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Cell Communication and Signaling : CCS Apr 2024Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The...
BACKGROUND
Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of β-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D-like receptors (DR, DR, DR), have suggested the existence of other desensitization mechanisms. The present study employed DR/DR variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT.
RESULTS
AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin β1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of β-Arr2. The deubiquitinated β-Arr2 then forms a complex with Gβγ, which serves as a biomarker for GPCRs desensitization. Like in DR, ubiquitination of AKT is also involved in the desensitization of β adrenoceptors.
CONCLUSION
Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.
Topics: TNF Receptor-Associated Factor 6; Proto-Oncogene Proteins c-akt; Receptors, G-Protein-Coupled; Ubiquitination; Phosphorylation; Karyopherins
PubMed: 38566235
DOI: 10.1186/s12964-024-01592-z -
BioRxiv : the Preprint Server For... Mar 2024Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the μ-opioid receptor. Opioid use is limited by...
Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the μ-opioid receptor. Opioid use is limited by side effects, including significant risk of opioid use disorder. Improvement of the effect/side effect profile of opioid medications is a key pursuit of opioid research, yet there is no consensus on how to achieve this goal. One hypothesis is that the degree of arrestin-3 recruitment to the μ-opioid receptor impacts therapeutic utility. However, it is not clear whether increased or decreased interaction of the μ-opioid receptor with arrestin-3 would reduce compulsive drug-seeking. To examine this question, we utilized three genotypes of mice with varying abilities to recruit arrestin-3 to the μ-opioid receptor in response to morphine in a novel longitudinal operant self-administration model. We demonstrate that arrestin-3 knockout and wild type mice have highly variable drug-seeking behavior with few genotype differences. In contrast, in mice where the μ-opioid receptor strongly recruits arrestin-3, drug-seeking behavior is much less varied. We created a quantitative method to define compulsivity in drug-seeking and found that mice lacking arrestin-3 were more likely to meet the criteria for compulsivity whereas mice with enhanced arrestin-3 recruitment did not develop a compulsive phenotype. Our data suggest that opioids that engage both G protein and arrestin-3, recapitulating the endogenous signaling pattern, will reduce abuse liability.
PubMed: 38562752
DOI: 10.1101/2023.03.30.534994 -
Molecular Psychiatry Apr 2024The intricate involvement of the serotonin 5-HT receptor (5-HTR) both in schizophrenia and in the activity of antipsychotic drugs is widely acknowledged. The currently...
The intricate involvement of the serotonin 5-HT receptor (5-HTR) both in schizophrenia and in the activity of antipsychotic drugs is widely acknowledged. The currently marketed antipsychotic drugs, although effective in managing the symptoms of schizophrenia to a certain extent, are not without their repertoire of serious side effects. There is a need for better therapeutics to treat schizophrenia for which understanding the mechanism of action of the current antipsychotic drugs is imperative. With bioluminescence resonance energy transfer (BRET) assays, we trace the signaling signature of six antipsychotic drugs belonging to three generations at the 5-HTR for the entire spectrum of signaling pathways activated by serotonin (5-HT). The antipsychotic drugs display previously unidentified pathway preference at the level of the individual Gα subunits and β-arrestins. In particular, risperidone, clozapine, olanzapine and haloperidol showed G protein-selective inverse agonist activity. In addition, G protein-selective partial agonism was found for aripiprazole and cariprazine. Pathway-specific apparent dissociation constants determined from functional analyses revealed distinct coupling-modulating capacities of the tested antipsychotics at the different 5-HT-activated pathways. Computational analyses of the pharmacological and structural fingerprints support a mechanistically based clustering that recapitulate the clinical classification (typical/first generation, atypical/second generation, third generation) of the antipsychotic drugs. The study provides a new framework to functionally classify antipsychotics that should represent a useful tool for the identification of better and safer neuropsychiatric drugs and allows formulating hypotheses on the links between specific signaling cascades and in the clinical outcomes of the existing drugs.
PubMed: 38561467
DOI: 10.1038/s41380-024-02531-7 -
European Journal of Pharmacology May 2024Synthetic cannabinoid receptor agonists (SCRAs) remain one the largest classes of new psychoactive substances, and are increasingly associated with severe adverse...
Synthetic cannabinoid receptor agonists (SCRAs) remain one the largest classes of new psychoactive substances, and are increasingly associated with severe adverse effects and death compared to the phytocannabinoid Δ-tetrahydrocannabinol (THC). In the attempt to circumvent the rapid emergence of novel SCRAs, several nations have implemented 'generic' legislations, or 'class-wide' bans based on common structural scaffolds. However, this has only encouraged the incorporation of new chemical entities, including distinct core and linker structures, for which there is a dearth of pharmacological data. The current study evaluated five emergent OXIZID SCRAs for affinity and functional activity at the cannabinoid CB receptor (CB) in HEK 293 cells, as well as pharmacological equivalence with THC in drug discrimination in mice. All OXIZID compounds behaved as agonists in Gα protein activation and β-arrestin 2 translocation assays, possessing low micromolar affinity at CB. All ligands also substituted for THC in drug discrimination, where potencies broadly correlated with in vitro activity, with the methylcyclohexane analogue BZO-CHMOXIZID being the most potent. Notably, MDA-19 (BZO-HEXOXIZID) exhibited partial efficacy in vitro, generating an activity profile most similar to that of THC, and partial substitution in vivo. Overall, the examined OXIZIDs were comparatively less potent and efficacious than previous generations of SCRAs. Further toxicological data will elucidate whether the moderate cannabimimetic activity for this series of SCRAs will translate to severe adverse health effects as seen with previous generations of SCRAs.
