Role Of Β-arrestins Research Articles

Insight into the regulatory mechanism of β-arrestin2 and its emerging role in diseases.

β-arrestin2, a member of the arrestin family, mediates the desensitization and internalization of most G protein-coupled receptors (GPCRs) and functions as a scaffold protein in signalling pathways. Previous studies have demonstrated that β-arrestin2 expression is dysregulated in malignant tumours, fibrotic diseases, cardiovascular diseases and metabolic diseases, suggesting its pathological roles. Transcription and post-transcriptional modifications can affect the expression of β-arrestin2. Furthermore, post-translational modifications, such as phosphorylation, ubiquitination, SUMOylation and S-nitrosylation affect the cellular localization of β-arrestin2 and its interaction with downstream signalling molecules, which further regulate the activity of β-arrestin2. This review summarizes the structure and function of β-arrestin2 and reveals the mechanisms involved in the regulation of β-arrestin2 at multiple levels. Additionally, recent studies on the role of β-arrestin2 in some major diseases and its therapeutic prospects have been discussed to provide a reference for the development of drugs targeting β-arrestin2.

β-arrestin1/2 are essential for embryonic lymphatic vessel development

Lymphatic dysfunction can lead to fetal loss due to interstitial edema known as hydrops fetalis during development and lymphedema in adulthood. Lymphedema affects an estimated 250 million individuals worldwide, yet there is no effective treatment available. Numerous studies have established G protein-coupled receptors (GPCRs) as key lymphangiogenic signaling pathways and ideal druggable therapeutical targets. β-arrestins (β-arrestin 1 and 2) are ubiquitously expressed cytosolic adaptor proteins which perform a wide range of functions in GPCR pathways, such as promoting agonist-induced internalization and forming signaling scaffolds with internalized GPCRs in endosomes. However, the roles of β-arrestins in lymphatic vessel development and function remain undefined. We hypothesize that β-arrestin1/2 are essential in embryonic lymphatic vessel development. To test this hypothesis, I used an inducible β-arrestin1fl/fl (Arrb1 fl/fl), β-arrestin2fl/fl (Arrb2 fl/fl);Prox1 CreERT2 mouse line. β-arrestin1 and 2 were deleted by oral gavage of tamoxifen at embryonic day 10.5 (E10.5) and E12.5, critical points for developmental lymphangiogenesis. Compared to Arrb1/2fl/flembryos, the Arrb1/2fl/fl;Prox1CreERT2 embryos exhibit profound hydrops fetalis, subcutaneous hemorrhages, and increased embryonic lethality between E14.5 and E16.5. At E15.5, the Arrb1/2fl/fl;Prox1CreERT2 embryos with edema show decreased weight indicating growth restriction compared to controls. Jugular lymphatic sacs are enlarged and filled with blood in Arrb1/2fl/fl;Prox1CreERT2 embryos compared to controls. Dermal lymphatic vessel length in the Arrb1/2fl/fl;Prox1CreERT2 embryos is shorter than controls. Collectively, our results demonstrate that loss of β-arrestin1/2 expression in lymphatics causes mid-gestational arrest in lymphatic development and fetal growth restriction, indicating that β-arrestins are required for developmental lymphangiogenesis in mammals. American Physiological Society postdoctoral Fellowship (to YT) and NIH R01 HL129086 (to KMC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

β-Arrestin-1 reduces acute myocardial infarction via promoting autophagy in cardiomyocytes

Background and purpose: β-Arrestins are key regulators of G protein–coupled receptor (GPCR) signaling. Through their function as scaffolding proteins, β-arrestins mediate a range of cellular signaling events. However, the role of β-arrestins during myocardial ischemia remains incompletely understood. In this study, we explored the regulatory effects of β-arrestin-1 on autophagy following myocardial infarction and sought to identify the underlying mechanism. Methods: Acute myocardial infarction was induced by permanent left anterior descending coronary artery ligation. Cardiac function was assessed using echocardiography. β-Arrestin-1, autophagy-related 5 (ATG5), and liver kinase B1 (LKB1) were overexpressed or knocked down using lentivirus-mediated transduction of the gene or short hairpin RNA (shRNA) in cultured primary cardiomyocytes. Oxygen-glucose deprivation (OGD) in cardiomyocytes was used to simulate cardiac ischemia in vitro. Autophagy and apoptosis were assessed by western blot, flow cytometry, and transmission electron microscopy. Cell survival and lactate dehydrogenase (LDH) release were evaluated using the respective kits. Results: β-Arrestin-1 knockout (KO) increased myocardial infarction size, an effect that was associated with decreased autophagy and deterioration of cardiac function. The overexpression of β-arrestin-1 significantly increased autophagy levels and decreased cell apoptosis in cardiomyocytes exposed to OGD, whereas the knockdown of β-arrestin-1 exerted the opposite effect. The protective effect of β-arrestin-1 overexpression was abrogated by ATG5 knockdown. β-Arrestin-1 KO attenuated the myocardial infarction–induced phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK). In cultured myocytes, the blockade of AMPK or the knockdown of LKB1 inhibited the β-arrestin-1–induced increase in the LC3-II/LC3-I ratio and beclin 1 expression levels and attenuated β-arrestin-1–mediated cardioprotective effects. Conclusions: Collectively, our findings suggested that β-arrestin-1 promotes cardiomyocyte survival under ischemic conditions via the regulation of LKB1/AMPK-dependent autophagy. These findings may be helpful in designing novel therapeutic strategies for myocardial ischemia.

