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Nature Communications Nov 2017A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP) is...
A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP) is a non-receptor activator of arrestin-3 and report the structure of IP-activated arrestin-3 at 2.4-Å resolution. IP-activated arrestin-3 exhibits an inter-domain twist and a displaced C-tail, hallmarks of active arrestin. IP binds to the arrestin phosphate sensor, and is stabilized by trimerization. Analysis of the trimerization surface, which is also the receptor-binding surface, suggests a feature called the finger loop as a key region of the activation sensor. We show that finger loop helicity and flexibility may underlie coupling to hundreds of diverse receptors and also promote arrestin-3 activation by IP. Importantly, we show that effector-binding sites on arrestins have distinct conformations in the basal and activated states, acting as switch regions. These switch regions may work with the inter-domain twist to initiate and direct arrestin-mediated signaling.
Topics: Amino Acid Sequence; Animals; Arrestins; Binding Sites; Cattle; Crystallography, X-Ray; Humans; Mitogen-Activated Protein Kinase 10; Models, Molecular; Phytic Acid; Protein Conformation; Protein Structure, Quaternary; Recombinant Proteins; Signal Transduction
PubMed: 29127291
DOI: 10.1038/s41467-017-01218-8 -
International Journal of Molecular... Nov 2017G protein-coupled receptors (GPCRs) are cell surface receptors that respond to a wide variety of stimuli, from light, odorants, hormones, and neurotransmitters to... (Review)
Review
G protein-coupled receptors (GPCRs) are cell surface receptors that respond to a wide variety of stimuli, from light, odorants, hormones, and neurotransmitters to proteins and extracellular calcium. GPCRs represent the largest family of signaling proteins targeted by many clinically used drugs. Recent studies shed light on the conformational changes that accompany GPCR activation and the structural state of the receptor necessary for the interactions with the three classes of proteins that preferentially bind active GPCRs, G proteins, G protein-coupled receptor kinases (GRKs), and arrestins. Importantly, structural and biophysical studies also revealed activation-related conformational changes in these three types of signal transducers. Here, we summarize what is already known and point out questions that still need to be answered. Clear understanding of the structural basis of signaling by GPCRs and their interaction partners would pave the way to designing signaling-biased proteins with scientific and therapeutic potential.
Topics: Animals; Arrestins; Humans; Protein Domains; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 29186792
DOI: 10.3390/ijms18122519 -
Phytomedicine : International Journal... Jun 2023Current treatments for lung cancer have their own deficiencies, such as severe adverse effect. Therefore, more safe and effective drugs are needed.
BACKGROUND
Current treatments for lung cancer have their own deficiencies, such as severe adverse effect. Therefore, more safe and effective drugs are needed.
PURPOSE
Fuzheng Kang-Ai (FZKA for short) has been applied as an adjuvant treatment in advanced Non-Small Cell Lung Cancer (NSCLC) patients for decades in China, showing a definitive effect with minimal toxicities. However, the underlying mechanism is yet to be identified.
STUDY DESIGN
Both in vitro and in vivo experiments were performed in this study to identify the exact mechanism by which FZKA inhibits NSCLC cell proliferation.
METHODS
MTT and CCK-8 assays were used to detect cell viability. Xenograft model was performed for in vivo experiments. CircRNA and miRNA sequencing were used to find the differentially expressed circRNAs and miRNAs, respectively. qRT-PCR was performed to check the expression levels of circRNA, miRNA and mRNA. BaseScope was carried out to observe the expression of circRNA in situ. Actinomycin D and RNase R experiments were done to show the stability of circRNA. Nuclear-cytoplasmic fractionation and FISH were used to identify the localization of circRNA and miRNA. Pull-down, RIP, and luciferase activity assays were performed to show the biding ability of circRNA, miRNA and target proteins. Flow cytometry was done to observe cell apoptosis. Western blot and IHC were done to detect the protein expression. TCGA database was used to analyze the survival rate.
RESULTS
FZKA inhibits NSCLC cell proliferation both in vitro and in vivo. Hsa_circ_0048091 and hsa-miR-378g were the most differentially expressed circRNA and miRNA, respectively, after FZKA treatment. Silencing hsa_circ_0048091 and overexpressing hsa-miR-378g promoted cell proliferation and reversed the inhibition effect of FZKA on NSCLC, respectively. Hsa-miR-378g was sponged by hsa_circ_0048091, and the overexpression of miR-378g reversed the inhibition effect of hsa_ circ_0048091 on NSCLC. ARRDC3, as a target of hsa-miR-378g, was increased by FZKA treatment. Silencing ARRDC3 reversed both the inhibition effect of FZKA and miR-378g inhibitor on NSCLC.
