β-arrestins in the Immune System

Summary

β-arrestins regulate G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) through receptor desensitization while also acting as signaling scaffolds to facilitate numerous effector pathways. Recent studies have provided evidence that β-arrestins play a key role in inflammatory responses. We here summarize these advances on the roles of β-arrestins in immune regulation and inflammatory responses under physiological and pathological conditions, with an emphasis on translational implications of β-arrestins on human diseases.

I. Introduction

β-arrestins are classically known to regulate GPCR signaling through receptor desensitization and internalization ¹. The structure and molecular characterization of β-arrestins have been established ^{2; 3; 4}. Collaborating with GPCR kinases (GRKs) ⁵, β-arrestins are known for their role in desensitizing the β2-adrenergic receptor ⁶. Much of the investigation on the role of β-arrestins has been associated with the deactivation and desensitization of the GPCRs. GPCR internalization is believed to occur through clathrin-mediated endocytosis, which consists of three processes: receptor desensitization, sequestration of receptors to clathrin-coated pits, and receptor internalization. These steps are each regulated by distinct signaling events and β-arrestins have a role in these processes. In addition, β-arrestins have been shown to mediate non-GPCR signaling pathways, such as through TGF-β receptor III ⁷.

Reports have also shown that β-arrestins facilitate the activation of numerous effector pathways, such as the mitogen-activated protein kinases (MAPKs) and Akt ⁸. β-arrestin 2 serves as s scaffold to enhance receptor tyrosine kinase (cRaf-1) and MAP kinase kinase (MEK)-dependent activation of extracellular signal-regulated kinase 2 (ERK2) ⁹ (also reviews ^{10; 11}). Furthermore, evidence suggests that β-arrestins mediate signaling pathways through transcriptional regulation (review ¹²). For example, β-arrestin 1 is able to translocate from the cytosol to the nucleus and recruit histone acetyltransferase p300, leading to enhanced local histone H4 acetylation and transcription of p27 and c-fos ¹³.

Extensive research demonstrates that β-arrestins have functions in development ^{14; 15}, cancer ^{16; 17}, and chemotaxis ^{18; 19}. During the last several years, roles of β-arrestins in innate and adaptive immunity, as well as in inflammatory responses have been reported. Studies in mice with targeted deletion β-arrestins have identified important roles for these adaptor molecules in immune responses and regulation of the pathogenesis of several disease processes have begun to been explored.

The immune system consists of innate and adaptive responses. Innate immune cells, receptor systems, and related signaling pathways defend the host from infections such as bacteria and virus, as well as from non-infectious tissue injuries. Defense by the innate immune system includes chemotaxis of inflammatory cells, release of cytokines, pathogen clearance, as well as the activation of the adaptive immune system through antigen presentation. The adaptive immune system, on the other hand, consists of highly specialized cells (such as T lymphocytes and B lymphocytes), proteins, and processes that aim to further eliminate pathogens. In addition to their role in pathogen clearance, both systems have a role in tissue injury, inflammation, and repair processes. Expression of β-arrestins has been found in innate immune cells such as macrophages, polymorphonuclear neutrophiles (PMNs), as well as in adaptive immune cells including T lymphocytes and B cells. β-arrestins have been suggested to have a role in both systems. In addition, β-arrestins may have a role in mammalian hematopoiesis, since β-arrestin 1 deficient mice demonstrate basal hematologic defects ²⁰. Similarly in zebrafish, β-arrestin 1 relieved polycomb group-mediated repression of the cdx4-hox pathway, regulating hematopoietic lineage specification during primitive hematopoiesis ²⁰.

II. β-arrestins in innate immunity

A. β-arrestins and macrophages

β-arrestins are expressed in macrophages. Both β-arrestin 1 and β-arrestin 2 mRNA and protein expression are found in both human and mouse mononuclear cells and macrophages ^{21; 22}. Lipopolysaccharides down regulates β-arrestin 2 expression in RAW264 macrophages ²³. Furthermore, β-arrestin 1 expression can be down-regulated by activation of TLR2 and TLR4 in primary mouse macrophages, and the regulation is both transcriptional and post-translational ²⁴. Expression of β-arrestin 1 and β-arrestin 2 by macrophages can be regulated by cytokines. Cytokine granulocyte-macrophage colony-stimulating factor-1 increased β-arrestin 1 expression, associated with down-regulation of surface chemokine receptor CXCR4 expression in human primary monocytes-macrophages ²⁵. Lectin phitoemagglutinin reduces β-arrestin 1 expression in mononuclear leukocytes, and interferon β-1a can quench the effect of phitoemagglutinin on the expression of β-arrestin 1 ²⁶. β-arrestin 2 mRNA and protein levels were significantly elevated in peripheral blood mononuclear cells of cryptococcal meningitis patients ²⁷. β-arrestin 1 and 2 expressions are reduced in blood leukocytes during acute kidney transplantation ²⁸. The modulation of β-arrestin expression by cytokines is involved in regulating related effector pathways.

β-arrestins regulate macrophage chemotaxis induced by chemokines and serine proteases. Chemokine CCL4 (MIP-1β) binding to CCR5 induces the translocation of β-arrestins, PI3K and Pyk2 to the membrane and forms a multi-kinase signaling complex mediating macrophage migration ²⁹. Knockdown of β-arrestin 1 and 2 by siRNA impairs complex formation and inhibits macrophage chemotaxis toward CCL4 ²⁹. Chemokine CCL2 (MCP-1) binds with the CCR2 receptor, leading to formation of a CCR2/β-arrestin/GRK2 complex in human monocytes/macrophages. This process promotes the rapid desensitization of CCL2-induced calcium flux responses, which are essential for chemotaxis in macrophages ³⁰. Protease-activated receptor-2 (PAR-2) activation has been shown to enhance β-arrestin and ERK1/2-regulated assembly of the actin cytoskeleton in pseudopodia, promoting polarized pseudopodia extension and facilitating macrophage chemotaxis ³¹. Collectively, these studies demonstrate that, β-arrestins are an integrated regulator of macrophage-mediated innate immune responses.

B. β-arrestins and polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) are essential for host defense against infection and the activation of PMNs is pivotal in the process of innate immunity ^{32; 33}. β-arrestin 1 is highly expressed in peripheral blood leukocytes including PMNs ²². β-arrestin 1 and 2 are critical modulators of inflammatory responses in PMNs, including PMN cytokine production, recruitment ³⁴, and granule release ³⁵. β-arrestins and GRK2 associated with the CCR2 receptor play an important role in CCL2 (MCP-1)-regulated leukocyte activation and migration following CCL2 binding ³⁰. PMNs from β-arrestin 2 deficient mice show enhanced release of inflammatory cytokines, including TNF-α and IL-6, compared to WT PMNs ³⁴. β-arrestin 2 deficiency, results in augmented PMN recruitment with increased expression of CD18 and CD62L, and enhanced PMN infiltration in the lung after cecal ligation and puncture (CLP) ³⁴. Furthermore, β-arrestins regulate interleukin (IL)-8-induced granule exocytosis in PMNs, which is critical for the innate immune response against infectious bacteria ³⁵. β-arrestin 2 plays a negative role in CXCR2 signaling in neutrophils. Neutrophils isolated from peritoneal cavity of β-arrestins 2-deficient mice show enhanced calcium mobilization, superoxide anion production, and GTPase activity, but decreased receptor internalization in CXCR2 signaling ³⁶. β-arrestin 2-deficient mice also demonstrate increased recruitment of PMNs in response to CXCL1 in the air pouch model and in excisional wound beds, leading to faster wound re-epithelialization ³⁶.