β-Arrestins: Structure, Function, Physiology, and Pharmacological Perspectives.

The two β-arrestins, β-arrestin-1 and -2 (systematic names: arrestin-2 and -3, respectively), are multifunctional intracellular proteins that regulate the activity of a very large number of cellular signaling pathways and physiologic functions. The two proteins were discovered for their ability to disrupt signaling via G protein-coupled receptors (GPCRs) via binding to the activated receptors. However, it is now well recognized that both β-arrestins can also act as direct modulators of numerous cellular processes via either GPCR-dependent or -independent mechanisms. Recent structural, biophysical, and biochemical studies have provided novel insights into how β-arrestins bind to activated GPCRs and downstream effector proteins. Studies with β-arrestin mutant mice have identified numerous physiologic and pathophysiological processes regulated by β-arrestin-1 and/or -2. Following a short summary of recent structural studies, this review primarily focuses on β-arrestin-regulated physiologic functions, with particular focus on the central nervous system and the roles of β-arrestins in carcinogenesis and key metabolic processes including the maintenance of glucose and energy homeostasis. This review also highlights potential therapeutic implications of these studies and discusses strategies that could prove useful for targeting specific β-arrestin-regulated signaling pathways for therapeutic purposes. SIGNIFICANCE STATEMENT: The two β-arrestins, structurally closely related intracellular proteins that are evolutionarily highly conserved, have emerged as multifunctional proteins able to regulate a vast array of cellular and physiological functions. The outcome of studies with β-arrestin mutant mice and cultured cells, complemented by novel insights into β-arrestin structure and function, should pave the way for the development of novel classes of therapeutically useful drugs capable of regulating specific β-arrestin functions.

PMON171 DRD2 Agonist Cabergoline Abolished the Escape Mechanism Induced by mTOR Inhibitor Everolimus in Pituitary Neuroendocrine Tumoral Cells

Abstract PI3K-AKT-mTOR inhibitors have been proposed as alternative therapeutic strategy for tumors resistant to conventional drugs. The mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to display antiproliferative effects on a wide spectrum of tumors. In vitro studies demonstrated that everolimus inhibited pituitary neuroendocrine tumors (PitNETs) cell growth and vitality in a subset of patients. Sensitivity to everolimus has been demonstrated to be reduced by an escape mechanism that increases AKT phosphorylation (p-AKT), leading to pro-survival pathway activation. This limited efficacy of everolimus due to AKT re-activation could be overtaken by upstream AKT phosphorylation inhibition. Given it was recently demonstrated that DRD2 mediates a reduction of p-AKT in a subset of NF-PitNETs and in rat prolactin-secreting tumor cells MMQ through a β-arrestin 2-dependent mechanism, the aim of the present work was to test the efficacy of adjuvant treatment with cabergoline in increasing sensitivity of human primary cultured NF-PitNET cells to antiproliferative effects of everolimus. In addition, to test a possible role of β-arrestin2. We found that 9 out of 14 NF-PitNETs were resistant to everolimus 1 nM, but the combined treatment with cabergoline inhibited cell proliferation in 7 out of 9 tumors (-31.4 ± 9.9%, p < 0.001 vs basal), increased p27 and reduced cyclin D3 expression. In everolimus unresponsive NF-PitNETs group, 3 h everolimus treatment determined a significant increase of p-AKT/total-AKT ratio (+2.1-fold, p < 0.01, vs basal), and this effect was reverted by cabergoline cotreatment in all tested samples. To investigate the molecular mechanism involved, we used MMQ cells as a model of everolimus escape mechanism. Indeed, 1nM everolimus did not affect MMQ cells proliferation due to an increase of p- AKT/total-AKT ratio (+1.53 ± 24%, p<0.001 vs basal), whereas cabergoline reduced cell proliferation (-22.8 ± 6.8%, p<0.001 vs basal) and AKT phosphorylation. The combined treatment of everolimus and cabergoline induced a significant reduction of both cell proliferation (-34.8 ± 18%, p<0.001 vs basal) and p-AKT/total-AKT ratio (-34.5 ± 14%, p<0.001 vs basal). To test a possible involvement of β-arrestin 2, silencing experiments were performed in MMQ cells. Our data showed that the lack of β-arrestin 2 prevented everolimus and cabergoline co-treatment inhibitory effects on both p-AKT and cells proliferation. In conclusion, these data revealed that cabergoline might overcome the everolimus escape mechanism in NF-PitNETs and tumoral lactotrophs, by inhibiting upstream AKT activation. The co-administration of cabergoline might improve mTOR inhibitors antitumoural activity, paving the way for a potential combined therapy in β-arrestin 2 expressing NF-PitNETs or other PitNETs resistant to conventional treatments. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

