Molecular Mechanisms Underlying Beta-Arrestin-Dependent Chemotaxis and Actin-Cytoskeletal Reorganization Kathryn W. McGovern and Kathryn A. DeFea

Contents 1 β-Arrestins as Regulators of Cell Migration: Receptor Turnover Versus Scaffolding of Actin Assembly Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Regulation of Receptor Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Regulation of Actin Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Regulation of the Cofilin Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2 Filamin and Other Actin-Bundling Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Chemokine Receptors: The Classic Chemotactic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 β-Arrestin-Dependent Signaling by Chemotactic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 β-Arrestin-Dependent ERK1/2 Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 β-Arrestin-Dependent Regulation of Src and Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 RhoA GTPases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Role of β-Arrestin-Dependent Chemotaxis in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . 5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract β-Arrestins play a crucial role in cell migration downstream of multiple G-protein-coupled receptors (GPCRs) through multiple mechanisms. There is considerable evidence that β-arrestin-dependent scaffolding of actin assembly proteins facilitates the formation of a leading edge in response to a chemotactic signal. Conversely, there is substantial support for the hypothesis that β-arrestins facilitate receptor turnover through their ability to desensitize and internalize GPCRs. This chapter discusses both theories for β-arrestin-dependent chemotaxis in the context K.W. McGovern Biochemistry and Molecular Biology Graduate Program, University of California, Riverside, CA, USA K.A. DeFea (*) Biochemistry and Molecular Biology Graduate Program, University of California, Riverside, CA, USA Biomedical Sciences Division, University of California, Riverside, CA, USA e-mail: [email protected] V.V. Gurevich (ed.), Arrestins - Pharmacology and Therapeutic Potential, Handbook of Experimental Pharmacology 219, DOI 10.1007/978-3-642-41199-1_17, © Springer-Verlag Berlin Heidelberg 2014

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of recent studies, specifically addressing known actin assembly proteins regulated by β-arrestins, chemokine receptors, and signaling by chemotactic receptors. Keywords Actin • Arrestins • Chemokine receptor • Chemotaxis • GPCR • Cell migration

1 β-Arrestins as Regulators of Cell Migration: Receptor Turnover Versus Scaffolding of Actin Assembly Proteins β-Arrestins are required for cell migration downstream of numerous receptors and have been implicated in a number of physiological scenarios involving cell migration, including tumor cell metastasis, inflammation, neuronal synapse remodeling, and developmental patterning (Min and Defea 2011). Given their essential roles in G-protein-coupled receptor (GPCR) signaling and signal termination, there are currently several hypotheses regarding their role in cell migration. Based on their ability to uncouple G-proteins from GPCRs and promote receptor internalization, some investigators propose that β-arrestins are crucial in receptor turnover at the leading edge, a process that is important for a cell’s ability to sense a gradient of agonist and migrate toward it. However, the ability of β-arrestins to scaffold signaling proteins and actin assembly machinery at the leading edge suggests that their signaling functions are also key factors in many cell migratory pathways. Thus, it is likely that the ability of β-arrestins to control both receptor turnover and actin assembly at the leading edge is crucial for cell migration (Fig. 1). Furthermore, the emergence of β-arrestin-biased agonists for a number of GPCRs suggests that it is possible to target β-arrestin signaling without G-protein engagement (see chapter “Quantifying Biased β-Arrestin Signaling”). Given the importance of β-arrestins in numerous diseases involving cell migration, the therapeutic implications of β-arrestin-biased drugs are very promising.

1.1

Regulation of Receptor Turnover

We will first look at the model wherein β-arrestins regulate turnover of receptor and chemokine to maintain a gradient of agonist and an active pool of receptors. The first argument in favor of this model is that β-arrestins play a well-known role in clathrin-mediated endocytosis of numerous GPCRs, acting as adaptors for clathrin (Goodman et al. 1996) and clathrin adaptor AP2 (Laporte et al. 1999) (see chapter “β-Arrestins and G Protein-Coupled Receptor Trafficking”). They also uncouple GPCRs from their cognate heterotrimeric G-proteins, thus rendering them