C. β-arrestins and natural killer (NK) cells

NK cells constitute a major component of the innate immune system that play a significant role in tumor rejection and viral clearance. NK cell receptors include activating NK receptors such as NKG2D and inhibitory receptors such as killer-cell immunoglobulin-like receptors. β-arrestin 2 has been reported to interact with inhibitory receptor KIR2DL1 and mediate the binding of tyrosine phosphatases to KIR2DL1 to facilitate the inhibitory signaling of NK cells ³⁷. KIR2DL1 with an arginine residue at position 245 in its transmembrane domain recruited more β-arrestin 2 and Src-homology-2 domain-containing protein tyrosine phosphatase 2 ³⁸. NK cells isolated from β-arrestin 2 deficient mice demonstrated higher cytotoxicity than WT mice, while NK cells from β-arrestin 2 transgenic mice showed reduced cytotoxicity which could be restored by β-arrestin 2 knockdown ³⁷. β-arrestin 2 is negative regulator NK cell cytotoxicity.

D. β-arrestins and mast cells

Mast cells contain granules rich in histamine and heparin, and play a role in allergy and anaphylaxis, as well as in tissue injury ⁷. Mast cells can be activated to degranulate by injury, cross-linking of Immunoglobulin E (IgE) receptors, and complement proteins ⁷. In the intestine, mast cells release tryptase to activate PAR2 in colonocytes. PAR2 then associates with β-arrestins to activate ERK1/2, which increases epithelial permeability by regulating the assembly of perijunctional F-actin ³⁹. β-arrestins may play a role in the increased epithelial permeability of the intestine during stress and inflammation through the mediation of mast cells activation.

E. β-arrestins and complement

The complement system contains more than 30 proteins, which regulate tissue injury and inflammation ⁴⁰. The complement system can be activated as part of the innate immune as well as adaptive immune responses. Complement fragments bind to specific receptors to induce cellular responses that trigger inflammatory and immune responses. β-arrestin 1 and β-arrestin 2 are involved in complement receptor C3aR desensitization, internalization, degranulation, NF-κB activation and chemokine generation in mast cells ⁴¹. Stable knockdown of β-arrrestin 2 expression attenuated C3aR desensitization, internalization and NF-κB activity as well as chemokine generation in human mast cell lines that endogenously express C3aR ⁴¹. However, silencing β-arrestin 1 decreased C3a–induced mast cell degranulation, not C3aR desensitization. Knockdown of β-arrestin 1, β-arrestin 2 or both enhanced the early response to C3a but inhibited G protein-dependent ERK1/2 phosphorylation ⁴¹. Others studies suggested that although C3aR interacts with β-arrestins, they do not appear to be involved in C3a–induced ERK activation in a leukemia cell line ⁴². Complement C5a binds to the C5a receptor (C5aR, a GPCR) and C5L2 receptor (a non-GPCR) to elicit its proinflammatory functions. A recent study suggested that C5a binding to C5a receptor recruits C5L2 and β-arrestin 2 ⁴³. Activation of C5L2 results in inhibition of C5aR-β-arrestin-mediated ERK1/2 activation, with no apparent alteration of G protein-mediated functions such as calcium influx and receptor endocytosis ⁴³. Bone marrow-derived macrophages devoid of β-arrestin 2 showed decreased complement C1q gene expression and enhanced factor-independent survival of CSF-1 through JNK/ERK signaling ⁴⁴. Therefore, β-arrestins appear to regulate complement functions through receptor desensitization as well as ERK signaling.

III. β-arrestins in adaptive immunity

A. β-arrestins and lymphocytes

Both β-arrestin 1 and β-arrestin 2 are expressed in lymphocytes ⁴⁵. In thymocytes, β-arrestin 1 expression was very low compared with that of splenocytes or from lymph nodes ⁴⁶. Compared with B cells, T cells contain substantially more β-arrestin 2 ⁴⁵. For both naive and activated cells, CD4⁺ T cells had much more nuclear β-arrestin 1 than CD8⁺ T cells ⁴⁶. CD4⁺ T lymphocytes of mice with allergic asthma expressed higher levels of β-arrestin 2 at both mRNA and protein levels compared to normal mice ⁴⁷. β-arrestin expression levels are dependent upon cell type, activation status, and disease states.

B. T cell activation

T lymphocytes are activated by the binding of the T cell receptor (TCR) to antigens bound by major histocompatibility complex (MHC) molecules on antigen presenting cells s(APC). The binding of co-stimulatory molecules to the CD28 receptor is also required for full T cell activation. In addition, the activation is mediated by kinases, phosphatases, and transcription factors. Engagement of the TCR is followed by rapid cAMP production in lipid rafts and activation of the cAMP-protein kinase A (PKA)-Csk pathway inhibiting proximal T-cell signaling ^{48; 49}. It appears that β-arrestins do not regulate the cAMP-PKA-Csk pathway ^{48; 49}. However, β-arrestins participate in CD28 receptor co-ligation signaling ⁴⁹. Upon TCR and CD28 co-ligation, β-arrestin was recruited to lipid rafts, and augmented TCR/CD28-stimulated cytokine production ^{48; 50}. β-arrestins form complexes with cAMP phosphodiesterase 4 and mediate cross-talk between phosphatidylinositol 3-kinase and cyclic AMP-protein kinase A signaling pathways ⁵¹. A PDE4/β-arrestin complex attenuates PKA phosphorylation of Csk ^{48; 52}, allowing full T-cell activation to proceed.

C. Migration and chemotaxis

The migration of T cells is important in adaptive immune responses. T cells must migrate to sites where antigen is found, because T cells respond to pathogens only on direct contact with pathogen-derived antigen ⁵³. β-arrestins function as fundamental adaptors connecting receptors to cell trafficking machinery ⁴⁸. β-arrestin-2 activation of the p38 MAPK cascade is required for CXCR4-mediated migration of human embryonic kidney 293 cells to CXCL12 (SDF-1α) ⁵⁴. It has been reported that lymphocytes devoid of β-arrestin-2 demonstrate impaired migration toward the chemotactic factor CXCL12 in vitro ⁴⁵. A subsequent study showed that β-arrestin 2 deficient mice have defective macrophage-derived chemokine-mediated CD4⁺ T cell migration to the lung ⁵⁵, and T cells in β-arrestin 2 deficient mice exhibit migration defects in a model of metastatic tumor growth ¹⁷.

