Biology subjects

Inoue, A.

Publications and source records attributed to Inoue, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Non-canonical internalization mechanisms of mGlu receptors

Cell surface density of G protein-coupled receptors (GPCRs) is tightly regulated through constitutive and agonist-induced internalization. Whereas the mechanisms of constitutive internalization remain elusive, agonist-induced internalization is accepted to involve receptor phosphorylation by GPCR kinases (GRKs), {beta}-arrestin binding and AP2 recruitment, targeting receptors to clathrin-coated pits. Dimeric class C metabotropic glutamate (mGlu1 to 8) receptors regulate synaptic transmission but their internalization process is ambiguous. Here, we used diffusion-enhanced energy transfer (DERET) to decipher their internalization kinetics. We showed that all mGlu receptors are constitutively internalized. However, only mGlu1, 5 and 3 homodimers are agonist-induced internalized, that require neither GRKs, nor {beta}-arrestins. In contrast, the constitutive internalization involves only {beta}-arrestins. This systematic study further illustrates how different class C receptors are relative to most other GPCRs, revealing non-canonical internalization mechanisms. These insights in mGlu receptor dynamics will help promoting the therapeutic action of drugs targeting mGlu receptors.

neuroscience↗

bioRxiv2025

Therapeutic ability of extracellular vesicles derived from angio-miRNA-modified mesenchymal stromal cells in ischemic animal models

BackgroundRegenerative therapy using extracellular vesicles (EVs) is expected to be an option for supportive therapy of chronic limb-threatening ischemia. In this study, we examined the angiogenic potential of EVs derived from genetically modified MSCs, focusing on the angio-miRNAs in these vesicles. MethodsBone marrow mesenchymal stromal cells (BM-MSCs) were transfected with a lentiviral vector containing specific angio-miRNAs (miRNA-126, -135b, or -210). EVs were separated from the culture medium of BM-MSCs via ultracentrifugation. The quality and quantity of the EVs were evaluated using nanoparticle tracking analysis, fluorometry, and an ExoScreen assay. Tube formation assays were conducted using human umbilical vein endothelial cells to assess the angiogenic potency of the EVs in vitro. The EVs were injected into the ischemic hind limb muscles of mice. The grades of limb ischemia, blood perfusion, and muscle tissues were analyzed to assess the angiogenic potency of the EVs in vivo. ResultsAngio-miRNAs were overexpressed in MSCs transfected with a lentiviral vector. Tube formation was significantly enhanced when endothelial cells were cultured with the EVs (P < 0.05). Compared to non-contact co-culture with BM-MSCs, EVs derived from modified BM-MSCs significantly promoted tube formation in endothelial cells (P < 0.05). The severity of limb ischemia on day 7 was alleviated when a combination of two or three EVs derived from modified BM-MSCs was injected compared with native EVs. Toe blood perfusion on day 14 significantly recovered when a combination of two or three EVs derived from modified BM-MSCs was injected (P < 0.05). Tissue analysis revealed that the number of newly formed endothelial cells in ischemic muscles increased when a combination of two or three EVs derived from modified BM-MSCs was injected. ConclusionsThe use of a combination of EVs derived from genetically modified MSCs stimulated angiogenesis both in vitro and in vivo.

cell biology↗

bioRxiv2024

Priming Epigenetic Landscape at Gene Promoters through Transcriptional Activation in Mammalian Germ Cells

In mammalian germ cells, a cycle of erasing and resetting epigenetic information is crucial for sex-specific gametogenesis and early embryogenesis. Specific chromatin states at promoters allow genes to be expressed at the appropriate time during germ cell development, however, the mechanism that establishes such characteristic chromatin states remains unclear. Here, we show that, in mouse male embryonic germ cells, promoters of developmental and housekeeping genes are selectively protected from de novo DNA methylation, coinciding with their transient upregulation and genome-wide erasure of H3K27me3. Importantly, a specific level of H3K4me3 density at the promoters serves as a potent probe that distinguishes between hypo- and hyper-DNA methylation states. Subsequent restoration of H3K27me3 contributes to establishing bivalent chromatin states that prime genes for activation at later developmental stages. These findings reveal a molecular framework shaping the epigenetic landscape at promoters with long-term effects beyond germ cell development.

