The role and mechanism of ß-arrestin2 in signal transduction.

ß-arrestin2 is a ubiquitously expressed scaffold protein localized on the cytoplasm and plasma membrane. It was originally found to bind to GPCRs, uncoupling G proteins and receptors' binding and inhibiting the signal transduction of the GPCRs. Further investigations have revealed that ß-arrestin2 not only mediates the desensitization of GPCRs but also serves as a multifunctional scaffold to mediate receptor internalization, kinase activation, and regulation of various signaling pathways, such as TLR4/NF-κB, MAPK, Wnt, TGF-ß, and AMPK/mTOR pathways. ß-arrestin2 regulates cell invasion, migration, autophagy, angiogenesis, and anti-inflammatory effects by regulating various signaling pathways, which play a vital role in many physiological and pathological processes. This paper reviews the structure and function of ß-arrestin2, the regulation of ß-arrestin2 based signaling pathways. The role and mechanism of ß-arrestin2 signaling have been delineated in sufficient detail. The prospect of regulating the expression and activity of ß-arrestin2 in multisystem diseases holds substantial therapeutic promise.

ß-Arrestin 2 and ERK1/2 Are Important Mediators Engaged in Close Cooperation between TRPV1 and µ-Opioid Receptors in the Plasma Membrane.

The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other's functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor's lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane ß-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of ß-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that ß-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that ß-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other's behavior and ß-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.

Dopamine Uses the DRD5-ARRB2-PP2A Signaling Axis to Block the TRAF6-Mediated NF-κB Pathway and Suppress Systemic Inflammation.

The functional relevance and mechanistic basis of the effects of the neurotransmitter dopamine (DA) on inflammation remain unclear. Here we reveal that DA inhibited TLR2-induced NF-κB activation and inflammation via the DRD5 receptor in macrophages. We found that the DRD5 receptor, via the EFD and IYX(X)I/L motifs in its CT and IC3 loop, respectively, can directly recruit TRAF6 and its negative regulator ARRB2 to form a multi-protein complex also containing downstream signaling proteins, such as TAK1, IKKs, and PP2A, that impairs TRAF6-mediated activation of NF-κB and expression of pro-inflammatory genes. Furthermore, the DA-DRD5-ARRB2-PP2A signaling axis can prevent S. aureus-induced inflammation and protect mice against S. aureus-induced sepsis and meningitis after DA treatment. Collectively, these findings provide the first demonstration of DA-DRD5 signaling acting to control inflammation and a detailed delineation of the underlying mechanism and identify the DRD5-ARRB2-PP2A axis as a potential target for future therapy of inflammation-associated diseases such as meningitis and sepsis.

Regulation of cardiac fibroblast-mediated maladaptive ventricular remodeling by ß-arrestins.

Cardiac fibroblasts (CF) play a critical role in post-infarction remodeling which can ultimately lead to pathological fibrosis and heart failure. Recent evidence demonstrates that remote (non-infarct) territory fibrosis is a major mechanism for ventricular dysfunction and arrhythmogenesis. ß-arrestins are important signaling molecules involved in ß-adrenergic receptor (ß-AR) desensitization and can also mediate signaling in a G protein independent fashion. Recent work has provided evidence that ß-arrestin signaling in the heart may be beneficial, however, these studies have primarily focused on cardiac myocytes and their role in adult CF biology has not been well studied. In this study, we show that ß-arrestins can regulate CF biology and contribute to pathological fibrosis. Adult male rats underwent LAD ligation to induce infarction and were studied by echocardiography. There was a significant decline in LV function at 2-12 weeks post-MI with increased infarct and remote territory fibrosis by histology consistent with maladaptive remodeling. Collagen synthesis was upregulated 2.9-fold in CF isolated at 8 and 12 weeks post-MI and ß-arrestin expression was significantly increased. ß-adrenergic signaling was uncoupled in the post-MI CF and ß-agonist-mediated inhibition of collagen synthesis was lost. Knockdown of ß-arrestin1 or 2 in the post-MI CF inhibited transformation to myofibroblasts as well as basal and TGF-ß-stimulated collagen synthesis. These data suggest that ß-arrestins can regulate CF biology and that targeted inhibition of these signaling molecules may represent a novel approach to prevent post-infarction pathological fibrosis and the transition to HF.