D. Lymphocyte survival and apoptosis

Several studies indicate that β-arrestins play a critical role in preventing apoptosis ^{56; 57}. For example, neuropeptide substance P inhibits apoptosis via a β-arrestin scaffolding complex ⁵⁸, and β-arrestins play a critical role in the suppression of apoptosis mediated by stimulated GPCRs using β-arrestin 1/2-knockout cells ⁵⁷. IGF1-mediated anti-apoptosis was diminished by knockdown of β-arrestin 1/2 and was restored by transfection of β-arrestin 1 ⁵⁶. β-arrestin 1 epigenetically regulates Bcl2 expression, regulating the survival and homeostasis of CD4⁺ T cells ⁴⁶. β-arrestin 1 promotes acetylation of histone H4 ⁵⁹ at the Bcl2 locus and Bcl2 expression in both naïve and activated CD4⁺ T cells ⁴⁶. Furthermore, β-arrestin 1 is highly expressed in CD4⁺ T cells from multiple sclerosis patients, and RNAi knockdown of β-arrestin 1 in those cells have increased apoptosis after induction by cytokine withdrawal ⁴⁶. β-arrestin 1 did not affect the homeostasis of CD8⁺ T cells ⁴⁶, possibly due to the difference in the subcellular distribution of β-arrestin 1 in CD4⁺ and CD8⁺ T cells. Moreover, β-arrestin 2 over-expression and interference can decrease and increase, respectively, the level of gp120/morphine-induced lymphocyte apoptosis ⁶⁰. Collectively, these studies support a role for β-arrestins in regulating lymphocyte survival.

E. Th1, Th2, and Th17 cells

Classically, helper T cells include Th1 and Th2 T cells. T cell antigen receptor-mediated activation of the Ras/MAP kinase pathway controls IL-4 receptor function and Th2 cell differentiation ⁶¹. Although the direct demonstration of β-arrestins in Th1 and Th2 skewing is lacking, it seems that β-arrestins play a role in both conditions. For example, β-arrestin 2 deficient mice showed a reduced CD4⁺ T cell accumulation in airway in an asthma model ⁵⁵. This may be a Th2 dependent event, since β-arrestin 2 deficient mice showed a normal inflammatory response in a LPS model ⁵⁵. Knocking down β-arrestin 2 with RNAi decreased expression of the Th2 cytokine IL-4 as well as the T cell specific transcription factor GATA3 in CD4⁺ T lymphocytes after terbutaline stimulation ⁴⁷. On the other hand, overexpression of β-arrestin 1 increased Th1 cytokine interferon-γ production ⁵⁹. The stress-induced suppression of Th1 cytokines and the increased production of Th2 cytokines were greatly enhanced in β-arrestin 2-deficient mice compared with wild-type mice ⁶². These studies suggest that β-arrestins may not have a role in T cell differentiation, but has a role in Th1 and Th2 responses during inflammation.

The role of β-arrestins in Th17 cell function has not been reported. Blocking CCL2-CCR2 interactions with a fusion peptide failed to recruit β−arrestin 2, leading to blocking IL-17 production in vitro ⁶³. Silencing β-arrestin 2 with specific siRNA reduced IL-17 expression ⁶⁴. Overexpression of β-arrestin 1 increased IL-17 expression, possibly by acetylation of histone H4 in the promoter region of IL-17 ⁵⁹. It would be interesting to determine if β-arrestins have any role in activation of Th17 cells.

IV. β-arrestins and structural cells

β-arrestins are expressed in epithelial cells ^{65; 66}, endothelial cells ^{22; 67; 68}, and smooth muscle ²². β-arrestin 1 has been found to be expressed in human endothelial cells and smooth muscle cells ²². In these cells, β-arrestin 1 expression can be upregulated by cAMP-inducing agents such as cholera toxin, forskolin, iloprost, and isoproterenol ²². β-arrestins mediate the infections of Streptococcus pneumonia ⁶⁷ and Neisseria meningitides ⁶⁹ through interactions wth endothelial cells. β-arrestin 2 regulates thrombin-PAR1 signaling in endothelial cell adhesion ⁶⁸.

β-arrestin influences CXCL12-mediated epithelial cell migration. CXCL12-stimulated cell migration is enhanced by CXCR7/CXCR4 co-expression in a β-arrestin dependent manner ⁶⁵. In a CXCR4/CXCR7-expressing MDA-MB-231 breast cancer epithelial cell line, inhibition of β-arrestin using either siRNA knockdown or a dominant negative mutant suppressed CXCL12-induced cell migration ⁶⁵.

V. β-arrestins regulate immune signaling pathways

A. β-arrestins and chemokine receptors

Chemokines are a family of small cytokines secreted by immune cells and inflammatory cells. Receptors for chemokines are typically seven-transmembrane GPCRs. Binding of chemokines and their cognate receptors regulate chemotaxis of leukocytes into inflammatory sites ⁷⁰ and cytokine release. β-arrestins are recruited to the receptors and mediate receptor desensitization and internalization (reviews ^{11; 18; 21; 71}).

CXC chemokines are one of the main subfamily of chemokines. β-arrestins have been shown to mediate the internalization and desensitization of IL-8 induced CXCR1 ^{72; 73}, CXCR2 ^{36; 73; 74}, and CXCL9- and CXCL10-induced CXCR3 ⁷⁵. β-arrestins regulate CXCR4 internalization in response to the specific ligand CXCL12/SDF-1 ⁷⁶. CXCR4 can also form a heteromeric complex with the CXCR7 chemokine receptor upon in response to CXCL12 ⁶⁵. Defects chemokine receptor-β-arrestin interactions may promote human disease states. WHIM (warts, hypogammaglobulinemia, infections, and myelokatexis) syndrome is an immunodeficiency syndrome linked to heterozygous mutations of the chemokine receptor CXCR4 resulting in truncations of its cytoplasmic tail. WHIM-associated mutant CXCR4 was unable to recruit β-arrestin 2, leading to the impaired CXCR4 internalization and enhanced chemotaxis in response to CXCL12 ⁷⁷.

CC chemokines are a second group of chemokines. Reports showed that β-arrestins regulate CC chemokine-induced CC chemokine receptor activation by desensitization and internalization. CCL2 (MCP-1) binds to CCR2 inducing the activation of CCR2 and, shortly after stimulation, recruits a protein complex including GRK2 and β-arrestin ³⁰. In addition, the actin binding protein filamin A binds CCR2B ⁷⁸ and together with β-arrestin regulates the internalization of the receptor complex. Chemokine CCL5 (RANTES) binding to the CCR5 induces the phosphorylation of CCR5. β-arrestin 1 is able to desensitize and internalize homo- and hetero-oligomers of CCR5 ^{79; 80}. β-arrestins thus regulate immune and inflammatory responses in part by desensitization and internalization of activated chemokine receptors.

