Constitutively active chemokine CXC receptors.

CHAPTER NINE

Constitutively Active Chemokine CXC Receptors Xinbing Han1 Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 1.1 GPCR signaling pathways 1.2 CXC receptor members, ligands, cellular location, and disease involvement 1.3 3D structure of CXC chemokine receptors 1.4 Constitutively active CXC chemokine receptors 2. Chemokine CXC Receptors 2.1 CXCR1 2.2 CXCR2 2.3 CXCR3 2.4 CXCR4 2.5 CXCR5 2.6 CXCR6 2.7 CXCR7 2.8 KSHV-GPCR 3. Conclusion Conflict of Interest References

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Abstract Chemokines are low-molecular-weight, secreted proteins that act as leukocyte-specific chemoattractants. The chemokine family has more than 40 members. Based on the position of two conserved cysteines in the N-terminal domain, chemokines can be divided into the CXC, C, CC, and CX3C subfamilies. The interaction of chemokines with their receptors mediates signaling pathways that play critical roles in cell migration, differentiation, and proliferation. The receptors for chemokines are G protein-coupled receptors (GPCRs), and thus far, seven CXC receptors have been cloned and are designated CXCR1–7. Constitutively active GPCRs are present in several human immunemediated diseases and in tumors, and they have provided valuable information in understanding the molecular mechanism of GPCR activation. Several constitutively active CXC chemokine receptors include the V6.40A and V6.40N mutants of CXCR1; the D3.49V variant of CXCR2; the N3.35A, N3.35S, and T2.56P mutants of CXCR3; the

Advances in Pharmacology, Volume 70 ISSN 1054-3589 http://dx.doi.org/10.1016/B978-0-12-417197-8.00009-2

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N3.35 mutation of CXCR4; and the naturally occurring KSHV-GPCR. Here, we review the regulation of CXC chemokine receptor signaling, with a particular focus on the constitutive activation of these receptors and the implications in physiological conditions and in pathogenesis. Understanding the mechanisms behind the constitutive activation of CXC chemokine receptors may aid in pharmaceutical design and the screening of inverse agonists and allosteric modulators for the treatment of autoimmune diseases and cancers.

ABBREVIATIONS GPCR G protein-coupled receptor TM transmembrane a helix COPD chronic obstructive pulmonary disease CXCL1 growth-related oncogene-a (GRO-a) CXCL2 growth-related oncogene-b (GRO-b) CXCL3 growth-related oncogene-g (GRO-g) CXCL4 platelet factor 4 (PF4) CXCL5 epithelial neutrophil-activating peptide-78 (ENA-78) CXCL6 granulocyte chemotactic protein-2 (GCP-2) CXCL7 neutrophil-activating peptide-2 (NAP-2) CXCL8 interleukin-8 (IL-8) CXCL9 monokine induced by interferon-g (MIG) CXCL10 interferon-g-inducible protein-10 (IP-10) CXCL11 IFN-inducible T-cell a-chemoattractant (I-TAC) CXCL12 stromal cell-derived factor 1a (SDF-1a) CXCL13 B-lymphocyte chemoattractant (BCA-1) CXCL16 Bonzo KS Kaposi’s sarcoma KSHV Kaposi’s sarcoma-associated herpesvirus ECL extracellular loop of GPCR ICL intracellular loop of GPCR CAM constitutively active mutants Tfh follicular B helper T cells

1. INTRODUCTION 1.1. GPCR signaling pathways The G protein-coupled receptor (GPCR) family comprises approximately 2–4% of the encoded human gene and has more than 800 members, representing the largest family of cell-surface receptors involved in signal transduction and the largest family of drug target proteins to date (Fredriksson & Schioth, 2005). Indeed, GPCRs are the target of more than 25–50% of