Topics: Humans; Mice; Animals; Cannabinoid Receptor Agonists; HEK293 Cells; Receptors, Cannabinoid; Ligands; Protein Processing, Post-Translational; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2
PubMed: 38561104
DOI: 10.1016/j.ejphar.2024.176549 -
International Journal of Molecular... Mar 2024Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of...
Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H receptor-mediated activation of ERK is dually regulated by G proteins and arrestins. In this study, we investigated the roles of G proteins and arrestins in the H receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H receptors, the G protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H receptors (ketotifen and diphenhydramine), G proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), β-arrestin2 (β-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H receptor-mediated phosphorylation of JNK is regulated by G-protein/Ca/PKC-dependent but GRK/arrestin/clathrin-independent pathways.
Topics: Animals; Cricetinae; Humans; Arrestin; Arrestins; beta-Arrestins; CHO Cells; Clathrin; Cricetulus; Extracellular Signal-Regulated MAP Kinases; G-Protein-Coupled Receptor Kinases; GTP-Binding Proteins; Histamine; Phosphorylation; Protein Kinase C; Receptors, Histamine H1; Signal Transduction
PubMed: 38542369
DOI: 10.3390/ijms25063395 -
Biosensors Mar 2024The human CC chemokine receptor 7 (CCR7) is activated by two natural ligands, CC chemokine ligand 19 (CCL19) and 21 (CCL21). The CCL19-CCL21-CCR7 axis has been...
The human CC chemokine receptor 7 (CCR7) is activated by two natural ligands, CC chemokine ligand 19 (CCL19) and 21 (CCL21). The CCL19-CCL21-CCR7 axis has been extensively studied in vitro, but there is still debate over whether CCL21 is an overall weaker agonist or if the axis displays biased signalling. In this study, we performed a systematic analysis at the transducer level using NanoBRET-based methodologies in three commonly used cellular backgrounds to evaluate pathway and ligand preferences, as well as ligand bias and the influence of the cellular system thereon. We found that both CCL19 and CCL21 activated all cognate G proteins and some non-cognate couplings in a cell-type-dependent manner. Both ligands recruited β-arrestin1 and 2, but the potency was strongly dependent on the cellular system. Overall, CCL19 and CCL21 showed largely conserved pathway preferences, but small differences were detected. However, these differences only consolidated in a weak ligand bias. Together, these data suggest that CCL19 and CCL21 share mostly overlapping, weakly biased, transducer profiles, which can be influenced by the cellular context.
Topics: Humans; Receptors, CCR7; Ligands; Signal Transduction
PubMed: 38534251
DOI: 10.3390/bios14030142 -
Briefings in Bioinformatics Jan 2024G-protein-coupled receptors (GPCRs) mediate diverse cell signaling cascades after recognizing extracellular ligands. Despite the successful history of known GPCR drugs,...
G-protein-coupled receptors (GPCRs) mediate diverse cell signaling cascades after recognizing extracellular ligands. Despite the successful history of known GPCR drugs, a lack of mechanistic insight into GPCR challenges both the deorphanization of some GPCRs and optimization of the structure-activity relationship of their ligands. Notably, replacing a small substituent on a GPCR ligand can significantly alter extracellular GPCR-ligand interaction patterns and motion of transmembrane helices in turn to occur post-binding events of the ligand. In this study, we designed 3D multilevel features to describe the extracellular interaction patterns. Subsequently, these 3D features were utilized to predict the post-binding events that result from conformational dynamics from the extracellular to intracellular areas. To understand the adaptability of GPCR ligands, we collected the conformational information of flexible residues during binding and performed molecular featurization on a broad range of GPCR-ligand complexes. As a result, we developed GPCR-ligand interaction patterns, binding pockets, and ligand features as score (GPCR-IPL score) for predicting the functional selectivity of GPCR ligands (agonism versus antagonism), using the multilevel features of (1) zoomed-out 'residue level' (for flexible transmembrane helices of GPCRs), (2) zoomed-in 'pocket level' (for sophisticated mode of action) and (3) 'atom level' (for the conformational adaptability of GPCR ligands). GPCR-IPL score demonstrated reliable performance, achieving area under the receiver operating characteristic of 0.938 and area under the precision-recall curve of 0.907 (available in gpcr-ipl-score.onrender.com). Furthermore, we used the molecular features to predict the biased activation of downstream signaling (Gi/o, Gq/11, Gs and β-arrestin) as well as the functional selectivity. The resulting models are interpreted and applied to out-of-set validation with three scenarios including the identification of a new MRGPRX antagonist.
