipulating linker length resulted in the identification of two bivalent noncompetitive D 3 R-selective antagonists, 18a and 25a, which further delineates SAR associated with allosterism at D 3 R and provides leads toward novel drug development.

The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The D3 dopamine receptor represents an important target in drug addiction in that reducing receptor activity may attenuate the self-administration of drugs and/or disrupt drug or cue-induced relapse. Medicinal chemistry efforts have led to the development of D3 preferring antagonists and partial agonists that are >100-fold selective vs. the closely related D2 receptor, as best exemplified by extended-length 4-phenylpiperazine derivatives. Based on the D3 receptor crystal structure, these molecules are known to dock to two sites on the receptor where the 4-phenylpiperazine moiety binds to the orthosteric site and an extended aryl amide moiety docks to a secondary binding pocket. The bivalent nature of the receptor binding of these compounds is believed to contribute to their D3 selectivity. In this study, we examined if such compounds might also be "bitopic" such that their aryl amide moieties act as allosteric modulators to further enhance the affinities of the full-length molecules for the receptor. First, we deconstructed several extended-length D3-selective ligands into fragments, termed "synthons", representing either orthosteric or secondary aryl amide pharmacophores and investigated their effects on D3 receptor binding and function. The orthosteric synthons were found to inhibit radioligand binding and to antagonize dopamine activation of the D3 receptor, albeit with lower affinities than the full-length compounds. Notably, the aryl amide-based synthons had no effect on the affinities or potencies of the orthosteric synthons, nor did they have any effect on receptor activation by dopamine. Additionally, pharmacological investigation of the full-length D3-selective antagonists revealed that these compounds interacted with the D3 receptor in a purely competitive manner. Our data further support that the 4-phenylpiperazine D3-selective antagonists are bivalent and that their enhanced affinity for the D3 receptor is due to binding at both the orthosteric site as well as a secondary binding pocket. Importantly, however, their interactions at the secondary site do not allosterically modulate their binding to the orthosteric site., (Published by Elsevier B.V.)

Rationale: (-)-Stepholidine is a tetrahydroberberine alkaloid that is known to interact with dopamine receptors and has also been proposed as a novel antipsychotic agent. Its suggested novelty lies in the fact that it has been proposed to have D1-like receptor agonist and D2-like receptor antagonist properties. Thus, it might be effective in treating both positive and negative (cognition) symptoms of schizophrenia. However, its activity on specific dopamine receptor subtypes has not been clarified, especially with respect to its ability to activate D1-like receptors., Objectives: We wished to examine the affinity and functional activity of (-)-stepholidine at each of the human dopamine receptor subtypes expressed in a defined cellular environment., Methods: D1-D5 dopamine receptors were stably expressed in cell lines and their interactions with (-)-stepholidine were examined using radioligand binding and various functional signaling assays. Radioligand binding assays were also performed using bovine striatal membranes., Results: (-)-Stepholidine exhibited high (nM) affinity for D1 and D5 receptors, somewhat lower (two- to four-fold) affinity for D2 and D3 receptors, and low micromolar affinity for D4 receptors. Functionally, (-)-stepholidine was ineffective in activating G protein-mediated signaling of D1-like and D2 receptors and was also ineffective in stimulating β-arrestin recruitment to any dopamine receptor subtype. It did, however, antagonize all of these responses. It also antagonized D1-D2 heteromer-mediated Ca(2+) mobilization. Radioligand binding assays of D1-like receptors in brain membranes also indicated that (-)-stepholidine binds to the D1 receptor with antagonist-like properties., Conclusions: (-)-Stepholidine is a pan-dopamine receptor antagonist and its in vivo effects are largely mediated through dopamine receptor blockade with potential cross-talk to other receptors or signaling proteins., Competing Interests: All authors declare no conflicts of interest.

In this manuscript we report a structure-activity relationship (SAR) study of analogs of 5/ 7-{[2-(4-Aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol. Our study is focused on introduction of various bioisoteric and aromatic substitutions on the piperazine ring of the hybrid template to further probe into the accessory binding domains on dopamine D2/D3 receptors. Specifically, the goal behind this study is to delineate the nature of the binding pockets for such substitutions on the piperazine ring to determine their influence on binding affinity (Ki), as measured with tritiated spiperone and HEK-293 cells expressing either D2 or D3 receptors. Functional activity of selected compounds was assessed with the GTPγS binding assay. Our data indicates that various N-substitution with substituted and unsubstituted benzene sulfonyl group produced varied affinity and potency for D2/D3. Compound D-660 produced highest selectivity for the D3 receptor in the binding assay. In general, presence of hydroxyl group improved overall activity for both D2/D3 receptors. One such compound D-668 produced exceptional potencies for both the receptors. Overall, our results suggest that binding to the sites removed from the orthosteric binding sites contribute significantly to enhance functional potencies of ligands. [ABSTRACT FROM AUTHOR]

A high-throughput screening campaign was conducted to interrogate a 380,000+ small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and β-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate β-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit β-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated β-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate β-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate β-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties., (U.S. Government work not protected by U.S. copyright.)

