Chemokine receptors intracellular trafficking

Chemokine receptors intracellular trafficking

Pharmacology & Therapeutics 127 (2010) 1–8 Contents lists available at ScienceDirect Pharmacology & Therapeutics j o u r n a l h o m e p a g e : w w...

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Pharmacology & Therapeutics 127 (2010) 1–8

Contents lists available at ScienceDirect

Pharmacology & Therapeutics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h a r m t h e r a

Associate Editor: M.M. Teixeira

Chemokine receptors intracellular trafficking Elena M. Borroni, Alberto Mantovani, Massimo Locati ⁎, Raffaella Bonecchi Department of Translational Medicine, University of Milan, I-20089 Rozzano (Milan), Italy IRCCS Istituto Clinico Humanitas, I-20089 Rozzano (Milan), Italy

a r t i c l e

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Keywords: Chemokine Chemokine receptors Chemokine decoy receptors Endocytosis Recycling Degradation

a b s t r a c t Chemokines coordinate leukocyte recruitment during inflammatory and immune responses through the interaction with a distinct subfamily of G protein-coupled receptors. The magnitude of the cellular response elicited by chemokines is dictated by the level of receptor expression at the plasma membrane, which is the balance of finely tuned endocytic and recycling pathways. Recent data have revealed that receptor trafficking properties can drive chemokine receptors to lysosomal degradation or recycling pathways, producing opposite effects on the strength of the intracellular signaling cascade. This review will cover recent advances on the molecular mechanisms underlying chemokine receptor internalization, recycling and degradation pathways, with particular attention to structural motifs present in receptor intracellular domains and their interacting adaptor proteins that modulate receptor trafficking and dictate proper biological response. © 2010 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . 2. CR internalization . . . 3. Post-endocytic sorting . 4. Receptor sequences and 5. Concluding remarks . . Acknowledgments . . . . . References . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . adapters involved . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . in trafficking . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Chemokine receptors (CRs) are members of the class A rhodopsinlike family of seven transmembrane domain G protein-coupled receptors (GPCRs) and recognize chemokines, a broad family of chemotactic cytokines that coordinate directional cell migration along ligand gradients during embryonic development and in adult life. In addition to their chemotactic eponymous function, these cytokines have effects on other biological functions, including cell proliferation and activation (Charo & Ransohoff, 2006; Bonecchi et al., 2009). Abbreviations: AIP4, athropin interacting protein 4; AP-2, adaptor protein beta2adaptin; CCP, clathrin-coated pits; CR, chemokine receptor; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HIP, HSC-70-interacting protein; HSC, heat shock cognate protein; HSP, heat shock protein; Myo, myosin; Rab11-FIP, Rab11 family interacting protein; RE, recycling endosome; WHIM, warts, hypogammaglobulinemia, infections, myelokathexis. ⁎ Corresponding author. IRCCS Istituto Clinico Humanitas, Via Manzoni 56, I-20089 Rozzano, Italy. Tel.: +39 02 8224 5116; fax: +39 02 8224 5101. E-mail address: [email protected] (M. Locati). 0163-7258/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2010.04.006

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CRs activation by their ligands induces conformational changes which lead to activation of an intracellular signaling cascade. CRs are associated with members of the Gi family of heterotrimeric G proteins (Thelen, 2001; Thelen & Stein, 2008). When the chemokine binds its cognate receptor the heterotrimeric Gi complex dissociates into its Gαi subunit, which inhibits adenylyl cyclase, and βγ dimer, which activates the signaling enzymes phospholipase C-β (Scandella et al., 2004) and phosphatidylinositol 3-kinase (Hirsch et al., 2000), leading to the activation of a complex and only partially characterized signaling cascade. Trafficking properties of CRs, represented by receptor internalization followed by either degradation in lysosomes (downmodulation) or termination of the activated state and recycling to the cell surface (resensitization), have strong influence on signaling properties. Furthermore, prolonged exposure to the ligand also leads to the regulatory process of desensitization, which results in diminished responsiveness of CRs to repeated exposure to the agonist. A family of CRs which do not support cell directional migration after chemokine recognition has also been described (Mantovani et al., 2006). These non-conventional CRs, which include DARC (Pruenster & Rot,

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2006), D6 (Locati et al., 2005; Graham, 2009), CCRL2 (Zabel et al., 2008), CCX CKR (Comerford et al., 2006), and CXCR7 (Boldajipour et al., 2008), are structurally related to signaling CRs but are apparently unable to activate transduction events. Though they do not directly induce cell migration, in vivo they play a non-redundant role in the leukocyte recruitment process by shaping the chemoattractant gradient, either by removing/scavenging, transporting, or concentrating their cognate ligands (Borroni & Bonecchi, 2009; Borroni et al., 2009). Intracellular trafficking properties have also emerged as a major determinant of nonconventional CR biological properties, which support continuous uptake, transport, and/or concentration, of the ligand.

2. CR internalization 2.1. Clatrin-coated pits and caveolae The best understood GPCR endocytosis mechanism is the clathrinand dynamin-dependent pathway. GPCRs initiate the internalization process through the binding of clathrin, which is usually mediated by β-arrestin and the adaptor protein beta2-adaptin (AP-2). The clathrin coats assemble to invaginate the plasma membrane, causing the budding of clathrin-coated pits (CCP), which are severed from the plasma membrane through the action of the GTPase dynamin, finally allowing the receptors to reach the endosomal compartment (Hanyaloglu & von Zastrow, 2008). For CR internalization, a role of clathrin-mediated pathway has been demonstrated for members of the CXC (CXCR1: (Barlic et al., 1999); CXCR2: (Yang et al., 1999); CXCR4: (Signoret et al., 1997)) and CC families (CCR5: (Signoret et al., 2005); CCR7: (Otero et al., 2006) (Fig. 1)). Consistent with this, the key proximal regulator of CCP assembly Eps15 is required for endocytosis of CXCR1, CXCR4, CCR5, and CCR7 (Venkatesan et al., 2003; Otero et al., 2006). The clathrin-mediated pathway has also been demonstrated for D6, whose internalization requires a dynamin I-, Rab5- and β-arrestin-dependent mechanism (Galliera et al., 2004; Bonecchi et al., 2008).