Loss of ß-arrestin-2 gene and possible functional consequences on Sezary Syndrome.

Sezary Syndrome is an aggressive T-cell Lymphoma involving blood, skin and lymphonodes Involvement of the CXCR4-SDF1 has been previously shown. We here present evidence also of the involvement of B-arrestin a downstream regulator of CXCR4, that is depleted and downregulated as well as a potential functional role for this depletion.

Indispensable role of ß-arrestin2 in the protection of remifentanil preconditioning against hepatic ischemic reperfusion injury.

Our previous study demonstrated that remifentanil, an opioid agonist, conferred profound liver protection during hepatic ischemia reperfusion injury (HIRI), in which Toll-like receptors (TLRs) played a crucial role in mediating the inflammatory responses. ß-arrestin2, a well-known mu opioid receptor desensitizer, is also a negatively regulator of Toll-like receptor 4 (TLR4)-mediated inflammatory reactions in a mitogen-activated protein kinase (MAPK)-dependent manner. Using the rodent models of hepatic ischemia reperfusion injury both in wild type and TLR4 knockout (TLR4 KO) mice, we found that remifentanil preconditioning could inhibit the expression of TLR4 and reduce the inflammatory response induced by HIRI in wild type but not in TLR4 KO mice. For the in-vitro study, LPS was used to treat RAW264.7 macrophage cells to mimic the inflammatory response induced by HIRI. Remifentanil increased ß-arrestin2 expression both in vivo and in vitro, while after silencing ß-arrestin2 RNA, the effect of remifentanil in reducing cell death and apoptosis, as well as decreasing phosphorylation of ERK and JNK were abolished in RAW264.7 cells. These data suggested that remifentanil could ameliorate mice HIRI through upregulating ß-arrestin2 expression, which may function as a key molecule in bridging opioid receptor and TLR4 pathway.

Free fatty acid receptor 4-ß-arrestin 2 pathway mediates the effects of different classes of unsaturated fatty acids in osteoclasts and osteoblasts.

Bone is a dynamic tissue that is constantly remodelled by bone resorbing osteoclasts and bone forming osteoblasts, respectively. A breakdown in the remodelling process underlies several bone diseases such as osteoporosis. Unsaturated fatty acids (UFAs) have been shown to have beneficial effects on bone health. However, the mechanism of action of UFAs in bone remains unclear. Free fatty acid receptor 4 (FFAR4) is expressed in bone cells and preferentially binds ω-3 and ω-7 UFAs. Therefore, we sought to determine if FFAR4 influenced the action of different classes of UFAs in bone cells. FFAR4 and potential signalling pathways, ß-arrestin 2 (ßarr2) and Gαq, were silenced in RAW264.7 murine macrophages (pre-osteoclasts) and MC3T3-E1 murine pre-osteoblasts. Cell differentiation, activation of signalling pathways and expression of regulatory genes were evaluated. The ω-3 UFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and the ω-7 UFA, palmitoleic acid (PLA), were shown to require the FFAR4/ßarr2 signalling pathway to inhibit osteoclast differentiation in RAW264.7 murine macrophages. The ω-6 UFA, arachidonic acid, and the ω-9 UFA, oleic acid (OA), were shown to inhibit osteoclast formation but did not use FFAR4. DHA, EPA, PLA and OA enhanced osteoblast signalling through the FFAR4/ßarr2 signalling axis. This study reveals that FFAR4/ßarr2 signalling may mediate the bone protective effects of different classes of UFAs in osteoclasts and osteoblasts.

ß-arrestin2 regulates the anti-inflammatory effects of Salmeterol in lipopolysaccharide-stimulated BV2 cells.