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (-)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.

In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal residue Thr(53). In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr(53) in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr(53) occurred with a stoichiometry of ~50% and was strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of the site by signaling pathways that impact DAT activity. Mutations of Thr(53) to prevent phosphorylation led to reduced dopamine transport V(max) and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.

Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with protein kinase C (PKC)- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues, we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCalpha, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCalpha-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses.

Palmitoylation, a lipid-based posttranslational protein modification, plays a crucial role in regulating various aspects of neuronal function through altering protein membrane-targeting, stabilities, and protein–protein interaction profiles. Disruption of palmitoylation has recently garnered attention as disease mechanism in neurodegeneration. Many proteins implicated in neurodegenerative diseases and associated neuronal dysfunction, including but not limited to amyloid precursor protein, β-secretase (BACE1), postsynaptic density protein 95, Fyn, synaptotagmin-11, mutant huntingtin, and mutant superoxide dismutase 1, undergo palmitoylation, and recent evidence suggests that altered palmitoylation contributes to the pathological characteristics of these proteins and associated disruption of cellular processes. In addition, dysfunction of enzymes that catalyze palmitoylation and depalmitoylation has been connected to the development of neurological disorders. This review highlights some of the latest advances in our understanding of palmitoylation regulation in neurodegenerative diseases and explores potential therapeutic implications. [ABSTRACT FROM AUTHOR]

Dopamine is a biologically active amine synthesized in the central and peripheral nervous system. This biogenic monoamine acts by activating five types of dopamine receptors (D 1-5 Rs), which belong to the G protein-coupled receptor family. Antagonists and partial agonists of D 2 Rs are used to treat schizophrenia, Parkinson's disease, depression, and anxiety. The typical pharmacophore with high D 2 R affinity comprises four main areas, namely aromatic moiety, cyclic amine, central linker and aromatic/heteroaromatic lipophilic fragment. From the literature reviewed herein, we can conclude that 4-(2,3-dichlorophenyl), 4-(2-methoxyphenyl)-, 4-(benzo[b]thiophen-4-yl)-1-substituted piperazine, and 4-(6-fluorobenzo[d]isoxazol-3-yl)piperidine moieties are critical for high D 2 R affinity. Four to six atoms chains are optimal for D 2 R affinity with 4-butoxyl as the most pronounced one. The bicyclic aromatic/heteroaromatic systems are most frequently occurring as lipophilic appendages to retain high D 2 R affinity. In this review, we provide a thorough overview of the therapeutic potential of D 2 R modulators in the treatment of the aforementioned disorders. In addition, this review summarizes current knowledge about these diseases, with a focus on the dopaminergic pathway underlying these pathologies. Major attention is paid to the structure, function, and pharmacology of novel D 2 R ligands, which have been developed in the last decade (2010-2021), and belong to the 1,4-disubstituted aromatic cyclic amine group. Due to the abundance of data, allosteric D 2 R ligands and D 2 R modulators from patents are not discussed in this review., (© 2022 Wiley Periodicals LLC.)

Somatostatin receptors (SSTs) are widely expressed in pituitary tumors and neuroendocrine neoplasms (NENs) of different origins, i.e. the gastrointestinal tract and the thorax (lungs and thymus), thus representing a well-established target for medical treatment with SST ligands (SRLs). However, the response to SRLs is highly heterogeneous between tumors. Two main factors can contribute to this variability: (i) the differential SST expression among tumor types and (ii) the differential expression/modulation of the SST-related intracellular machinery. In this literature review, we provide an overview of available data on the variable expression of SSTs in pituitary tumors and NENs, together with the resulting clinical implications. Moreover, we aim to describe the complex intracellular machinery involved in SST signaling and trafficking. Particularly, we will focus on ß-arrestins and describe their role in receptor internalization and recycling, as well as the various functions of these scaffold molecules in tumor pathogenesis and progression. This review highlights the interplay between membrane receptors and intracellular machinery, together with its role in determining the clinical behavior of the tumor and the response to treatment in patients with pituitary tumors or NENs. [ABSTRACT FROM AUTHOR]