A second pathway of internalization depends on caveolae (Anderson, 1998), cholesterol-rich and highly organized membrane structures whose shape and structural organization depend upon the presence of caveolins, a specific set of proteins which self-assemble in high mass oligomers and form a cytoplasmic coat on membrane invaginations. The caveosome intracellular compartment then fuses with early endosomes, converging to the clathrin-dependent pathway. Among CRs, CCR2 (Garcia Lopez et al., 2009), CCR4 (Mariani et al., 2004), and CCR5 (Mueller et al., 2002; Fraile-Ramos et al., 2003) use a combination of clathrin- and caveolae-dependent pathways to internalize (Fig. 1). The ability to use a clathrin-independent pathway by CCR5 has been associated to the ability of palmitoylated cysteine residues in C-terminal tail to insert in cholesterol-enriched raft microdomains (Venkatesan et al., 2003), though these results have been recently questioned by the finding that CCR5 internalization requires caveolae but not cholesterol (Cardaba et al., 2008). Conversely, CXCR3 internalization has been described as a process independent from both clathrin and caveolae (Meiser et al., 2008). The caveolae-dependent pathway can also be engaged by nonconventional CRs. DARC is targeted into caveolae after internalization in polarized MDCK cells (Pruenster et al., 2009), and results in conditions of caveolin-1 over-expression strongly suggest that CCX CKR internalizes through a pathway mediated by caveolae and dynamin, and not arrestins and CCP (Comerford et al., 2006). 2.2. Differential effects of ligands on CR internalization Different chemokines active at a given receptor may have different effects on its internalization (Neel et al., 2005). CCR4 is efficiently internalized by CCL22 but not CCL17 (Mariani et al., 2004), and CCR5 is efficiently internalized when engaged by CCL3 and CCL5, but not CCL4 (Mueller & Strange, 2004). Similarly, CXCR2 is internalized by CXCL8 but significantly less after engagement by CXCL6 and CXCL7 (Feniger-Barish et al., 2000). Molecular mechanisms accounting for these differential effects are largely unknown. An exception is CCR7, which efficiently internalizes after binding with CCL19 because this

Fig. 1. CR intracellular trafficking pathways. In the absence of ligand some CRs undergo constitutive internalization, followed by degradation (CXCR3) or recycling (CXCR4 and D6) (left panel). Most CRs are internalized after agonist engagement through clathrin-mediated or caveolae-dependent mechanisms. Some CRs take advantage of both of these pathways (CCR2, CCR4, and CCR5), while others display preferential usage of a specific pathway (clathrin-dependent: CXCR1, CXCR2, CXCR4, and CCR7; caveolae-dependent: DARC, and CCX CKR). The mechanism of CXCR3 internalization is still unknown. After endocytosis the sorting endosome plays a mayor role in determining the fate of the internalized receptor. There are three known destinations after the sorting endosome: the rapid (CCR5) and slow (CXCR2) recycling endosomes, from which CRs recycle back to the plasma membrane, and the late endosomes/lysosome, where receptors are targeted to degradation (CCR2b, CXCR2, CXCR3, and CXCR4).

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chemokine, differently from the other ligand CCL21, induces strong receptor phosphorylation by GRK3 and GRK6 and recruitment of βarrestin 2 (Byers et al., 2008; Zidar et al., 2009). For CXCR3, CXCL11 is the most potent agonist in inducing receptor internalization and, differently from the weak agonists CXCL9 and CXCL10, it uses a dynamin and β-arrestin independent pathway that requires the third intracellular loop and not the C-terminal domain of the receptor (Xanthou et al., 2003; Colvin et al., 2004; Dagan-Berger et al., 2006). On the other side, even though a chemokine is able to internalize in the same way different target receptors, it may have divergent effects on the receptors recycling properties (Elsner et al., 2000). CCR1, CCR3 and CCR5 are efficiently internalized by CCL5, but the trafficking of the receptors follows different pathways: CCR5 is entirely recycled back to the cell surface (Signoret et al., 2000); CCR3 is partially degraded, but a proportion is able to recycle (Zimmermann et al., 1999) while CCR1 is not more able to be recycled (Elsner et al., 2000). A modified version of CCL5 named aminooxypentane-RANTES (AOP-RANTES) has been developed and works as a CCR5 antagonist by means of inhibition of receptor recycling (Mack et al., 1998). Receptor internalization is generally considered an agonist-dependent event, but recent evidence suggests that CRs can also be internalized in a constitutive manner. Of note, constitutive internalization affects receptor distribution, in that receptors that cycle constitutively are mostly detected in intracellular compartments in steady state conditions and undergo redistribution to the cell membrane after agonist engagement. On this basis, ligand-independent internalization has been proposed as a mechanism used by some chemokine scavenger receptors (D6: (Bonecchi et al., 2004); CCX CKR: (Weber et al., 2004); the virus-encoded CR US28: (Fraile-Ramos et al., 2003)) to rapidly upregulate their expression in order to cope with the immediate needs of the tissue (Prevo et al., 2004; Schaer et al., 2006). Constitutive internalization has also been demonstrated for the conventional CR CXCR4 (Y. Zhang et al., 2004) and CXCR3 (Meiser et al., 2008). Though its biological significance is not evident at present, it is interesting to note that CXCR4 constitutive endocytosis is a CCP-dependent process requiring the S(E/D)S motif in the C-terminal domain (Futahashi et al., 2007), which is on the contrary not required for CXCL12-induced endocytosis and chemotactic response. CXCR3 ligand-independent internalization requires the YXXL motif at the extreme of the C-terminal domain (Meiser et al., 2008). Finally, CR internalization may also be the result of activation of distinct receptors, in a process known as heterologous desensitization. As an example, CCR5 undergoes heterologous internalization as a result of C-terminal domain phosphorylation sustained by either PKCε activation after CXCR1 engagement (Nasser et al., 2005) or PKA activation after adenosine 2A receptors engagement (N. Zhang et al., 2006). 2.3. Endocytosis and signaling Although cell signaling and endocytosis have been considered as distinct processes, it is now recognized that they are under the control of the same molecular mechanisms, adaptor proteins and enzymes, that play a dual function by interacting with signaling an/or endocytic molecules (Sorkin & von Zastrow, 2009). It is well assessed that ligandinduced endocytosis is a mechanism of negative regulation of receptor signaling activities, reducing the number of membrane receptors available for ligand activation. This regulation is of functional relevance, preventing excessive ligand-induced activation of downstream effectors. At the same time, it has been recently demonstrated that signaling can also occurs from endosomes, where receptors can interact with intracellular proteins through their exposed intracellular domains. Thus, internalized receptors can continue to transduce signal events initiated at the plasma membrane or they can assemble in newly formed signaling complexes generating specific signals (Sadowski et al., 2009). GPCRs in particular can activate ERK both from the plasma membrane