DRD2 Agonist Cabergoline Abolished the Escape Mechanism Induced by mTOR Inhibitor Everolimus in Tumoral Pituitary Cells.

The mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to display antiproliferative effects on a wide spectrum of tumors. In vitro studies demonstrated that everolimus inhibited pituitary neuroendocrine tumor (PitNET) cell growth in a subset of patients. Sensitivity to everolimus is reduced by an escape mechanism that increases AKT phosphorylation (p-AKT), leading to pro-survival pathway activation. Dopamine receptor type 2 (DRD2) mediates a reduction of p-AKT in a subgroup of non-functioning PitNETs (NF-PitNETs) and in prolactin-secreting tumor cells (MMQ cells) through a β-arrestin 2-dependent mechanism. The aim of this study was to investigate the efficacy of everolimus combined with DRD2 agonist cabergoline in reducing NF-PitNET primary cells and MMQ cell proliferation and to evaluate AKT phosphorylation and a possible role of β-arrestin 2. We found that 9 out of 14 NF-PitNETs were resistant to everolimus, but the combined treatment with cabergoline inhibited cell proliferation in 7 out of 9 tumors (-31.4 ± 9.9%, p < 0.001 vs. basal) and reduced cyclin D3 expression. In the everolimus-unresponsive NF-PitNET group, everolimus determined a significant increase of p-AKT/total-AKT ratio (2.1-fold, p < 0.01, vs. basal) that was reverted by cabergoline cotreatment. To investigate the molecular mechanism involved, we used MMQ cells as a model of everolimus escape mechanism. Indeed everolimus did not affect MMQ cell proliferation and increased the p-AKT/total-AKT ratio (+1.53 ± 0.24-fold, p < 0.001 vs. basal), whereas cabergoline significantly reduced cell proliferation (-22.8 ± 6.8%, p < 0.001 vs. basal) and p-AKT. The combined treatment of everolimus and cabergoline induced a reduction of both cell proliferation (-34.8 ± 18%, p < 0.001 vs. basal and p < 0.05 vs. cabergoline alone) and p-AKT/total-AKT ratio (-34.5 ± 14%, p < 0.001 vs. basal and p < 0.05 vs. cabergoline alone). To test β-arrestin 2 involvement, silencing experiments were performed in MMQ cells. Our data showed that the lack of β-arrestin 2 prevented the everolimus and cabergoline cotreatment inhibitory effects on both p-AKT and cell proliferation. In conclusion, this study revealed that cabergoline might overcome the everolimus escape mechanism in NF-PitNETs and tumoral lactotrophs by inhibiting upstream AKT activation. The co-administration of cabergoline might improve mTOR inhibitor antitumoral activity, paving the way for a potential combined therapy in β-arrestin 2-expressing NF-PitNETs or other PitNETs resistant to conventional treatments.

Multiple roles of β‐Arrestin2 in initiating and sustaining D3R‐mediated ERK1/2 phosphorylation by G protein‐biased and unbiased D3R agonists