genomics↗

bioRxiv2024

Design of allosteric modulators that change GPCR G protein subtype selectivity

G protein-coupled receptors (GPCRs), the largest family of drug targets, can signal through 16 subtypes of G proteins. Biased compounds that selectively activate therapy-relevant pathways promise to be safer, more effective medications. The determinants of bias are poorly understood, however, and rationally-designed, G protein-subtype-selective compounds are lacking. Here, using the prototypical class A GPCR neurotensin receptor 1 (NTSR1), we find that small molecules binding the intracellular GPCR-transducer interface change G protein coupling by subtype-specific and predictable mechanisms, enabling rational drug design. We demonstrate that the compound SBI-553 switches NTSR1 G protein preference by acting both as a molecular bumper and a molecular glue. Structurally, SBI-553 occludes G protein binding determinants on NTSR1, promoting association with select G protein subtypes for which an alternative, shallow-binding conformation is energetically favorable. Minor modifications to the SBI-553 scaffold produce allosteric modulators with distinct G protein subtype selectivity profiles. Selectivity profiles are probe-independent, conserved across species, and translate to differences in in vivo activity. These studies demonstrate that G protein selectivity can be tailored with small changes to a single chemical scaffold targeting the receptor-transducer interface and, as this pocket is broadly conserved, present a strategy for pathway-selective drug discovery applicable to the diverse GPCR superfamily.

pharmacology and toxicology↗

bioRxiv2024

Amylin receptor subunit interactions are modulated by agonists and determine signaling

Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMY1R, AMY2R, and AMY3R are heterodimers consisting of the G protein-coupled calcitonin receptor (CTR) paired with a RAMP1, -2, or -3 accessory subunit, respectively, which increases amylin potency. Little is known about AMYR subunit interactions and their role in signaling. Here, we show that the AMYRs have distinct basal subunit equilibriums that are modulated by peptide agonists and determine the cAMP signaling phenotype. Using a novel biochemical assay that resolves the AMYR heterodimers and free subunits, we found that the AMY1/2R subunit equilibriums favored free CTR and RAMP1/2, and rat amylin and CGRP agonists promoted subunit association. A stronger CTR-RAMP3 transmembrane domain interface yielded a more stable AMY3R, and human and salmon calcitonin agonists promoted AMY3R dissociation. Similar changes in subunit association-dissociation were observed in live cell membranes, and G protein coupling and cAMP signaling assays showed how these altered signaling. Our findings reveal regulation of heteromeric GPCR signaling through subunit interaction dynamics.

biochemistry↗

bioRxiv2024

Four-color single-molecule imaging system for tracking GPCR dynamics with fluorescent HiBiT peptide

Single-molecule imaging provides information on diffusion dynamics, oligomerization, and protein-protein interactions in living cells. To simultaneously monitor different types of proteins at the single-molecule level, orthogonal fluorescent labeling methods with different photostable dyes are required. G-protein-coupled receptors (GPCRs), a major class of drug targets, are prototypical membrane receptors that have been studied using single-molecule imaging techniques. Here we developed a method for labeling cell-surface GPCRs inspired by the HiBiT system, which utilizes the high affinity complementation between LgBiT and HiBiT fragments of the NanoLuc luciferase. We synthesized four fluorescence-labeled HiBiT peptides (F-FiBiTs) with a different color dye (Setau-488, TMR, SaraFluor 650 and SaraFluor 720). We constructed a multicolor total internal reflection fluorescence microscopy system that allows us to track four color dyes simultaneously. As a proof-of-concept experiment, we labeled an N-terminally LgBiT-fused GPCR (Lg-GPCR) with a mixture of the four F-FiBiTs and successfully tracked each dye within a cell at the single molecule level. The F-FiBiT-labeled Lg-GPCRs showed agonist-dependent changes in the diffusion dynamics and accumulation into the clathrin-coated pits as observed with a conventional method using a C-terminally HaloTag-fused GPCR. Taking advantage of luciferase complementation by the F-FiBiT and Lg-GPCRs, the F-FiBiT was also applicable to bioluminescence plate-reader-based assays. By combining existing labeling methods such as HaloTag, SNAP-tag, and fluorescent proteins, the F-FiBiT method will be useful for multicolor single-molecule imaging and will enhance our understanding of GPCR signaling at the single-molecule level.