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and endosomes, and it has been reported that a prolonged permanence of the GPCR/β-arrestin complex in the early endosome gives rise to prolonged ERK signaling. Consistently with this, for some CR inhibition of endocytosis correlates with the lack of activation of a functional signaling cascade, as in the case of CCR2, whose ability to activate the ERK pathway is impaired by inhibition of dynamin (Garcia Lopez et al., 2009), suggesting that prolonged endosomal signaling is required. Other CRs are still able to signal despite the inhibition of internalization. The best example of this dissociation between signaling and internalization is given by the C-terminal tail truncated CXCR4 variants associated with the immunodeficiency WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome, which are still able to signal and to interact with β-arrestin, but are refractory to internalization and are not desensitized (Lagane et al., 2008). As mentioned, chemokine binding to non-conventional CRs does not trigger known receptor signaling events, possibly due to modifications in structural determinants of key relevance for signaling of conventional CRs (Mantovani et al., 2006). However, several nonconventional CRs have been demonstrated to possess peculiar intracellular trafficking properties which support chemokine degradation, transport, or concentration, and recent data have revealed that in the D6 case receptor upregulation and increased degradation properties require the presence of a proline residue in position 2 of the ligand N-terminal domain, suggesting that this non-conventional CRs may be able to transmit an intracellular signal in order to optimize chemokine degradation (Savino et al., 2009) (Fig. 2). 3. Post-endocytic sorting Endosomes are key control organelles for CR sorting, where the decision is taken whether receptors are directed to late endosomes and lysosomes for degradation, leading to long-term attenuation of signaling, or are recycled back to the plasma membrane, resulting in functional resensitization (Marchese et al., 2008). Primary vesicles that have been generated by both clathrin-dependent and -independent endocytosis processes are transported to the early endosome, a cargosorting station where the mildly acidic lumen facilitates conformational changes leading to ligand release from the receptor (Hanyaloglu & von Zastrow, 2008). The molecular events that are involved in vesicle carrier formation, movement and fusion with target membranes are under the control of low-molecular-mass GTP-binding proteins called Rab, that cycle between an inactive GDP-bound state and an active GTP-bound state in which they interact with and activate several effector proteins (Zerial & McBride, 2001). In particular, the rapid recycling pathway of cargo proteins directly back to the cell surface from the early endosome is supported by Rab4/Rab5 and PI3K-dependent processes (Hunyady et al., 2002), the slow recycling pathway is associated to the perinuclear brefeldin-A sensitive recycling endosomes (RE) and the trans-Golgi network under the control of Rab11 (van Dam et al., 2002), and lysosomal sorting of GPCRs is dependent upon Rab7 activity (Marchese et al., 2008). Referring to CRs, it has been reported that some are preferentially transported to the sorting endosome and recycled back to the cell surface (CCR5: (Mueller et al., 2002); CCR7: (Otero et al., 2006)), others are targeted to lysosome for degradation (CXCR3: (Meiser et al., 2008); CCR2B: (Garcia Lopez et al., 2009)), and some can undergo recycling or degradation on the basis of the duration of the chemokine stimulation (CXCR2: (Fan et al., 2003); CXCR4: (Y. Zhang et al., 2004)). 3.1. Rapid and slow recycling pathways Few data are present in the literature regarding the mechanisms that control the post-endocytic recycling pathways of CRs (Fig. 1). A significant portion of CXCR2 colocalizes with Rab11-positive endosomes at plasma membrane before induction of internalization, followed by

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Fig. 2. Trafficking properties of the chemokine decoy receptor D6. Under homeostatic conditions, a minority of D6 molecules undergoes a constitutive internalization process from the cell membrane into clathrin-coated pits vesicles and are transported to Rab5-positive early endosomes through a dynamin-dependent process, while most D6 are detected in both Rab4 and Rab11-positive recycling endosomes (left panel). Chemokine stimulation does not affect D6 internalization rate, but receptor engagement accelerates the Rab11dependent recycling pathway thus mobilizes the intracellular pools to plasma membrane (right panel).

complete cytoplasmic colocalization of the two molecules after elicitation of receptor internalization and CXCR2 recycling to the membrane (Fan et al., 2003). CXCR2 recycling is inhibited by overexpression of a dominant-negative mutant of Rab11, suggesting that it plays a regulatory role in control receptor trafficking at plasma membrane through a slow recycling pathway (Fan et al., 2003, 2004). Conversely, CCR5 accumulate in perinuclear transferrin-positive RE following ligand stimulation (Signoret et al., 2000; Pollok-Kopp et al., 2003). However, since the inhibition of Rab11-positive vesicles formation by brefeldin-A does not affect the recovery rate of the internalized CCR5, it seems likely that the receptor is recycled back to the cell surface through a rapid recycling pathway without passing through the Rab11-positive endosomes (Mueller et al., 2002). Finally, CXCR4 constitutive trafficking is supported by both rapid and slow recycling pathways (Y. Zhang et al., 2004). Among non-conventional CRs, under basal conditions D6 is localized in both the rapid and slow recycling pathways (Bonecchi et al., 2008), and is not detected within lysosomes (Weber et al., 2004). After chemokine engagement, D6 optimizes its degradative activity by increasing its expression on cell surface through a Rab11-dependent mechanism (Bonecchi et al., 2008) (Fig. 2). Once internalized, the ligand becomes trapped in endocytic compartments and targeted to degradation, while the receptor is recycled back to the plasma membrane through both rapid and slow recycling pathways (Bonecchi et al., 2008) with a mechanism that is strictly dependent on cytoskeleton dynamics (Borroni et al., in preparation). As for D6, also CXCR7 is predominantly detected in intracellular compartments under resting conditions, but the precise compartments involved are unknown (Boldajipour et al., 2008). Conversely, CCRL2 is predominantly expressed on cell surface and is not internalized upon ligand binding (Zabel et al., 2008). 3.2. Degradative pathways Agonist-induced ubiquitin-dependent targeting to lysosomes is emerging as a general mechanism supporting receptor degradation. Lysosomal degradation and trafficking through Lamp-1 positive endo-

somes have been demonstrated for several CRs after prolonged exposure to their ligands (CXCR2: (Fan et al., 2003); CXCR4: (Y. Zhang et al., 2004); CXCR3: (Meiser et al., 2008); CCR2B: (Garcia Lopez et al., 2009)). Ubiquitination has been demonstrated only for CXCR4 (Marchese et al., 2003). On the contrary, CXCR3 (Meiser et al., 2008) and CXCR2 (Baugher & Richmond, 2008) have been demonstrated to be degraded in lysosomes through an ubiquitin-independent mechanism. Interestingly, CXCR3 also undergoes constitutive degradation in the absence of ligand (Meiser et al., 2008). Finally, lysosomal sorting via Rab7 activity has been demonstrated for CXCR2. Over-expression of a dominant-negative mutant of Rab7 inhibits CXCR2 degradation by tethering internalized receptor in Rab5- and Rab11-positive endosomes and blocking its correct trafficking to Lamp-1-positive lysosomes (Fan et al., 2003). 4. Receptor sequences and adapters involved in trafficking CR internalization and sorting to recycling endosomes or lysosomal degradation involve complex protein–protein interactions. Short peptide “sorting” sequences residing in the cytosolic domains of CRs serve as recognition signals mediating interactions with several endocytic adaptor proteins. These interactions control the rate of CR internalization, recycling, and lysosomal receptor degradation, and hence the magnitude and duration of cellular signaling. 4.1. The di-leucine motif and the adaptor protein AP-2 The C-terminal tail of several GPCRs presents a di-leucin (I/L–I/L) motif that interacts with the clathrin adaptor protein AP-2. This interaction is required for the formation of CCP that ‘pinch off’ from the membrane through the action of dynamin and become clathrincoated vesicles. A di-leucine motif is present in several CRs (Fig. 3), and a direct association with AP-2 has been shown for CXCR2 (Fan et al., 2001) and CXCR4 (Lindsay & McCaffrey, 2005). Furthermore, mutations in this motif also affect CCR5 internalization in a phosphorylation-independent manner (Kraft et al., 2001). On the contrary, mutations of the di-leucin motif LLLRL sequence in the CXCR3 C-terminal domain do not affect its

