Structure of active ß-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ß-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ß-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ß-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ß-arrestin-1. The structure of the ß-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ß-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of ß-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ß-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

ß-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity.

The Toll signaling pathway plays an important role in the innate immunity ofDrosophila melanogasterand mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense againstStaphylococcus aureusby regulating expression of antimicrobial peptides in shrimp. We then found that ß-arrestins negatively regulate Toll signaling in two different ways. ß-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of ß-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. ß-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser(276)that impairs Dorsal transcriptional activity. Our study suggests that ß-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.

ß-arrestin1 is critical for the full activation of NLRP3 and NLRC4 inflammasomes.

Inflammasomes are multiprotein complexes that trigger the activation of caspase-1 and the maturation of IL-1ß, which are critical for inflammation and control of pathogen infection. Although the function of inflammasomes in immune response and disease development is well studied, the molecular mechanism by which inflammasomes are activated and assembled remains largely unknown. In this study, we found that ß-arrestin1, a key regulator of the G protein-coupled receptor signaling pathway, was required for nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing protein 4 (NLRC4) inflammasome-mediated IL-1ß production and caspase-1 activation, but it had no effect on absent in melanoma 2 (AIM2) inflammasome activation. Moreover, apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome, which is ASC aggregation mediating caspase-1 activation, was also impaired in ß-arrestin1-deficient macrophages upon NLRP3 or NLRC4, but not AIM2 inflammasome activation. Mechanistic study revealed that ß-arrestin1 specifically interacted with NLRP3 and NLRC4 and promoted their self-oligomerization. In vivo, in a monosodium urate crystal (MSU)-induced NLRP3-dependent peritonitis model, MSU-induced IL-1ß production and neutrophil flux were significantly reduced in ß-arrestin1 knockout mice. Additionally, ß-arrestin1 deficiency rescued the weight loss of mice upon log-phase Salmonella typhimurium infection, with less IL-1ß production. Taken together, our results indicate that ß-arrestin1 plays a critical role in the assembly and activation of two major canonical inflammasomes, and it may provide a new therapeutic target for inflammatory diseases.

Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function.

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (ß-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.

A biased non-Gαi OXE-R antagonist demonstrates that Gαi protein subunit is not directly involved in neutrophil, eosinophil, and monocyte activation by 5-oxo-ETE.

Gαi-coupled chemoattractant receptors, such as the 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) receptor (OXE-R), are able to switch on Gαißγ protein-dependent and ß-arrestin-related signaling traits. However, which of these signaling pathways are truly important for the chemoattractant functions in leukocytes is not clarified yet. As we recently reported, Gue1654 is a unique Gßγ-biased OXE-R antagonist having no inhibitory activity on Gαi-related signaling, which makes Gue1654 an unprecedented tool for assessing the involvement of G protein subunits in chemoattractant receptor function. ß-arrestin2 recruitment was studied in OXE-R-overexpressing HEK293 cells using bioluminescence resonance energy transfer assays. Activation of leukocytes was assessed by flow cytometric assays and by immunofluorescence microscopy. Leukocyte capture to endothelial cells was addressed under physiological flow conditions. We found that Gue1654 blocks ß-arrestin2 recruitment in HEK293 cells overexpressing OXE-R and ERK1/2 phosphorylation in human eosinophils and neutrophils. Furthermore, Gue1654 was able to prevent several 5-oxo-ETE-triggered functional events in eosinophils and neutrophils, such as activation of CD11b/CD18 integrins, oxidative burst, actin polymerization, and interaction with endothelial cells. In addition, Gue1654 completely prevented 5-oxo-ETE-induced Ca(2+) flux and chemotaxis of human primary monocytes. All of these leukocyte responses to 5-oxo-ETE, except ERK1/2 phosphorylation and oxidative burst, were likewise prevented by pertussis toxin. Therefore, we conclude that chemoattractant receptors require Gαi subunits only as adaptors to transactivate the Gßγ heteromers, which then act responsible for cell activation. Finally, our data characterize Gue1654 as a non-Gαi-biased antagonist of OXE-R that provides a new basis for therapeutic intervention in inflammatory diseases that involve activation of eosinophils, neutrophils, and monocytes.

Multifaceted role of ß-arrestins in inflammation and disease.

Arrestins are intracellular scaffolding proteins known to regulate a range of biochemical processes including G protein-coupled receptor (GPCR) desensitization, signal attenuation, receptor turnover and downstream signaling cascades. Their roles in regulation of signaling network have lately been extended to receptors outside of the GPCR family, demonstrating their roles as important scaffolding proteins in various physiological processes including proliferation, differentiation and apoptosis. Recent studies have demonstrated a critical role for arrestins in immunological processes including key functions in inflammatory signaling pathways. In this review, we provide a comprehensive analysis of the different functions of the arrestin family of proteins especially related to immunity and inflammatory diseases.