β‐arrestins play key roles in both terminating G protein signaling and activating G protein‐independent signaling across many G protein coupled receptors (GPCRs); however, the roles of β‐arrestins in dopamine D3 receptor (D3R)‐mediated ERK phosphorylation/signaling are poorly characterized. Our previous studies showed that novel compounds SK609 and its analog SK608 are selective D3R agonists and display Gi/o biased signaling without significant recruitment of β‐arrestin2 (βarr2) compared with dopamine which can potently activate G proteins and recruit β‐arrestins. We hypothesize that βarr2 plays multiple roles in initiating and sustaining D3R‐mediated ERK1/2 phosphorylation by G protein‐biased and unbiased D3R agonists. We tested this hypothesis using neuron‐like SH‐SY5Y cells stably expressing D3R (SH‐SY5Y‐D3R cells) with βarr2 knockdown (βarr2KD) in vitro. Our results show: SK609 and SK608 or pramipexole (PRX) time‐dependently elicited both the early and late phases of mono phosphorylation of ERK1/2; SK608 or SK609 only elicited the early phase of dual phosphorylation, whereas PRX induced both the early and late phases and sustained for 1‐4 h; βarr2KD partially reduced mono ERK1/2 phosphorylation by PRX but not SK608 or SK609; βarr2KD significantly enhanced both the early and late phases of dual ERK1/2 phosphorylation by SK608 or SK609 whereas it partially reduced the early phase by PRX and completely abolished the late phase; Gi/o inhibitor PTX abolished PRX‐ or SK608 and SK609‐induced mono ERK1/2 phosphorylation and SK608‐ and SK609‐induced the early phase of dual ERK1/2 phosphorylation, whereas the early phase of dual phosphorylation by PRX was abolished but the late phase was not affected; Gbg inhibitor Galllein had no effect on PRX‐ induced dual phosphorylation of ERK1/2 but inhibited SK609‐ or SK608‐indued dual phosphorylation; PKC inhibitor Go6983 greatly reduced or completely abolished PRX‐ or SK608‐ and SK609‐induced the mono phosphorylation, and blocked SK608‐ or SK609‐induced the dual phosphorylation in SH‐SY5Y‐D3R cells. In contrast, Go6983 abolished PRX‐induced late phase of dual phosphorylation, it only partially reduced PRX‐induced early phase; PTX plus Go6983 completely abolished PRX‐ or SK608‐ and SK609‐induced mono or dual ERK1/2 phosphorylation in SH‐SY5Y‐D3R/βarr2KD cells. In summary, βarr2 not only participates in inhibiting G protein‐biased D3R agonist‐induced early and late phases of dual ERK1/2 phosphorylation and activating unbiased D3R agonists‐induced the early and late phases of the dual phosphorylation, but also is required for Gi/o‐ or PKCs‐dependent the early phase of the mono and dual ERK1/2 phosphorylation. These findings support multiple roles in which βarr2 acts as both signal terminator for G protein biased agonist‐induced ERK1/2 signaling and signal transducer for unbiased D3R agonist‐induced signaling interactions among βarr2 and Gi/o and PKCs which collaboratively regulate ERK1/2 signaling.

Sex- and β-arrestin-dependent effects of kappa opioid receptor-mediated ethanol consumption

The kappa opioid receptor is a known regulator of ethanol consumption, but the molecular mechanisms behind its actions have been underexplored. The scaffolding protein β-arrestin 2 has previously been implicated in driving ethanol consumption at the related delta opioid receptor and has also been suggested to be a driver behind other negative kappa opioid receptor mediated effects. Here, we used kappa opioid agonists with different efficacies for recruiting β-arrestin 2 and knockout animals to determine whether there is a role for β-arrestin 2 in the modulation of voluntary ethanol consumption by the kappa opioid receptor. We find that an agonist with low β-arrestin 2 efficacy more consistently lowers ethanol consumption than agonists with high efficacy for β-arrestin 2. However, knockdown of β-arrestin 2 amplifies the ethanol consumption-promoting effects of the arrestin-recruiting kappa agonists U50,488 and nalfurafine. We control for potentially confounding sedative effects at the kappa opioid receptor and find that β-arrestin 2 is not necessary for kappa opioid receptor-mediated sedation, and that sedation does not correlate with effects on ethanol consumption. Overall, the results suggest a complex relationship between agonist profile, sex, and kappa opioid receptor modulation of ethanol consumption, with little role for kappa opioid receptor-mediated sedation.

Deficiency of β-arrestin2 alleviates apoptosis through GRP78-ATF6-CHOP signaling pathway in primary Sjögren's syndrome