β-arrestins also regulate chemokine signaling non-canonical G protein pathways. β-arrestin 2 and GRK6 play positive regulatory roles, mediating the chemotactic responses of T and B lymphocytes to CXCL12 ⁴⁵. β-arrestin 2 strengthened CXCR4 activation through the ASK1-p38MAPK pathway ⁵⁴. In addition to the formation of a CXCR4 homodimer, CXCL12 also binds to CXCR7 ^{81; 82}, and induces CXCR4-CXCR7 heterodimers ⁶⁵. The CXCR4-CXCR7 heterodimer complex upon CXCL12 binding recruits β-arrestin, resulting in preferential activation of β-arrestin-linked signaling pathways over canonical G protein pathways ⁶⁵. This activation was dependent on β-arrestin-mediated cell signaling pathways, such as ERK1/2, p38 MAPK, and SAPK, leading to enhanced cell migration in response to CXCL12 stimulation ⁶⁵. The CC chemokine CCL4 (MIP-1β) binds to CCR5 regulating macrophage migration. Chemotaxis upon CCR5 stimulation by CCL4 requires activation of Pyk2, PI3K p85, and Lyn, and ERK. β-arrestins regulate the receptor complex formation and enhance macrophage chemotaxis toward the ligand ²⁹.

B. β-arrestins and other GPCRs in inflammation

Angiotensin II receptor Type 1A

Angiotensin II is a potent vasopressor peptide hormone, binds its receptor, the angiotensin II type 1A receptor (AGTR1, AT1R, or AT1AR), a GPCR, to mediate the formation of reactive oxygen species and inflammatory responses ⁸³, in addition to its role as an important effector controlling blood pressure. For example in liver, angiotensin II-dependent NF-κB activation leads to proinflammatory cytokine release from hepatocytes and stellate cells that is implicated in the development of hepatic fibrosis ⁸⁴. Studies suggested that β-arrestins promote desensitization and sequestration of AGTR1 ⁸⁵. Both β-arrestin 1 and β-arrestin 2 showed similar roles in agonist-stimulated receptor desensitization, while the β-arrestin 2 may have more profound effects on angiotensin II-stimulated internalization of the AGTR1 ⁸⁵. Furthermore, β-arrestin 2 also mediates chemotaxis through an AGTR1-dependent and G-protein-independent mechanism ⁸⁶.

However, β-arrestins also facilitate GPCR-mediated ERK activation but inhibit ERK-dependent transcription to target activated ERK1/2 to non-nuclear substrates following angiotensin AT1a receptor stimulation ⁸⁷. Furthermore, β-arrestin 2 binds JNK and mediates ASK1 and MKK4 activity ⁸⁸.

The angiotensin II receptor has been shown to play a role in immune-mediated renal injury ^{89; 90}. Deletion of AGTR1 in mice markedly attenuated glomerular expression of CCL2 (MCP-1), proteinuria, and tissue damage in an anti-glomerular basement membrane nephritis model ⁸⁹. Angiotensin II binds its receptors on immune cells, triggering the proliferation of splenic lymphocytes and leading to the potency of cellular alloimmune responses ⁹⁰. The absence of AGTR1 signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine ⁹⁰. The manipulation of β-arrestin-mediated renin-angiotensin signaling can be used as a way to regulate immune responses.

β2-adrenergic receptor (β2AR)

β-arrestin attenuates agonist-induced β2-adrenergic receptor signaling by GRK-mediated desensitization ⁹¹ and by clathrin-mediated receptor internalization ⁹². It is reported that both isoforms of β-arrestins promote desensitization of β2-adrenergic receptors ⁸⁵, however, deficiency in β-arrestin 2, not in β-arrestin 1, significantly compromised the sequestration of β2-adrenergic receptors ⁸⁵. A cAMP phosphodiesterase PDE4 is recruited to the receptor-β-arrestin complex and PDE4 plays a role in the regulation of G protein switching by the β2-adrenergic receptor in cardiac myocytes ⁹³.

Lipid signaling receptors

Prostaglandins are lipid compounds derived from fatty acids. Prostaglandins bind to their respective receptors to elicit functions including inflammatory regulation. Prostaglandin E2 receptor (EP4) is a typical GPCR. Studies suggested that β-arrestins have both positive and negative effects on the EP4 receptor. β-arrestin 1 is necessary for maximal agonist-induced internalization through the interaction with a Ser/Thr cluster in the C-terminal domain of EP4 receptor ⁹⁴.

Leukotrienes are arachidonic acid metabolites, binding to their respective receptors to elicit their role in inflammatory responses. β-arrestin 1 is required for internalization of the leukotriene B4-activated leukotriene B4 receptor ⁹⁵.

Lysophosphatidylcholines are phospholipids that bind to G2A receptors on PMNs recruiting clathrin, β-arrestin-1 and GRK6 for receptor de-sensitization. Phospholipids cause acute lung injury ⁹⁶ and are involved in transfusion-related acute lung injury ⁹⁷. β-arrestin 1 both propagates and terminates inflammatory signals ⁹⁷.

Lysophosphatidic acid binds to lysophosphatidic acid receptors, including LPAR1 and mediates diverse biologic roles such as proliferation, platelet aggregation, smooth muscle contraction, chemotaxis, fibroblast migration, and tumor cell invasion. β-arrestin 1 and GRK2 mediate lysophosphatidic acid receptor LPAR1 desensitization ⁹⁸. Furthermore, β-arrestin 2 promotes a distinct subset of ERK1/2-mediated responses to LPA receptor activation ⁹⁹ and lysophosphatidic acid-induced NF-κB activation ¹⁰⁰. Suppression of β-arrestin 2 expression using small interfering RNA abolished chemotaxis induced by lysophosphatidic acid ⁸⁶. β-arrestin signaling regulates lysophosphatidic acid-mediated migration and invasion of human breast tumor cells ¹⁰¹

Phospholipid platelet-activating factor (PAF) is an effective chemoattractant that primes PMN oxidases. The binding of PAF with the PAF receptor (PAFR, or PTAFT) recruits β-arrestin 1 and desensitizes the receptor ¹⁰². β-arrestin recruitment blocks both NF-κB activation and Ca2+/calcineurin-dependent signaling pathways, leading to reduced cytokine production ¹⁰³. Platelet-activating factor-induced clathrin-mediated endocytosis requires β-arrestin 1-MKK3-p38 MAPK complex and p38 MAPK activation ¹⁰⁴. Finally, the same scaffolding complex regulates actin rearrangement in human neutrophils ^{104; 105}.

Protease-activated receptors

Both protease-activated receptor-1 (PAR-1) and PAR-2 mediate leukocyte recruitment to sites of injury and infection. β-arrestins may play a dual role in both in PAR-1 and PAR-2 desensitization and scaffolding. A report suggested that only β-arrestin 1 is capable of PAR-1 receptor desensitization, but receptor internalization is independent of either β-arrestin ¹⁰⁶. In contrast, β-arrestins function as scaffolds for PAR-2 activation. A recent study reported that upon PAR-2 activation, β-arrestins scaffolding aggregates the intracellular actin-modulating protein cofilin and activator chronophin, to facilitate the localized generation of free actin barbed ends, leading to the reorganization of the actin cytoskeleton and chemotaxis ¹⁰⁷. The formation of the signaling complex of β-arrestins and activated ERK1/2 is required to mediate PAR-2 signaling ¹⁰⁸.

C. β-arrestins and TLR signaling

Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogen-associated molecular patterns such as bacterial coat components ¹⁰⁹ as well as endogenous ligands ¹¹⁰. TLRs belong to the interleukin-1 receptor/Toll-like receptor superfamily that play a key role in the innate immune system ¹⁰⁹. β-arrestins are implicated in TLR4 signaling in several studies^{111; 112; 113; 114}. β-arrestin 2 attenuates TLR4-initiated apoptosis by controlling the activation and inactivation of GSK-3β ¹¹⁵. β-arrestin 2 modulates immune responses induced by stress through TLR2-mediated signaling ¹¹⁶.