T-cell immunoglobulin and ITIM domain (TIGIT) receptor/poliovirus receptor (PVR) ligand engagement suppresses interferon-γ production of natural killer cells via ß-arrestin 2-mediated negative signaling.

Natural killer (NK) cell activation is well orchestrated by a wide array of NK cell receptor repertoire. T-cell immunoglobulin and ITIM domain (TIGIT) receptor was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. TIGIT receptor/poliovirus receptor (PVR) ligand engagement signaling inhibits cytotoxicity mediated by NK and CD8(+) T cells. However, it is unclear how TIGIT/PVR signaling regulates cytokine secretion in NK cells. Here we show that TIGIT/PVR engagement suppresses interferon-γ (IFN-γ) production of NK cells. TIGIT transgenic NK cells generate less IFN-γ undergoing TIGIT/PVR ligation. Moreover, TIGIT knock-out NK cells produce much more IFN-γ. TIGIT/PVR ligation signaling mediates suppression of IFN-γ production via the NF-κB pathway. We identified a novel adaptor ß-arrestin 2 that associates with phosphorylated TIGIT for further recruitment of SHIP1 (SH2-containing inositol phosphatase 1) through the ITT-like motif. Importantly, SHIP1, but not other phosphatases, impairs the TNF receptor-associated factor 6 (TRAF6) autoubiquitination to abolish NF-κB activation, leading to suppression of IFN-γ production in NK cells.

Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

Beta-arrestin-1 (ß-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1ß (IL-1ß) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As ß-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of ß-arrestin-1 in a model of IL-1ß-induced inflammatory response in mouse chondrocytes. IL-1ß treatment significantly increases chondrocytes TLR-4, CD44, ß-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1ß-stimulated chondrocytes with ß-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of ß-arrestin-1. ß-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1ß-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1ß-induced activation of ß-arrestin-1.

Discovery of Novel CXCR2 Inhibitors Using Ligand-Based Pharmacophore Models.

The chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. CXCR2 and its ligand, CXCL8, are implicated in a number of inflammation-mediated diseases such as chronic obstructive pulmonary disease, cystic fibrosis, and cancer. Though the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents has been pursued by pharmaceutical companies within the past decade, there are currently no clinically approved CXCR2 inhibitors. A pharmacophore model based on previously reported CXCR2 antagonists was developed to screen a database of commercially available compounds. Small-molecule compounds identified from the pharmacophore screening were selected for in vitro screening in a cell-based CXCR2-mediated ß-arrestin-2 recruitment assay and further characterized in several cell-based assays and lipopolysaccharide (LPS)-induced lung inflammation studies in mice. CX compounds identified from pharmacophore modeling inhibited cell migration, lung and colon cancer cell proliferation, and colony formation. Mechanistic studies of CX4152 showed that this compound inhibits CXCR2 signaling through downregulation of surface CXCR2. Additionally, CX4152 significantly inhibits CXCL8-mediated neutrophil migration and LPS-induced lung inflammation in mice. Using a CXCR2-inhibitor-based pharmacophore model, we identified a novel set of sulfonamides from a diverse library of small molecules. These compounds inhibit CXCR2/ß-arrestin-2 association, cell migration and proliferation, and acute inflammation in mouse models. CX compounds identified from our pharmacophore models are potential leads for further optimization and development as anti-inflammatory and anticancer agents.

Disruption of the complement anaphylatoxin receptor C5L2 exacerbates inflammation in allergic contact dermatitis.

The complement anaphylatoxin C5a is a critical mediator of allergic contact dermatitis, bridging essential aspects of innate and adaptive immunity. This anaphylatoxin functions by interacting with two 7-transmembrane segment receptors, the C5aR and C5L2. The C5aR is a classical G protein coupled receptor, whereas C5L2 is deficient in coupling to G proteins because of variations in the sequence. Our previous work in human neutrophils revealed a unique role for C5L2 in negatively modulating anaphylatoxin receptor mediated cellular activation through interactions with ß-arrestin. When C5L2 is deficient, C5aR-mediated ß-arrestin signaling is greatly enhanced. The work described in this study was undertaken first to determine the effect of C5L2 deficiency in a murine model of contact sensitivity, and second to determine whether the resultant exacerbation of inflammatory parameters reflects a negative modulatory function of C5L2 on the C5aR. First, we find dramatic increases in inflammation in C5L2(-/-) animals compared with wild type mice. Second, these increases are completely reversed following administration of mAb against the C5aR. Thus, in allergic contact sensitivity, as in isolated human neutrophils, C5L2 functions to suppress C5a-C5aR-mediated responses, further underscoring its role as a negative regulator of anaphylatoxin activity.