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therapeutic drugs on the market (O’Hayre et al., 2013; Pierce, Premont, & Lefkowitz, 2002). GPCRs are characterized by a seven-transmembrane domain structure with an extracellular amino terminus, an intracellular carboxyl terminus, and three interhelical loops to the extracellular space and three interhelical loops into the cytoplasm. GPCRs are present in virtually all eukaryotic cells and have a broad repertoire of ligands that include light, lipids, nucleotides, polypeptides, and proteins. GPCRs function as key transducers of signals from the extracellular milieu to the inside of the cell. Recent discoveries in GPCR biology support the concept that GPCRs can exhibit different conformational states that are stabilized by different classes of ligands, leading to the activation of variable intracellular signaling pathways (O’Hayre et al., 2013; Pierce et al., 2002). The fact that several ligands can bind to the same chemokine receptors and the same ligand can interact with different chemokine receptors suggests temporal and spatial complexity and a variety of signaling activity initiated by chemokine receptors and their ligands. Based on their efficacy, GPCR ligands are divided into four categories: full agonists, partial agonists, inverse agonists, and allosteric modulators. Of note, ligand efficacy is independent of affinity to receptor. Although it was postulated that there were a limited number of defined receptor conformations representing inactive, active, and some intermediate states of activation, more and more evidence indicates that GPCRs can possess multiple biding sites and multiple conformations that modulate multiple signal pathways. It is well established that different ligands selectively stabilize different “active” conformations of GPCRs to modulate different pathways selectively (Paavola & Hall, 2012). This concept has drawn considerable attention because selective activation of beneficial signal pathways without activating other pathways may offer a therapeutic advantage and may help to assess the efficacy of inverse agonists of GPCRs. The activation of GPCRs is due to the disruption of key interhelical contacts. This activation involves the rotation of TM3 and TM6 domains and affects the conformational structure of G protein-interacting cytoplasmic loops (CLs) of the receptor, thereby uncovering previously masked G protein-binding sites on the intracellular loops. The result is a conformational shift of the hydrophobic core and cytoplasmic domains to a state permissive for the formation of a high-affinity ternary complex with Ga subunits (Vauquelin & Van Liefde, 2005). Among the identified motifs critical for G protein coupling and receptor activation, one example is the polar

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interaction between the highly conserved E/DRY motif on TM3 and a glutamate residue on TM6, which forms an “ionic lock” to bridge intracellular TM helices (Rosenbaum, Rasmussen, & Kobilka, 2009). On ligand binding, TM6 moves outward from the center of the bundle, forming a new pocket between TM3, TM5, and TM6 that binds to the C-terminus of a Ga subunit (Rasmussen, Choi, et al., 2011). It is believed that the mutation of multiple residues at the interhelical interface of TM3, TM5, and TM6 might alter conformation states and facilitate coupling of GPCR to G proteins, leading to ligand-independent GPCR constitutive activity. This concept was supported by the constitutive activation of chemokine receptors CXCR1 (V247 on TM6.40 position of CXCR1) (Han, Tachado, Koziel, & Boisvert, 2012), CXCR4 (N114A and N114S) (Zhang et al., 2002), and Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor (KSHV-GPCR).

1.2. CXC receptor members, ligands, cellular location, and disease involvement Chemokines are secreted small cytokines with low molecular mass (8–10 kDa); they are important mediators with chemotactic and proactivatory effects on different leukocyte lineages in inflammation. More than 40 chemokines have been identified. Based on the number and position of conserved N-terminal cysteine residues, chemokines are classified into four families, that is, CC, CXC, CX3C, and C chemokines (Murphy et al., 2000). The presence of four conservative cysteine residues in chemokines is key to forming three-dimensional structure. The CC chemokines have two adjacent cysteine residues in their amino terminus, and there are at least 27 members of this subgroup named CC chemokine ligands (CCL) 1 to 28. The two N-terminal cysteines of CXC chemokines are separated by one amino acid (named “X”). There are 17 different CXC chemokines that are subdivided into two groups, depending on whether they contain a glutamic acid-leucine-arginine (ELR) motif before the first cysteine of the CXC motif: ELR-positive CXC chemokines, which specifically induce neutrophil migration by interacting with CXCR1 and CXCR2, and ELR-negative CXC chemokines, which tend to be chemoattractants for lymphocytes. CX3C chemokines have thee amino acids between the two cysteines. C chemokine has only two cysteines, making it distinct from other chemokines, which have four cysteines. All of these chemokines exert their biological effects by interacting with GPCRs called chemokine receptors. Approximately 21 receptors have been identified:


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10 for CC chemokines, 7 for CXC chemokines, 1 for C chemokines, 1 for the CX3C chemokine, and 2 for nonsignaling binding heptahelical proteins. There is significant redundancy in the repertoire of chemokine- and receptor-binding activities. Seven CXC chemokine receptors have been discovered to date, designated CXCR1–7. The chemokine CXC receptors are primarily located on the surface of various immune cells, and they are involved in autoimmune diseases, cancer, and many other diseases (Table 9.1). The central roles of CXC chemokine receptors in a number of physiological and pathological situations, including leukocyte responses, hematopoiesis, homeostasis, angiogenesis, tissue maintenance or development, and tumor metastasis and tumor cell survival, have been well established. In this review, we will only focus on the CXC chemokine receptor family.