biophysics↗

bioRxiv2024

Molecular mechanism of the endothelin receptor type B interactions with Gs, Gi, and Gq

The endothelin receptor type B (ETB) exhibits promiscuous coupling with various G protein subtypes including Gs, Gi/o, Gq/11, and G12/13, leading to multi-modal signal transduction. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi and Gq triggered by ETB activation. ETB coupled to Gi and Gq but not with Gs. We underscored the critical roles of specific regions, including the C-terminus of G and intracellular loop 2 (ICL2) of ETB in ETB-Gi1 or ETB-Gq coupling. Although The C-terminus of G is essential for ETB-Gi1 and ETB-Gq coupling, ETB ICL2 influences Gq-coupling but not Gi1-coupling. Our results suggest a differential coupling efficiency of ETB with Gs, Gi1, and Gq, accompanied by distinct conformational changes in G proteins upon ETB-induced activation.

molecular biology↗

bioRxiv2024

OrgaMeas: A pipeline that integrates all the processes of organelle image analysis

Although image analysis has emerged as a key technology in the study of organelle dynamics, the commonly used image-processing methods, such as threshold-based segmentation and manual setting of regions of interests (ROIs), are error-prone and laborious. Here, we present a highly accurate high-throughput image analysis pipeline called OrgaMeas for measuring the morphology and dynamics of organelles. This pipeline mainly consists of two deep learning-based tools: OrgaSegNet and DIC2Cells. OrgaSegNet quantifies many aspects of different organelles by precisely segmenting them. To further process the segmented data at a single-cell level, DIC2Cells automates ROI settings through accurate segmentation of individual cells in differential interference contrast (DIC) images. This pipeline was designed to be low cost and require less coding, to provide an easy-to-use platform. Thus, we believe that OrgaMeas has potential to be readily applied to basic biomedical research, and hopefully to other practical uses such as drug discovery.

cell biology↗

bioRxiv2024

Structural and Dynamic Insights into the Biased Signaling Mechanism of the Human Kappa Opioid Receptor

The {kappa}-opioid receptor (KOR) is a member of the G protein-coupled receptor (GPCR) family, responsible for modulating cellular responses through transducers such as G proteins and arrestins. G protein-biased KOR agonists hold promise due to their potential to mitigate side effects such as drug aversion and sedation while preserving analgesic and antipruritic effects. Here, we shed light on the structural dynamics of the human KOR-Gi signaling complex bound with either nalfurafine (a G-protein-biased agonist) or U-50,488H (a balanced agonist) using cryo-electron microscopy (cryo-EM). Cryo-EM structures of the KOR-Gi signaling complexes identify the ligand binding mode in the activated state. Vibrational spectroscopy analysis reveals changes in the ligand-binding pocket upon binding to these ligands. Cell-based mutant experiments pinpoint four amino acids (K2275.40, C2866.47, H2916.52, and Y3127.34; Ballesteros-Weinstein numbering is shown in superscript) that play crucial roles in arrestin recruitment. Among these four amino acids, H2916.52 and Y3127.34 are also implicated in G-protein coupling. Our findings pave the way for targeting specific residues in the KOR ligand-binding pocket to enhance KOR-mediated therapeutic effects while mitigating unwanted side effects.

biophysics↗

bioRxiv2024

Structural insights into sphingosine 1-phosphate receptor 4 activation

S1PR4 is one of five subtypes of sphingosine 1-phosphate receptors (S1PRs) that regulate immune cell functioning, with functional distinctions to other subtypes. S1PR1-targeted modulators caused serious cardiac and vascular adverse effects because S1PR1 was expressed throughout the whole body. Since S1PR4 was only expressed in lung and lymphoid cells, S1PR4-targeted modulators might not trigger these side effects. However, the development of S1PR4-specific agonists is greatly hindered because of the lack of activated S1PR4 structure. Here, we resolved cryo-EM structures of activated S1PR4 and revealed the structural mechanism of ligand recognition, receptor activation, and Gi coupling. Our results offered structural templates for the development of selective S1PR4 agonists with improved safety profiles.