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Fig. 1 Models for β-arrestin-dependent chemotaxis. As a cell senses a chemotactic gradient (depicted as red circles), it polarizes in the direction of the gradient. Current models predict that at low concentrations of agonist, β-arrestin-dependent receptor recycling and ligand degradation, paired with scaffolding and activation of actin assembly proteins, allows the cell to sense the chemotactic gradient and form a leading edge to direct chemotaxis. At high concentrations of agonist, receptor and ligand are degraded, ceasing the migratory process

insensitive to further agonist stimulation (DeWire et al. 2007; Shenoy and Lefkowitz 2011). During chemotaxis (directed cell migration) in vivo, a cell senses gradient of a chemotactic agonist; it essentially migrates up the gradient and stops when it reaches the source, i.e., where the concentration is highest. A second important factor in successful cell migration in vivo is the ability of the cell to stop migrating when it reaches a uniform concentration of agonist, a phenomenon that prevents cells from migrating back away from their destination (Iglesias and Devreotes 2008). Because prolonged exposure to any agonist usually results in downregulation of receptors, it has been proposed that gradient-sensing chemotactic receptors are able to rapidly turnover at the leading edge such that the cell remains responsive to the chemoattractant (Fig. 2). This receptor recycling appears to require β-arrestins in many cases. Upon internalization, β-arrestins either dissociate in the early endosomes allowing recycling of the receptor and degradation of the agonist or remain associated and target the receptor to lysosomes for degradation. In some cases, β-arrestins can also remain bound to the receptor and recruit signaling proteins to form a “signalosome” (discussed in the next section; see also chapters “Arrestin-Dependent Activation of ERK and Src Family Kinases,” “Arrestin-Dependent Activation of JNK Family Kinases,” and “Arrestin-Mediated Activation of p38 MAPK: Molecular Mechanisms and Behavioral Consequences”) or facilitate their degradation, through interaction with ubiquitin ligases (chapter “Arrestin Interaction with E3 Ubiquitin Ligases and Deubiquitinases: Functional and Therapeutic Implications”) or targeting to lysosomes. By allowing receptors to recycle back to the membrane and facilitating removal of agonists, it is predicted that transient interactions with β-arrestins contribute to the maintenance of both a chemotactic gradient and a pool of responsive receptors. At very high concentrations of agonist (such as would be seen at the cells “destination”) (Fig. 2), it is proposed that β-arrestins target endocytosed receptors for degradation through ubiquitin- and lysosomal-mediated processes. This model would predict that β-arrestin/receptor interactions vary in an agonist dose-dependent fashion.

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Fig. 2 Trafficking of receptor and chemokine by β-arrestins. In the receptor turnover/ligand degradation model, β-arrestins bind chemotactic receptors, to promote clathrin-dependent internalization of the receptor. In the early endosome, receptors dissociate from ligand and β-arrestin, allowing for the receptor to be returned to the surface and the ligand degraded. This model predicts that because β-arrestins are required to target chemokine-bound receptors to clathrin-coated pits, in their absence, receptors remain at the surface and chemokine levels accumulate. This results in the cell losing polarity and being unable to migrate

There are considerable data to support this receptor turnover/agonist degradation model for β-arrestin-dependent chemotaxis. Early studies investigating the role of β-arrestins in chemotaxis revealed that many of the receptors that require β-arrestins for internalization also show impaired chemotaxis in their absence (DeFea 2007). Receptors that were allowed to signal constitutively, such as they would in the absence of β-arrestins, also showed impaired chemotaxis. Recent proteomics screens for β-arrestin-binding proteins have led to the identification of more clathrin adaptor proteins, such as AGEP and ARF, with additional evidence suggesting that chemokine receptor turnover via β-arrestins, through association with endocytotic machinery, is required for migrating cells to respond to an agonist gradient (Bouschet et al. 2007; DeFea 2007; Xiao et al. 2007). Biochemical isolation of pseudopodia (actin-rich extensions formed at the front of a migrating cell) reveals that β-arrestins and other components of the endocytotic machinery are enriched at the leading edge (Ge et al. 2003; Parisis et al. 2013). Furthermore, early data suggested that coupling to Gα12 or Gαq was required for chemotaxis downstream of many of these chemotactic receptors. Collectively, these data have led to the hypothesis that G-protein signaling mediates the chemotactic response, while β-arrestin-dependent turnover of receptors and chemokine at the leading edge is important for maintaining responsiveness to the chemoattractant gradient.

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However, a number of receptors can promote β-arrestin-dependent cell migration in the absence of prior G-protein coupling, suggesting a more complex paradigm is necessary to explain this process. Since most commonly used assays for quantifying chemotaxis do not differentiate effectively between differences in migration speed, persistence, and directionality, it is likely that these more subtle aspects of cell migration require the cooperation between β-arrestin signaling, G-protein signaling, and β-arrestin-dependent receptor turnover.