The emerging roles of ß-arrestins in fibrotic diseases.

ß-Arrestins and ß-arrestin2 are important adaptor proteins and signal transduction proteins that are mainly involved in the desensitization and internalization of G-protein-coupled receptors. Fibrosis is characterized by accumulation of excess extracellular matrix (ECM) molecules caused by chronic tissue injury. If highly progressive, the fibrotic process leads to organ malfunction and, eventually, death. The incurable lung fibrosis, renal fibrosis and liver fibrosis are among the most common fibrotic diseases. Recent studies show that ß-arrestins can activate signaling cascades independent of G-protein activation and scaffold many intracellular signaling networks by diverse types of signaling pathways, including the Hedgehog, Wnt, Notch and transforming growth factor-ß pathways, as well as downstream kinases such as MAPK and PI3K. These signaling pathways are involved in the pathological process of fibrosis and fibrotic diseases. This ß-arrestin-mediated regulation not only affects cell growth and apoptosis, but also the deposition of ECM, activation of inflammatory response and development of fibrotic diseases. In this review, we survey the involvement of ß-arrestins in various signaling pathways and highlight different aspects of their regulation of fibrosis. We also discuss the important roles of ß-arrestins in the process of fibrotic diseases by regulating the inflammation and deposit of ECM. It is becoming more evident that targeting ß-arrestins may offer therapeutic potential for the treatment of fibrotic diseases.

Sphingosine-1-phosphate receptors control B-cell migration through signaling components associated with primary immunodeficiencies, chronic lymphocytic leukemia, and multiple sclerosis.

BACKGROUND: Five different G protein-coupled sphingosine-1-phosphate (S1P) receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic processes, including lymphocyte circulation, multiple sclerosis (MS), and cancer. Although B-lymphocyte circulation plays an important role in these processes and is essential for normal immune responses, little is known about S1P receptors in human B cells. OBJECTIVE: To explore their function and signaling, we studied B-cell lines and primary B cells from control subjects, patients with leukemia, patients with S1P receptor inhibitor-treated MS, and patients with primary immunodeficiencies. METHODS: S1P receptor expression was analyzed by using multicolor immunofluorescence microscopy and quantitative PCR. Transwell assays were used to study cell migration. S1P receptor internalization was visualized by means of time-lapse imaging with fluorescent S1P receptor fusion proteins expressed by using lentiviral gene transfer. B-lymphocyte subsets were characterized by means of flow cytometry and immunofluorescence microscopy. RESULTS: Showing that different B-cell populations express different combinations of S1P receptors, we found that S1P1 promotes migration, whereas S1P4 modulates and S1P2 inhibits S1P1 signals. Expression of CD69 in activated B lymphocytes and B cells from patients with chronic lymphocytic leukemia inhibited S1P-induced migration. Studying B-cell lines, normal B lymphocytes, and B cells from patients with primary immunodeficiencies, we identified Bruton tyrosine kinase, ß-arrestin 2, LPS-responsive beige-like anchor protein, dedicator of cytokinesis 8, and Wiskott-Aldrich syndrome protein as critical signaling components downstream of S1P1. CONCLUSION: Thus S1P receptor signaling regulates human B-cell circulation and might be a factor contributing to the pathology of MS, chronic lymphocytic leukemia, and primary immunodeficiencies.

Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo.

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced ß-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

C5L2: a controversial receptor of complement anaphylatoxin, C5a.

C5a is the paramount proinflammatory mediator of the complement cascade, and has been previously thought to act only through a single, G-protein-coupled, C5a receptor (C5aR; also termed CD88). In 2000, a second C5a receptor, C5L2 (previously known as GPR77), was discovered; yet, despite 12 yr of intensive research, its biological, or pathophysiological, function is both enigmatic and controversial. Unlike C5aR, this receptor does not couple to G proteins, and early studies promoted the hypothesis that C5L2 functions as a decoy receptor. However, recent data have provided other evidence for more complicated and conflicting interactions between C5L2 and other inflammatory mediators. C5L2 has been recently demonstrated to physically interact with both C5aR and ß-arrestin to negatively regulate C5aR signaling toward an anti-inflammatory manner, and to reduce pathology, in several disease models in vivo. In direct contrast, other groups have demonstrated that C5L2 stimulation caused release of HMGB1 both in vitro and in vivo, and enhanced pathology in sepsis models, suggesting a clear proinflammatory signaling role. These astoundingly contradictory data challenge our precepts and complicate the foundational bases for the possible targeting of C5L2 as a therapeutic option in inflammatory disease. C5L2 may be the great masquerader in complement biology; its function dependent on the cell type, species, and disease context. Because of these unusual and unforeseen complexities, we present the current state of knowledge on C5L2 structure, expression and, most controversially, its putative functions.-Li, R., Coulthard, L.G., Wu, M. C. L., Taylor, S. M., Woodruff, T. M. C5L2: a controversial receptor of complement anaphylatoxin, C5a.