Dopamine D2 receptor (D2R) has been shown to activate extracellular signal‐regulated kinases (ERKs) via distinct pathways dependent on either G‐protein or β‐arrestin. However, there has not been a systematic study of the regulatory process of D2R‐mediated ERKs activation by G protein‐ versus β‐arrestin‐dependent signaling since D2R stimulation of ERKs reflects the simultaneous action of both pathways. Here, we investigated that differential regulation of D2R‐mediated ERKs activation via these two pathways. Our results showed that G protein‐dependent ERKs activation was transient, rapid, reached maximum level at around 2 min, and importantly, the activated ERKs were entirely confined to the cytoplasm. In contrast, β‐arrestin‐dependent ERKs activation was more sustained, slower, reached maximum level at around 10 min, and phosphorylated ERKs translocated into the nucleus. Src was found to be commonly involved in both the G protein‐ and β‐arrestin‐dependent pathway‐mediated ERKs activation. Pertussis toxin Gi/o inhibitor, GRK2‐CT, AG1478 epidermal growth factor receptor inhibitor, and wortmannin phosphoinositide 3‐kinase inhibitor all blocked G protein‐dependent ERKs activation. In contrast, GRK2 and β‐Arr2 played a main role in β‐arrestin‐dependent ERKs activation. Receptor endocytosis showed minimal effect on the activation of ERKs mediated by both pathways. Furthermore, we found that the formation of a complex composed of phospho‐ERKs, β‐Arr2, and importinβ1 promoted the nuclear translocation of activated ERKs. The differential regulation of various cellular components, as well as temporal and spatial patterns of ERKs activation via these two pathways, suggest the existence of distinct physiological outcomes. [ABSTRACT FROM AUTHOR]

The increasing prevalence of screen time among modern citizens has raised concerns regarding its potential impact on neuroinflammation and overall brain health. This review examines the complex interconnections between screen time and neuroinflammatory processes, particularly in children and adolescents. We analyze existing literature that explores how excessive digital media use can lead to alterations in neurobiological pathways, potentially exacerbating inflammatory responses in the brain. Key findings suggest that prolonged exposure to screens may contribute to neuroinflammation through mechanisms such as disrupted sleep patterns, diminished cognitive engagement, and increased stress levels. Similarly, we discuss the implications of these findings for mental health and cognitive development, emphasizing the need for a balanced approach to screen time. This review highlights the necessity for further research to elucidate the causal relationships and underlying mechanisms linking screen time and neuroinflammation, thereby informing guidelines for healthy media consumption., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.16177. G protein‐coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are essential cell membrane signaling molecules and represent the most important class of drug targets. Some signaling pathways downstream of a GPCR may be responsible for drug adverse effects, while others mediate therapeutic efficacy. Biased ligands preferentially activate only a subset of all GPCR signaling pathways. They hold great potential to become next-generation GPCR drugs with less side effects due to their potential to exclusively activate desired signaling pathways. However, the molecular basis of biased agonism is poorly understood. GPCR activation occurs through allosteric coupling, the propagation of conformational changes from the extracellular ligand-binding pocket to the intracellular G protein-binding interface. Comparison of GPCR structures in complex with G proteins or β-arrestin reveals that intracellular transducer coupling results in closure of the ligand-binding pocket trapping the agonist inside its binding site. Allosteric coupling appears to be transducer-specific offering the possibility of harnessing this mechanism for the design of biased ligands. Here, we review the biochemical, pharmacological, structural, and biophysical evidence for allosteric coupling and delineate that biased agonism should be a consequence of preferential allosteric coupling from the ligand-binding pocket to one transducer-binding site. As transducer binding leads to large structural rearrangements in the extracellular ligand-binding pocket, we survey biased ligands with an extended binding mode that interact with extracellular receptor domains. We propose that biased ligands use ligand-specific triggers inside the binding pocket that are relayed through preferential allosteric coupling to a specific transducer, eventually leading to biased signaling., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The dopamine transporter (DAT) has been implicated in a variety of arousal-related processes including the regulation of motor activity, learning, motivated behavior, psychostimulant abuse, and, more recently, sleep/wake state. We previously demonstrated that DAT uptake regulates fluctuations in extracellular dopamine (DA) in the striatum across the light/dark cycle with DA levels at their highest during the dark phase and lowest during the light phase. Despite this evidence, whether fluctuations in DA uptake across the light/dark cycle are associated with changes in sleep/wake has not been tested. To address this, we employed a combination of sleep/wake recordings, fast scan cyclic voltammetry, and western blotting to examine whether sleep/wake state and/or light/dark phase impact DA terminal neurotransmission in male rats. Further, we assessed whether variations in plasma membrane DAT levels and/or phosphorylation of the threonine 53 site on the DAT accounts for fluctuations in DA neurotransmission. Given the extensive evidence indicating that psychostimulants increase DA through interactions with the DAT, we also examined to what degree the effects of cocaine at inhibiting the DAT vary across sleep/wake state. Results demonstrated a significant association between individual sleep/wake states and DA terminal neurotransmission, with higher DA uptake rate, increased phosphorylation of the DAT, and enhanced cocaine potency observed after periods of sleep. These findings suggest that sleep/wake state influences DA neurotransmission in a manner that is likely to impact a host of DA-dependent processes including a variety of neuropsychiatric disorders.