1.2

Regulation of Actin Assembly

We will now examine the model in which β-arrestins facilitate chemotaxis by sequestering and activating actin assembly proteins at the leading edge. A number of studies have demonstrated a role for β-arrestins in the modulation of actin assembly, the process that provides the primary driving force behind the initial formation of a leading edge. Actin assembly can be regulated, both directly and indirectly, by various proteins, some of which have been identified as β-arrestinbinding partners or as targets of β-arrestin-dependent phosphorylation (Xiao et al. 2007, 2010; Christensen et al. 2010; Min and Defea 2011). To understand the role of β-arrestins in the initiation of cell migration, it is important to understand the mechanics of actin assembly. Actin polymerization from monomers is a spontaneous but slow process, and the rate-limiting step is the formation of a stable nucleus, or actin seed, consisting of three or more actin monomers. Addition of actin monomers is always at the ATP-binding or barbed end of the actin molecule (Campellone and Welch 2010). Provided that the barbed end of a filament is free from capping proteins, addition of monomers onto preassembled filaments or actin seeds is very rapid. These seeds can be created in two major ways: (1) activation of proteins that sever existing filaments into smaller fragments creating a free barbed end at each break or (2) activation of nucleators, i.e., proteins that overcome the rate-limiting step in actin assembly by facilitating association of actin monomers into filament seeds. Actin nucleators can also bind to the sides of existing filaments, an action that in turn facilitates branching of actin filaments. These branched filaments are found in the broad lamellipodia of the leading edge (Firat-Karalar and Welch 2011). Together, these processes provide directionality during migration, as well as assemble and maintain cortical actin filaments. Cortical actin filaments interact with contractile proteins and allow the cell to contract against the substrate over which it is migrating (Campellone and Welch 2010). These actin structures must be dynamically remodeled, requiring an intricate balance of input from various signaling pathways.

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Fig. 3 Scaffolding of signaling molecules by β-arrestins at the leading edge. Many signaling molecules are sequestered at the leading edge by β-arrestins in response to a chemotactic signal. β-Arrestins have been shown to bind and inactivate LIMK while scaffolding cofilin with its upstream phosphatases. Together these events result in increased actin filament severing, creating of free actin barbed ends and actin polymerization. Proteins associated with ARP2/3-mediated nucleation have been identified as targets of β-arrestin-dependent phosphorylation. Active cofilin and Arp2/3 work together to create branched lamellipodia that push the membrane forward during chemotaxis. β-Arrestins have also been demonstrated to bind filamin and regulate activation of the G-protein Ral, which is important for membrane protrusion formation. Finally, β-arrestins regulate the cellular pool up PIP3 through activation and inactivation of PI3K. This controls the activation of many guanine exchange factors for RhoA, Cdc42, and Rac, providing a spatiotemporal regulation of these small G-proteins and, in turn, their ability to promote stress fiber, lamellipodia, and filopodia formation

1.2.1

Regulation of the Cofilin Pathway

A requirement for β-arrestin in the creation of free barbed ends through cofilinmediated actin filament severing strongly supports the notion that β-arrestins regulate actin assembly (Zoudilova et al. 2007, 2010). Cofilin is one of the primary actin filament-severing proteins and its activation is often an early event in cell migration. Cofilin is activated by dephosphorylation on serine 3, carried out by the phosphatases slingshot and chronophin, and inactivated by LIM kinase (LIMK). Its activity is crucial to the formation of a leading edge, downstream of multiple receptors (Wang et al. 2007; Oser and Condeelis 2009). Several lines of evidence indicate that proteins of the cofilin pathway (cofilin, chronophin, slingshot, and LIMK) can interact with β-arrestins. β-Arrestins can facilitate cofilin dephosphorylation by scaffolding it with its activating phosphatases at the leading edge or by binding and inhibiting LIMK-induced phosphorylation (Zoudilova et al. 2007,

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2010; Xiao et al. 2010). A popular model for actin remodeling at the leading edge predicts that cofilin rapidly disassembles existing filaments, providing free barbed ends for elongation and, in coordination with activation of nucleating proteins, drives the direction of cell migration (Oser and Condeelis 2009). Spatial control over cofilin activity is thus essential to allow for treadmilling of filaments. This model, combined with data demonstrating β-arrestin-dependent cofilin activation and sequestration (Zoudilova et al. 2007, 2010), suggests that β-arrestins spatially regulate cofilin activity. By restricting cofilin activity to the front of the migrating cell, they can facilitate protrusion formation while preventing severing of the more stable cortical actin filaments. Several studies showing β-arrestin-dependent localization of cofilin and cofilin activity support this hypothesis (Zoudilova et al. 2010; Pontrello et al. 2012). A scaffold containing β-arrestin, cofilin, and chronophin has been demonstrated in primary leukocytes and is essential for their migration (Zoudilova et al. 2010). When cofilin activity is not spatially controlled, protrusions can form randomly and cells lose their ability to move toward a chemotactic gradient (Mouneimne et al. 2004). Thus, it is likely that β-arrestins contribute to the formation of a leading edge, through the regulation of cofilin activity (Fig. 3).

