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Pharmacological Research Nov 2023Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs,... (Review)
Review
Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs, antidepressants and SSRI's. Within the opioid family mu and kappa opioid receptor are commonly targeted. However, activation of the mu opioid receptor has side effects of constipation, tolerance, dependence, euphoria, and respiratory depression; activation of the kappa opioid receptor leads to dysphoria and sedation. The side effects of mu opioid receptor activation have led to mu receptor drugs being widely abused with great overdose risk. For these reasons, newer safer opioid analgesics are in high demand. For many years a focus within the opioid field was finding drugs that activated the G protein pathway at mu opioid receptor, without activating the β-arrestin pathway, known as biased agonism. Recent advances have shown that this may not be the way forward to develop safer analgesics at mu opioid receptor, though there is still some promise at the kappa opioid receptor. Here we discuss recent novel approaches to develop safer opioid drugs including efficacy vs bias and fine-tuning receptor activation by targeting sub-pockets in the orthosteric site, we explore recent works on the structural basis of bias, and we put forward the suggestion that Gα subtype selectivity may be an exciting new area of interest.
Topics: Humans; Analgesics, Opioid; Receptors, Opioid, mu; Receptors, Opioid, kappa; Receptors, Opioid; Acute Pain
PubMed: 37844653
DOI: 10.1016/j.phrs.2023.106961 -
The Journal of Biological Chemistry Nov 2023β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G...
β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gα, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gα and β-arrestins in controlling gene expression. We found that Gα is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gα dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gα-KO cells. These results were validated by re-expressing Gα in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gα-driven nuclear transcriptional activity.
Topics: Humans; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation; GTP-Binding Proteins; Mitogen-Activated Protein Kinases; Phosphorylation; Receptors, Adrenergic, beta-2; HEK293 Cells; GTP-Binding Protein alpha Subunits; Protein Structure, Tertiary; Protein Isoforms; Enzyme Activation
PubMed: 37774973
DOI: 10.1016/j.jbc.2023.105293 -
Nature Communications Dec 2023Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT receptor activation mediates psychedelic effects, prototypical psychedelics activate...
Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT-Gq/11 and β-arrestin2 transducers, making their respective roles unclear. To elucidate this, we develop a series of 5-HT-selective ligands with varying Gq efficacies, including β-arrestin-biased ligands. We show that 5-HT-Gq but not 5-HT-β-arrestin2 recruitment efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT Gq-efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT agonists. We also demonstrate that β-arrestin-biased 5-HT receptor agonists block psychedelic effects and induce receptor downregulation and tachyphylaxis. Overall, 5-HT receptor Gq-signaling can be fine-tuned to generate ligands distinct from classical psychedelics.
Topics: Male; Animals; Mice; Hallucinogens; Receptor, Serotonin, 5-HT2A; Serotonin; Signal Transduction; beta-Arrestins; Ligands
PubMed: 38102107
DOI: 10.1038/s41467-023-44016-1 -
Proceedings of the National Academy of... Oct 2023Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor and...
Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor and glucagon receptor in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 13 reported splice variants (SVs), more than half of which have sequence variations at either C or N terminus. To explore their roles in endogenous peptide-mediated GIPR signaling, we determined the cryoelectron microscopy (cryo-EM) structures of the two N terminus-altered SVs (referred as GIPR-202 and GIPR-209 in the Ensembl database, SV1 and SV2 here, respectively) and investigated the outcome of coexpressing each of them in question with GIPR in HEK293T cells with respect to ligand binding, receptor expression, cAMP (adenosine 3,5-cyclic monophosphate) accumulation, β-arrestin recruitment, and cell surface localization. It was found that while both N terminus-altered SVs of GIPR neither bound to the hormone nor elicited signal transduction per se, they suppressed ligand binding and cAMP accumulation of GIPR. Meanwhile, SV1 reduced GIPR-mediated β-arrestin 2 responses. The cryo-EM structures of SV1 and SV2 showed that they reorganized the extracellular halves of transmembrane helices 1, 6, and 7 and extracellular loops 2 and 3 to adopt a ligand-binding pocket-occupied conformation, thereby losing binding ability to the peptide. The results suggest a form of signal bias that is constitutive and ligand-independent, thus expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-independent (e.g., SV1 of the growth hormone-releasing hormone receptor).