Dopamine is a biologically active amine synthesized in the central and peripheral nervous system. This biogenic monoamine acts by activating five types of dopamine receptors (D1–5Rs), which belong to the G protein‐coupled receptor family. Antagonists and partial agonists of D2Rs are used to treat schizophrenia, Parkinson's disease, depression, and anxiety. The typical pharmacophore with high D2R affinity comprises four main areas, namely aromatic moiety, cyclic amine, central linker and aromatic/heteroaromatic lipophilic fragment. From the literature reviewed herein, we can conclude that 4‐(2,3‐dichlorophenyl), 4‐(2‐methoxyphenyl)‐, 4‐(benzo[b]thiophen‐4‐yl)‐1‐substituted piperazine, and 4‐(6‐fluorobenzo[d]isoxazol‐3‐yl)piperidine moieties are critical for high D2R affinity. Four to six atoms chains are optimal for D2R affinity with 4‐butoxyl as the most pronounced one. The bicyclic aromatic/heteroaromatic systems are most frequently occurring as lipophilic appendages to retain high D2R affinity. In this review, we provide a thorough overview of the therapeutic potential of D2R modulators in the treatment of the aforementioned disorders. In addition, this review summarizes current knowledge about these diseases, with a focus on the dopaminergic pathway underlying these pathologies. Major attention is paid to the structure, function, and pharmacology of novel D2R ligands, which have been developed in the last decade (2010–2021), and belong to the 1,4‐disubstituted aromatic cyclic amine group. Due to the abundance of data, allosteric D2R ligands and D2R modulators from patents are not discussed in this review. [ABSTRACT FROM AUTHOR]

A major obstacle in treating opioid use disorder is the persistence of drug seeking or craving during periods of abstinence, which is believed to contribute to relapse. Dopamine transmission in the mesolimbic pathway is posited to contribute to opioid reinforcement, but the processes by which dopamine influences drug seeking have not been completely elucidated. To examine whether opioid seeking during abstinence is associated with alterations in dopamine transmission, female and male rats self-administered oxycodone under an intermittent access schedule of reinforcement. Following self-administration, rats underwent a forced abstinence period, and cue-induced seeking tests were conducted to assess oxycodone seeking. One day following the final seeking test, rats were sacrificed to perform ex vivo fast scan cyclic voltammetry and western blotting in the nucleus accumbens. Rats displayed reduced dopamine uptake rate on abstinence day 2 and abstinence day 15, compared to oxycodone-naïve rats. Further, on abstinence day 15, rats had reduced phosphorylation of the dopamine transporter. Additionally, local application of oxycodone to the nucleus accumbens reduced dopamine uptake in oxycodone-naïve rats and in rats during oxycodone abstinence, on abstinence day 2 and abstinence day 15. These observations suggest that abstinence from oxycodone results in dysfunctional dopamine transmission, which may contribute to sustained oxycodone seeking during abstinence. [ABSTRACT FROM AUTHOR]

Aberrant dopamine (DA) signalling has been implicated in various neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), schizophrenia, bipolar disorder (BPD) and addiction. The availability of extracellular DA is sculpted by the exocytotic release of vesicular DA and subsequent transporter-mediated clearance, rendering the presynaptic DA transporter (DAT) a crucial regulator of DA neurotransmission. D2-type DA autoreceptors (D2ARs) regulate multiple aspects of DA homeostasis, including (i) DA synthesis, (ii) vesicular release, (iii) DA neuron firing and (iv) the surface expression of DAT and DAT-mediated DA clearance. The DAT Val559 variant, identified in boys with ADHD or ASD, as well as in a girl with BPD, supports anomalous DA efflux (ADE), which we have shown drives tonic activation of D2ARs. Through ex vivo and in vivo studies of the DAT Val559 variant using transgenic knock-in mice, we have uncovered a circuit and sex-specific capacity of D2ARs to regulate DAT, which consequently disrupts DA signalling and behaviour differently in males and females. Our studies reveal the ability of the construct-valid DAT Val559 model to elucidate endogenous mechanisms that support DA signalling, findings that may be of translational and/or therapeutic importance., (© 2023 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society). Published by John Wiley & Sons Ltd.)