1.2.2

Filamin and Other Actin-Bundling Proteins

The actin-binding protein, filamin, has been identified in a complex with β-arrestins and ERK1/2. The role of filamin in cell migration is a compound one, and the manner in which it is controlled by β-arrestins is still not completely clear. It plays an important role in turnover of adhesion proteins such as integrins during cell migration (Kim and McCulloch 2011), a process that is important for cell attachment and contraction. It has also been reported to regulate the internalization of GPCRs such as the dopamine receptor (Kim et al. 2005; Cho et al. 2007) and CXCR4 (Kim and McCulloch 2011). Downstream of angiotensin II receptor (AT1AR), filamin associates with β-arrestin-2 and MAPK and this complex is thought to play a role in membrane ruffling (Scott et al. 2006). These studies support both models of β-arrestin involvement in chemotaxis. First, the studies on dopamine receptor and CXCR4 support a model where β-arrestin-dependent regulation of filamin contributes to both focal adhesion remodeling and receptor turnover, both of which are proposed to be important for gradient sensing. However, the studies on AT1AR suggest that β-arrestin-bound filament is involved in the sequestration of ERK1/2 activity at the leading edge (Ge et al. 2003), which likely facilitates localized phosphorylation of key substrates involved in chemotaxis. While a number of actin nucleators and accessory proteins have been identified as either putative β-arrestin-binding partners or substrates for β-arrestin-dependent phosphorylation, regulation of actin nucleation has not yet been demonstrated for β-arrestins. However, it remains possible that some of these proteins may be among the long sought-after substrates for β-arrestin-sequestered MAPK activity. This possibility is discussed in the sections below.

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2 Chemokine Receptors: The Classic Chemotactic Receptors The most well-characterized mediators of chemotaxis are the chemokine receptors, a family of G-protein-coupled receptors that play crucial roles in immune cell recruitment and tumor cell metastasis. β-Arrestins are required for the desensitization and internalization of many chemokine receptors, including CXCR4, CXCR7, CXCR1 and 2, CXCR5, CCR2, CCR5, and CCR7. For CCR2, CCR5, and CXCR5, β-arrestin recruitment has been reported, but little is known about the role of β-arrestins in their chemotactic responses. Because they are well-established mediators of cell migration, we will examine some examples of signaling through β-arrestins downstream of some of these chemokine receptors. In the case of CXCR4 and 7, CXCR1 and 2, and CCR7, recent studies have revealed multiple roles for β-arrestins in their signaling pathways. CXCR4 is a crucial mediator of cell migration in many cells: monocytes/ macrophages, T cells, B cells, plasma cells, neutrophils, and dendritic cells, as well as tumor cells and germ cells. It was one of the first to show impaired cell migration in the absence of β-arrestin-2 (Fong et al. 2002). CXCR4 cooperates with another chemokine receptor CXCR7 in a unique fashion. CXCR7 is considered a “scavenger” receptor for CXCR4 ligands, as it binds CXCL12 without promoting G-protein signaling. Both CXCR4 and CXCR7 recruit β-arrestins, and internalization of both is impaired in their absence (Malik and Marchese 2010; Rajagopal et al. 2010; De´caillot et al. 2011). Studies have predicted that the main role of CXCR7 in chemotaxis is the internalization of CXCL12, something that is dependent upon the presence of β-arrestins. Recent in vivo studies examining primordial germ cell migration in zebrafish suggested that, in the absence of β-arrestins, CXCR7 did not recycle to the membrane, but was targeted to lysosomes. As a result the gradient of the agonist CXCL12 was not maintained and germ cell migration was also inhibited (De´caillot et al. 2011). Thus, CXCR7 is a prime example of a receptor system that utilizes β-arrestins for gradient sensing by reducing the immediate concentration of CXCL12, thus perpetuating the gradient. However, another recent study reveals that CXCR4 can also form heterodimers with CXCR7 and that this heterodimer preferentially promotes β-arrestin-dependent ERK1/2 phosphorylation and chemotaxis over G-protein signaling (Drury et al. 2011). This study would suggest that β-arrestin-dependent signaling is associated with chemotaxis. Thus, the CXCR4/CXCR7 system stands out as an argument for the importance of both ERK1/2 scaffolding and internalization functions of β-arrestins in chemotaxis. CXCR1 and CXCR2 are activated by CXCL8 (aka IL-8) in humans and CCL-1 (GRO/KC) in mice to promote recruitment primarily of neutrophils but also basophils and CD8+ T cells. It is also expressed on endothelial cells where it may play a role in extravasation. Both receptors promote chemotaxis but CXCR2 is internalized more rapidly in response to IL-8 (Barlic et al. 1999; Richardson et al. 2003). Internalization and degranulation are all induced by IL-8 via a