CONCLUSION
This study, for the first time, has established the function of hsa_circ_0048091, hsa- miR-378g, and ARRDC3 in lung cancer. It also shows that FZKA inhibits NSCLC cell proliferation through hsa_circ_0048091/hsa-miR-378g/ARRDC3 pathway, uncovering a novel mechanism by which FZKA controls human NSCLC cell growth.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; RNA, Circular; Cell Line, Tumor; MicroRNAs; Cell Proliferation; Arrestins
PubMed: 37062135
DOI: 10.1016/j.phymed.2023.154819 -
Proceedings of the National Academy of... Sep 2021Arrestins were initially identified for their role in homologous desensitization and internalization of G protein-coupled receptors. Receptor-bound arrestins also...
Arrestins were initially identified for their role in homologous desensitization and internalization of G protein-coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange-mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.
Topics: Animals; Arrestins; COS Cells; Chlorocebus aethiops; Extracellular Signal-Regulated MAP Kinases; Fluorescence Resonance Energy Transfer; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mass Spectrometry; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nuclear Magnetic Resonance, Biomolecular; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteins; Rats; Signal Transduction; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins
PubMed: 34507982
DOI: 10.1073/pnas.2026491118 -
Structure (London, England : 1993) Jul 2019Arrestins, in addition to desensitizing GPCR-induced G protein activation, also mediate G protein-independent signaling by interacting with various signaling proteins....
Arrestins, in addition to desensitizing GPCR-induced G protein activation, also mediate G protein-independent signaling by interacting with various signaling proteins. Among these, arrestins regulate MAPK signal transduction by scaffolding mitogen-activated protein kinase (MAPK) signaling components such as MAPKKK, MAPKK, and MAPK. In this study, we investigated the binding mode and interfaces between arrestin-3 and JNK3 using hydrogen/deuterium exchange mass spectrometry, F-NMR, and tryptophan-induced Atto 655 fluorescence-quenching techniques. Results suggested that the β1 strand of arrestin-3 is the major and potentially only interaction site with JNK3. The results also suggested that C-lobe regions near the activation loop of JNK3 form the potential binding interface, which is variable depending on the ATP binding status. Because the β1 strand of arrestin-3 is buried by the C-terminal strand in its basal state, C-terminal truncation (i.e., pre-activation) of arrestin-3 facilitates the arrestin-3/JNK3 interaction.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Arrestins; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Gene Expression; Genetic Vectors; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 10; Models, Molecular; Phosphorylation; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Rats; Recombinant Proteins
PubMed: 31080119
DOI: 10.1016/j.str.2019.04.002 -
Science Signaling Aug 2021G protein-coupled receptors (GPCRs) are implicated in the regulation of fear and anxiety. GPCR signaling involves canonical G protein pathways but can also engage...
G protein-coupled receptors (GPCRs) are implicated in the regulation of fear and anxiety. GPCR signaling involves canonical G protein pathways but can also engage downstream kinases and effectors through scaffolding interactions mediated by β-arrestin. Here, we investigated whether β-arrestin signaling regulates anxiety-like and fear-related behavior in mice in response to activation of the GPCR δ-opioid receptor (δOR or DOR). Administration of β-arrestin-biased δOR agonists to male C57BL/6 mice revealed β-arrestin 2-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the dorsal hippocampus and amygdala and β-arrestin 1-dependent activation of ERK1/2 in the nucleus accumbens. In mice, β-arrestin-biased agonist treatment was associated with reduced anxiety-like and fear-related behaviors, with some overlapping and isoform-specific input. In contrast, applying a G protein-biased δOR agonist decreased ERK1/2 activity in all three regions as well as the dorsal striatum and was associated with increased fear-related behavior without effects on baseline anxiety. Our results indicate a complex picture of δOR neuromodulation in which β-arrestin 1- and 2-dependent ERK signaling in specific brain subregions suppresses behaviors associated with anxiety and fear and opposes the effects of G protein-biased signaling. Overall, our findings highlight the importance of noncanonical β-arrestin-dependent GPCR signaling in the regulation of these interrelated emotions.
Topics: Animals; Anxiety; Fear; Male; Mice; Mice, Inbred C57BL; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins
PubMed: 34344831
DOI: 10.1126/scisignal.aba0245 -
Cell Research Jul 2021Compelling evidence has revealed that biased activation of G protein-coupled receptor (GPCR) signaling, including angiotensin II (AngII) receptor type 1 (AT1) signaling,...
Compelling evidence has revealed that biased activation of G protein-coupled receptor (GPCR) signaling, including angiotensin II (AngII) receptor type 1 (AT1) signaling, plays pivotal roles in vascular homeostasis and injury, but whether a clinically relevant endogenous biased antagonism of AT1 signaling exists under physiological and pathophysiological conditions has not been clearly elucidated. Here, we show that an extracellular matrix protein, cartilage oligomeric matrix protein (COMP), acts as an endogenous allosteric biased modulator of the AT1 receptor and its deficiency is clinically associated with abdominal aortic aneurysm (AAA) development. COMP directly interacts with the extracellular N-terminus of the AT1 via its EGF domain and inhibits AT1-β-arrestin-2 signaling, but not Gq or Gi signaling, in a selective manner through allosteric regulation of AT1 intracellular conformational states. COMP deficiency results in activation of AT1a-β-arrestin-2 signaling and subsequent exclusive AAA formation in response to AngII infusion. AAAs in COMP or ApoE mice are rescued by AT1a or β-arrestin-2 deficiency, or the application of a peptidomimetic mimicking the AT1-binding motif of COMP. Explorations of the endogenous biased antagonism of AT1 receptor or other GPCRs may reveal novel therapeutic strategies for cardiovascular diseases.