Studies suggest that endosomal trafficking of the LPS-TLR receptor complex is essential for signal termination and LPS-associated antigen presentation ¹¹⁷. Reports have shown that β-arrestins may have a direct role in regulating TLR sensitization and internalization. β-arrestin 2 directly interacts with TRAF6 in response to LPS or IL-1β stimulation, preventing TRAF6 autoubiquitination and oligomerization ¹¹¹. β-arrestin 2 also inhibits AP-1 and NF-κB activation and inhibits LPS- and IL-1-induced inflammatory cytokine production ¹¹¹. Moreover, β-arrestin 2 and GRK5 negatively regulate TLR4 signaling through NF-κB and ERK phosphorylation ¹¹⁸. On the other hand, a study suggested that β-arrestins regulate cytokine production following LPS-induced chromatin modification and not MAPK and IκBα-NFκB pathways ¹¹². In contrast, in drosophila, β-arrestin Kurtz inhibits MAPK and Toll signaling in Drosophila development ¹¹⁹.

β-arrestins may have a role in regulating cross-talk between TLRs and other receptor families (Figure 1). A recent study showed that there was cross-talk between the β2-adrenergic receptor and TLR4, and the suppression of β-arrestin 2 eliminated the anti-inflammatory effects of β2-adrenergic receptor activation in monocytes ¹²⁰. β-arrestin-2 mediated redistribution of CD14 and TLR4/CD14 complex ¹²⁰. β2-adrenergic receptor regulates TLR4-induced late-phase NF-κB activation ¹²¹. Furthermore, recent studies also suggest there is cross-talk between TLRs and chemokine receptors. TLR2 activation leads to β-arrestin-mediated endocytosis of CCR1, CCR2, and CCR5 on human blood monocytes, providing a molecular mechanism for inhibiting monocyte migration after pathogen recognition ¹²². Moreover, it seems that TLRs also directly regulate β-arrestins. For instance, activation of TLR2 and TLR4 significantly decrease β-arrestin 2 protein in macrophages ²⁴.

Figure 1.

β-arrestins regulate GPCR and TLR signaling during inflammation. Upon binding of chemokines and their respective receptors, β-arrestins are recruited to the receptors and regulate chemotaxis of leukocytes into inflammatory sites and cytokine release in two ways: mediating receptor desensitization and internalization, and acting as signaling scaffolds to facilitate numerous effector pathways. Classically, LPS-TLR interaction induces signaling cascade including TRAF6 and NF-κB activation, leading inflammatory response. The LPS-TLR binding also recruits β-arrestins and the latter mediates TLR signaling through regulating TRAF6, NF-κB, as well as chromatin modification. Furthermore, β-arrestins are able to mediate the crosstalk to chemokine receptors.

D. Other non-GPCR signaling pathways

Peroxisome proliferator-activated receptors are nuclear receptor proteins, regulating metabolism and inflammation. β-arrestin 1 binds peroxisome proliferator-activated receptor-γ (PPARγ) to elicit the repression of PPARγ/9-cis-retinoic acid receptor-α function and to promote PPARγ/nuclear receptor corepressor function in PPARγ-mediated adipogenesis and inflammatory cytokine expression ¹²³.

TNFα binds to its TNF receptor, a type I transmembrane protein, recruiting death domain containing proteins and leading to apoptosis pathways. As TNFα is such a pleiotropic cytokine, the interaction of TNF and its cognate receptor regulates an array of biological functions including apoptosis, immune regulation, and inflammatory response. β-arrestin 1 can function as a signaling molecule in the TNFα action cascade that stimulates ERK activation and lipolysis, mediates phosphatidylinositol 3-kinase activation and inflammatory gene expression ⁶⁰. β-Arrestin 1 and 2 also bind and prevent degradation of IκBα-inhibiting NF-κB activation ^{124; 125}, leading to the suppression of TNFα-induced proinflammatory cytokines. A subsequent report confirmed that although TNF receptor-1 is a non-GPCR, it functions through a Gα (q/11) signaling complex and through ERK activation, mediating TNFα-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression ¹²⁶. β-arrestin 1 was found to interact with STAT1 upon interferon-γ stimulation ¹²⁷. β-arrestin 1 accelerates STAT1 tyrosine dephosphorylation by recruiting T cell protein tyrosine phosphatase (TC45) and negatively regulates interferon-γ induced gene transcription ¹²⁷. Interestingly, β-arrestin 2 did not have a similar effect in regulating interferon-γ signaling, although β-arrestin 2 also binds to STAT1.

TGF-β binds to TGF-β receptors, regulating cell growth, differentiation, and immune responses. β-arrestin 2, but not β-arrestin 1, binds to the phosphorylated type III TGF-β receptor and mediates endocytosis of the type II TGF-β receptor/type III TGF-β receptor complex ¹²⁸. Further study suggested that β-arrestin 2 modulates the association of type III TGF-β receptor with ALK6 and ALK3 and enhances ALK6-mediated bone morphogenetic protein 2 signaling ¹²⁹. TGF-β signaling mediates immune regulation, inflammatory responses, and tissue fibrosis. However, functional studies on β-arrestins-mediating TGF-β signaling are largely lacking. Interaction of β-arrestin 2 and type III TGF-β receptors regulate epithelial cell migration through activation of Cdc42 ¹³⁰. In a tissue fibrosis model, our initial finding suggests that deficiency in β-arrestins may not affect TGF-β-induced matrix production ¹³¹. Detailed analysis such as prolonged treatment with TGF-β may shed some light on the role of TGF-β and β-arrestins in inflammatory and fibrotic diseases. In addition, β-arrestins have been shown to regulate the orexin-1 receptor ¹³² and T cell receptor and co-receptor ¹³³. Through these interactions with these signaling pathways, β-arrestins regulate inflammatory responses.

VI. Role of β-arrestins in human diseases

A. Pathogen recognition and clearance

β-arrestins play a role in bacterial-induced inflammation and clearance, such as in sepsis ^{34; 134; 135}, and Cryptococcus infections ²⁷. For example, deficiency in β-arrestin-2 in mice showed decreased mortality and reduced infections. Therefore, inhibition of β-arrestins may be used to limit bacterial and fungal infections in some instances ^{27; 69}.

Neisseria meningitides

Neisseria meningitides, a commensal bacterium causing cerebrospinal meningitis, is an intracellular human-specific pathogen that functions through Type IV pilus-mediated adhesion to human brain endothelial cells, leading to the opening of intercellular junctions and allowing meningeal colonization ¹³⁶. The signaling receptor activated by the pathogen may be the β2-adrenoceptor ⁶⁹. Studies have suggested that the β2-adrenergic receptor/β-arrestin signaling pathway may mediate bacterial colonization ^{136; 137}. Type IV pili of N. meningitidis are able to activate the β2-adrenergic receptor/β-arrestin signaling pathway, thus recruiting the polarity complex and the cell junction proteins and allowing the opening of a paracellular route in endothelial cells ¹³⁸. By this process, Neisseria meningitidis is able to cross of the blood-brain barrier and affect the central nervous system ¹³⁸. Activation of β-adrenoceptor endocytosis with specific agonists prevents signaling events downstream of N. meningitidis adhesion and inhibits bacterial crossing of the endothelial barrier ⁶⁹. Therefore, targeting the β2-adrenergic receptor/β-arrestin signaling pathway may be used for treatment and prevention of meningococcal infection ⁶⁹.