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β-arrestin-dependent mechanism; however, the role of β-arrestins CXCR1- or CXCR2-stimulated migration depends upon the organism and the system studied. In mice, CXCL1-mediated neutrophil migration is enhanced, in vivo, in β-arrestin2 / mice (Su et al. 2005). In vitro, human neutrophil recruitment in response to IL-8 is abolished in the absence of β-arrestins (Barlic et al. 2000). Based on these opposite findings with respect to the role of β-arrestins in CXCR1- and CXCR2induced chemotaxis, one might conclude that CXCR1- and CXCR2-induced migration in response to CXCL1 differs from that observed with the human CXCR1 and 2 agonist, IL-8. Alternatively, since the CXCL1 experiments were based on recruitment of neutrophils in vivo, while the IL-8 experiments were performed on isolated neutrophils, it is possible that additional factors contributing to chemotaxis may be differentially affected by β-arrestins in vivo. CCR5 is widely expressed in immune cells, especially memory T cells and has an array of physiologically relevant agonists (RANTES, MIP-1α, MIP-1β, and MCP-2). In response to RANTES, CCR5 internalizes via a β-arrestin-dependent mechanism (Kraft et al. 2001). Another physiological agonist of CCR5, MIP1β, induces the formation of a membrane-associated multiprotein complex including β-arrestin-2, PI3K, and a number of non-receptor tyrosine kinases. Scaffolding of these kinases by β-arrestin-2 appears to be crucial for MIP1β-induced chemotaxis (Cheung et al. 2009). It is tempting to assume that RANTES and other CCR5 agonists would induce a similar β-arrestin scaffold-dependent mechanism of chemotaxis; however, given the emergence of naturally occurring biased agonists, it remains possible that different CCR5 agonists might use β-arrestin in distinct manners during chemotaxis. CCR7 is a well-established homing receptor, expressed on mature dendritic cells (DCs), naı¨ve and memory T cells, and naı¨ve B cells. CCR7 has two ligands, CCL-19 and CCL-21, which bind to their receptor with equal affinity, and are equally efficacious at promoting Ca2+ mobilization (Yoshida et al. 1998; Sullivan et al. 1999). Despite their apparent similarities with respect to G-protein signaling, subtle differences elicited by CCL-19 and CCL-21 have been reported (Yoshida et al. 1998; Sullivan et al. 1999; Ott et al. 2004). In contrast to Ca2+ mobilization, ERK1/2 activation in response to CCL-19 is more robust than in response to CCL-21. Knockdown of β-arrestin-2 reduces ERK1/2 phosphorylation in response to CCL-19; whether it does the same in response to CCL-21 is not known. Similar to what was observed for ERK1/2 phosphorylation, both CCL-19 and CCL-21 can promote β-arrestin-2 recruitment to CCR7 but CCL-21 does so to a lesser degree than CCL-19 (Zidar et al. 2009). Further differences are observed with respect to receptor endocytosis. Depending on the cell type studied, very little or no endocytosis in response to CCL-21 is observed. In contrast, CCL-19 promotes receptor endocytosis in a variety of cell types (Bardi et al. 2001; Byers et al. 2008). Further, phosphorylation of CCR7 by GRK6 occurs in response to either ligand, but phosphorylation by GRK3 only occurs after stimulation with CCL-19 (Zidar et al. 2009). Thus, differences in GRK-mediated phosphorylation of CCR7 at the carboxy-terminal serine/threonine cluster in response to each agonist may underlie these differences in β-arrestin recruitment and subsequent receptor internalization

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and MAPK activation (Kohout et al. 2004). However, whether differential phosphorylation of CCR7 recruits separates functional pools of arrestins has not been definitely proven.

3 β-Arrestin-Dependent Signaling by Chemotactic Receptors A number of other GPCRs that promote β-arrestin-dependent chemotaxis, and while they do not fall into the category of chemokine receptors, they have provided important insights into how β-arrestins regulate various aspects of cell migration. In fact, the majority of what we know regarding β-arrestin-dependent signaling and reorganization of the cytoskeleton (a crucial factor in cell migration) has been mapped out using less typical chemotactic receptors. We have discussed, earlier in this chapter, how β-arrestins scaffold and regulate some actin assembly proteins to influence actin-cytoskeletal remodeling, but they also regulate a number of other signaling networks that impact the actin cytoskeleton indirectly. These include MAPKs, RhoA GTPases, and PI3K. MAPK was the first “β-arrestin-dependent signal” to be identified and there is considerable evidence that, when active MAPKs are sequestered by β-arrestins, they can phosphorylate substrates to facilitate actin reorganization (Ge et al. 2003, 2004).