Microglial activation contributes to chronic inflammation and neuronal loss in progressive neurodegenerative disorders such as Parkinson's disease (PD). Thus, treatments suppressing microglial activation may have therapeutic benefits to prevent neuronal loss in neurodegenerative diseases. Our previous findings show that Salmeterol, a long-acting ß2-adrenergic receptor (ß2-AR) agonist, is neuroprotective in two distinct animal models of PD, including where lipopolysaccharide (LPS) from E. coli was used to initiate chronic neurodegeneration. Salmeterol was found to be a potent inhibitor of dopaminergic neurodegeneration by regulating the production of pro-inflammatory mediators from activated microglial cells. In the present study, we investigated the molecular basis of the anti-inflammatory effects of Salmeterol on LPS-activated murine microglial BV2 cells. BV2 cells were pretreated with Salmeterol and followed by stimulation with LPS. Salmeterol inhibited LPS-induced release of the pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and nitric oxide from BV2 cells. Additionally, Salmeterol suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) p65 by inhibiting the IκB-α degradation and TAK1 (transforming growth factor-beta-activated kinase1) phosphorylation. We have also found that Salmeterol increases the expression of ß-arrestin2 and enhances the interaction between ß-arrestin2 and TAB1 (TAK1-binding protein), reduced TAK1/TAB1 mediated activation of NFκB and expression of pro-inflammatory genes. Furthermore, silencing of ß-arrestin2 abrogates the anti-inflammatory effects of Salmeterol in LPS-stimulated BV2 cells. Our findings suggest that the anti-inflammatory properties of Salmeterol is ß-arrestin2 dependent and also offers novel therapeutics targeting inflammatory pathways to prevent microglial cell activation and neuronal loss in neuroinflammatory diseases like PD.

Differential regulation of ß2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle.

Generation of cAMP through Gs-coupled G protein-coupled receptor (GPCR) [e.g. ß2-adrenoceptor (ß2AR), adenosine A2B receptor (A2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. Gs-coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that ß2AR and A2BR can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for ß2AR- and A2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged ß2AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A2BR was regulated by GRK5, but not GRK2. ß2AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A2BR-AC signalling and attenuated A2BR desensitization, while ß2AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle.

ßArrestin2 Mediates Renal Cell Carcinoma Tumor Growth.

Renal Cell Carcinoma (RCC) is one of the most lethal urological cancers worldwide. The disease does not present early clinical symptoms and is commonly diagnosed at an advanced stage. Limited molecular drivers have been identified for RCC, resulting in the lack of effective treatment for patients with progressive disease. Ubiquitous ßArrestin2 (ßArr2) is well established for its function in the desensitization and trafficking of G protein-coupled receptors. More recently, ßArr2 has been implicated in the regulation of fundamental cellular functions, including proliferation and invasion. We used bioinformatic and genetic approaches to determine role of ßArr2 in RCC tumor growth. Analysis of published human datasets shows that ARRB2 (gene encoding ßArr2) expression is increased in RCC tumor compared to normal tissue and that high levels of ARRB2 correlate with worse patient survival. Experimentally, we show that knockout of ARRB2 decreases rate of RCC cell proliferation and migration in vitro and xenograft tumor growth in animals. Mechanistically, ßArr2 regulates c-Src activity, Cyclin A expression and cell cycle progression that are involved in tumor growth. These results show that ßArr2 is a critical regulator of RCC tumor growth and suggest its utility as a potential marker and drug target to treat advanced disease.

ß-Arrestin-biased ß-adrenergic signaling promotes extinction learning of cocaine reward memory.

Extinction learning of cocaine-associated contextual cues can help prevent cocaine addicts from relapsing. Pharmacological manipulation of ß-adrenergic receptor (ß-AR) during extinction learning is being developed as a potential strategy to treat drug addiction. We demonstrated that the extinction learning of cocaine-associated memory was mediated by ß-arrestin2-biased but not heterotrimeric guanine nucleotide-binding protein (G protein)-dependent ß-adrenergic signaling. We found that administration of the nonbiased ß-AR antagonist propranolol, but not the G protein-biased ß-AR antagonist carvedilol, blocked extinction learning of cocaine-conditioned place preference and the associated ERK activation in the infralimbic prefrontal cortex. Overexpression of ß-arrestin2 in the infralimbic prefrontal cortex promoted extinction learning, which was blocked by propranolol. Knockout of ß-arrestin2 in the infralimbic prefrontal cortex, specifically in excitatory neurons, impaired extinction learning of cocaine-conditioned place preference, which was not rescued by carvedilol. ß-Arrestin2 signaling in infralimbic excitatory neurons was also required for the extinction learning in the cocaine self-administration model. Our results suggest that ß-arrestin-biased ß-adrenergic signaling in the infralimbic prefrontal cortex regulates extinction learning of cocaine-associated memories and could be therapeutically targeted to treat addiction.