Background: Although clozapine is an effective option for treatment-resistant schizophrenia (TRS), there are still 1/3 to 1/2 of TRS patients who do not respond to clozapine. The main purpose of this randomized, double-blind, placebo-controlled trial was to explore the amisulpride augmentation efficacy on the psychopathological symptoms and cognitive function of clozapine-resistant treatment-refractory schizophrenia (CTRS) patients. Methods: A total of 80 patients were recruited and randomly assigned to receive initial clozapine plus amisulpride (amisulpride group) or clozapine plus placebo (placebo group). Positive and Negative Syndrome Scale (PANSS), Scale for the Assessment of Negative Symptoms (SANS), Clinical Global Impression (CGI) scale scores, Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), Treatment Emergent Symptom Scale (TESS), laboratory measurements, and electrocardiograms (ECG) were performed at baseline, at week 6, and week 12. Results: Compared with the placebo group, amisulpride group had a lower PANSS total score, positive subscore, and general psychopathology subscore at week 6 and week 12 (PBonferroni < 0.01). Furthermore, compared with the placebo group, the amisulpride group showed an improved RBANS language score at week 12 (PBonferroni < 0.001). Amisulpride group had a higher treatment response rate (P = 0.04), lower scores of CGI severity and CGI efficacy at week 6 and week 12 than placebo group (PBonferroni < 0.05). There were no differences between the groups in body mass index (BMI), corrected QT (QTc) intervals, and laboratory measurements. This study demonstrates that amisulpride augmentation therapy can safely improve the psychiatric symptoms and cognitive performance of CTRS patients. Conclusion: This study indicates that amisulpride augmentation therapy has important clinical significance for treating CTRS to improve clinical symptoms and cognitive function with tolerability and safety. Trial registration Clinicaltrials.gov identifier- NCT03652974. Registered August 31, 2018, https://clinicaltrials.gov/ct2/show/NCT03652974 [ABSTRACT FROM AUTHOR]

Background: S100B is an established biomarker of brain development and damage. Lutein (LT) is a naturally occurring xanthophyll carotenoid mainly concentrated in the central nervous system (CNS), but its neurotrophic role is still debated. We investigated whether LT cord blood concentrations correlate with S100B in a cohort of preterm and term healthy newborns. Methods: We conducted a prospective study on the distribution of LT and S100B in arterial cord blood of healthy preterm (n = 50) and term (n = 50) newborns. Results: S100B and LT showed a pattern of concentration characterized by higher levels (P < 0.01, for all) at 33-36 weeks gestation (GA) followed by a progressive decrease (P < 0.01, for all) from 37 onwards with a dip at term. Both S100B and LT were gender-dependent with significantly (P < 0.01, for all) higher levels in females in preterm and term groups. S100B (R = 0.68; P < 0.001) and LT (R = 0.40; P = 0.005) correlated with GA at sampling. A positive significant correlation (R = 0.87; P < 0.001) between S100B and LT was found. Conclusions: The present data showing a correlation between S100B and LT supports the notion of a LT trophic role in the CNS. Further investigations in high-risk infants are needed to elucidate LT involvement in the pathophysiological cascade of events leading to CNS development and damage. [ABSTRACT FROM AUTHOR]

Dopamine D 2 receptor (D 2 R) has been shown to activate extracellular signal-regulated kinases (ERKs) via distinct pathways dependent on either G-protein or β-arrestin. However, there has not been a systematic study of the regulatory process of D 2 R-mediated ERKs activation by G protein- versus β-arrestin-dependent signaling since D 2 R stimulation of ERKs reflects the simultaneous action of both pathways. Here, we investigated that differential regulation of D 2 R-mediated ERKs activation via these two pathways. Our results showed that G protein-dependent ERKs activation was transient, rapid, reached maximum level at around 2 min, and importantly, the activated ERKs were entirely confined to the cytoplasm. In contrast, β-arrestin-dependent ERKs activation was more sustained, slower, reached maximum level at around 10 min, and phosphorylated ERKs translocated into the nucleus. Src was found to be commonly involved in both the G protein- and β-arrestin-dependent pathway-mediated ERKs activation. Pertussis toxin Gi/o inhibitor, GRK2-CT, AG1478 epidermal growth factor receptor inhibitor, and wortmannin phosphoinositide 3-kinase inhibitor all blocked G protein-dependent ERKs activation. In contrast, GRK2 and β-Arr2 played a main role in β-arrestin-dependent ERKs activation. Receptor endocytosis showed minimal effect on the activation of ERKs mediated by both pathways. Furthermore, we found that the formation of a complex composed of phospho-ERKs, β-Arr2, and importinβ1 promoted the nuclear translocation of activated ERKs. The differential regulation of various cellular components, as well as temporal and spatial patterns of ERKs activation via these two pathways, suggest the existence of distinct physiological outcomes., (© 2023 Federation of European Biochemical Societies.)