Topics: Humans; Gastric Inhibitory Polypeptide; Ligands; Cryoelectron Microscopy; HEK293 Cells; Signal Transduction; Receptors, Gastrointestinal Hormone; Peptides; Glucagon-Like Peptide-1 Receptor
PubMed: 37792509
DOI: 10.1073/pnas.2306145120 -
Cell Communication and Signaling : CCS Sep 2023G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand...
BACKGROUND
G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA heteromer and characterized the unique molecular features and function of this heteromer.
METHODS
We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA heteromer, we performed various assays, including cAMP, BRET for G protein activation, β-arrestin recruitment, ligand binding, and transwell migration assays.
RESULTS
We observed that CXCR4 forms heteromers with LPA in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gα activation, and β-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gα recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA-selective antagonists enhanced CXCL12-induced Gα signaling and cell migration in the parental MDA-MB-231 cells but not in LPA-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA antagonists.
CONCLUSIONS
The presence and impact of CXCR4-LPA heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA antagonists alone for such conditions. Video Abstract.
Topics: Humans; Calcium Signaling; Cell Movement; Chemokine CXCL12; Ligands; Prospective Studies; Receptors, CXCR4; Receptors, Lysophosphatidic Acid
PubMed: 37749552
DOI: 10.1186/s12964-023-01261-7 -
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 Death & Disease Jul 2023β-arrestin 2 (ARRB2) is functionally implicated in cancer progression via various signaling pathways. However, its role in lung cancer remains unclear. To obtain...
β-arrestin 2 (ARRB2) is functionally implicated in cancer progression via various signaling pathways. However, its role in lung cancer remains unclear. To obtain clinical insight on its function in lung cancer, microarray data from lung tumor tissues (LTTs) and matched lung normal tissues (mLNTs) of primary non-small cell lung cancer (NSCLC) patients (n = 37) were utilized. ARRB2 expression levels were markedly decreased in all 37 LTTs compared to those in matched LNTs of NSCLC patients. They were significantly co-related to enrichment gene sets associated with oncogenic and cancer genes. Importantly, Gene Set Enrichment Analysis (GSEA) between three LTTs with highly down-regulated ARRB2 and three LTTs with lowly down-regulated ARRB2 revealed significant enrichments related to toll-like receptor (TLR) signaling and autophagy genes in three LTTs with highly down-regulated ARRB2, suggesting that ARRB2 was negatively involved in TLR-mediated signals for autophagy induction in lung cancer. Biochemical studies for elucidating the molecular mechanism revealed that ARRB2 interacted with TNF receptor-associated factor 6 (TRAF6) and Beclin 1 (BECN1), thereby inhibiting the ubiquitination of TRAF6-TAB2 to activate NF-κB and TRAF6-BECN1 for autophagy stimulated by TLR3 and TLR4, suggesting that ARRB2 could inhibit the TRAF6-TAB2 signaling axis for NF-κB activation and TRAF6-BECN1 signaling axis for autophagy in response to TLR3 and TLR4. Notably, ARRB2-knockout (ARRB2KO) lung cancer cells exhibited marked enhancements of cancer migration, invasion, colony formation, and proliferation in response to TLR3 and TLR4 stimulation. Altogether, our current data suggest that ARRB2 can negatively regulate lung cancer progression by inhibiting TLR3- and TLR4-induced autophagy.
Topics: Humans; NF-kappa B; TNF Receptor-Associated Factor 6; Lung Neoplasms; Toll-Like Receptor 4; Toll-Like Receptor 3; beta-Arrestin 2; Carcinoma, Non-Small-Cell Lung; Toll-Like Receptors; Lung; Autophagy; Adaptor Proteins, Signal Transducing
PubMed: 37443143
DOI: 10.1038/s41419-023-05945-3 -
Proceedings of the National Academy of... Jul 2023E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, β-arrestins...