Streptococcus pneumoniae

β-arrestin 1 has been reported to be involved in pneumococci infection. A report showed that upon infection with Streptococcus pneumoniae, β-arrestin 1 moved to the plasma membrane of endothelial cells ⁶⁷. By transfection of β-arrestin 1, pneumococcal invasion was enhanced in a cell line. β-arrestin 1 was shown to interact with the platelet-activating factor receptor and contribute to successful infection by Streptococcus pneumoniae ⁶⁷.

Polymicrobial sepsis

β-arrestin 2 protein expression was reduced in a murine model of CLP-induced polymicrobial sepsis ¹³⁴. β-arrestin 2 deficient mice exhibit decreased mortality, increased IL-6 and elevated caecal myeloperoxidase in a mouse model of polymicrobial sepsis by caecal ligation and puncture (CLP). Splenocytes devoid of β-arrestin 2 produced higher levels of TNF-α, IL-6 and IL-10 ¹³⁵. This is reminiscent of another study showing that CLP-induced myeloperoxidase was increased in β-arrestin 2 deficient mouse lung ³⁴. β-arrestin 1 and β-arrestin 2 deficient mice were protected from TLR4-mediated endotoxic shock and lethality ¹¹². These studies suggest that β-arrestin 2 is a negative regulator in polymicrobial sepsis. Therefore, treatment with flavocoxid preserved β-arrestin 2 expression and reduced cytokine production, leading to protection against tissue damage induced by CLP presumably by decreasing myeloperoxidase activity in the lung and liver ¹³⁴.

Since chemokine receptors play key roles in pathogen recognition and β-arrestins regulate chemokine receptor internalization, it is not surprising that β-arrestins regulate chemokine receptor-mediated pathogen clearance. For example, during Staphylococcus aureus associated lipoteichoic acid induced pathogen recognition and monocyte migration, β-arrestins mediate endocytosis of CCR5 into recycling endosomes. Thus facilitating the TLR2 negative regulation of CCR1, CCR2 and CCR5 on monocytes ¹²².

Cryptococcal meningitis

β-arrestin 2 mRNA and protein levels were significantly elevated in peripheral blood mononuclear cells of patients with cryptococcal meningitis. The increased β-arrestin 2 levels were concurrent with reduced cytotoxic activity against Cryptococcus, increased IL-10 levels and reduced IFN-γ expression in patient serum ²⁷. This study suggests that increasing INF-γ by inhibition of β-arrestin 2 could be a therapeutic strategy in Cryptococcus infections²⁷.

Viral responses

Reports suggest that β-arrestins mediate antiviral responses. β-arrestin 1 accelerates STAT1 tyrosine dephosphorylation by recruiting T cell protein tyrosine phosphatase (TC45) and negatively regulates IFN-γ induced gene transcription in IFN-γ-induced cellular antiviral responses ¹²⁷. β-arrestin 1 knockdown promotes the antiviral immune response in vesicular stomatitis virus-infected cells ¹²⁷. β-arrestin 2 plays a negative role in HIV-1 gp120 and opioids-induced lymphocyte cell death ⁶⁰. Increased or decreased β-arrestin 2 expression inhibited or enhanced gp120/morphine-induced apoptosis, respectively ⁶⁰. β-arrestin 1 and 2 can function as both positive and negative regulators of adenovirus-induced innate immune responses both in vivo and in vitro ¹¹⁴.

B. Fibrosis

The molecular mechanisms that control progressive tissue fibrosis remain poorly understood. Idiopathic pulmonary fibrosis (IPF) is a progressive disease and a major cause of morbidity and mortality. Progressive pulmonary fibrosis results from numerous micro-injuries to the alveolar epithelia that lead to detonation of excessive fibroblast activity. Lung epithelial cell apoptosis is believed to be responsible for initiation of fibrogenesis. β-arrestins are known to regulate cell apoptosis (reviews ^{19; 57; 139}). Some GPCRs are reported to have a role in lung epithelial apoptosis. For instance, angiotensin II receptors are expressed by lung alveolar epithelial cells, and the interaction of angiotensin II and its receptors mediate alveolar epithelial cell apoptosis ^{140; 141}. However, it is unclear whether β-arrestins mediate type II epithelial cell apoptosis during lung fibrosis. During tissue injury, TGF-β is released by fibroblasts and the epithelium. There is abundant evidence that TGF-β is critical for the progression of pulmonary fibrosis due to its role in regulating collagen synthesis, fibroblast proliferation, apoptosis, and myofibroblast differentiation ^{142; 143; 144}. β-arrestin 2, but not β-arrestin 1, mediates endocytosis of type II TGF-β receptor/type III TGF-β receptor complex and down-regulates TGF-β signaling ¹²⁸ and bone morphogenetic protein 2 signaling ¹²⁹. In a lung fibrosis model, our initial findings suggest that deficiency in β-arrestins may not affect TGF-β-induced matrix production ¹³¹. Detailed analysis such as prolonged treatment with TGF-β may shed some light on the role of β-arrestins on TGF-β signaling in inflammatory and fibrotic diseases (Figure 2).

Figure 2.

β-arrestins mediate signaling pathways during fibrogenesis. A simplified model of tissue fibrosis depicts that epithelial cell injury and apoptosis leads to dysregulated inflammatory responses and fibroblast/myofibroblast activation resulting to excessive matrix production and eventually fibrosis. β-arrestins regulate signaling pathways mediating cell injury and apoptosis in epithelial cells, inflammatory responses in immune cells, and fibroblast migration and invasiveness, matrix production in effector cells.

Fibroblast and myofibroblast migration is an important process during fibrogenesis. Chemokines and chemokine receptors regulate fibroblast migration ^{145; 146}. Furthermore, lysophosphatidic acid in bronchoalveolar lavage fluid interacts with its receptor LPAR1 regulating fibroblast migration during fibrogenesis ¹⁴⁷. β-arrestins regulate chemokine signaling as well as LPAR1 signaling. The role of β-arrestins in fibroblast migration has not been explored. We have shown that deletion of β-arrestins did not significantly affect fibroblast migration ¹³¹.