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Fig. 3. Amino acid sequence of chemokine and chemokine decoy receptors C-terminus domain. Alignment of conventional and non-conventional CR C-terminal tails was performed using the program ClustalW2 (available on at http://www.ebi.ac.uk/Tools/clustalw2/index.html). Conserved motifs are color-coded: blue, di-leucine motif; purple, palmytoilation motif; red, PDZ ligand motif; green, ser/thr motif; yellow, ubiquitination sites.

ligand-induced internalization (Meiser et al., 2008). The functional role of di-leucine motif present in other CRs has not been investigated. 4.2. C-terminal domain ser/thr residues and β-arrestin The arrestin gene family consists of four members that play a crucial role in the regulation of GPCR desensitization, intracellular signaling, and trafficking. Arrestins 1 and 4 are expressed only by visual sensory tissue cells, where they regulate rhodopsin photoreceptor signaling. Arrestins 2 and 3 are ubiquitously expressed and interact with the vast majority of other GPCRs (Reiter & Lefkowitz, 2006). It was proposed that the affinity of the interaction between the GPCR and β-arrestin dictates the intracellular trafficking fate of the receptor. Class A receptors preferentially bind β-arrestin 3 with low affinity and are rapidly recycled to the plasma membrane, while class B receptors which display strong affinity interactions with both β-arrestins 2 and 3, are targeted to endocytic vesicles and slowly recycle back to the cell surface or are targeted to lysosomes for degradation (Oakley et al., 2000). Binding of β-arrestin to GPCR exerts multiple roles on their activity. In the classical view, β-arrestins act as a negative regulator of GPCR activity, desensitizing them through the uncoupling of G proteins. On the other hand, β-arrestins also act as scaffold protein for several members of the c-Src family of tyrosine kinases, allowing internalized receptors to continue signaling. CRs interacts with β-arrestin through phosphorylated serine and threonine residues in their C-terminal tail. Though a direct interaction with β-arrestins has been demonstrated only for CCR5, CXCR3, and CXCR4 (Oppermann et al., 1999; Colvin et al., 2004; Lagane et al., 2008), these residues are required for the internalization of most CRs (CCR1: (Richardson et al., 2000); CCR2: (Arai et al., 1997); CCR5: (Oppermann, 2004); CXCR3: (Colvin et al., 2004); CXCR4: (Lagane et al., 2008)). It was initially supposed that serine/threonine residues in the C-terminal tail

were the only CR residues involved in β-arrestins recruitment. More recently, it has been shown that binding can also involve other CR domains (the DRY motif in the second intracellular loop for CCR5: (Huttenrauch et al., 2002); the SHSK motif in the third intracellular loop for CXCR4: (Cheng et al., 2000)). Interestingly, β-arrestin interaction with different intracellular domains of CR appears to have different functional effects, with the interaction on the C-terminal tail supporting receptor internalization and desensitization and the binding on intracellular loops being required for β-arrestin-mediated signaling events. Despite these general considerations, it has to be remarked that some CRs can internalize in a β-arrestin-independent way (CXCR3 after CXCL11 engagement: (Colvin et al., 2004)) and that truncation of the Cterminal serine/threonine-rich domain in CXCR2 and CXCR4, which blocked arrestin binding to the mutant receptors, did not completely prevent their internalization (Richardson et al., 2003; Lagane et al., 2008). As far as non-conventional CR is concerned, CXCR7 interacts with β-arrestin 3 in basal conditions and this interaction is significantly enhanced by treatment with its ligands CXCL11 and CXCL12. Furthermore, β-arrestin 3 is necessary for CXCR7-dependent uptake of CXCL12 from the extracellular space, as demonstrated by β-arrestin 3 loss of function mutants (Luker et al., 2009). The association of D6 with β-arrestin is still under debate. Galliera et al. reported that D6 associates β-arrestin 2 and 3 in basal conditions and that this interaction is required for its constitutive internalization (Galliera et al., 2004). Conversely, McCulloch et al. demonstrated that β-arrestins re-localization is not required for D6 internalization but the receptor uses both β-arrestins to enhance its stability rather than direct internalization (McCulloch et al., 2008). Despite these discrepancies, the two studies agree that D6 has the potential to drive re-localization of β-arrestins through a presently unknown mechanism, suggesting that D6 interaction with β-arrestins might be of functional relevance, as demonstrated for CXCR7.

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4.3. Chaperone proteins Chaperone molecules, including Heat Shock Proteins (HSP) and Heat Shock Cognate (HSC) proteins, participate in the regulation of protein folding and transport from the endoplasmic reticulum to the cell surface. After ligand engagement CXCR4 binds HSC-73 with its C-terminal tail and the two proteins colocalize in endosomal compartments. CXCR4 interaction with HSC-73 is required for receptor internalization, as assessed by HSC-73 silencing (Ding et al., 2006). Furthermore, CXCR2 and CXCR4 C-terminal domains interact with HSC-70-Interacting protein (HIP) and this interaction is increased by ligand stimulation. HIP binds to the C-terminal leucine-rich domain (KILAIHGLI) of CXCR2, and mutation of the Ile–Leu motif in the C-terminal domain of CXCR2 blocks the agonistdependent association of the mutant receptor with HIP. Inhibition of HIP activity by over-expression of a mutant version unable to activate HSP-70 resulted in partial inhibition of CXCR2 and CXCR4 internalization (Fan et al., 2002). The interpretation given to these results is that HSC-73 and HIP, besides their main chaperone activity, may facilitate receptor conformational change required to undergo internalization. HSC-73 might also modulate clathrin dynamics acting in the release of coat proteins from the nascent vesicles, a process required for the correct fusion with endosomes (Hannan et al., 1998). 4.4. Motor and cytoskeleton proteins Most vesicular trafficking events are dependent on cytoskeleton and motor proteins. CCR5 and CXCR4 C-terminal domains interact with nonmuscle conventional myosin (Myo) IIA (Rey et al., 2002), also involved in cell motility and adhesion (Vicente-Manzanares et al., 2009), and this interaction is required for their ligand-induced internalization. MyoIIA also constitutively interacts with β-arrestin, and CR stimulation promotes a rapid MyoIIA/β-arrestin dissociation. It has been proposed that MIIA works as an adapter molecule linking CR to both β-arrestin and the cytoskeleton (Rey et al., 2007). Myo V is a non-conventional myosin widely implicated in vesicle and organelle trafficking (Desnos et al., 2007). Myo-Vb in particular associates with RE and regulates the trafficking of a variety of receptors from RE to the plasma membrane due to its interaction with Rab11 and its effector Rab11-Family Interacting Protein 2 (Rab11-FIP2) (Lapierre et al., 2001). Myo-Vb activity and its interaction with Rab11-FIP2 and Rab11 are required for the correct recycling and resensitization of CXCR2 (Fan et al., 2004), but its role for other CRs is presently unknown. Besides motor proteins, the Rho family of small GTPases is critical regulators of actin dynamics and has been involved in the control of endocytosis and recycling. While RhoA has been mainly implicated in the regulation of receptor internalization, RhoB appears to play an important role in sorting decision and intracellular trafficking of GPCR (Wheeler & Ridley, 2004). Referring to CRs, RhoB is involved in CXCR2 sorting as inhibition of its activity causes CXCR2 missorting to the Rab4dependent rapid recycling pathway rather than targeting to lysosomes or Rab11 RE after chemokine engagement (Neel et al., 2007). Other CR-interacting proteins related to cytoskeleton have been identified, including VASP (Neel et al., 2009) and filamin A (JimenezBaranda et al., 2007), but their role in CR internalization and recycling has not been addressed. 4.5. PDZ ligand motif and interacting proteins An internal PDZ ligand motif serves as a post-endocytic trafficking motif for several GPCRs. Based on the consensus sequence, PDZ ligands can be classified into type I (X–S/T–X–Φ) and type II (X–Φ– X–Φ), where X is any amino acid and Φ is a hydrophobic amino acid (Trejo, 2005). Both types of PDZ ligands are present in the C-terminal tail of some CR. The C-terminal tip of CCR5 contains the type II PDZ ligand motif SVGL which is required for receptor recycling to the cell surface (Delhaye et al., 2007). Conversely, the CXCR2 C-terminal tail