3.1

β-Arrestin-Dependent ERK1/2 Activity

There is a significant body of evidence pointing to an essential role of for β-arrestin sequestration of ERK1/2 in chemotaxis; however, information as to what it these kinases are phosphorylating at the leading edge has been slow to emerge. Many of the receptors first demonstrated to promote β-arrestin-dependent ERK1/2 activation also require β-arrestins and/or ERK1/2 for chemotaxis. PAR2-stimulated ERK1/2 activation requires β-arrestin-dependent endocytosis, and chemotaxis is dependent on both β-arrestins and ERK1/2 (DeFea et al. 2000; Ge et al. 2003). AT1AR also requires β-arrestins for both ERK1/2 activation and chemotaxis (Tohgo et al. 2002; Hunton et al. 2005). CXCR4 can form heterodimers with the decoy receptor CXCR7, leading to dominance of β-arrestin-dependent ERK1/2 phosphorylation and chemotaxis over G-protein signaling (De´caillot et al. 2011). Thus, CXCR4 and PAR2 stand out as an argument for the importance of both ERK1/2 scaffolding and internalization functions of β-arrestins in chemotaxis. A number of proteins involved in actin assembly have been identified as β-arrestin-dependent ERK1/2 phosphorylation targets; however, for most of these a definitive role in chemotaxis downstream of β-arrestins has not been proven.

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Regulation of actin nucleation by β-arrestins has not been directly shown, although several proteins involved in actin nucleation have been identified as β-arrestin-dependent ERK1/2 targets. Arp2/3 complex components and Wiskott–Adrich syndrome protein (WASp) family proteins have been identified in a proteomics screen as potential β-arrestin-interacting proteins (Xiao et al. 2007) and phosphorylation targets of MAPK (Christensen et al. 2010) and activation of WASp family proteins is enhanced by ERK1/2 phosphorylation (Mendoza Michelle et al. 2011). The Arp2/3 complex, along with formins and p150spir, is the primary nucleation factor in mammalian cells (Campellone and Welch 2010; Firat-Karalar and Welch 2011). Arp2/3 is crucial for the formation of branched actin filaments such as are observed in the leading edge of migrating cells. Activation by WASps, which bind Arp2/3 and induce a conformational change resulting in apposition of Arp2 and 3, is essential to formation of branched filaments. Patients with Wiskott–Aldrich syndrome have defective lymphocyte trafficking and function, due in part to the disruption of actin nucleation. The actin-nucleating proteins, formins, and p150spir are primarily responsible for de novo assembly of unbranched actin filaments (Firat-Karalar and Welch 2011). Recently, a forminlike protein was also identified as a putative β-arrestin-dependent phosphorylation target (Christensen et al. 2010). Thus, β-arrestins may sequester kinases such as ERK1/2 at the leading edge where they could promote actin nucleation through the phosphorylation of Arp2/3 and formins or by the phosphorylation and activation of WASp family proteins. So far we have discussed actin assembly proteins that create free actin barbed ends by regulating actin nucleation or increasing filament severing. However, the pool of free barbed ends, available for polymerization, can also be increased by removing capping proteins. Another protein identified as a putative β-arrestindependent ERK1/2 substrate in two separate proteomics screens is the barbed-end capping protein adducin. Phosphorylation of adducin by Ser/Thr kinases, PKC, and Rho-activated kinase (ROCK) diminishes its affinity for actin, increasing the pool of free barbed ends and facilitating actin polymerization. Thus, phosphorylation by ERK1/2 may have a similar effect. Cofilin (discussed above) was also identified as a β-arrestin-dependent ERK1/2 substrate. The known regulatory site on cofilin (S3) was not the site identified in the phosphoproteomics screen. Thus, if ERK1/2 phosphorylation contributes to β-arrestin-dependent cofilin dephosphorylation, it would be through a distinct mechanism, possibly by stabilizing the active form.

3.2

β-Arrestin-Dependent Regulation of Src and Chemotaxis

Src was identified as a β-arrestin-binding partner downstream of the several GPCRs, linking them to ERK1/2 activation (Luttrell and Gesty-Palmer 2010) (see chapter “Arrestin-Dependent Activation of ERK and Src Family Kinases”). Downstream of prostaglandin E2 (PGE2) receptor, cell migration involves β-arrestindependent recruitment of Src into a signaling complex that then transactivates the

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EGF receptor (Kim et al. 2010). Src and other non-receptor tyrosine kinases also impact cell migration through multiple mechanisms, including stabilization of nucleation-promoting factors in their active forms. In an example discussed earlier, we saw that activation of CCR5 with MIP1α led to recruitment of Pyk2, the p85 regulatory subunit of PI3K, and the tyrosine kinase, Lyn, into a complex with β-arrestin-2 that appears to be essential for macrophage chemotaxis (Cheung et al. 2009). There are a number of putative functions for membrane-recruited PYK2 and Lyn tyrosine kinase activities. Both have been implicated in activation of nucleation-promoting factors (Guinamard et al. 1998; Chellaiah et al. 2007). PI3K generates PIP3, which is necessary for activation of Cdc42 and Rac activators (Hall 1998). Just as β-arrestins sequester and activate MAPKs at the membrane, sequestration of Src-like kinases by β-arrestins may allow for phosphorylation of actin nucleation proteins and other components of cytoskeletal machinery to facilitate cell migration.