Gene dosage-dependent negative regulatory role of ß-arrestin-2 in polymicrobial infection-induced inflammation.

ß-arrestin-2 (ß-arr2) is a scaffolding protein of the arrestin family with a wide variety of cellular functions. Recent studies have demonstrated differential roles for ß-arr2 in inflammation following endotoxemia and cecal ligation and puncture (CLP) models of sepsis. Because CLP-induced inflammation involves response to fecal contents and necrotic cecum in addition to microbial challenge, in this study, we examined the role of ß-arr2 in an exclusively polymicrobial infection (PMI) model. In addition, we examined the role of gene dosage of ß-arr2 in polymicrobial sepsis. Our studies demonstrate that ß-arr2 is a negative regulator of systemic inflammation in response to polymicrobial infection and that one allele is sufficient for this process. Our results further reveal that loss of ß-arr2 leads to increased neutrophil sequestration and overt inflammation specifically in the lungs following polymicrobial infection. Consistent with this, specific NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways were differentially activated in the ß-arr2 knockout (KO) mice lungs compared to the wild type (WT) following PMI. Associated with enhanced inflammation in the KO mice, PMI-induced mortality was also significantly higher in KO mice than in WT mice. To understand the differential role of ß-arr2 in different sepsis models, we used cell culture systems to evaluate inflammatory cytokine production following endotoxin and polymicrobial stimulation. Our results demonstrate cell-type- as well as stimulus-specific roles for ß-arr2 in inflammation. Taken together, our results reveal a negative regulatory role for ß-arr2 in polymicrobial infection-induced inflammation and further demonstrate that one allele of ß-arr2 is sufficient to mediate most of these effects.

Effects of beta-arrestin 2 on cytokine production of CD4+ T lymphocytes of mice with allergic asthma.

Beta-arrestin 2 has been shown to participate in the pathogenesis of asthma by inducing Th2 cell migration to the lungs. Whether beta-arrestin 2 regulates cytokine production of CD4+ T cells is still unknown. The aim of the present study was to investigate the effect of beta-arrestin 2 on the cytokine production of CD4+ T lymphocytes and the mechanism involved in a mouse model for asthma. After silencing beta-arrestin 2 expression in CD4+ T lymphocytes from asthmatic mice by RNA interference (RNAi), the interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) levels in CD4+ T lymphocyte culture supernatants with or without terbutaline stimulation were determined. Cell-surface beta2 adrenergic receptor (beta2AR) as well as GATA3 expression of CD4+ T lymphocytes were also measured. CD4+ T lymphocytes of mice with allergic asthma expressed higher levels of beta-arrestin 2 on both mRNA and protein levels. beta-arrestin 2 RNAi decreased IL-4 (43.16%) and GATA3 (protein 77.21%, mRNA 62.98%) expression after terbutaline stimulation. Cell-surface beta2AR of CD4+ T lymphocytes decreased (15.27%) after terbutaline treatment, but recovered after beta-arrestin 2 RNAi down-modulation. These findings demonstrate that beta-arrestin 2 regulates IL-4 production and GATA3 expression of CD4+ T lymphocytes partly through the beta2AR signaling pathway in an allergic asthma model.

Beta2-adrenergic receptor regulates Toll-like receptor-4-induced nuclear factor-kappaB activation through beta-arrestin 2.