contains the type I PDZ ligand motif STLL, which prevents its association with the lysosomal sorting machinery, thus delaying its sorting to lysosomes and degradation (Baugher & Richmond, 2008). Interestingly, CXCR3 is constitutively degraded in the absence of ligand through a process that is mediated by the type I PDZ ligand motif YSGL present at the extreme of its C-terminal domain (Meiser et al., 2008). Several proteins have been reported to interact with PDZ ligand domains and control the fate of internalized GPCRs. As CRs are concerned, CXCR2 has been reported to interact with GASP, a protein implicated in selective lysosomal sorting (Heydorn et al., 2004), and 143-3 scaffolding proteins (Baugher & Richmond, 2008). However, the functional implications of these interactions have not been elucidated. 4.6. Ubiquitin and ubiquitination motifs Ubiquitination is a post-transcriptional modification of a protein that consists in the covalent binding of one or more ubiquitin monomers. The main function of this modification is to target protein to degradation to both proteasome and lysosomes. It is also being increasingly appreciated that deubiquitination also regulates the fate and function of ubiquitinconjugated receptors, though mechanisms and functional consequences of protein deubiquitination are less understood than ubiquitination mechanisms (Komada, 2008). While CXCR2 and CXCR3 have been demonstrated to be degraded in lysosomes through a ubiquitinindependent mechanism (Baugher & Richmond, 2008; Meiser et al., 2008), ubiquitin-mediated degradation has been demonstrated for CXCR4 (Marchese & Benovic, 2001). CXCR4 ubiquitination is an agonistdependent process occurring at the plasma membrane on lysine residues present in the SSLKILSKGK motif in its C-terminal tail by the activity of the E3 ubiquitin ligase atrophin interacting protein 4 (AIP4), which directly interacts with the phosphorylated serine residues 324 and 325 within the C-terminal tail of the receptor (Bhandari et al., 2009). Upon internalization, ubiquitinated CXCR4 and AIP4 traffic to endosomes where they colocalize with the ubiquitin-binding endosomal protein HRS. AIP4 also mediates CXCR4-dependent ubiquitination of HRS and the recruiting of VPS-4, an AAA ATPase possibly involved in CXCR4 deubiquination (Marchese et al., 2003). Ubiquitination-dependent CXCR4 degradation is inhibited by the cytokine-independent survival kinase CISK, which phosphorylates AIP4 (Slagsvold et al., 2006), and by the deubiquitinating enzyme USP14, which binds CXCR4 and prevents its degradation without affecting its internalization and signaling (Mines et al., 2009). It has also been reported that the AIP4mediated CXCR4 degradation is facilitated by β-arrestin 1, which binds the phosphorylated receptor at the plasma membrane, co-internalizes as a complex, and once on endosomes interacts with the endosomal pool of AIP4 (Bhandari et al., 2007, 2009). Thus, CXCR4 undergoes a ligand-modulated ubiquitination–deubiquitination cycle that not only modulates receptor degradation but also CXCL12-evoked chemotaxis. 5. Concluding remarks Chemokines and their receptors are involved in almost all diseases and are ideal targets for therapeutic intervention, and indeed the first CR antagonists have been recently approved. Similarly to other GPCRs, CR signaling and intracellular traffic properties appear to be strictly interconnected, and fragmentary information present in the literature indicate that every CR has unique trafficking mechanisms that rely on different sets of interacting molecules. Despite the enormous interest in CRs for pharmaceutical intervention, receptor trafficking has not yet been targeted as a therapeutic approach despite the fact that inhibition of CCR5 recycling exerted by AOP-RANTES has been the starting point for the development of HIV inhibitors as microbiocidal drugs. We believe that better understanding of their trafficking properties and of the interplay with receptor signaling will be beneficial for the development of new therapeutic strategies.