3.3

RhoA GTPases

RhoA GTPases are often upstream regulators of both cofilin and other actin assembly activities. The three most commonly studied members of the Rho family are RhoA, Rac-1, and Cdc42, and each is associated with a different actin structure: RhoA typically causes stress-fiber formation; Cdc42 induces filopodia formation; and Rac-1 is important for membrane ruffling and lamellipodia formation (Hall 1998). β-Arrestins can regulate the activity of many monomeric GTPases including those of the Rho family (Bhattacharya et al. 2002, 2006; Barnes et al. 2005) (see chapter “Arrestin Regulation of Small GTPases”). For example, knockdown of β-arrestin-1 but not β-arrestin-2 with siRNA significantly reduces RhoA activation and stress-fiber formation by the angiotensin receptor, AT1AR (Barnes et al. 2005). β-Arrestin-1-dependent p38 MAPK activation can elicit F-actin rearrangement via a Rac-1-dependent mechanism, downstream of β2-adrenergic receptors (Gong et al. 2008), raising the possibility that Rac-1 could be a β-arrestin-dependent p38 MAPK target. β-Arrestins can also negatively regulate RhoA GTPases. The type III TGF-β receptor (TβRIII) forms a β-arrestin-2 scaffolding complex with Cdc42, leading to inhibition of lamellipodia formation in both cancer and epithelial cells (Mythreye and Blobe 2009a, b). How is Rho GTPase activity affected by β-arrestins? To answer this question, one must consider how monomeric GTPases are regulated in general. Their activity is increased by binding to guanine exchange factors (GEFs) and turned off by association with GTPase-activating proteins (GAPs). β-Arrestin-1 directly binds and inhibits the RhoGAP (ARHGAP21) downstream of AT1AR activation. Disruption of this complex inhibits AT1AR-mediated stress-fiber formation and RhoA activation (Anthony et al. 2011). Phosphatidylinositol-3-phosphate (PIP3), which is generated by PI3K, can also activate a number of GEFs for RhoA, Cdc42, and Rac. β-Arrestins have been shown to regulate PI3K activity both positively and

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negatively depending on the activating receptor (Povsic et al. 2003; Wang and DeFea 2006) (Fig. 3). β-Arrestin-1-dependent p38 MAPK activation was reported to elicit F-actin rearrangement via a Rac-1-dependent mechanism, downstream of β2-adrenergic receptors (Gong et al. 2008), and so regulation of RhoA GTPases may also lie downstream of the MAPK scaffolds. β-Arrestin-dependent regulation of RhoA GTPases has also been implicated in inhibition of cell migration. The type III TGF-β receptor (TβRIII) inhibits migration and alters actin cytoskeleton via forming a β-arrestin-2 scaffolding complex with Cdc42 in both cancer and epithelial cells (Mythreye and Blobe 2009a, b). Another GTPase, RalA, induces membrane blebbing in response to the fMLP receptor in neutrophils and LPA in cancer cells (Bhattacharya et al. 2002, 2006; Li et al. 2009). RalGDS is a guanine exchange factor that activates RalA and can exist in an inactive complex with β-arrestin-1 in the cytosol of resting cells. Activation of the fMLP or LPA receptor recruits the β-arrestin-1/RalGDS complex to the membrane. Upon receptor/β-arrestin-1 binding, RalGDS is released and activates RalA. fMLP receptor-induced membrane ruffling is blocked by a mutant RalGDS which cannot bind β-arrestin-1, suggesting that the ability of β-arrestin-1 to traffic it to the membrane is crucial for its activity (Fig. 3). Subsequent studies have demonstrated that expression of RalA and β-arrestin-1 and β-arrestin-2 is increased in metastatic cancers and expression of a RalGDS mutant that is deficient in β-arrestin binding inhibits RalA activation in tumor cells, leading to decreased cell migration. RalA itself is important for targeting another actin-binding protein regulated by β-arrestins, filamin, to the membrane (Ohta et al. 1999).