E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, β-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the β-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the β-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of β-arrestin1 in complex with Mdm2 peptide. The acidic residues of Mdm2 bind to the positively charged concave side of the β-arrestin1 N-domain. The C-tail of β-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of β-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate β-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on β-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2 binding to β-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP-induced oligomer of β-arrestin1. These results show how the E3 ligase, Mdm2, interacts with β-arrestins to promote the internalization of GPCRs.
Topics: beta-Arrestins; Ubiquitin-Protein Ligases; Arrestins; beta-Arrestin 1; Ubiquitination; Receptors, G-Protein-Coupled; beta-Arrestin 2; Phosphorylation
PubMed: 37399373
DOI: 10.1073/pnas.2301934120 -
The Journal of Biological Chemistry Dec 2023G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3...
G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3 ubiquitin ligases. E3 ligases have been linked to agonist-stimulated ubiquitination of GPCRs via simultaneous binding to βarrestins. In addition, βarrestins have been suggested to assist E3 ligases for ubiquitination of key effector molecules, yet mechanistic insight is lacking. Here, we developed an in vitro reconstituted system and show that βarrestin1 (βarr1) serves as an adaptor between the effector protein signal-transducing adaptor molecule 1 (STAM1) and the E3 ligase atrophin-interacting protein 4. Via mass spectrometry, we identified seven lysine residues within STAM1 that are ubiquitinated and several types of ubiquitin linkages. We provide evidence that βarr1 facilitates the formation of linear polyubiquitin chains at lysine residue 136 on STAM1. This lysine residue is important for stabilizing the βarr1:STAM1 interaction in cells following GPCR activation. Our study identifies atrophin-interacting protein 4 as only the second E3 ligase known to conjugate linear polyubiquitin chains and a possible role for linear ubiquitin chains in GPCR signaling and trafficking.
Topics: Lysine; Polyubiquitin; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; beta-Arrestin 1
PubMed: 37981209
DOI: 10.1016/j.jbc.2023.105474 -
ELife Jan 2024The α-arrestins form a large family of evolutionally conserved modulators that control diverse signaling pathways, including both G-protein-coupled receptor...
The α-arrestins form a large family of evolutionally conserved modulators that control diverse signaling pathways, including both G-protein-coupled receptor (GPCR)-mediated and non-GPCR-mediated pathways, across eukaryotes. However, unlike β-arrestins, only a few α-arrestin targets and functions have been characterized. Here, using affinity purification and mass spectrometry, we constructed interactomes for 6 human and 12 α-arrestins. The resulting high-confidence interactomes comprised 307 and 467 prey proteins in human and , respectively. A comparative analysis of these interactomes predicted not only conserved binding partners, such as motor proteins, proteases, ubiquitin ligases, RNA splicing factors, and GTPase-activating proteins, but also those specific to mammals, such as histone modifiers and the subunits of V-type ATPase. Given the manifestation of the interaction between the human α-arrestin, TXNIP, and the histone-modifying enzymes, including HDAC2, we undertook a global analysis of transcription signals and chromatin structures that were affected by TXNIP knockdown. We found that TXNIP activated targets by blocking HDAC2 recruitment to targets, a result that was validated by chromatin immunoprecipitation assays. Additionally, the interactome for an uncharacterized human α-arrestin ARRDC5 uncovered multiple components in the V-type ATPase, which plays a key role in bone resorption by osteoclasts. Our study presents conserved and species-specific protein-protein interaction maps for α-arrestins, which provide a valuable resource for interrogating their cellular functions for both basic and clinical research.
Topics: Animals; Humans; Arrestin; Histones; Drosophila; Vacuolar Proton-Translocating ATPases; Arrestins; Mammals
PubMed: 38270169
DOI: 10.7554/eLife.88328