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Acknowledgments This work was supported by research grants of the European Community (INNOCHEM project 518167), the Ministero dell'Istruzione dell'Università e della Ricerca (Progetti di Ricerca di Interesse Nazionale projects 2002061255 and 2007ENYMAN_003), and the Fondazione Cariplo (NOBEL project and #2008/2279). This work was conducted with the support of the Fondazione Humanitas per la Ricerca and the Italian Association for Cancer Research (AIRC). EMB is the recipient of an AIRC fellowship. References Anderson, R. G. (1998). The caveolae membrane system. Annu Rev Biochem 67, 199−225. Arai, H., Monteclaro, F. S., Tsou, C. L., Franci, C., & Charo, I. F. (1997). Dissociation of chemotaxis from agonist-induced receptor internalization in a lymphocyte cell line transfected with CCR2B. Evidence that directed migration does not require rapid modulation of signaling at the receptor level. J Biol Chem 272, 25037−25042. Barlic, J., Khandaker, M. H., Mahon, E., Andrews, J., DeVries, M. E., Mitchell, G. B., et al. (1999). beta-arrestins regulate interleukin-8-induced CXCR1 internalization. J Biol Chem 274, 16287−16294. Baugher, P. J., & Richmond, A. (2008). The carboxyl-terminal PDZ ligand motif of chemokine receptor CXCR2 modulates post-endocytic sorting and cellular chemotaxis. J Biol Chem 283, 30868−30878. Bhandari, D., Robia, S. L., & Marchese, A. (2009). The E3 ubiquitin ligase atrophin interacting protein 4 binds directly to the chemokine receptor CXCR4 via a novel WW domain-mediated interaction. Mol Biol Cell 20, 1324−1339. Bhandari, D., Trejo, J., Benovic, J. L., & Marchese, A. (2007). Arrestin-2 interacts with the ubiquitin–protein isopeptide ligase atrophin-interacting protein 4 and mediates endosomal sorting of the chemokine receptor CXCR4. J Biol Chem 282, 36971−36979. Boldajipour, B., Mahabaleshwar, H., Kardash, E., Reichman-Fried, M., Blaser, H., Minina, S., et al. (2008). Control of chemokine-guided cell migration by ligand sequestration. Cell 132, 463−473. Bonecchi, R., Borroni, E. M., Anselmo, A., Doni, A., Savino, B., Mirolo, M., et al. (2008). Regulation of D6 chemokine scavenging activity by ligand- and Rab11-dependent surface up-regulation. Blood 112, 493−503. Bonecchi, R., Galliera, E., Borroni, E. M., Corsi, M. M., Locati, M., & Mantovani, A. (2009). Chemokines and chemokine receptors: An overview. Front Biosci 14, 540−551. Bonecchi, R., Locati, M., Galliera, E., Vulcano, M., Sironi, M., Fra, A. M., et al. (2004). Differential recognition and scavenging of native and truncated macrophagederived chemokine (macrophage-derived chemokine/CC chemokine ligand 22) by the D6 decoy receptor. J Immunol 172, 4972−4976. Borroni, E. M., & Bonecchi, R. (2009). Shaping the gradient by nonchemotactic chemokine receptors. Cell Adh Migr 3, 146−147. Borroni, E. M., Bonecchi, R., Mantovani, A., & Locati, M. (2009). Chemoattractant receptors and leukocyte recruitment: More than cell migration. Sci Signal 2, pe10. Borroni, E. M., Savino, B., Cancellieri, C., Mirolo, M., Buracchi, C., Anselmo, A., Mantovani, A., Locati, M., Bonecchi, R. Myosin Vb mobilizes the atypical chemokine receptor D6 from recycling endosomes for adaptive up-regulation and chemokine degradation. Manuscript in preparation. Byers, M. A., Calloway, P. A., Shannon, L., Cunningham, H. D., Smith, S., Li, F., et al. (2008). Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21. J Immunol 181, 4723−4732. Cardaba, C. M., Kerr, J. S., & Mueller, A. (2008). CCR5 internalisation and signalling have different dependence on membrane lipid raft integrity. Cell Signal 20, 1687−1694. Charo, I. F., & Ransohoff, R. M. (2006). The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med 354, 610−621. Cheng, Z. J., Zhao, J., Sun, Y., Hu, W., Wu, Y. L., Cen, B., et al. (2000). beta-arrestin differentially regulates the chemokine receptor CXCR4-mediated signaling and receptor internalization, and this implicates multiple interaction sites between beta-arrestin and CXCR4. J Biol Chem 275, 2479−2485. Colvin, R. A., Campanella, G. S., Sun, J., & Luster, A. D. (2004). Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function. J Biol Chem 279, 30219−30227. Comerford, I., Milasta, S., Morrow, V., Milligan, G., & Nibbs, R. (2006). The chemokine receptor CCX–CKR mediates effective scavenging of CCL19 in vitro. Eur J Immunol 36, 1904−1916. Dagan-Berger, M., Feniger-Barish, R., Avniel, S., Wald, H., Galun, E., Grabovsky, V., et al. (2006). Role of CXCR3 carboxyl terminus and third intracellular loop in receptormediated migration, adhesion and internalization in response to CXCL11. Blood 107, 3821−3831. Delhaye, M., Gravot, A., Ayinde, D., Niedergang, F., Alizon, M., & Brelot, A. (2007). Identification of a postendocytic sorting sequence in CCR5. Mol Pharmacol 72, 1497−1507. Desnos, C., Huet, S., & Darchen, F. (2007). ‘Should I stay or should I go?’: Myosin V function in organelle trafficking. Biol Cell 99, 411−423. Ding, Y., Li, M., Zhang, J., Li, N., Xia, Z., Hu, Y., et al. (2006). The 73-kDa heat shock cognate protein is a CXCR4 binding protein that regulates the receptor endocytosis and the receptor-mediated chemotaxis. Mol Pharmacol 69, 1269−1279. Elsner, J., Mack, M., Bruhl, H., Dulkys, Y., Kimmig, D., Simmons, G., et al. (2000). Differential activation of CC chemokine receptors by AOP-RANTES. J Biol Chem 275, 7787−7794. Fan, G. H., Lapierre, L. A., Goldenring, J. R., & Richmond, A. (2003). Differential regulation of CXCR2 trafficking by Rab GTPases. Blood 101, 2115−2124.