The etiology of primary Sjögren's syndrome (pSS) remains unknown, and there is no ideal drug for the specific treatment of pSS. β-arrestin2 is a key protein that mediates desensitization and internalization of G protein-coupled receptors (GPCRs) and it participates in inflammatory and immune responses that have been found to mediate apoptosis in autoimmune disease. In this study, we established an experimental Sjögren's syndrome (ESS) mouse model to elucidate the molecular mechanisms of β-arrestin2 in pSS. First, excessive activation of β-arrestin2 and GRP78-ATF6-CHOP apoptosis signaling were detected in specimens from pSS patients. In vivo, we found that inhibition of GRP78-ATF6-CHOP apoptosis signaling improved ESS symptoms, and the targeted deletion of β-arrestin2 significantly increased saliva flow, alleviated salivary gland indices, and improved tissue integrity in the ESS model by downregulating GRP78-ATF6-CHOP apoptosis signaling. In vitro, we used IFNα to stimulate human salivary gland epithelial cells (HSGECs), and the results showed that IFNα activated GRP78-ATF6-CHOP apoptosis signaling, decreased cell viability, and induced apoptosis, which were negatively regulated by the ERS inhibitor 4-PBA. In addition, β-arrestin2 depletion downregulated GRP78-ATF6-CHOP apoptosis signaling to alleviate cell apoptosis, and the effect depended on the interaction between GRP78 and β-arrestin2. In summary, our results suggest that excessive activation of GRP78-ATF6-CHOP apoptosis signaling is involved in the pathogenesis of pSS and that β-arrestin2 encourages inflammation-induced epithelial apoptosis through GRP78-ATF6-CHOP apoptosis signaling. This research further clarified the underlying role of β-arrestin2 and provided an experimental foundation for β-arrestin2 depletion in the treatment of the human autoimmune disorder pSS.

β-Arrestin 2 Regulates Inflammatory Responses against Mycobacterium tuberculosis Infection through ERK1/2 Signaling.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, exhibits complex host-pathogen interactions. Pattern recognition receptors and their downstream signaling pathways play crucial roles in determining the outcome of infection. In particular, the scaffold protein β-arrestin 2 mediates downstream signaling of G protein-coupled receptors. However, the role of β-arrestin 2 in conferring immunity against M. tuberculosis has not yet been explored. We found that β-arrestin 2 was upregulated in the lesioned regions of lung tissues in patients with tuberculosis. M. tuberculosis infection upregulated β-arrestin 2 expression in human macrophages, and silencing of β-arrestin 2 significantly enhanced bactericidal activity by enhancing the expression of proinflammatory cytokines such as TNF-α. β-Arrestin 2 was shown to inhibit the activation of the TLR2/ERK1/2 pathway and its transcriptional regulation activity upon M. tuberculosis infection. Furthermore, β-arrestin 2 transcriptionally regulates TNF-α by binding to CREB1. These observations revealed that the upregulation of β-arrestin 2 is critical for M. tuberculosis to escape immune surveillance through an unknown mechanism. Our research offers a novel interference modality to enhance the immune response against tuberculosis by targeting β-arrestin 2 to modulate the TLR2-β-arrestin 2-ERK1/2-CREB1-TNF-α regulatory axis.

β-arrestin 2 is essential for fluoxetine-mediated promotion of hippocampal neurogenesis in a mouse model of depression.

Over the last decade, the roles of β-arrestins in the treatment of neuropsychological diseases have become increasingly appreciated. Fluoxetine is the first selective serotonin reuptake inhibitor developed and is approved for the clinical treatment of depression. Emerging evidence suggests that fluoxetine can directly combine with the 5-HT receptor, which is a member of the G protein-coupled receptor (GPCR) family, in addition to suppressing the serotonin transporter. In this study, we prepared a chronic mild stress (CMS)-induced depression model with β-arrestin2-/- mice and cultured adult neural stem cells (ANSCs) to investigate the involvement of the 5-HT receptor-β-arrestin axis in the pathogenesis of depression and in the therapeutic effect of fluoxetine. We found that β-arrestin2 deletion abolished the fluoxetine-mediated improvement in depression-like behaviors and monoamine neurotransmitter levels, although β-arrestin2 knockout did not aggravate CMS-induced changes in mouse behaviors and neurotransmitters. Notably, the β-arrestin2-/- mice had a shortened dendritic length and reduced dendritic spine density, as well as decreased neural precursor cells, compared to the WT mice under both basal and CMS conditions. We further found that β-arrestin2 knockout decreased the number of proliferating cells in the hippocampal dentate gyrus and suppressed the proliferative capability of ANSCs in vitro. Moreover, β-arrestin2 knockout aggravated the impairment of cell proliferation induced by corticosterone and further blocked the fluoxetine-mediated promotion of mouse hippocampal neurogenesis. Mechanistically, we found that the 5-HT2BR-β-arrestin2-PI3K/Akt axis is essential to maintain the modulation of hippocampal neurogenesis in depressed mice. Our study may provide a promising target for the development of new antidepressant drugs.

The Role of β-Arrestins in Regulating Stem Cell Phenotypes in Normal and Tumorigenic Cells.