The destruction of alveolar basement membrane was observed in experimental lung fibrosis and the biopsies of patients with lung fibrosis ¹⁴⁸. Moreover, fibroblasts and myofibroblasts from patients with progressive lung fibrosis have been shown to have the ability to invade extracellular matrix in the manner of metastatic cancer cells ¹⁴⁸. β-arrestins have been shown to play a role in tumor metastasis ^{16; 17}. For example, β-arrestin signaling mediates lysophosphatidic acid-induced cancer cell migration and invasion ¹⁰¹. We found that fibroblasts from bleomycin-treated β-arrestin deficient mice and human IPF fibroblasts with β-arrestin 2 knockdown appear failed to invade extracellular matrix ¹³¹. Furthermore, we showed that loss of β-arrestin 1 or β-arrestin 2 in primary lung fibroblasts results in altered expression of genes involved in matrix production, basement membrane degradation, and cell adhesion ¹³¹. Therefore, this study suggests that inhibiting β-arrestin expression in lung fibroblasts would be a potential therapy for IPF and other types of pulmonary fibrosis.

β-arrestin 1 expression is increased in T cells from primary biliary cirrhosis patients. There is also a positive correlation between the levels of β-arrestin 1 mRNA in peripheral blood mononuclear cells and progression of cirrhosis ⁵⁹. β-arrestin 1 was overexpressed in autoreactive T cells from cirrhosis patients augmented cell proliferation, increased interferon release, and regulated autoimmune-related gene expression ⁵⁹. The study suggests that β-arrestin 1 overexpression contributes to the pathogenesis of primary biliary cirrhosis. Angiotensin II binds its receptor angiotensin II type 1A receptor to mediate inflammatory and fibrotic responses ⁸³. β-arrestins regulate desensitization and sequestration of AGTR1 ^{85, 149}. In cirrhosis, impaired vascular reactivity leads to vasodilation and contributes to portal hypertension. Vascular hyporesponsiveness to angiotensin II in CCl4-treated rats is due to enhanced interaction of the AGTR1 with β-arrestin 2 and alterations in receptor function ¹⁵⁰. Silencing β-arrestins might be a therapeutic strategy for cirrhosis.

C. Arthritis

Rheumatoid arthritis is an autoimmune disease characterized by joint inflammation and destruction. GPCRs such as chemokine receptors, G-protein-coupled receptor kinases, and β-arrestin1 and 2 are involved in the pathogenesis of rheumatoid arthritis. Reduction in GRK activity with a decreased level of GRK-2 and GRK-6 protein expression was found in patients with rheumatoid arthritis, while β-arrestin-1 expression was unchanged between rheumatoid arthritis patients and controls ¹⁵¹. In a rat adjuvant arthritis model, the expression of β-arrestin 1 in splenocytes was increased at the most severe period of the disease ¹⁵². Using collagen-induced arthritis and human TNFα transgenic mouse models, another group demonstrated that expression of β-arrestin 1 and 2 was elevated in joint tissue ¹⁵³. Furthermore, β-arrestins differentially regulated inflammatory cytokine production by fibroblast-like synoviocytes, in which β-arrestin 1 increased TNFα and IL-6 production while β-arrestin decreased their production by synoviocytes ¹⁵³. Finally, mice deficient in β-arrestin 2 exhibited more severe arthritis in the collagen-induced arthritis model ¹⁵³.

The role of chemokine receptor CCR2 and β-arrestins has been investigated in inflammatory arthritis, with some studies suggesting that blocking CCR2 signaling may ameliorate arthritis in animal models. A fusion protein using GM-CSF and an N-terminal truncated version of CCL2 binding to CCR2 led to altered conformational changes in the CCR2 homodimer and failed to induce the recruitment of β-arrestin 2 to the receptor ⁶³. The fusion peptide was capable of blocking IL-17 production in vitro and attenuated inflammatory arthritis in vivo in mice ⁶³. This studysuggests that suppressing expression of β-arrestins may protect joints from rheumatoid arthritis.

Thus, strategies that regulate β-arrestin expression or activity may be developed for patients with rheumatoid arthritis. For example, Paeoniflorin, a monoterpene glucoside, decreased expression of β-arrestin 1 and β-arrestin 2, and increased β2-adrenergic receptor and GRK2 in the mesenteric lymph node lymphocytes, significantly attenuated arthritis scores, and reversed the changes of cytokines ¹⁵⁴.

D. Multiple sclerosis

Multiple sclerosis is characterized by the presence of multiple plaques of demyelination throughout the central nervous system. The destruction of myelin is believed to involve autoimmune mechanisms. β-arrestin 1 was autoantibodies have been found in sera from multiple sclerosis patients ¹⁵⁵. Multiple sclerosis patients had a greater prevalence of positive T-cell proliferative responses to β-arrestin than healthy controls ¹⁵⁶. These studies suggest a role for β-arrestin 1 in the pathogenesis of multiple sclerosis. β-arrestin 1 expression was increased in brains of multiple sclerosis patients relative to non-multiple sclerosis brains ¹⁵⁷. This suggests β-arrestin 1 may be a negative regulator in multiple sclerosis. In contrast, another study showed β-arrestin 1 mRNA levels were reduced by phitoemagglutinin (PHA), but, increased in interferon β-1a-treated mononuclear leukocytes? ²⁶. Since IFNβ-1a is known to ameliorate the course of multiple sclerosis, β-arrestin 1 may be a downstream mediator in multiple sclerosis. Additional studies suggest that β-arrestin 1 is critical for CD4⁺ T cell survival and may be a factor in susceptibility to autoimmunity ⁴⁶.

E. Encephalomyelitis

β-arrestin protein expression was significantly increased in splenocytes following myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE), while G-protein-coupled receptor kinases (GRK) 2, GRK 6, and A1 adenosine receptor were decreased during the disease ^{157; 158}. Mice deficient in β-arrestin 1 were much more resistant to experimental autoimmune encephalomyelitis, whereas overexpression of β-arrestin 1 increased susceptibility to the disease ⁴⁶. Glucocorticoid treatment eliminated EAE-induced neuroinflammation and neurobehavioral deficits, and also reduced β-arrestin 1 expression and enhanced A1 adenosine receptor expression ¹⁵⁷. These suggest that β-arrestins may play a pathogenic role in EAE.

F. Acute rejection in organ transplant

β-arrestin 1 and 2 are expressed by intravascular renal graft monocytes and T cells in a rat model of kidney transplantation, and β-arrestin 1 and 2 expression is decreased in monocytes during rejection. Decreased β-arrestin 2 expression was concurrent with depression of I-κB and augmentation of NF-κB ²⁸. These data suggest that the up-regulation of β-arrestin 2 in renal isografts may suppress the activation of blood monocytes via NF-κB activation. Ischemic injury following heart transplantation reduced β-arrestin 1 expression ¹⁵⁹. Immunomodulatory peptide α-melanocyte stimulating hormone was able to preserve β-arrestin 1 protein levels ¹⁵⁹ and reduced damage in transplanted heart grafts.