1.3. 3D structure of CXC chemokine receptors Over the past few years, the three-dimensional structures of several GPCRs in various activation states have been revealed, a finding that has thrown light onto the understanding of GPCR structure and function. To date, the 3D structures of two chemokine receptors, CXCR4 (Wu et al., 2010) and CXCR1 (Park et al., 2012), have been identified. The structure information of GPCRs could be useful for the development of therapeutic agents and the generation of lead compounds as a strategy for the so-called rational drug design.

1.4. Constitutively active CXC chemokine receptors Human chemokine receptors generally do not show high levels that signal in the absence of ligands. However, several constitutively active chemokine receptor mutants have been described that signal in the absence of ligands. Examples are naturally occurring KSHV-GPCR, CXCR1 V247A, and V247N mutants (Han et al., 2012); CXCR2 D3.49V (Burger et al., 1999); CXCR3 N3.35A (Verzijl et al., 2008); and a mutation of N3.35 of CXCR4 (Zhang et al., 2002) (Table 9.2). Single-point mutations can change the receptor conformation leading to constitutive activation, thereby mimicking the active state of the wild-type receptor. Indeed, receptor mutants have proved to be very useful to derive conformational models of receptor activation indirectly from pharmacological and biochemical data (Berchiche et al., 2007).

Table 9.1 CXC chemokine receptors involved in disease and their expression in immune cells CXC chemokine receptors Ligands Cellular location Disease involvement

CXCR1

6, 8

N, M, T, NK, Bs, Ms, En

Chronic obstructive pulmonary disease (COPD), asthma, inflammatory bowel diseases, and Crohn’s disease, and tumor

CXCR2

1, 2, 3, 5, 6, 7, 8

N, Eo, M, T, NK, COPD, cystic fibrosis, emphysema, Ms, As, Nn, Ms, En ischemia, psoriasis, transplantation, chronic inflammation, sepsis, atherosclerosis, neuroinflammation, and tumor

CXCR3

4, 9, 10, T, M, NK, B 11

CXCR4

12

My, T, N, B, Ep, HIV, tumor, non-Hodgkin’s En, DC, Ms, Eo, Bs, lymphoma, stem cell transplant, multiple myeloma M

CXCR5

13

B, Tfh

Autoimmune disease (rheumatoid disease, multiple sclerosis, Sj€ ogren’s syndrome, autoimmune thyroid disease)

CXCR6

16

T, NK, En, BM

Tumor, psoriasis, system sclerosis, liver fibrosis

CXCR7

11, 12

T, B, M, DC, NK, Tumor, multiple sclerosis, G, P, Nn, As, En, rheumatoid arthritis, tumor MSC

KSHVGPCR

1, 6, 7, B, En 8, 9, 10, 11

Rheumatoid arthritis, multiple sclerosis, transplantation rejection, atherosclerosis, and inflammatory skin diseases, asthma, diabetes, psoriasis