Dysfunctions of the dopaminergic system are believed to play a major role in the core symptoms of schizophrenia such as positive, negative, and cognitive symptoms. The first line of treatment of schizophrenia are antipsychotics, a class of medications that targets several neurotransmitter receptors in the brain, including dopaminergic, serotonergic, adrenergic and/or muscarinic receptors, depending on the given agent. Although the currently used antipsychotics display in vitro activity at several receptors, majority of them share the common property of having high/moderate in vitro affinity for dopamine D2 receptors (D2Rs) and D3 receptors (D3Rs). In terms of mode of action, these antipsychotics are either antagonist or partial agonist at the above-mentioned receptors. Although D2Rs and D3Rs possess high degree of homology in their molecular structure, have common signaling pathways and similar in vitro pharmacology, they have different in vivo pharmacology and therefore behavioral roles. The aim of this review, with summarizing preclinical and clinical evidence is to demonstrate that while currently used antipsychotics display substantial in vitro affinity for both D3Rs and D2Rs, only very few can significantly occupy D3Rs in vivo. The relative importance of the level of endogenous extracellular dopamine in the brain and the degree of in vitro D3Rs receptor affinity and selectivity as determinant factors for in vivo D3Rs occupancy by antipsychotics, are also discussed. [ABSTRACT FROM AUTHOR]

The lipid post‐translational modification S‐palmitoylation is a vast developing field, with the modification itself and the enzymes that catalyse the reversible reaction implicated in a number of diseases. In this review, we discuss the past and recent advances in the experimental tools used in this field, including pharmacological tools, animal models and techniques to understand how palmitoylation controls protein localisation and function. Additionally, we discuss the obstacles to overcome in order to advance the field, particularly to the point at which modulating palmitoylation may be achieved as a therapeutic strategy. [ABSTRACT FROM AUTHOR]

The serotonin (5-HT) transporter (SERT) is a key regulator of 5-HT signaling and is a major target for antidepressants and psychostimulants. Human SERT coding variants have been identified in subjects with obsessive–compulsive disorder (OCD) and autism spectrum disorder (ASD) that impact transporter phosphorylation, cell surface trafficking and/or conformational dynamics. Prior to an initial description of a novel mouse line expressing the non-phosphorylatable SERT substitution Thr276Ala, we review efforts made to elucidate the structure and conformational dynamics of SERT with a focus on research implicating phosphorylation at Thr276 as a determinant of SERT conformational dynamics. Using the high-resolution structure of human SERT in inward- and outward-open conformations, we explore the conformation dependence of SERT Thr276 exposure, with results suggesting that phosphorylation is likely restricted to an inward-open conformation, consistent with prior biochemical studies. Assessment of genotypes from SERT/Ala276 heterozygous matings revealed a deviation from Mendelian expectations, with reduced numbers of Ala276 offspring, though no genotype differences were seen in growth or physical appearance. Similarly, no genotype differences were evident in midbrain or hippocampal 5-HT levels, midbrain and hippocampal SERT mRNA or midbrain protein levels, nor in midbrain synaptosomal 5-HT uptake kinetics. Behaviorally, SERT Ala276 homozygotes appeared normal in measures of anxiety and antidepressant-sensitive stress coping behavior. However, these mice displayed sex-dependent alterations in repetitive and social interactions, consistent with circuit-dependent requirements for Thr276 phosphorylation underlying these behaviors. Our findings indicate the utility of SERT Ala276 mice in evaluation of developmental, functional and behavioral consequences of regulatory SERT phosphorylation in vivo. [ABSTRACT FROM AUTHOR]

HIV-1 transactivator of transcription (Tat) has a great impact on the development of HIV-1 associated neurocognitive disorders through disrupting dopamine transmission. This study determined the mutational effects of human dopamine transporter (hDAT) on basal and Tat-induced inhibition of dopamine transport. Compared to wild-type hDAT, the maximal velocity (Vmax) of [3H]dopamine uptake was decreased in D381L and Y88F/D206L/H547A, increased in D206L/H547A, and unaltered in D206L. Recombinant TatR1 − 86 inhibited dopamine uptake in wild-type hDAT, which was attenuated in either DAT mutants (D206L, D206L/H547A, and Y88F/D206L/H547A) or mutated TatR1 − 86 (K19A and C22G), demonstrating perturbed Tat-DAT interaction. Mutational effects of hDAT on the transporter conformation were evidenced by attenuation of zinc-induced increased [3H]WIN35,428 binding in D206L/H547A and Y88F/D206A/H547A and enhanced basal MPP+ efflux in D206L/H547A. H547A-induced outward-open transport conformational state was further validated by enhanced accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of an inserted cysteine (I159C) on a hDAT background.. Furthermore, H547A displayed an increase in palmitoylation inhibitor-induced inhibition of dopamine uptake relative to wide-type hDAT, indicating a change in basal palmitoylation in H547A. These results demonstrate that Y88F, D206L, and H547A attenuate Tat inhibition while preserving DA uptake, providing insights into identifying targets for improving DAT-mediated dopaminergic dysregulation. HIV-1 Tat inhibits dopamine uptake through human dopamine transporter (hDAT) on the presynaptic terminal through a direct allosteric interaction. Key hDAT residues D-H547, D-Y88, and D-D206 are predicted to be involved in the HIV-1 Tat-DAT binding. Mutating these residues attenuates this inhibitory effect by disrupting the Tat-hDAT interaction [ABSTRACT FROM AUTHOR]