7

Fan, G. H., Lapierre, L. A., Goldenring, J. R., Sai, J., & Richmond, A. (2004). Rab11-family interacting protein 2 and myosin Vb are required for CXCR2 recycling and receptormediated chemotaxis. Mol Biol Cell 15, 2456−2469. Fan, G. H., Yang, W., Sai, J., & Richmond, A. (2001). Phosphorylation-independent association of CXCR2 with the protein phosphatase 2A core enzyme. J Biol Chem 276, 16960−16968. Fan, G. H., Yang, W., Sai, J., & Richmond, A. (2002). Hsc/Hsp70 interacting protein (hip) associates with CXCR2 and regulates the receptor signaling and trafficking. J Biol Chem 277, 6590−6597. Feniger-Barish, R., Belkin, D., Zaslaver, A., Gal, S., Dori, M., Ran, M., et al. (2000). GCP-2induced internalization of IL-8 receptors: Hierarchical relationships between GCP-2 and other ELR(+)–CXC chemokines and mechanisms regulating CXCR2 internalization and recycling. Blood 95, 1551−1559. Fraile-Ramos, A., Kohout, T. A., Waldhoer, M., & Marsh, M. (2003). Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway. Traffic 4, 243−253. Futahashi, Y., Komano, J., Urano, E., Aoki, T., Hamatake, M., Miyauchi, K., et al. (2007). Separate elements are required for ligand-dependent and -independent internalization of metastatic potentiator CXCR4. Cancer Sci 98, 373−379. Galliera, E., Jala, V. R., Trent, J. O., Bonecchi, R., Signorelli, P., Lefkowitz, R. J., et al. (2004). beta-Arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6. J Biol Chem 279, 25590−25597. Garcia Lopez, M. A., Aguado Martinez, A., Lamaze, C., Martinez, A. C., & Fischer, T. (2009). Inhibition of dynamin prevents CCL2-mediated endocytosis of CCR2 and activation of ERK1/2. Cell Signal 21, 1748−1757. Graham, G. J. (2009). D6 and the atypical chemokine receptor family: novel regulators of immune and inflammatory processes. Eur J Immunol 39, 342−351. Hannan, L. A., Newmyer, S. L., & Schmid, S. L. (1998). ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: A dual role for Hsc70. Mol Biol Cell 9, 2217−2229. Hanyaloglu, A. C., & von Zastrow, M. (2008). Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu Rev Pharmacol Toxicol 48, 537−568. Heydorn, A., Sondergaard, B. P., Ersboll, B., Holst, B., Nielsen, F. C., Haft, C. R., et al. (2004). A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor–associated sorting protein (GASP). J Biol Chem 279, 54291−54303. Hirsch, E., Katanaev, V. L., Garlanda, C., Azzolino, O., Pirola, L., Silengo, L., et al. (2000). Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science 287, 1049−1053. Hunyady, L., Baukal, A. J., Gaborik, Z., Olivares-Reyes, J. A., Bor, M., Szaszak, M., et al. (2002). Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor. J Cell Biol 157, 1211−1222. Huttenrauch, F., Nitzki, A., Lin, F. T., Honing, S., & Oppermann, M. (2002). Beta-arrestin binding to CC chemokine receptor 5 requires multiple C-terminal receptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif. J Biol Chem 277, 30769−30777. Jimenez-Baranda, S., Gomez-Mouton, C., Rojas, A., Martinez-Prats, L., Mira, E., Ana Lacalle, R., et al. (2007). Filamin-A regulates actin-dependent clustering of HIV receptors. Nat Cell Biol 9, 838−846. Komada, M. (2008). Controlling receptor downregulation by ubiquitination and deubiquitination. Curr Drug Discov Technol 5, 78−84. Kraft, K., Olbrich, H., Majoul, I., Mack, M., Proudfoot, A., & Oppermann, M. (2001). Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization. J Biol Chem 276, 34408−34418. Lagane, B., Chow, K. Y., Balabanian, K., Levoye, A., Harriague, J., Planchenault, T., et al. (2008). CXCR4 dimerization and beta-arrestin-mediated signaling account for the enhanced chemotaxis to CXCL12 in WHIM syndrome. Blood 112, 34−44. Lapierre, L. A., Kumar, R., Hales, C. M., Navarre, J., Bhartur, S. G., Burnette, J. O., et al. (2001). Myosin vb is associated with plasma membrane recycling systems. Mol Biol Cell 12, 1843−1857. Lindsay, A. J., & McCaffrey, M. W. (2005). Purification and functional properties of Rab11-FIP2. Methods Enzymol 403, 491−499. Locati, M., Torre, Y. M., Galliera, E., Bonecchi, R., Bodduluri, H., Vago, G., et al. (2005). Silent chemoattractant receptors: D6 as a decoy and scavenger receptor for inflammatory CC chemokines. Cytokine Growth Factor Rev 16, 679−686. Luker, K. E., Gupta, M., Steele, J. M., Foerster, B. R., & Luker, G. D. (2009). Imaging liganddependent activation of CXCR7. Neoplasia 11, 1022−1035. Mack, M., Luckow, B., Nelson, P. J., Cihak, J., Simmons, G., Clapham, P. R., et al. (1998). Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity. J Exp Med 187, 1215−1224. Mantovani, A., Bonecchi, R., & Locati, M. (2006). Tuning inflammation and immunity by chemokine sequestration: Decoys and more. Nat Rev Immunol 6, 907−918. Marchese, A., & Benovic, J. L. (2001). Agonist-promoted ubiquitination of the G proteincoupled receptor CXCR4 mediates lysosomal sorting. J Biol Chem 276, 45509−45512. Marchese, A., Paing, M. M., Temple, B. R., & Trejo, J. (2008). G protein-coupled receptor sorting to endosomes and lysosomes. Annu Rev Pharmacol Toxicol 48, 601−629. Marchese, A., Raiborg, C., Santini, F., Keen, J. H., Stenmark, H., & Benovic, J. L. (2003). The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G proteincoupled receptor CXCR4. Dev Cell 5, 709−722. Mariani, M., Lang, R., Binda, E., Panina-Bordignon, P., & D'Ambrosio, D. (2004). Dominance of CCL22 over CCL17 in induction of chemokine receptor CCR4 desensitization and internalization on human Th2 cells. Eur J Immunol 34, 231−240.

8

E.M. Borroni et al. / Pharmacology & Therapeutics 127 (2010) 1–8

McCulloch, C. V., Morrow, V., Milasta, S., Comerford, I., Milligan, G., Graham, G. J., et al. (2008). Multiple roles for the C-terminal tail of the chemokine scavenger D6. J Biol Chem 283, 7972−7982. Meiser, A., Mueller, A., Wise, E. L., McDonagh, E. M., Petit, S. J., Saran, N., et al. (2008). The chemokine receptor CXCR3 is degraded following internalization and is replenished at the cell surface by de novo synthesis of receptor. J Immunol 180, 6713−6724. Mines, M. A., Goodwin, J. S., Limbird, L. E., Cui, F. F., & Fan, G. H. (2009). Deubiquitination of CXCR4 by USP14 is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK ativation. J Biol Chem 284, 5742−5752. Mueller, A., Kelly, E., & Strange, P. G. (2002). Pathways for internalization and recycling of the chemokine receptor CCR5. Blood 99, 785−791. Mueller, A., & Strange, P. G. (2004). Mechanisms of internalization and recycling of the chemokine receptor, CCR5. Eur J Biochem 271, 243−252. Nasser, M. W., Marjoram, R. J., Brown, S. L., & Richardson, R. M. (2005). Crossdesensitization among CXCR1, CXCR2, and CCR5: Role of protein kinase C-epsilon. J Immunol 174, 6927−6933. Neel, N. F., Barzik, M., Raman, D., Sobolik-Delmaire, T., Sai, J., Ham, A. J., et al. (2009). VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis. J Cell Sci 122, 1882−1894. Neel, N. F., Lapierre, L. A., Goldenring, J. R., & Richmond, A. (2007). RhoB plays an essential role in CXCR2 sorting decisions. J Cell Sci 120, 1559−1571. Neel, N. F., Schutyser, E., Sai, J., Fan, G. H., & Richmond, A. (2005). Chemokine receptor internalization and intracellular trafficking. Cytokine Growth Factor Rev 16, 637−658. Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G., & Barak, L. S. (2000). Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J Biol Chem 275, 17201−17210. Oppermann, M. (2004). Chemokine receptor CCR5: Insights into structure, function, and regulation. Cell Signal 16, 1201−1210. Oppermann, M., Mack, M., Proudfoot, A. E., & Olbrich, H. (1999). Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus. J Biol Chem 274, 8875−8885. Otero, C., Groettrup, M., & Legler, D. F. (2006). Opposite fate of endocytosed CCR7 and its ligands: Recycling versus degradation. J Immunol 177, 2314−2323. Pollok-Kopp, B., Schwarze, K., Baradari, V. K., & Oppermann, M. (2003). Analysis of ligand-stimulated CC chemokine receptor 5 (CCR5) phosphorylation in intact cells using phosphosite-specific antibodies. J Biol Chem 278, 2190−2198. Prevo, R., Banerji, S., Ni, J., & Jackson, D. G. (2004). Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/ homing receptor stabilin-1 (FEEL-1/CLEVER-1). J Biol Chem 279, 52580−52592. Pruenster, M., Mudde, L., Bombosi, P., Dimitrova, S., Zsak, M., Middleton, J., et al. (2009). The Duffy antigen receptor for chemokines transports chemokines and supports their promigratory activity. Nat Immunol 10, 101−108. Pruenster, M., & Rot, A. (2006). Throwing light on DARC. Biochem Soc Trans 34, 1005−1008. Reiter, E., & Lefkowitz, R. J. (2006). GRKs and beta-arrestins: Roles in receptor silencing, trafficking and signaling. Trends Endocrinol Metab 17, 159−165. Rey, M., Valenzuela-Fernandez, A., Urzainqui, A., Yanez-Mo, M., Perez-Martinez, M., Penela, P., et al. (2007). Myosin IIA is involved in the endocytosis of CXCR4 induced by SDF-1alpha. J Cell Sci 120, 1126−1133. Rey, M., Vicente-Manzanares, M., Viedma, F., Yanez-Mo, M., Urzainqui, A., Barreiro, O., et al. (2002). Cutting edge: Association of the motor protein nonmuscle myosin heavy chain-IIA with the C terminus of the chemokine receptor CXCR4 in T lymphocytes. J Immunol 169, 5410−5414. Richardson, R. M., Marjoram, R. J., Barak, L. S., & Snyderman, R. (2003). Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J Immunol 170, 2904−2911. Richardson, R. M., Pridgen, B. C., Haribabu, B., & Snyderman, R. (2000). Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2. J Biol Chem 275, 9201−9208. Sadowski, L., Pilecka, I., & Miaczynska, M. (2009). Signaling from endosomes: Location makes a difference. Exp Cell Res 315, 1601−1609. Savino, B., Borroni, E. M., Torres, N. M., Proost, P., Struyf, S., Mortier, A., et al. (2009). Recognition versus adaptive up-regulation and degradation of CC chemokines by