Kaposi’s sarcoma, lymphoproliferative disorder

Chemokines are represented by only their ligand number. For example, the “6” adjacent to “CXCR1” represents CXCL6. As, astrocyte; B, B -lymphocyte; BM, bone marrow stromal cells; Bs, basophil; DC, dendritic cell; En, endothelial cell; Eo, eosinophil; Ep, epithelial cell; G, granulocytes; Hp, hepatocyte; M, monocyte/macrophage; MB, macrophage; Ms, mast cell; MSC, mesenchymal stem cells; My, myeloid; N, neutrophil; NK, natural killer cell; Nn, neuron; P, platelet; T, T -lymphocyte; Tfh, follicular B helper T cells. CXCL1, growth-related oncogene-a (GRO-a); CXCL2, growth-related oncogene-b (GRO-b); CXCL3, growth-related oncogene-g (GRO-g); CXCL4, platelet factor 4 (PF4), which interacts with a splice variant of the chemokine receptor CXCR3, known as CXCR3B; CXCL5, epithelial neutrophil-activating peptide-78 (ENA-78); CXCL6, granulocyte chemotactic protein-2 (GCP-2); CXCL7, neutrophil-activating peptide-2 (NAP-2); CXCL8, interleukin-8 (IL-8); CXCL9, monokine induced by interferon-g (MIG); CXCL10, interferon-g-inducible protein-10 (IP-10); CXCL11, IFNinducible T cell-a chemoattractant (I-TAC); CXCL12, stromal cell-derived factor 1a (SDF-1a); CXCL13, B-lymphocyte chemoattractant (BCA-1); CXCL16, Bonzo.

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Table 9.2 Locations and clinical implications of constitutively active mutants of CXC chemokine receptors Location of constitutively CXC active receptors mutants Clinical implications References

Initiation and progression of Kaposi’s sarcoma (KS), recruitment of inflammatory cells and induced endothelial angiogenesis

Mesri, Cesarman, and Boshoff (2010), Montaner, Kufareva, Abagyan, and Gutkind (2013)

CXCR1 V6.40A, V6.40N

Increased chemotaxis

Han et al. (2012)

CXCR2 D3.49V

Cell transformation

Burger et al. (1999)

CXCR3 N3.35A, N3.35S, T2.56P

Inverse agonist identification

Verzijl et al. (2008)

CXCR4 N3.35A

Inverse agonist identification

Zhang et al. (2002), Zhang, Navenot, Frilot, Fujii, and Peiper (2007), Jahnichen et al. (2010)

KSHVGPCR

Naturally occurred

2. CHEMOKINE CXC RECEPTORS 2.1. CXCR1 2.1.1 Clinical significance of CXCR1 CXCR1 and CXCR2 are two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8) (CXCL8), a major mediator of immune and inflammatory responses implicated in many diseases and tumor growths (Sallusto & Baggiolini, 2008; Waugh & Wilson, 2008). IL-8 is released in response to inflammatory stimuli by almost all types of cells. Although CXCR1 and CXCR2 both bind IL-8, they have distinct physiological activities. Compared with CXCR2, CXCR1 is generally more resistant to desensitization and downregulation; it is also important in the generation of antimicrobial responses and in the respiratory burst upon neutrophil activation (Sabroe & Whyte, 2007). Binding of IL-8 to CXCR1 activates intracellular signaling pathways, leading to neutrophil migration to the site of inflammation

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(Sallusto & Baggiolini, 2008). Furthermore, the constitutive formation of CXCR1 and CXCR2 homo- and heterodimers (Wilson, Wilkinson, & Milligan, 2005) suggests that the active interaction between these two close members may affect the immune response to IL-8. Excessive inflammation caused by the recruited neutrophils is thought, at least partially, to be responsible for COPD, asthma, inflammatory bowel diseases, and Crohn’s disease. Hence, targeting CXCR1 using structural and biochemical approaches to develop specific antagonists is a promising therapeutic strategy to modulate the receptor activity to combat these diseases (Bizzarri et al., 2006; Panina, Mariani, & D’Ambrosio, 2006; Snelgrove, 2011). Additionally, CXCR1 promotes IL-8-mediated tumor growth such as prostate cancer (Shamaladevi, Lyn, Escudero, & Lokeshwar, 2009), breast cancer (Ginestier et al., 2010), colorectal cancer, and melanoma (Sharma, Singh, Varney, & Singh, 2010), and the CXCR1 blockade provides a possible therapeutic intervention point in targeting the tumor microenvironment. 2.1.2 Structural features of CXCR1 and regulation of CXCR1 signaling Recently, NMR spectroscopy determined the three-dimensional structure of human CXCR1 (Park et al., 2012). Only CXCR1 and CXCR4 have had structures determined thus far, and these two chemokine receptors have a number of similarities (Park et al., 2012; Wu et al., 2010). Three features for intracellular G protein activation and signal transduction are worth noting. First, the NMR data show that two disulfide bonds are significant for ligand binding, shaping the receptor’s extracellular structure, and establishing important restraints for structure determination (Park et al., 2012). One bond connects the N-terminus to the extracellular start of TM7 (Cys30–Cys277); the other connects the extracellular end of TM3 to ECL2 (Cys110–Cys187). Second, in both CXCR1 and CXCR4, charged residues close to the membrane–water interface and negative charges clustered in the extracellular loops take part in ligand binding and receptor activation. The NMR data also revealed that four charged residues form a polar cluster in the core of the helical bundle of CXCR1. These residues, contributed by TM2 (Asp85), TM3 (Lys117), and TM7 (Asp288 and Glu291), may be relevant to ligand binding and receptor signal transduction (Park et al., 2012). Third, as with most other GPCR members, intracellular loop 3 (ICL3) of CXCR1 (from Thr228 to Gln236, connecting helices TM5 and TM6) is significant for CXCR1 coupling to G proteins, calcium mobilization, chemokine-mediated migration, and cell adhesion (Park et al., 2012). The discovery of new compounds that interact with CXCR1 and


