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Molecular and Cellular Endocrinology Aug 2024Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the...
Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the activation of a complex signaling network. We recently identified iPRC1, the first variable fragment from heavy-chain-only antibody (VHH) interacting with intracellular loop 3 (ICL3) of the follicle-stimulating hormone receptor (FSHR). Because of the high sequence similarity of the human FSHR and LHR (LHCGR), here we examined the ability of the iPRC1 intra-VHH to modulate LHCGR activity. In this study, we demonstrated that iPRC1 binds LHCGR, to a greater extent when the receptor was stimulated by the hormone. In addition, it decreased LH-induced cAMP production, cAMP-responsive element-dependent transcription, progesterone and testosterone production. These impairments are not due to Gs nor β-arrestin recruitment to the LHCGR. Consequently, iPRC1 is the first intra-VHH to bind and modulate LHCGR biological activity, including steroidogenesis. It should help further understand signaling mechanisms elicited at this receptor and their outcomes on reproduction.
Topics: Receptors, LH; Humans; Signal Transduction; Luteinizing Hormone; Animals; Cyclic AMP; Protein Binding; Progesterone; Receptors, FSH; Testosterone; HEK293 Cells; GTP-Binding Proteins; Steroids
PubMed: 38621656
DOI: 10.1016/j.mce.2024.112235 -
Biochemical Pharmacology Jun 2024Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can...
Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of 'phosphorylation barcode,' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB receptor (CB) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.
Topics: Humans; Receptor, Cannabinoid, CB1; Signal Transduction; HEK293 Cells; Arrestins; Protein Transport; GTP-Binding Proteins; G-Protein-Coupled Receptor Kinases; Animals; beta-Arrestin 2
PubMed: 38604257
DOI: 10.1016/j.bcp.2024.116190 -
Nature May 2024The µ-opioid receptor (µOR) is an important target for pain management and molecular understanding of drug action on µOR will facilitate the development of better...
The µ-opioid receptor (µOR) is an important target for pain management and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of β-arrestin-1 with the μOR core binding site appears less specific and occurs with much lower affinity than binding of G.
Topics: Humans; beta-Arrestin 1; Binding Sites; Fluorescence Resonance Energy Transfer; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Diphosphate; Ligands; Models, Molecular; Protein Binding; Protein Conformation; Receptors, Opioid, mu; Single Molecule Imaging
PubMed: 38600384
DOI: 10.1038/s41586-024-07295-2 -
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 -
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