the chemokine decoy receptor D6 are determined by their N-terminal sequence. J Biol Chem 284, 26207−26215. Scandella, E., Men, Y., Legler, D. F., Gillessen, S., Prikler, L., Ludewig, B., et al. (2004). CCL19/ CCL21-triggered signal transduction and migration of dendritic cells requires prostaglandin E2. Blood 103, 1595−1601. Schaer, D. J., Schaer, C. A., Buehler, P. W., Boykins, R. A., Schoedon, G., Alayash, A. I., et al. (2006). CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin. Blood 107, 373−380. Signoret, N., Hewlett, L., Wavre, S., Pelchen-Matthews, A., Oppermann, M., & Marsh, M. (2005). Agonist-induced endocytosis of CC chemokine receptor 5 is clathrin dependent. Mol Biol Cell 16, 902−917. Signoret, N., Oldridge, J., Pelchen-Matthews, A., Klasse, P. J., Tran, T., Brass, L. F., et al. (1997). Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4. J Cell Biol 139, 651−664. Signoret, N., Pelchen-Matthews, A., Mack, M., Proudfoot, A. E., & Marsh, M. (2000). Endocytosis and recycling of the HIV coreceptor CCR5. J Cell Biol 151, 1281−1294. Slagsvold, T., Marchese, A., Brech, A., & Stenmark, H. (2006). CISK attenuates degradation of the chemokine receptor CXCR4 via the ubiquitin ligase AIP4. Embo J 25, 3738−3749. Sorkin, A., & von Zastrow, M. (2009). Endocytosis and signalling: intertwining molecular networks. Nat Rev Mol Cell Biol 10, 609−622. Thelen, M. (2001). Dancing to the tune of chemokines. Nat Immunol 2, 129−134. Thelen, M., & Stein, J. V. (2008). How chemokines invite leukocytes to dance. Nat Immunol 9, 953−959. Trejo, J. (2005). Internal PDZ ligands: Novel endocytic recycling motifs for G proteincoupled receptors. Mol Pharmacol 67, 1388−1390. van Dam, E. M., Ten Broeke, T., Jansen, K., Spijkers, P., & Stoorvogel, W. (2002). Endocytosed transferrin receptors recycle via distinct dynamin and phosphatidylinositol 3-kinase-dependent pathways. J Biol Chem 277, 48876−48883. Venkatesan, S., Rose, J. J., Lodge, R., Murphy, P. M., & Foley, J. F. (2003). Distinct mechanisms of agonist-induced endocytosis for human chemokine receptors CCR5 and CXCR4. Mol Biol Cell 14, 3305−3324. Vicente-Manzanares, M., Ma, X., Adelstein, R. S., & Horwitz, A. R. (2009). Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol 10, 778−790. Weber, M., Blair, E., Simpson, C. V., O'Hara, M., Blackburn, P. E., Rot, A., et al. (2004). The chemokine receptor D6 constitutively traffics to and from the cell surface to internalize and degrade chemokines. Mol Biol Cell 15, 2492−2508. Wheeler, A. P., & Ridley, A. J. (2004). Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res 301, 43−49. Xanthou, G., Williams, T. J., & Pease, J. E. (2003). Molecular characterization of the chemokine receptor CXCR3: Evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation. Eur J Immunol 33, 2927−2936. Yang, W., Wang, D., & Richmond, A. (1999). Role of clathrin-mediated endocytosis in CXCR2 sequestration, resensitization, and signal transduction. J Biol Chem 274, 11328−11333. Zabel, B. A., Nakae, S., Zuniga, L., Kim, J. Y., Ohyama, T., Alt, C., et al. (2008). Mast cellexpressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis. J Exp Med 205, 2207−2220. Zerial, M., & McBride, H. (2001). Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2, 107−117. Zhang, N., Yang, D., Dong, H., Chen, Q., Dimitrova, D. I., Rogers, T. J., et al. (2006). Adenosine A2a receptors induce heterologous desensitization of chemokine receptors. Blood 108, 38−44. Zhang, Y., Foudi, A., Geay, J. F., Berthebaud, M., Buet, D., Jarrier, P., et al. (2004). Intracellular localization and constitutive endocytosis of CXCR4 in human CD34+ hematopoietic progenitor cells. Stem Cells 22, 1015−1029. Zidar, D. A., Violin, J. D., Whalen, E. J., & Lefkowitz, R. J. (2009). Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands. Proc Natl Acad Sci U S A 106, 9649−9654. Zimmermann, N., Conkright, J. J., & Rothenberg, M. E. (1999). CC chemokine receptor-3 undergoes prolonged ligand-induced internalization. J Biol Chem 274, 12611−12618.