β-Arrestins (ARRBs) are ubiquitously expressed scaffold proteins that mediate inactivation of G-protein-coupled receptor signaling, and in certain circumstances, G-protein independent pathways. Intriguingly, the two known ARRBs, β-arrestin1 (ARRB1) and β-Arrestin2 (ARRB2), seem to have opposing functions in regulating signaling cascades in several models in health and disease. Recent evidence suggests that ARRBs are implicated in regulating stem cell maintenance; however, their role, although crucial, is complex, and there is no universal model for ARRB-mediated regulation of stem cell characteristics. For the first time, this review compiles information on the function of ARRBs in stem cell biology and will discuss the role of ARRBs in regulating cell signaling pathways implicated in stem cell maintenance in normal and malignant stem cell populations. Although promising targets for cancer therapy, the ubiquitous nature of ARRBs and the plethora of functions in normal cell biology brings challenges for treatment selectivity. However, recent studies show promising evidence for specifically targeting ARRBs in myeloproliferative neoplasms.

Depletion of β-arrestin 2 protects against CCl4-induced liver injury in mice

Acute liver injury can be caused by oxidative stress within a short period and is a common pathway to many liver diseases. The liver is vulnerable to reactive oxygen species (ROS) and free radical-mediated disorders. β-arrestin2 was initially discovered to be a negative regulator of G protein-coupled receptor signaling. Recently, β-arrestin2 has been found to act as a multifunctional adaptor protein and play new roles in regulating intracellular signaling networks. However, the role of β-arrestin2 in the pathogenesis of acute liver injury is unclear. In this study, we hypothesize that β-arrestin2 regulates acute liver injury via modulation of oxidative stress. β-arrestin2 knockout mice were used to investigate the impacts of β-arrestin2 on carbon tetrachloride (CCl4)-induced acute liver injury and oxidative stress. Results here suggested that β-arrestin2 deficiency decreased serum activities of aminotransferase and alleviated liver injury induced by CCl4 injection as compared with wildtype mice. β-arrestin2 knockout mice exhibited stronger tolerance in oxidative stress compared with wild-type mice, which was demonstrated by decreased ROS level and increased superoxide dismutase (SOD) and glutathione (GSH) in the liver. Furthermore, β-arrestin2 deficiency significantly inhibited NOX4 (a major source of ROS) expression and the activation of the extracellular regulated kinase (ERK) and, c-Jun NH2-terminal kinase (JNK) pathways. These results suggest that β-arrestin2 deficiency protects against CCl4-induced acute liver injury through attenuating oxidative damage and decreased ERK and JNK phosphorylation.

Arrb1 Ameliorates Liver Ischemia/Reperfusion Injury Via Interacting with TRAF6 in Hepatocytes

Background: The hallmarks of hepatic ischemia/reperfusion (I/R) injury, a common clinical problem that occurs during liver surgical procedures, include severe cell death and inflammatory responses that contribute to early graft failure and a higher incidence of organ rejection. Unfortunately, effective therapeutic strategies are limited. β-arrestins are multifunctional proteins that mediate receptor desensitization and serve as important signaling scaffolds in numerous physiopathological processes. However, the role of β-arrestins in hepatic I/R remains largely unknow. Methods: We overexpressed Arrb1 in the hepatocyte of C57BL/6 mice using an Arrb1 adeno-associated virus. Wide-type, Arrb1 overexpressing and knockout mice were subjected to a partial (70%) hepatic ischemia for 60 minutes, followed by various periods of reperfusion. Isolated hepatocytes from different group of mice were subjected to hypoxia-reoxygenation (H/R) injury to determine the in vitro effects of Arrb1. The molecular mechanisms underlying Arrb1-regulatd hepatic I/R injury were further explored and verified in mice. Findings: Here, we showed that only Arrb1 was downregulated during I/R-injured liver injury. Hepatocyte-specific overexpression of Arrb1 significantly ameliorated liver damage, as demonstrated by decreases in serum aminotransferases, hepatocellular necrosis and apoptosis, infiltrating inflammatory cells and secretion of pro-inflammatory cytokines relative to control mice. Whereas, experiments with Arrb1 knockout mice reveled opposite effects. Mechanistically, Arrb1 directly interacts with TRAF6 in hepatocyte, and inhibits the ubiquitination of TRAF6, which then prevents the activation of the downstream MAPK and NF-κB signaling pathways during hepatic I/R injury. Interpretation: The Arrb1-TRAF6 interaction inhibited the ubiquitination of TRAF6 and the activation of MAPK and NF-κB signaling, and then protected against cell death and inflammation, suggesting Arrb1 is a potential therapeutic target for hepatic I/R injury. Funding: This research was supported by grants from the 481 National Key Research and Development Program of China (2016YFC0905900), The State Key Program of National Natural Science Foundation of China (81430062), and the Innovative Research Groups of the National Natural Science Foundation (81521004 to B.S.). The authors declare no conflicts of interest. Declaration of Interest: The authors declare no potential conflicts of interest. Ethical Approval: Animal protocols were approved by the Animal Care and Use Committee of Nanjing Medical University, and experiments were conducted in adherence with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). We gained permission from all the patients and healthy volunteers, and the procedures of our study on the basis of the basic principles of Helsinki Declaration were approved by the Ethics Committee of Nanjing Drum Tower Hospital.