Dopamine (DA) transporters (DATs) are regulated by trafficking and modulatory processes that probably rely on stable and transient interactions with neighboring proteins and lipids. Using proximity-dependent biotin identification (BioID), we found novel potential partners for DAT, including several membrane proteins, such as the transmembrane chaperone 4F2hc, the proteolipid M6a and a potential membrane receptor for progesterone (PGRMC2). We also detected two cytoplasmic proteins: a component of the Cullin1-dependent ubiquitination machinery termed F-box/LRR-repeat protein 2 (FBXL2), and the enzyme inositol 5-phosphatase 2 (SHIP2). Immunoprecipitation (IP) and immunofluorescence studies confirmed either a physical association or a close spatial proximity between these proteins and DAT. M6a, SHIP2 and the Cullin1 system were shown to increase DAT activity in coexpression experiments, suggesting a functional role for their association. Deeper analysis revealed that M6a, which is enriched in neuronal protrusions (filopodia or dendritic spines), colocalized with DAT in these structures. In addition, the product of SHIP2 enzymatic activity (phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]) was tightly associated with DAT, as shown by co-IP and by colocalization of mCherry-DAT with a specific biosensor for this phospholipid. PI(3,4)P2 strongly stimulated transport activity in electrophysiological recordings, and conversely, inhibition of SHIP2 reduced DA uptake in several experimental systems including striatal synaptosomes and the dopaminergic cell line SH-SY5Y. In summary, here we report several potential new partners for DAT and a novel regulatory lipid, which may represent new pharmacological targets for DAT, a pivotal protein in dopaminergic function of the brain. [ABSTRACT FROM AUTHOR]

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate. [ABSTRACT FROM AUTHOR]

Anorexia Nervosa (AN) is a complex disease that impairs the metabolic, mental and physiological health of affected individuals in a severe and sometimes lethal way. Many of the common symptoms in AN patients, such as reduced food intake, anxiety, impaired gut motility or overexercising are connected to both the orexigenic gut hormone ghrelin and the dopaminergic system. Targeting the ghrelin receptor (GhrR) to treat AN seems a promising possibility in current research. However, GhrR signaling is highly complex. First, the GhrR can activate four known intracellular pathways Gαq, Gαi/o, Gα12/13 and the recruitment of β-arrestin. Biased signaling provides the possibility to activate or inhibit only one or a subset of the intracellular pathways of a pleiotropic receptor. This allows specific targeting of physiological functions without adverse effects. Currently little is known on how biased signaling could specifically modulate GhrR effects. Second, GhrR signaling has been shown to be interconnected with the dopaminergic system, particularly in the context of AN symptoms. This review highlights that a biased agonist for the GhrR may be a promising target for the treatment of AN, however extensive and systematic translational studies are still needed and the connection to the dopaminergic system has to be taken into account. [ABSTRACT FROM AUTHOR]

More recently, there has been a paradigm shift toward selective drug targeting in the treatment of neurological disorders, including drug addiction, schizophrenia, and Parkinson's disease mediated by the different dopamine receptor subtypes. Antagonists with higher selectivity for D3 dopamine receptor (D3DR) over D2 dopamine receptor (D2DR) have been shown to attenuate drug‐seeking behavior and associated side effects compared to non‐subtype selective antagonists. However, high conservations among constituent residues of both proteins, particularly at the ligand‐binding pockets, remain a challenge to therapeutic drug design. Recent studies have reported the discovery of two small‐molecules R‐VK4‐40 and Y‐QA31 which substantially inhibited D3DR with >180‐fold selectivity over D2DR. Therefore, in this study, we seek to provide molecular and structural insights into these differential binding mechanistic using meta‐analytic computational simulation methods. Findings revealed that R‐VK4‐40 and Y‐QA31 adopted shallow binding modes and were more surface‐exposed at D3DR while on the contrary, they exhibited deep hydrophobic pocket binding at D2DR. Also, two non‐conserved residues; Tyr361.39 and Ser18245.51 were identified in D3DR, based on their crucial roles and contributions to the selective binding of R‐VK4‐40 and Y‐QA31. Importantly, both antagonists exhibited high affinities in complex with D3DR compared to D2DR, while van der Waals energies contributed majorly to their binding and stability. Structural analyses also revealed the distinct stabilizing effects of both compounds on D3DR secondary architecture relative to D2DR. Therefore, findings herein pinpointed the origin and mechanistic of selectivity of the compounds, which may assist in the rational design of potential small molecules of the D2‐like dopamine family receptor subtype with improved potency and selectivity. [ABSTRACT FROM AUTHOR]

