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Journal of Neuroinflammation May 2023Postoperative delirium (POD) is a frequent and debilitating complication, especially amongst high risk procedures, such as orthopedic surgery. This kind of...
Postoperative delirium (POD) is a frequent and debilitating complication, especially amongst high risk procedures, such as orthopedic surgery. This kind of neurocognitive disorder negatively affects cognitive domains, such as memory, awareness, attention, and concentration after surgery; however, its pathophysiology remains unknown. Multiple lines of evidence supporting the occurrence of inflammatory events have come forward from studies in human patients' brain and bio-fluids (CSF and serum), as well as in animal models for POD. β-arrestins are downstream molecules of guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). As versatile proteins, they regulate numerous pathophysiological processes of inflammatory diseases by scaffolding with inflammation-linked partners. Here we report that β-arrestin1, one type of β-arrestins, decreases significantly in the reactive astrocytes of a mouse model for POD. Using β-arrestin1 knockout (KO) mice, we find aggravating effect of β-arrestin1 deficiency on the cognitive dysfunctions and inflammatory phenotype of astrocytes in POD model mice. We conduct the in vitro experiments to investigate the regulatory roles of β-arrestin1 and demonstrate that β-arrestin1 in astrocytes interacts with the dynamin-related protein 1 (Drp1) to regulate mitochondrial fusion/fission process. β-arrestin1 deletion cancels the combination of β-arrestin1 and cellular Drp1, thus promoting the translocation of Drp1 to mitochondrial membrane to provoke the mitochondrial fragments and the subsequent mitochondrial malfunctions. Using β-arrestin1-biased agonist, cognitive dysfunctions of POD mice and pathogenic activation of astrocytes in the POD-linked brain region are reduced. We, therefore, conclude that β-arrestin1 is a promising target for the understanding of POD pathology and development of POD therapeutics.
Topics: Humans; Mice; Animals; Arrestins; Mitochondrial Dynamics; Astrocytes; Emergence Delirium; beta-Arrestins; Dynamins; Mice, Knockout
PubMed: 37170230
DOI: 10.1186/s12974-023-02794-x -
International Journal of Molecular... Jan 2019The adenosine monophosphate-activated protein kinase (AMPK) plays a central role in the regulation of cellular metabolism. Recent studies reveal a novel role for AMPK in... (Review)
Review
The adenosine monophosphate-activated protein kinase (AMPK) plays a central role in the regulation of cellular metabolism. Recent studies reveal a novel role for AMPK in the regulation of glucose and other carbohydrates flux by controlling the endocytosis of transporters. The first step in glucose metabolism is glucose uptake, a process mediated by members of the GLUT/SLC2A (glucose transporters) or HXT (hexose transporters) family of twelve-transmembrane domain glucose transporters in mammals and yeast, respectively. These proteins are conserved from yeast to humans, and multiple transporters-each with distinct kinetic properties-compete for plasma membrane occupancy in order to enhance or limit the rate of glucose uptake. During growth in the presence of alternative carbon sources, glucose transporters are removed and replaced with the appropriate transporter to help support growth in response to this environment. New insights into the regulated protein trafficking of these transporters reveal the requirement for specific α-arrestins, a little-studied class of protein trafficking adaptor. A defining feature of the α-arrestins is that each contains PY-motifs, which can bind to the ubiquitin ligases from the NEDD4/Rsp5 (Neural precursor cell Expressed, Developmentally Down-regulated 4 and Reverses Spt- Phenotype 5, respectively) family. Specific association of α-arrestins with glucose and carbohydrate transporters is thought to bring the ubiquitin ligase in close proximity to its membrane substrate, and thereby allows the membrane cargo to become ubiquitinated. This ubiquitination in turn serves as a mark to stimulate endocytosis. Recent results show that AMPK phosphorylation of the α-arrestins impacts their abundance and/or ability to stimulate carbohydrate transporter endocytosis. Indeed, AMPK or glucose limitation also controls α-arrestin gene expression, adding an additional layer of complexity to this regulation. Here, we review the recent studies that have expanded the role of AMPK in cellular metabolism to include regulation of α-arrestin-mediated trafficking of transporters and show that this mechanism of regulation is conserved over the ~150 million years of evolution that separate yeast from man.
Topics: AMP-Activated Protein Kinases; Animals; Arrestin; Binding Sites; Endocytosis; Fungal Proteins; Glucose; Glucose Transport Proteins, Facilitative; Humans; Protein Transport; Ubiquitination
PubMed: 30691068
DOI: 10.3390/ijms20030515 -
Nature May 2018β-arrestins are critical regulator and transducer proteins for G-protein-coupled receptors (GPCRs). β-arrestin is widely believed to be activated by forming a stable...