4 Role of β-Arrestin-Dependent Chemotaxis in Health and Disease β-arrestin-dependent chemotaxis has been implicated in both tumor cell metastasis and inflammation. Tumor metastasis requires migration of malignant cells from the original tumor to other sites within the body. This process requires a number of chemotactic signals that allow the cells to migrate to nearby vessels, enter the vasculature, and extravasate at distal sites. Similarly, inflammation involves recruitment of leukocytes and other inflammatory cells from the vasculature to injured tissue and numerous changes within the tissue such as epithelial proliferation, extracellular matrix deposition, and functional alterations in both the invading and host cells. Several studies over the last decade in tumor cell lines have demonstrated that constitutive migration of many cancer cells is dependent upon β-arrestin (Ge et al. 2004; Zabel et al. 2009). Likewise, studies have demonstrated that certain inflammatory processes are impaired in the absence of β-arrestin-2. Furthermore, β-arrestin-dependent regulation of actin-cytoskeletal proteins and signaling pathways that affect cell migration has been demonstrated in human cancer cell lines and leukocytes (Zoudilova et al. 2007, 2010).

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Recent in vivo studies have shed more light on the multiple mechanisms by which β-arrestins can promote tumor cell migration and metastasis. Interestingly, these studies suggest that both tumor cell and host β-arrestin play important roles in this process and that β-arrestins are pro-metastatic in some scenarios and antimetastatic in others. In many cases, β-arrestin complexes described in the previous sections play a major role in the metastatic progression of cancers in vivo. CXCR4, which we discussed earlier in the chapter, is upregulated in numerous malignant cancers and CXCL12 is expressed in many of the tissues to which tumor cells commonly metastasize. While CXCL12 monomers promote metastasis, CXCL12 dimers have tumor suppressor functions, effectively inhibiting cell migration through a β-arrestin-independent pathway (Drury et al. 2011). Thus, development of biased ligands that act like CXCL12 dimers may have therapeutic value for the treatment of some cancers. Several reports have also indicated that β-arrestins are required for the recruitment of immune cells to the airways during asthma (see chapter “GPCRs and Arrestins in Airways: Implications for Asthma”). Influx of leukocytes to the lungs was not only decreased in β-arrestin-2 knockout mice but in wild type mice receiving knockout bone marrow. Thus, the role of β-arrestins in asthma appears to be mediating the chemotaxis of invading inflammatory cells (Walker et al. 2003; Hollingsworth et al. 2010; Nichols et al. 2012). In another inflammatory process, pulmonary fibrosis, a major contributing factor is the uncontrolled invasion of the extracellular matrix by fibroblasts and the secretion of matrix components. In a mouse model of pulmonary fibrosis, β-arrestin-1 or β-arrestin-2 knockout mice were protected from excessive matrix deposition, resulting in protected lung function and increased survival. Knockdown of either β-arrestin in fibroblasts from patients with pulmonary fibrosis inhibited their migration and invasive behavior (Lovgren et al. 2011). Thus, in this context β-arrestins were negatively regulating an inflammatory process. However, a resolution to these disparate findings is found when one looks at the underlying role of chemotaxis in both processes. β-arrestindependent chemotaxis in vivo contributes to recruitment of inflammatory cells, which can result in chronic inflammation. In contrast, in the sepsis studies in which β-arrestin-2 appears to negatively regulate tissue damage and mortality resulting from sepsis (Fan et al. 2010), the focus was not on β-arrestin-stimulated chemotaxis, but rather production of inflammatory mediators important for resolving bacterial infections. Release of these cytokines may be dependent upon the G-protein signaling arm of chemokine receptors and thus inhibited by β-arrestins. It is important to bear in mind that the role of β-arrestins in processes requiring chemotaxis in vivo is far more complicated than their role in chemotaxis in vitro. Depending on the physiological scenario and the receptor being activated, the classical role of β-arrestins as terminators of G-protein signaling dominate or their role as facilitators of chemotaxis may dominate.

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5 Concluding Remarks Although it has been over a decade since the demonstration that β-arrestins are important for chemotaxis downstream of numerous GPCRs, much remains to be elucidated regarding the underlying molecular mechanisms. Clearly receptor turnover and ligand degradation via β-arrestin-dependent endocytosis is important, but evidence suggests that the story is far more complicated than this. β-Arrestins are capable of regulating, both spatially and temporally, a wide array of cellular activities essential for cell migration. Since the spatiotemporal regulation of signals generated during cell migration must be tightly controlled, it stands to reason that the role of β-arrestins as signaling scaffolds is equally important in cell migration as is their role in gradient sensing. This chapter summarizes the experimental evidence supporting multiple roles for β-arrestins in chemotaxis. Given the plethora of biological responses—from inflammation to cancer—that are controlled by β-arrestin-dependent regulation of actin assembly and chemotaxis, it is clear that much more needs to be learned regarding the molecular mechanisms underlying this process.

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