Topics: Receptors, G-Protein-Coupled; Signal Transduction; Ligands; Structure-Activity Relationship
PubMed: 38517694
DOI: 10.1093/bib/bbae105 -
Investigative Ophthalmology & Visual... Mar 2024Variants in the ARR3 gene have been linked to early-onset high myopia (eoHM) with a unique X-linked female-limited inheritance. However, the clinical validity of this...
PURPOSE
Variants in the ARR3 gene have been linked to early-onset high myopia (eoHM) with a unique X-linked female-limited inheritance. However, the clinical validity of this gene-disease association has not been systematically evaluated.
METHODS
We identified two Chinese families with novel ARR3 splicing variants associated with eoHM. Minigene constructs were generated to assess the effects of the variants on splicing. We integrated previous evidence to curate the clinical validity of ARR3 and eoHM using the ClinGen framework.
RESULTS
The variants c.39+1G>A and c.100+4A>G were identified in the two families. Minigene analysis showed both variants resulted in abnormal splicing and introduction of premature termination codons. Based on genetic and experimental evidence, the ARR3-eoHM relationship was classified as "definitive."
CONCLUSIONS
Our study identified two novel splicing variants of the ARR3 gene linked to eoHM and confirmed their functional validity via minigene assay. This research expanded the mutational spectrum of ARR3 and confirmed the minigene assay technique as an effective tool for understanding variant effects on splicing mechanisms.
Topics: Female; Humans; Mutation; Myopia; RNA Splicing; Arrestins; East Asian People
PubMed: 38517428
DOI: 10.1167/iovs.65.3.32 -
Physical Chemistry Chemical Physics :... Apr 2024Biased ligands selectively activating specific downstream signaling pathways (termed as biased activation) exhibit significant therapeutic potential. However, the...
Biased ligands selectively activating specific downstream signaling pathways (termed as biased activation) exhibit significant therapeutic potential. However, the conformational characteristics revealed are very limited for the biased activation, which is not conducive to biased drug development. Motivated by the issue, we combine extensive accelerated molecular dynamics simulations and an interpretable deep learning model to probe the biased activation features for two complex systems constructed by the inactive μOR and two different biased agonists (G-protein-biased agonist TRV130 and β-arrestin-biased agonist endomorphin2). The results indicate that TRV130 binds deeper into the receptor core compared to endomorphin2, located between W293 and D114, and forms hydrogen bonding with D114, while endomorphin2 binds above W293. The G protein-biased agonist induces greater outward movements of the TM6 intracellular end, forming a typical active conformation, while the β-arrestin-biased agonist leads to a smaller extent of outward movements of TM6. Compared with TRV130, endomorphin2 causes more pronounced inward movements of the TM7 intracellular end and more complex conformational changes of H8 and ICL1. In addition, important residues determining the two different biased activation states were further identified by using an interpretable deep learning classification model, including some common biased activation residues across Class A GPCRs like some key residues on the TM2 extracellular end, ECL2, TM5 intracellular end, TM6 intracellular end, and TM7 intracellular end, and some specific important residues of ICL3 for μOR. The observations will provide valuable information for understanding the biased activation mechanism for GPCRs.
Topics: Molecular Dynamics Simulation; Spiro Compounds; GTP-Binding Proteins; beta-Arrestins; Machine Learning; Ligands; Thiophenes
PubMed: 38512140
DOI: 10.1039/d3cp05050e -
Biochemical and Biophysical Research... Apr 2024Secretory myeloid-derived growth factor (MYDGF) exerts beneficial effects on organ repair, probably via a plasma membrane receptor; however, the identity of the expected...
Secretory myeloid-derived growth factor (MYDGF) exerts beneficial effects on organ repair, probably via a plasma membrane receptor; however, the identity of the expected receptor has remained elusive. In a recent study, MYDGF was reported as an agonist of the sphingosine-1-phosphate receptor 2 (S1PR2), an A-class G protein-coupled receptor that mediates the functions of the signaling lipid, sphingosine-1-phosphate (S1P). In the present study, we conducted living cell-based functional assays to test whether S1PR2 is a receptor for MYDGF. In the NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment assay and the cAMP-response element (CRE)-controlled NanoLuc reporter assay, S1P could efficiently activate human S1PR2 overexpressed in human embryonic kidney (HEK) 293T cells; however, recombinant human MYDGF, overexpressed either from Escherichia coli or HEK293 cells, had no detectable effect. Thus, the results demonstrated that human MYDGF is not a ligand of human S1PR2. Considering the high conservation of MYDGF and S1PR2 in evolution, MYDGF is also probably not a ligand of S1PR2 in other vertebrates.
Topics: Animals; Humans; Sphingosine-1-Phosphate Receptors; Receptors, Lysosphingolipid; Ligands; Granulocyte Colony-Stimulating Factor; HEK293 Cells; Lysophospholipids; Sphingosine
PubMed: 38484568
DOI: 10.1016/j.bbrc.2024.149766