β-arrestins are critical regulator and transducer proteins for G-protein-coupled receptors (GPCRs). β-arrestin is widely believed to be activated by forming a stable and stoichiometric GPCR-β-arrestin scaffold complex, which requires and is driven by the phosphorylated tail of the GPCR. Here we demonstrate a distinct and additional mechanism of β-arrestin activation that does not require stable GPCR-β-arrestin scaffolding or the GPCR tail. Instead, it occurs through transient engagement of the GPCR core, which destabilizes a conserved inter-domain charge network in β-arrestin. This promotes capture of β-arrestin at the plasma membrane and its accumulation in clathrin-coated endocytic structures (CCSs) after dissociation from the GPCR, requiring a series of interactions with membrane phosphoinositides and CCS-lattice proteins. β-arrestin clustering in CCSs in the absence of the upstream activating GPCR is associated with a β-arrestin-dependent component of the cellular ERK (extracellular signal-regulated kinase) response. These results delineate a discrete mechanism of cellular β-arrestin function that is activated catalytically by GPCRs.
Topics: Animals; Biocatalysis; COS Cells; Cell Membrane; Chlorocebus aethiops; HEK293 Cells; Humans; Phosphatidylinositols; Protein Transport; Receptors, G-Protein-Coupled; beta-Arrestins
PubMed: 29720660
DOI: 10.1038/s41586-018-0079-1 -
Nature Communications Mar 2023The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may...
The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR.
Topics: Receptors, Opioid, kappa; Morphinans; GTP-Binding Proteins; Arrestin; Analgesics, Opioid
PubMed: 36906681
DOI: 10.1038/s41467-023-37041-7 -
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 -
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 -
British Journal of Pharmacology Feb 2023Arrestin or G protein bias may be desirable for novel cannabinoid therapeutics. Arrestin-2 and arrestin-3 translocation to CB receptor have been suggested to mediate...
BACKGROUND AND PURPOSE
Arrestin or G protein bias may be desirable for novel cannabinoid therapeutics. Arrestin-2 and arrestin-3 translocation to CB receptor have been suggested to mediate different functions that may be exploited with biased ligands. Here, the requirement of a recently described phosphorylation motif 'pxxp' (where 'p' denotes phosphorylatable serine or threonine and 'x' denotes any other amino acid) within the CB receptor C-terminus for interaction with different arrestin subtypes was examined.
EXPERIMENTAL APPROACH
Site-directed mutagenesis was conducted to generate nine different phosphorylation-impaired CB receptor C-terminal mutants. Bioluminescence resonance energy transfer (BRET) was employed to measure arrestin-2/3 translocation and G protein dissociation of a high efficacy agonist for each mutant. Immunocytochemistry was used to quantify receptor expression.
KEY RESULTS
The effects of each mutation were shared for arrestin-2 and arrestin-3 translocation to CB receptor pxxp motifs are partially required for arrestin-2/3 translocation, but translocation was not completely inhibited until all phosphorylation sites were mutated. The rate of arrestin translocation was reduced with simultaneous mutation of S425 and S429. Desensitisation of G protein dissociation was inhibited in different mutants proportional to the extent of their respective loss of arrestin translocation.
CONCLUSIONS AND IMPLICATIONS
These data do not support the existence of an 'essential' pxxp motif for arrestin translocation to CB receptor. These data also identify that arrestin-2 and arrestin-3 have equivalent phosphorylation requirements within the CB receptor C-terminus, suggesting arrestin subtype-selective biased ligands may not be viable and that different regions of the C-terminus contribute differently to arrestin translocation.