G. Asthma

Asthma is characterized by chronic airway inflammation and airway hyperresponsiveness. Studies have showed that Th2 cytokines such as IL-4 and IL-13 play a key role in the pathogenesis. β-arrestin 2 regulated T cell function in human asthma and animal models of allergic asthma. β-arrestin 2 knockout mice are protected from OVA induced allergic-asthmatic inflammation but not LPS mediated non-allergic inflammation suggesting that neutrophil recruitment is not dependent on. β-arrestin 2 β-arrestin 2 deficient mice are competent in their ability to present antigen, to generate antigen-specific T cell responses, and to undergo Ig isotype switching, but they have defective macrophage-derived chemokine-mediated CD4⁺ T lymphocytes migration to sites of inflammation ⁵⁵. A subsequent study determined the cell types required for β-arrestin 2-dependent allergic pathology and inflammation. By using bone marrow chimera mice, the same group was able to show expression of β-arrestin 2 in eosinophilic and lymphocytic cells contributes to the airway inflammation, while expression of β-arrestin 2 in structural cells contributes to the airway hyperresponsiveness ⁶⁶. Another study investigated the role of β-arrestin 2 in CD4⁺ T lymphocytes, and found that β-arrestin 2 regulated IL-4 production and GATA3 expression ⁴⁷.

β-arrestins can regulate beta-agonist-induced desensitization of airway smooth muscle beta-2-adrenergic receptors, which is the cause of loss of bronchoprotective effect and deterioration of asthma control by beta-agonists. β-arrestin 2 deficient mice show augmented beta-agonist-mediated airway smooth muscle relaxation, and increased beta-agonist-stimulated cyclic adenosine monophosphate production. Knockdown of β-arrestins in cultured human airway smooth muscle cells demonstrated increased beta-agonist-stimulated cyclic adenosine monophosphate production. Collectively, these studies suggest that β-arrestins regulate beta-2-adrenergic receptor signaling and function in airway smooth muscle ^{160; 161; 162}.

Interleukin-17 is a pro-inflammatory cytokine that plays a critical role in the pathogenesis of allergic asthma. A recent study found that β-arrestin2 and IL-17 expression in CD4⁺ T cells from a murine asthma model were increased compared with those from wild-type mice ⁶⁴. Moreover, β-arrestin2 stimulated IL-17 production and expression of CD4⁺ T lymphocytes in a murine asthma model ⁶⁴.

H. Cystic fibrosis

β-arrestin 2 protein level is increased in cystic fibrosis cell models, Cftr^−/− mouse nasal epithelia, and nasal scrapes obtained from cystic fibrosis patients ¹⁶³. Elevated β-arrestin 2 expression leads to increased cAMP response element binding protein (CREB) activation and ERK activation in βarr2-GFP expressing tracheal epithelial cells. Inhibition of ERK resulted in diminished cAMP response element (CRE) activation in both cells with and without βarr2-GFP expressiion. Nasal epithelium excised from Cftr and β-arrestin 2 double knockout mice exhibit reduced pCREB and pERK levels compared to Cftr^−/− mice, but similar to WT mice. Thus, β-arrestin 2 directly regulates cystic fibrosis induced CREB activation through the ERK signaling pathway ¹⁶⁴.

I. Cardioprotection

In addition to regulating the PKA-apoptosis pathway, β1AR activation also signals through the β-arrestin-Src-MMP pathway, leading to transactivation of the EGF receptor and promoting cardiomyocyte survival ¹⁶⁵. Both β-arrestin 1 and β-arrestin 2 are required for β1AR transactivation of the EGF receptor ¹⁶⁵. Mechanical stretch in the heart triggers activation of AT1aR and β-arrestin recruitment to the receptor in the absence of ligand ¹⁶⁶. β-arrestin deficient hearts failed to induce a cardioprotective response to mechanical stretch and demonstrated enhanced myocyte apoptosis ¹⁶⁶, suggesting that the heart responds to mechanical stress by activating β-arrestin-mediated cell survival signals ^{167; 168}.

VII. β-arrestins in therapeutic development for inflammatory diseases

Understanding β-arrestin biology offers the potential to accelerate drug development in targeting GPCR functions (reviews ^{169; 170}). Several assay systems have been proposed and tested to target β-arrestins. For example, β-arrestin conformational changes were used to develop a biased agonist to the GPR109A receptor, leading to a therapeutic targeting lipolytic effect, but devoid of potential cutaneous side effects¹⁷¹.

RNA interference targeting β-arrestins has been used to demonstrate the role of arrestins in desensitization, internalization, and signaling functions ^{125; 172}. Furthermore, these specific siRNAs have been used in vitro as well as in vivo settings. Silencing β-arrestin 2 with RNA interference in allergic asthmatic mice reduced Th2 cytokines such as IL-4 ⁴⁷. Silencing β-arrestin 2 reduced fibroblast invasiveness ¹³¹. Suppression of beta-arrestin 2 expression using small interfering RNA eliminated AT(1A)R- and LPAR1-mediated chemotaxis ⁸⁶.

There are few reports exploring the interplay between microRNAs and β-arrestins. For example, β-arrestin 2-mediated ERK phosphorylation is required for the down-regulation of miR-190 ¹⁷³. Expression of miR-326 is decreased in human glioblastomas and correllated with decreased expression of host gene β-arrestin 1 ¹⁷⁴. Although there are no reports identifying β-arrestins as targets of any microRNAs thus far, microRNAs regulating β-arrestin expression may prove to be a powerful strategy for therapeutic development.

Therapeutic targeting of β-arrestins has been challenging thus far. β-arrestins have been shown to interact with an array of receptors as well as downstream proteins. For example, there are over a hundred proteins interacting with either β-arrestin when angiotensin II type 1a receptor is activated, by proteomic analysis ¹⁷⁵. Furthermore, there are 171 proteins with increased phosphorylation, and 53 with decreased phosphorylation, which represent a β-arrestin-mediated kinase-substrate network involved in a number of cellular pathways ¹⁷⁶. However, the protean impact of β-arrestins in the pathobiology of a variety of diseases processes suggest future efforts at developing agonists or antagonists will continue.

Table 1.

Disease models with β-arrestin 1 and β-arrestin 2 deficient mice

Disease	Disease model	Mouse model	Key phenotype	References
Lung fibrosis	Bleomycin-induced lung fibrosis	βarr1−/− or βarr2−/−	Reduced susceptibility and lung fibrosis	131
Asthma	Ovalbumin sensitization model of asthma	βarr2−/−	Reduced airyway hyperresponsiveness	55,66
Infection	Cytomegalovirus infection	βarr2−/−	Reduced cytomegalovirus infection	37
Sepsis	Galactosamine-D sensitized model of endotoxic shock	βarr2−/−	More susceptible to endotoxic shock	111
Sepsis	LPS injection or Caecal ligation and puncture	βarr1−/− or βarr2−/−	Protected from endotoxic shock and lethality.	112,135
Wound healing	Skin air pouch model of inflammation	βarr2−/−	Increased re-epithelialization	36
Encephalomyelitis	Experimental autoimmune encephalomyelitis	βarr1−/−	Resistant to experimental autoimmune encephalomyelitis	46
Stress	Chronic physical restraint	βarr2−/−	Augments stress-induced immune suppression	62; 116
Cutaneous flushing	Nicotinic acid induced-cutaneous flushing	βarr1−/−	Reduced cutaneous flushing in response to nicotinic acid	171
Lung cancer	Teterotopic murine Lewis lung cancer and tail vein metastasis tumor model	βarr2−/−	Increase in Lewis lung cancer tumor growth and metastasis	17
Rheumatoid arthritis	Collagen-induced arthritis	βarr2−/−	More severe arthritis in collagen-induced arthritis	153