β-Arrestin-2-ERK1/2 cPLA2α axis mediates TLR4 signaling to influence eicosanoid induction in ischemic brain.

LPS has been shown to elicit neuroinflammation associated with the up-regulation of the eicosanoid pathway in animal models; however, the regulatory mechanisms of TLR4 in brain neuroinflammatory conditions remain elusive. β-Arrestins are key regulators of the GPCR signaling pathway and are involved in the leukotriene B4-induced leukocyte migration to initiate inflammatory response. However, the roles of β-arrestins in eicosanoid regulation and related diseases are not clear. To address this issue, we conducted a study to investigate the effect of TLR4 on the eicosanoid pathway in ischemic stroke brain and to explore the underlying molecular regulation mechanism. Cerebral ischemia was produced by occlusion of the middle cerebral artery, followed by reperfusion for 24 h. We demonstrated that knockout of TLR4 improves ischemic stroke brain associated with eicosanoid down-regulation. Interestingly, genetic disruption of β-arrestin-2 failed to decrease neuroinflammation in the damaged brain of TLR4-/- mice, which indicates the requirement of β-arrestin-2 for TLR4 knockdown protection. Further study showed that the negative regulation of phosphorylated (phospho-)ERK1/2 and phospho-cytosolic phospholipase A2 α (cPLA2α) by TLR4 deficiency was eliminated by genetic disruption of β-arrestin-2. In addition, β-arrestin-2 deficiency reversed the reduction of colocalization of phospho-ERK1/2 with phospho-cPLA2α in TLR4-/- mice following ischemic stroke. Mechanistic studies indicated that β-arrestin-2 specifically colocalized and associated with ERK1/2 to prevent ERK1/2-dependent cPLA2α activation following ischemic injury, and β-arrestin-2 deficiency blocked the negative regulation of phospho-ERK1/2, revived the association of phospho-ERK1/2 with phospho-cPLA2α, and subsequently increased the prostaglandin E2 and thromboxane A2 production remarkably. Our findings may provide novel insights that β-arrestin-2 is responsible for ischemic brain improvement in TLR4-/- mice via negative regulation of eicosanoid production.-Xiang, Y., Wei, X., Du, P., Zhao, H., Liu, A., Chen, Y. β-Arrestin-2-ERK1/2 cPLA2α axis mediates TLR4 signaling to influence eicosanoid induction in ischemic brain.

CCR2 Signaling Selectively Regulates IFN-α: Role of β-Arrestin 2 in IFNAR1 Internalization.

An integral component of the antiviral response, type I IFNs require regulation to modulate immune activation. We identify β-arrestin 2 as a key modulator of type I IFN in primary human macrophages, an essential component of the innate immune response. β-Arrestin 2 was selectively activated by CCL2/CCR2 signaling, which induced a decrease in IFN-α, but not IFN-β expression. Small interfering RNA knockdown of β-arrestin 2 demonstrated its role in IFNAR1 internalization, as well as STAT1 and IRF3 activation. As a result, cytokine responses were not propagated following HIV infection and TLR3 activation. However, remnants of IFN signaling remained intact, despite β-arrestin 2 activation, as IFN-β, IFN-γ, IFN-λ1, IRF7, TRAIL, and MxA expression were sustained. Similar effects of β-arrestin 2 on IFN signaling occurred in hepatocytes, suggesting that arrestins may broadly modulate IFN responses in multiple cell types. In summary, we identify a novel role of β-arrestin 2 as an integral regulator of type I IFN through its internalization of IFNAR1 and a subsequent selective loss of downstream IFN signaling.

Methods to Study the Roles of β-Arrestins in Meningococcal Signaling.