Topics: Arrestins; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins; Cannabinoids; GTP-Binding Proteins; Phosphorylation; Receptor, Cannabinoid, CB1; Humans
PubMed: 36250246
DOI: 10.1111/bph.15973 -
Neuropharmacology Jul 2019G protein-coupled receptors (GPCR) have a long history of being considered a prime target for drug development to treat a plethora of diseases and disorders. In fact in... (Review)
Review
G protein-coupled receptors (GPCR) have a long history of being considered a prime target for drug development to treat a plethora of diseases and disorders. In fact in 1827, the first approved therapeutic in the United States was morphine, a drug that targets a GPCR, namely the mu opioid receptor. However, with the rise in biologics over the last two decades, the market share of small molecules targeting GPCRs has declined. Still, two phenomena concerning GPCR pharmacology, specifically heteromerization and biased signaling, have bolstered new interests in this particular class of drug targets. Heteromerization, the process by which two distinct GPCRs come together to form a unique signaling complex, has been demonstrated between many different GPCRs and has spurred efforts to discover heteromer selective drugs. Additionally, the discovery of biased signaling, a concept by which a GPCR can transduce intracellular signaling by favoring a specific pathway (e.g. G-protein) over another pathway (e.g. arrestin), has led to the development of signal-biased drugs with potentially fewer side effects. Our goal for this review is to highlight studies that have investigated the interplay of these two phenomena by providing an overview of the current literature describing instances where GPCR heteromers have distinct arrestin recruitment profiles when compared to the individual GPCRs, with a focus on those GPCRs expressed in the central nervous system. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.
Topics: Allosteric Regulation; Arrestins; Protein Multimerization; Receptors, G-Protein-Coupled; Signal Transduction; beta-Arrestins
PubMed: 30419245
DOI: 10.1016/j.neuropharm.2018.11.010 -
Science Advances May 2023The glucagon-like peptide-1 receptor (GLP-1R) is a major type 2 diabetes therapeutic target. Stimulated GLP-1Rs are rapidly desensitized by β-arrestins, scaffolding...
The glucagon-like peptide-1 receptor (GLP-1R) is a major type 2 diabetes therapeutic target. Stimulated GLP-1Rs are rapidly desensitized by β-arrestins, scaffolding proteins that not only terminate G protein interactions but also act as independent signaling mediators. Here, we have assessed in vivo glycemic responses to the pharmacological GLP-1R agonist exendin-4 in adult β cell-specific β-arrestin 2 knockout (KO) mice. KOs displayed a sex-dimorphic phenotype consisting of weaker acute responses that improved 6 hours after agonist injection. Similar effects were observed for semaglutide and tirzepatide but not with biased agonist exendin-phe1. Acute cyclic adenosine 5'-monophosphate increases were impaired, but desensitization reduced in KO islets. The former defect was attributed to enhanced β-arrestin 1 and phosphodiesterase 4 activities, while reduced desensitization co-occurred with impaired GLP-1R recycling and lysosomal targeting, increased trans-Golgi network signaling, and reduced GLP-1R ubiquitination. This study has unveiled fundamental aspects of GLP-1R response regulation with direct application to the rational design of GLP-1R-targeting therapeutics.
Topics: Animals; Mice; beta-Arrestin 2; Diabetes Mellitus, Type 2; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Mice, Knockout
PubMed: 37134170
DOI: 10.1126/sciadv.adf7737 -
Proceedings of the National Academy of... Apr 2015A long-standing hypothesis posits that a G protein-coupled signaling pathway mediates β-adrenergic nervous system functions, including learning and memory. Here we...
A long-standing hypothesis posits that a G protein-coupled signaling pathway mediates β-adrenergic nervous system functions, including learning and memory. Here we report that memory retrieval (reactivation) induces the activation of β1-adrenergic β-arrestin signaling in the brain, which stimulates ERK signaling and protein synthesis, leading to postreactivation memory restabilization. β-Arrestin2-deficient mice exhibit impaired memory reconsolidation in object recognition, Morris water maze, and cocaine-conditioned place preference paradigms. Postreactivation blockade of both brain β-adrenergic Gs protein- and β-arrestin-dependent pathways disrupts memory reconsolidation. Unexpectedly, selective blockade of the Gs/cAMP/PKA signaling but not the β-arrestin/ERK signaling by the biased β-adrenergic ligands does not inhibit reconsolidation. Moreover, the expression of β-arrestin2 in the entorhinal cortex of β-arrestin 2-deficient mice rescues β1-adrenergic ERK signaling and reconsolidation in a G protein pathway-independent manner. We demonstrate that β-arrestin-biased signaling regulates memory reconsolidation and reveal the potential for β-arrestin-biased ligands in the treatment of memory-related disorders.
Topics: Alprenolol; Animals; Arrestins; Brain; Brain Mapping; Carbazoles; Carvedilol; Cocaine; Cyclic AMP-Dependent Protein Kinases; Extracellular Signal-Regulated MAP Kinases; Male; Maze Learning; Memory; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Propanolamines; Propranolol; Receptors, Adrenergic, beta-1; Signal Transduction; Time Factors; beta-Arrestin 2; beta-Arrestins
PubMed: 25831532
DOI: 10.1073/pnas.1421758112