biochemistry↗

bioRxiv2024

Multi-faceted roles of β-arrestins in G protein-coupled receptors endocytosis

Internalization plays a crucial role in regulating the density of cell surface receptors and has been demonstrated to regulate intracellular signaling. Dysregulation of this process has been implicated in various diseases. The vast majority of GPCRs were considered to adopt one way for internalization. We challenged this conventional view by showing that multiple pathways converge to regulate the internalization of a specific receptor, based on an unparalleled characterization of 60 GPCR internalization profiles, both in the absence and presence of individual {beta}-arrestins ({beta}arrs). Furthermore, we revealed the internalization mechanism of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR pivotal in promoting insulin secretion from pancreatic beta cells to maintain glucose homeostasis. GLP-1R can undergo agonist-induced internalization without {beta}arrs, but can recruit and form stable complexes with {beta}arrs. We found that GLP-1R recruits clathrin adaptor protein-2 for agonist-induced internalization in both {beta}arr-dependent and -independent manners. These results provide a valuable resource for GPCR signaling and reveal the plasticity of different GPCRs to employ or not {beta}arrs in the clathrin-mediated internalization.

cell biology↗

bioRxiv2023

Characterization of Gαs and Gαolf activation by catechol and non-catechol dopamine D1 receptor agonists

The dopamine D1 receptor (D1R) couples to Gs and Golf and plays a crucial role in regulating voluntary movement and other cognitive functions, making it a potential therapeutic target for several neurological and neuropsychiatric disorders, such as Parkinsons disease and schizophrenia. In the central nervous system, Gs is widely expressed in the cortex and Golf is predominantly found in the striatum. We used two different configurations of bioluminescence resonance energy transfer (BRET) assays and a fluorescence-based cyclic AMP (cAMP) production functional assay to test a series of tetracyclic catechol (dihydrexidine, methyl-dihydrexidine, doxanthrine) and non-catechol (tavapadon, PF-8294, PF-6142) D1R agonists for their activity at these G proteins. We discovered that these tetracyclic catechol compounds, PF-8294 and PF-6142 exerted full agonism when D1R coupled to Gs but partial agonism when D1R coupled to Golf. In contrast, tavapadon acted as a full agonist at Golf and a partial agonist at Gs. The effects of these compounds on the cortical and nigral electrophysiological events agree with their selectivity profiles. This suggests the possibility of achieving region-specific pharmacology and opens new directions for developing D1R drugs to treat relevant neurological and neuropsychiatric disorders.

pharmacology and toxicology↗

bioRxiv2023

ArreSTick Motif is Responsible for GPCR-beta-Arrestin Binding Stability and Extends Phosphorylation-Dependent beta-arrestin Interactions to Non-Receptor Proteins

The binding and function of {beta}-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. To investigate this pattern, we employed a 1D sequence convolution model trained on a dataset of GPCRs that have established {beta}-arrestin binding properties. This approach allowed us to identify the amino acid pattern required for GPCRs to form stable interactions with {beta}-arrestins. This motif was named "arreSTick." Our data show that the model predicts the strength of the coupling between GPCRs and {beta}-arrestins with high accuracy, as well as the specific location of the interaction within the receptor sequence. Furthermore, we show that the arreSTick pattern is not limited to GPCRs, and is also present in numerous non-receptor proteins. Using a proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between numerous non-receptor proteins and {beta}-arrestins. For example, the HIV-1 Tat Specific Factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and our data show that its subcellular localization is influenced by its coupling to {beta}-arrestin2. Our findings unveil a broader regulatory role for {beta}-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.