Topics: Animals; Cartilage Oligomeric Matrix Protein; HEK293 Cells; Humans; Mice; Receptor, Angiotensin, Type 1; Vascular System Injuries; beta-Arrestin 2; beta-Arrestins
PubMed: 33510386
DOI: 10.1038/s41422-020-00464-8 -
Advances in Protein Chemistry and... 2020Most vertebrates express four arrestin subtypes: two visual ones in photoreceptor cells and two non-visuals expressed ubiquitously. The latter two interact with hundreds... (Review)
Review
Most vertebrates express four arrestin subtypes: two visual ones in photoreceptor cells and two non-visuals expressed ubiquitously. The latter two interact with hundreds of G protein-coupled receptors, certain receptors of other types, and numerous non-receptor partners. Arrestins have no enzymatic activity and work by interacting with other proteins, often assembling multi-protein signaling complexes. Arrestin binding to every partner affects cell signaling, including pathways regulating cell survival, proliferation, and death. Thus, targeting individual arrestin interactions has therapeutic potential. This requires precise identification of protein-protein interaction sites of both participants and the choice of the side of each interaction which would be most advantageous to target. The interfaces involved in each interaction can be disrupted by small molecule therapeutics, as well as by carefully selected peptides of the other partner that do not participate in the interactions that should not be targeted.
Topics: Animals; Arrestins; Binding Sites; Gene Expression Regulation; Genetic Therapy; Humans; Leber Congenital Amaurosis; Molecular Targeted Therapy; Mutation; Protein Binding; Receptors, G-Protein-Coupled; Retinal Rod Photoreceptor Cells; Signal Transduction; Small Molecule Libraries
PubMed: 32312421
DOI: 10.1016/bs.apcsb.2019.11.011 -
The FEBS Journal Apr 2021G protein-coupled receptors (GPCRs) play critical roles in the regulation of human physiology in response to a wide array of different extracellular stimuli and thus... (Review)
Review
G protein-coupled receptors (GPCRs) play critical roles in the regulation of human physiology in response to a wide array of different extracellular stimuli and thus represent one of the largest groups of therapeutic drug targets. Recent advances in the structural characterization of GPCRs in different conformations and in complex with G proteins and arrestins have provided important insights into the mechanism and function of GPCRs. However, in order to truly understand the molecular basis of the functional versatility of GPCRs, the structural snapshots obtained by X-ray crystallography or cryo-EM need to be complimented with information about the conformational dynamics of receptors and their signaling complexes. In the last decade, a combination of biophysical approaches and computational studies has been utilized to examine the molecular motions of GPCRs and their transducer complexes and how they are regulated by ligands of different efficacy and bias. These studies revealed that GPCRs are highly dynamic allosteric proteins that can sample multiple conformational states. Ligands with distinct signaling profiles not only impact the conformational landscape of GPCRs but also of the receptor-engaged G proteins and arrestins. The conformational dynamics of GPCRs and their signaling complexes and the ligand-dependent bias sampling of distinct functional states are important underlying principles behind the complex signaling behavior of GPCRs.
Topics: Arrestins; Crystallography, X-Ray; GTP-Binding Proteins; Humans; Ligands; Protein Conformation; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 33871923
DOI: 10.1111/febs.15841 -
Molecular and Cellular Endocrinology Mar 2019G protein-coupled receptors (GPCRs) are the largest family of signaling proteins targeted by more clinically used drugs than any other protein family. GPCR signaling via... (Review)
Review
G protein-coupled receptors (GPCRs) are the largest family of signaling proteins targeted by more clinically used drugs than any other protein family. GPCR signaling via G proteins is quenched (desensitized) by the phosphorylation of the active receptor by specific GPCR kinases (GRKs) followed by tight binding of arrestins to active phosphorylated receptors. Thus, arrestins engage two types of receptor elements: those that contain GRK-added phosphates and those that change conformation upon activation. GRKs attach phosphates to serines and threonines in the GPCR C-terminus or any one of the cytoplasmic loops. In addition to these phosphates, arrestins engage the cavity that appears between trans-membrane helices upon receptor activation and several other non-phosphorylated elements. The residues that bind GPCRs are localized on the concave side of both arrestin domains. Arrestins undergo a global conformational change upon receptor binding (become activated). Arrestins serve as important hubs of cellular signaling, emanating from activated GPCRs and receptor-independent.
Topics: Animals; Arrestin; Binding Sites; G-Protein-Coupled Receptor Kinases; Humans; Models, Molecular; Phosphorylation; Protein Binding; Protein Conformation; Protein Domains; Receptors, G-Protein-Coupled
PubMed: 30703488
DOI: 10.1016/j.mce.2019.01.019