Neisseria meningitidis is a Gram-negative diplococcus restricted to humans that causes severe septicemia and/or meningitidis. Initial adhesion to human endothelial cells is mediated through the interaction of type IV pili with the hetero-oligomeric complexes formed by the human receptors CD147 and the β2-adrenergic receptor. Interaction with this complex heterodimer activates a β-arrestin-biased signaling pathway leading to actin polymerization and accumulation of ezrin and ezrin-binding partners. These signaling events promote the formation of cell plasma membrane protrusions in endothelial cells, which are crucial for N. meningitidis colonies to resist shear stress and colonize blood vessels. Here we provide detailed protocols to evaluate the role of β-arrestins in actin and ezrin signaling downstream of G protein-coupled receptor activation.

Role of βarrestin1 in AT1 R-mediated mitogen-activated protein kinase activation in Wistar and SHR brainstem astrocytes.

βarrestin (βarr)-1 and -2 are ubiquitously (outside the retina) expressed G-protein-coupled receptor adapter proteins. They uncouple G-protein-coupled receptors from G proteins, internalize the receptor, and subsequently initiate their own wave of signaling independently of G proteins. Angiotensin (Ang) II type 1 receptor (AT1 R) is a well-established example of a receptor signaling through βarrs. Despite the pivotal role of brain AT1 Rs in the regulation of blood pressure, the involvement of βarr-dependent signaling, mediated by AT1 Rs is not well studied. Particularly, in brain astrocytes very little is known about the effects of βarrs in AT1 R signaling. Herein, we utilized a combination of pharmacological and gene manipulation approaches to investigate the role of βarrs in AT1 R-mediated signaling in isolated brainstem astrocytes from spontaneously hypertensive rats (SHRs) and Wistar rats. We observed that βarr1 is the predominant arrestin isoform at the protein level in these cells. Its expression was down-regulated in SHR astrocytes compared to Wistar rat astrocytes. Ang II, contrary to observations in SHR astrocytes where it had no effect, up-regulates βarr1 protein in Wistar rat astrocytes. We observed differential involvement of βarr1 in MAPK activation in brainstem astrocytes of SHR versus Wistar rats. The βarr-biased agonist peptide [Sar1 , Il4 , Il8] Ang-II (SII), induced AT1 R-mediated ERK and p38 activation in Wistar rat astrocytes. SII had no effect on ERK and p38 activation in SHRs brainstem astrocytes. Our results indicate, reduced involvement of βarr1 in dampening Ang II-induced MAPKs activation and diminished βarr1-mediated ERK and p38 activation in SHR brainstem astrocytes. These findings might be mechanistically related to the development of the brain renin-angiotensin-aldosterone system hyperactivity, which leads to pathogenesis of the hypertensive state of the SHR model.

β-Arrestin1 enhances liver fibrosis through autophagy-mediated Snail signaling.

GPCR mediator β-arrestins (β-arr1 and β-arr2) regulate a variety of physiologic and pathologic processes, including hepatocellular carcinogenesis. However, the role of β-arrestins in liver fibrosis remains unknown. β-arr1, but not β-arr2, was upregulated in liver fibrotic tissues in both humans and mice; moreover, autophagy was increased. β-arr1 deficiency or autophagic inhibitor 3-methyladenine (3-MA) significantly blocked autophagy and downregulated liver fibrosis. Furthermore, β-arr1 enhanced hepatocyte compensatory proliferation, and hepatic stellate cell growth with activation via autophagy resulted in liver fibrosis. In the fibrosis, β-arr1 promoted nuclear translocation of Snail by downregulation of glycogen synthase kinase-3β, and the nuclear translocation of Snail was abrogated either by β-arr1 deficiency or by 3-MA. These results suggest that β-arr1 promotes liver fibrosis via autophagy-mediated Snail signaling, and β-arr1 may be a therapeutic target for liver fibrosis.-Tan, S., Lu, Y., Xu, M., Huang, X., Liu, H., Jiang, J., Wu, B. β-Arrestin1 enhances liver fibrosis through autophagy-mediated Snail signaling.

The role of β-arrestins in G protein-coupled receptor heterologous desensitization: A brief story.

G protein-coupled receptors (GPCRs) are transmembrane proteins that have an important impact in a myriad of cellular functions. Posttranslational modifications on GPCRs are a key processes that allow these proteins to recruit other intracellular molecules. Among these modifications, phosphorylation is the most important way of desensitization of these receptors. Several research groups have described two different desensitization mechanisms: heterologous and homologous desensitization. The first one involves the phosphorylation of the receptors by protein kinases, such as PKC, following the desensitization and internalization of the receptor, while the second one involves the phosphorylation of the receptors by GRKs, allowing for the receptor to recruit β-arrestins to be desensitized and internalized. Interestingly, a few number of studies have described the participation of β-arrestins during the heterologous desensitization process. Hence, the aim of this review is to briefly explore the role that β-arrestins play during the heterologous desensitization of several GPCRs.