ß-Arrestin2 directly or through GRK2 inhibits PKCßII activation in a ubiquitination-dependent manner.

The GRK/ß-arrestin and PKC/PKA mediate the homologous and heterologous regulation of G protein-coupled receptors (GPCRs), respectively. Interaction between the two pathways is one of the most important issues in understanding the regulation of GPCRs. The present study investigated the regulatory effect of GRK2 and ß-arrestins on PKC activation. The roles of GRK2 and ß-arrestins in the functional regulation of PKC were assessed by determining their influence on PKC autophosphorylation and intracellular translocation. Radioligand binding assay was utilized to characterize intracellular trafficking of dopamine D2R, D3R, and ß2 adrenergic receptor (ß2AR). The subdomains involved in the mutual interactions among GRK2, ß-arrestin2, and PKCßII were determined by in vitro binding assay. Various point mutants of key regulatory players were combined with knockdown cells of GRK2, ß-arrestins, and Mdm2 to functionally correlate the biochemical changes with functional outcomes. GRK2 and ß-arrestin2 mutually inhibited the PKCßII autophosphorylation, a hallmark of PKCßII activation. ß-Arrestin2 ubiquitination was required for the inhibitory activities of GRK2 as well as ß-arrestin2. Furthermore, GRK2 facilitated ß-arrestin2 ubiquitination, thus to enhance the inhibitory actions of ß-arrestin2 on PKCßII activity. Aforementioned processes were also involved in the GRK2/ß-arrestin2-mediated inhibition of the D2R, D3R, and ß2AR endocytosis. The present study provides new insights into the intricate interactions between the homologous and heterologous GPCR regulation pathways. In addition, a novel regulatory role of GRK2 was proposed for the ubiquitination of ß-arrestin in the context of the PKC-mediated heterologous regulation of GPCRs.

ß-Arrestin2 mediates progression of murine primary myelofibrosis.

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. ß-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, ß-arrestin2 (ßarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between ßarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor ßarr2-knockout (ßarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted ßarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of ßarr2-/- cells. In order to assess the effect of acute loss of ßarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional ßarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic ßarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that ßarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

ß-arrestin-2 is involved in irisin induced glucose metabolism in type 2 diabetes via p38 MAPK signaling.

Type 2 diabetes mellitus (T2DM) is a common metabolic disease worldwide. It has been reported that irisin play regulatory role in glucose metabolism in T2DM. However, the underlying mechanism involved in that is not completely known. Herein, we determined the novel role of ß-arrestin-2 in irisin-induced glucose utilization in diabetes. Effects of irisin and ß-arrestin-2 on glucose utilization were investigated in a rat model of diabetes and in diabetic C2C12 cells in vitro. Results showed that irisin had positive role in glucose metabolism via regulating glucose tolerance as well as uptake in cardiac and skeletal muscle tissues, as evidenced by IPGTT, 2-deoxyglucose uptake and plasma membrane GLUT-4 assay. ß-arrestin-2 also improved glucose utilization in diabetes by increasing the glucose uptake and insulin sensitivity, as shown in mice overexpressing ß-arrestin-2. In diabetic C2C12 myocytes, irisin-induced GLUT4 and glucose uptake were restrained by ß-arrestin-2 inhibition, but was enhanced by ß-arrestin-2 overexpression. Additionally, irisin and ß-arrestin-2 increased the activation of p38 MAPK in diabetic C2C12 cells, and the repression of p38 MAPK activation decreased the glucose uptake and plasma membrane GLUT-4 was enhanced by irisin and ß-arrestin-2 overexpression in diabetic C2C12 cells. In conclusion, we demonstrated that ß-arrestin-2 has a crucial role in irisin induced glucose metabolism in T2DM by regulating the p38 MAPK signaling. This might present a novel therapeutic target of treatment for human diabetes.