Toll-like receptors (TLRs) play an important role in innate immunity while, beta(2)-adrenergic receptors (beta(2)AR) provide the key linkages for the sympathetic nervous system to regulate the immune system. However, their role in macrophages remains uncertain. Here, we demonstrate the cross-talk between beta(2)AR and TLR signalling pathways. Expression of beta(2)AR was down-regulated by TLR4 ligand lipopolysaccharide (LPS) stimulation. To investigate the physiological consequence of this down-regulation RAW264 cells, a macrophage cell line, were transfected with a beta(2)AR expression vector (RAWar). Both LPS-stimulated inducible nitric oxide synthase (NOS II) expression and NO production were markedly suppressed in the RAWar cells. The activation of nuclear factor-kappaB (NF-kappaB) and degradation of the inhibitor of NF-kappaB (IkappaBalpha) in response to LPS were markedly decreased in these cells. The level of beta-arrestin 2, which regulates beta(2)AR signalling, was also reduced in RAW264 cells after stimulation with LPS, but not in RAWar cells. Overexpression of beta-arrestin 2 (RAWarr2) also inhibited NO production and NOS II expression. Furthermore, we demonstrated that beta-arrestin 2 interacted with cytosolic IkappaBalpha and that the level of IkappaBalpha coimmunoprecipitated by anti-beta-arrestin 2 antibodies was decreased in the RAW264 cells but not in RAWar or RAWarr2 cells. These findings suggest that LPS-stimulated signals suppress beta(2)AR expression, leading to down-regulation of beta-arrestin 2 expression, which stabilizes cytosolic IkappaBalpha and inhibits the NF-kappaB activation essential for NOS II expression, probably to ensure rapid and sufficient production of NO in response to microbial attack.

GRKs and arrestins: regulators of migration and inflammation.

In the immune system, signaling by G protein-coupled receptors (GPCRs) is crucial for the activity of multiple mediators, including chemokines, leukotrienes, and neurotransmitters. GPCR kinases (GRKs) and arrestins control GPCR signaling by mediating desensitization and thus, regulating further signal propagation through G proteins. Recent evidence suggests that the GRK-arrestin desensitization machinery fulfills a vital role in regulating inflammatory processes. First, GRK/arrestin levels in immune cells are dynamically regulated in response to inflammation. Second, in animals with targeted deletion of GRKs or arrestins, the progression of various acute and chronic inflammatory disorders, including autoimmunity and allergy, is profoundly affected. Third, chemokine receptor signaling in vitro is known to be tightly regulated by the GRK/arrestin machinery, and even small changes in GRK/arrestin expression can have a marked effect on cellular responses to chemokines. This review integrates data about the role of GRKs and arrestins in inflammation, with results on the molecular mechanism of action of GRKs/arrestins, and describes the pivotal role of GRKs/arrestins in inflammatory processes, with a special emphasis on regulation of chemokine responsiveness.

Chemokine receptor CCR5: insights into structure, function, and regulation.

CC chemokine receptor 5 (CCR5) is a seven-transmembrane, G protein-coupled receptor (GPCR) which regulates trafficking and effector functions of memory/effector T-lymphocytes, macrophages, and immature dendritic cells. It also serves as the main coreceptor for the entry of R5 strains of human immunodeficiency virus (HIV-1, HIV-2). Chemokine binding to CCR5 leads to cellular activation through pertussis toxin-sensitive heterotrimeric G proteins as well as G protein-independent signalling pathways. Like many other GPCR, CCR5 is regulated by agonist-dependent processes which involve G protein coupled receptor kinase (GRK)-dependent phosphorylation, beta-arrestin-mediated desensitization and internalization. This review discusses recent advances in the elucidation of the structure and function of CCR5, as well as the complex mechanisms that regulate CCR5 signalling and cell surface expression.

Protective Role of ß-arrestin2 in Colitis Through Modulation of T-cell Activation.

ß-arrestin2 (ß-arr2), identified as a scaffolding protein in G-protein-coupled receptor desensitization, is a negative regulator of inflammation in polymicrobial sepsis. In this study, we wanted to investigate the role of ß-arr2 in intestinal inflammation, a site of persistent microbial stimulation. In the absence of ß-arr2, mice exhibited greater extent of mucosal inflammation determined by cellular infiltration and expression of inflammatory mediators even under homeostatic conditions. Furthermore, ß-arr2-deficient mice were more susceptible to dextran sulfate sodium-induced colitis as demonstrated by greater body weight loss, higher disease activity index, and shortened colon as compared with wild-type mice. We also show that T cells from ß-arr2 knockout mice exhibit altered activation status under both basal and colitic conditions, implicating their involvement in disease induction. Further assessment of the role of ß-arr2 in intrinsic T-cell differentiation confirmed its importance in T-cell polarization. Using the T-cell transfer model of colitis, we demonstrate that T-cell-specific ß-arr2 is important in limiting colitic inflammation; however, it plays a paradoxical role in concurrent systemic wasting disease. Together, our study highlights a critical negative regulatory role of ß-arr2 in intestinal inflammation and demonstrates a distinct role of T-cell-specific ß-arr2 in systemic wasting disease.