Cariprazine is a novel antipsychotic drug that exerts partial agonism of dopamine D2/D3 receptors with preferential binding to the D3 receptor, antagonism of 5HT2B receptors, and partial agonism of 5HT1A. Currently, cariprazine has shown clinical efficacy in patients with schizophrenia and with bipolar disorder, as well as adjunctive treatment in patients with Major Depressive Disorder (MDD) and drug-resistant MDD. In the present case series, we report on two patients with treatment-resistant schizophrenia and partial response to clozapine who benefit from combination with cariprazine. The effects of cariprazine combination were remarkable also concerning the adverse metabolic effects of clozapine. [ABSTRACT FROM AUTHOR]

The reinforcing efficacy of cocaine is largely determined by its capacity to inhibit the dopamine transporter (DAT), and emerging evidence suggests that differences in cocaine potency are linked to several symptoms of cocaine use disorder. Despite this evidence, the neural processes that govern cocaine potency in vivo remain unclear. In male rats, we used chemogenetics with intra-VTA microinfusions of the agonist clozapine-n-oxide to bidirectionally modulate dopamine neurons. Using ex vivo fast scan cyclic voltammetry, pharmacological probes of the DAT, biochemical assessments of DAT membrane availability and phosphorylation, and cocaine self-administration, we tested the effects of chemogenetic manipulations on cocaine potency at distal DATs in the nucleus accumbens as well as the behavioral economics of cocaine self-administration. We discovered that chemogenetic manipulation of dopamine neurons produced rapid, bidirectional modulation of cocaine potency at DATs in the nucleus accumbens. We then provided evidence that changes in cocaine potency are associated with alterations in DAT affinity for cocaine and demonstrated that this change in affinity coincides with DAT conformation biases and changes in DAT phosphorylation state. Finally, we showed that chemogenetic manipulation of dopamine neurons alters cocaine consumption in a manner consistent with changes in cocaine potency at distal DATs. Based on the spatial and temporal constraints inherent to our experimental design, we posit that changes in cocaine potency are driven by alterations in dopamine neuron activity. When considered together, these observations provide a novel mechanism through which GPCRs regulate cocaine's pharmacological and behavioral effects. [ABSTRACT FROM AUTHOR]

Abstract There is increasing appreciation that G-protein-coupled receptors (GPCRs) can initiate diverse cellular responses by activating multiple G proteins, arrestins, and other biochemical effectors. Structurally different ligands targeting the same receptor are thought to stabilize the receptor in multiple distinct active conformations such that specific subsets of signaling effectors are engaged at the exclusion of others, creating a bias toward a particular outcome, which has been referred to as ligand-induced selective signaling, biased agonism, ligand-directed signaling, and functional selectivity, among others. The potential involvement of functional selectivity in mammalian olfactory signal transduction has received little attention, notwithstanding the fact that mammalian olfactory receptors comprise the largest family of mammalian GPCRs. This position review considers the possibility that, although such complexity in G-protein function may have been lost in the specialization of olfactory receptors to serve as sensory receptors, the ability of olfactory receptor neurons (ORNs) to function as signal integrators and growing appreciation that this functionality is widespread in the receptor population suggest otherwise. We pose that functional selectivity driving 2 opponent inputs have the potential to generate an output that reflects the balance of ligand-dependent signaling, the direction of which could be either suppressive or synergistic and, as such, needs to be considered as a mechanistic basis for signal integration in mammalian ORNs. [ABSTRACT FROM AUTHOR]

The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease1 and antipsychotic drugs2. DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine3, leading to stimulation of Gi and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2–Gi complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular Gi-binding site. The DRD2–Gi structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor–G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders. The structure of the D2 dopamine receptor in complex with its G protein reveals how dopamine receptors are activated and, importantly, how a G-protein-coupled receptor can interact with its G protein in a phospholipid membrane. [ABSTRACT FROM AUTHOR]