Dopamine D2 receptor and ß-arrestin 2 mediate Amyloid-ß elevation induced by anti-parkinson's disease drugs, levodopa and piribedil, in neuronal cells.

Although levodopa is the first-line medication for the treatment of Parkinson's disease (PD) showing unsurpassable efficiency, its chronic use causes dyskinesia. Accordingly, dopamine agonists are increasingly employed as monotherapy or in combination with levodopa to reduce the risk of motor complications. It is well recognized that patients with PD often exhibit cognitive deficits. However, clinical and animal studies assessing the effects of dopaminergic medications on cognition are controversial. Amyloid-ß (Aß) is one of the major hallmarks of Alzheimer's disease (AD), leading to progressive memory loss and cognitive deficit. Interestingly, the abnormal accumulation of Aß is also detected in PD patients with cognitive deficits. Evidence indicated that levodopa induced a mild increase of Aß plaque number and size in the brain of AD mouse. However, the underlying mechanism is unclear. Here we present that both levodopa and piribedil enhance the generation of Aß and the activity of γ-secretase in human neuronal cells and primary neurons isolated from AD mouse. This effect was reduced by either the antagonism or the knockdown of dopamine D2 receptor (D2R). We further showed that in the cells expressing ß-arrestin 2-biased D2R mutant, piribedil promoted cellular Aß production to the extent comparable to the wild-type D2R whereas this activity was absent in those with G protein-biased D2R mutant. Moreover, the knockdown of ß-arrestin 2 attenuated the increases of Aß generation and γ-secretase activity mediated by levodopa or piribedil. Thus, our study suggests that targeting D2R-mediated ß-arrestin function may have potential risk in the modulation of Aß pathology.

ß-Arrestin mediates the Frank-Starling mechanism of cardiac contractility.

The Frank-Starling law of the heart is a physiological phenomenon that describes an intrinsic property of heart muscle in which increased cardiac filling leads to enhanced cardiac contractility. Identified more than a century ago, the Frank-Starling relationship is currently known to involve length-dependent enhancement of cardiac myofilament Ca2+ sensitivity. However, the upstream molecular events that link cellular stretch to the length-dependent myofilament Ca2+ sensitivity are poorly understood. Because the angiotensin II type 1 receptor (AT1R) and the multifunctional transducer protein ß-arrestin have been shown to mediate mechanosensitive cellular signaling, we tested the hypothesis that these two proteins are involved in the Frank-Starling mechanism of the heart. Using invasive hemodynamics, we found that mice lacking ß-arrestin 1, ß-arrestin 2, or AT1R were unable to generate a Frank-Starling force in response to changes in cardiac volume. Although wild-type mice pretreated with the conventional AT1R blocker losartan were unable to enhance cardiac contractility with volume loading, treatment with a ß-arrestin-biased AT1R ligand to selectively activate ß-arrestin signaling preserved the Frank-Starling relationship. Importantly, in skinned muscle fiber preparations, we found markedly impaired length-dependent myofilament Ca2+ sensitivity in ß-arrestin 1, ß-arrestin 2, and AT1R knockout mice. Our data reveal ß-arrestin 1, ß-arrestin 2, and AT1R as key regulatory molecules in the Frank-Starling mechanism, which potentially can be targeted therapeutically with ß-arrestin-biased AT1R ligands.

RalA employs GRK2 and ß-arrestins for the filamin A-mediated regulation of trafficking and signaling of dopamine D2 and D3 receptor.

Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D2 and D3 receptors (D2R, D3R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D2R and D3R, comparative studies were conducted on the signaling and intracellular trafficking of the D2R and D3R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D2R and D3R. Lowering the level of cellular FLNA caused an elevation in RalA activity and resulted in selective interference with the normal intracellular trafficking and signaling of the D2R and D3R, through GRK2 and ß-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D2R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D2R. Knockdown of FLNA or coexpression of active RalA prevented D3R from coupling with G protein. The selective involvement of GRK2- and ß-arrestins in the RalA-mediated cellular processes of the D2R and D3R was achieved via their different modes of interactions with the receptor and their distinct functional roles in receptor regulation. Our results show that FLNA is a multi-functional protein that acts as a platform on which D2R and D3R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and ß-arrestins.