biochemistry↗

bioRxiv2023

Structure of the human Duffy antigen receptor

The Duffy antigen receptor, also known as FY glycoprotein or CD234, is a seven transmembrane protein expressed primarily at the surface of red blood cells, which displays promiscuous binding to multiple chemokines. Not only does it serve as the basis of the Duffy blood group system but it also acts as the primary attachment site for malarial parasite Plasmodium vivax on erythrocytes and as one of the nucleating receptors for the pore forming toxins secreted by Staphylococcus aureus. Despite a predicted 7TM architecture and efficient binding to a spectrum of chemokines, it fails to exhibit canonical second messenger response such as calcium release, likely due to a lack of G protein coupling. Unlike prototypical GPCRs and {beta}-arrestin-biased atypical chemokine receptors, the Duffy antigen receptor also appears to lack {beta}-arrestin binding, making it an enigmatic 7TM chemokine receptor. In order to decipher the molecular mechanism of this intriguing functional divergence exhibited by the Duffy antigen receptor, we have determined its cryo-EM structure in complex with a C-C type chemokine, CCL7. The structure reveals a relatively superficial binding mode of CCL7, with the N-terminus of the receptor serving as the key interaction interface, and a partially formed orthosteric binding pocket lacking the second site for chemokine recognition compared to prototypical chemokine receptors. The structural framework allows us to employ HDX-MS approach to uncover ligand-induced structural changes in the receptor and draw important insights into the promiscuous nature of chemokine binding. Interestingly, we also observe a dramatic shortening of TM5 and 6 on the intracellular side, compared to prototypical GPCRs, which precludes the coupling of canonical signal-transducers namely G proteins, GRKs and {beta}-arrestins, as demonstrated through extensive cellular assays. Taken together, our study uncovers a previously unknown structural mechanism that imparts unique functional divergence on the 7TM fold encoded in the Duffy antigen receptor while maintaining its scavenging function and should facilitate the designing of novel therapeutics targeting this receptor.

biochemistry↗

bioRxiv2023

Cell swelling enhances GPCR ternary complex formation, underpinning the potentiation of beta2 adrenergic receptor-mediated cAMP response

G protein-coupled receptors conformational landscape can be affected by their local, microscopic interactions within the cell plasma membrane. We employ here a pleiotropic stimulus, namely osmotic swelling, to alter the cortical environment within intact cells and monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the {beta}2-adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a model system where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the {beta}2-adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism.

cell biology↗

bioRxiv2023

Receptor endocytosis orchestrates the spatiotemporal bias of β-arrestin signaling

The varying efficacy of biased and balanced agonists is generally explained by the stabilization of different active receptor conformations. In this study, systematic profiling of transducer activation of AT1 angiotensin receptor agonists revealed that the extent and kinetics of {beta}-arrestin binding exhibit substantial ligand-dependent differences, which however completely disappear upon the inhibition of receptor internalization. Even weak partial agonists for the {beta}- arrestin pathway acted as full or near full agonists, if receptor endocytosis was prevented, indicating that receptor conformation is not an exclusive determinant of {beta}-arrestin recruitment. The ligand-dependent variance in {beta}-arrestin translocation at endosomes was much larger than it was at the plasma membrane, showing that ligand efficacy in the {beta}-arrestin pathway is spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shape the effects of agonists on endosomal receptor-{beta}-arrestin binding and thus determine the extent of bias. Among others, ligand dissociation rate and G protein activity have particularly strong impact on receptor-{beta}-arrestin interaction, and their effects are integrated at endosomes. Our results highlight that endocytosis forms a key spatiotemporal platform for biased GPCR signaling and can aid the development of more efficacious functionally-selective compounds. One Sentence summaryAgonist-specific differences in {beta}-arrestin recruitment are mainly determined by the ligand dissociation rate and G protein activation at the endosomes.

cell biology↗

bioRxiv2023

Phosphorylation barcodes direct biased chemokine signaling at CXCR3

G protein-coupled receptor (GPCR) biased agonism, the activation of some signaling pathways over others, is thought to largely be due to differential receptor phosphorylation, or "phosphorylation barcodes." At chemokine receptors, ligands act as "biased agonists" with complex signaling profiles, which contributes to the limited success in pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of CXCR3 phosphosites altered {beta}-arrestin conformation in cellular assays and was confirmed by molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes and lead to distinct physiological processes.

biochemistry↗

bioRxiv2022

The dynamic nature of neurotensin receptor 1 (NTS1) allostery and signaling bias

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13C{varepsilon}H3-methionine NMR spectroscopy to show that phosphatidylinositol-4,5-bisphosphate (PIP2) promotes transducer complexation not by dramatically altering the receptor structure but by strengthening long-range allosteric connections, in the form of correlated conformational kinetics, between the orthosteric pocket and highly-conserved activation motifs. {beta}-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on the rate. A {beta}-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

biochemistry↗