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combat diseases such as breast cancer is possible through understanding the NMR structure of human CXCR1 (Ginestier et al., 2010). CXCR1 couples to both pertussis toxin-sensitive Gai and pertussis toxin-resistant Ga15 (Wu, LaRosa, & Simon, 1993) upon activation. This triggers several cell responses such as phosphoinositide (PI)3 hydrolysis; intracellular Ca2+ mobilization; activation of effectors phosphatidylinositol-3kinase and phospholipase C (PLC); Akt, PKC, and MAPK signaling cascades; and chemotaxis (Waugh & Wilson, 2008). The human CXCR1 consists of several critical amino acid residues and functional motifs/domains. One is the N-terminal region, which determines the receptor subtype selectivity (Rajagopalan & Rajarathnam, 2004) and receptor activation ( Joseph et al., 2010). Another is the C-terminal tail, which is responsible for IL-8-induced internalization (Feniger-Barish, Ran, Zaslaver, & BenBaruch, 1999) and ERK1/2 activation (Prado et al., 2007), migration, and cell activation (Richardson, Marjoram, Barak, & Snyderman, 2003). 2.1.3 Constitutively active CXCR1 mutation (V6.40A and V6.40N) The constitutive activity of CXCR1 was first reported by Han et al. (2012). Their studies demonstrated that Val2476.40(V247, a residue that corresponds to Baldwin location on TM6.40) of CXCR1 is related to the inactive conformational structure of CXCR1. More specifically, the selective mutation of residue V247 resulted in the constitutive activation of the receptor, either by transforming the receptor from inactive to active conformation or by stabilizing the receptor in its active state (Han et al., 2012). The supposition is that cytoplasmic peptide sequences of GPCR—prevented from interacting with G proteins through concealment in the inactive, constrained GPCR conformation—would be exposed. They can then bind and activate the relevant G proteins to an active conformation by constraint disruption and receptor stabilization (Vauquelin & Van Liefde, 2005). Amino acid V247 is therefore critical to maintaining CXCR1 in an inactive state. It is likely that substituting V247 with A or N, which would switch the transmembrane domains to convert the receptor to an active state, disrupts the constraint of intramolecular bond stabilizing CXCR1 in an inactive state. Structural level disruption of TM6, which is believed to play an important role in the signaling mechanism, would likely affect G protein binding and activation. Several recent observations in the crystal structure of GPCRs support this idea. For example, the movement of cytoplasmic ends of TM5 and TM6 away from the light-activated rhodopsin core opens up a cleft in the center of the helix bundle, which allows for the binding of the carboxy terminus of a G protein

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(Scheerer et al., 2008). In addition, comparing the agonist-bound and inverse agonist-bound b2AR structure identified the largest change to be in TM6 via the outward movement of the cytoplasmic end of TM6 and the rearrangements of TM5 and TM7, which are remarkably similar to those observed in the active form of rhodopsin (Rasmussen, DeVree, et al., 2011; Rosenbaum et al., 2011; Scheerer et al., 2008; Standfuss et al., 2011). The NMR structure of the chemokine receptor CXCR1 reveals the importance of transmembrane domain dynamics, especially the helical kink angle on the transmembrane helix 6. This underscores the significance of that region in CXCR1 activation (Vaidehi, Bhattacharya, & Larsen, 2014). Consistent with these findings, agonist-independent constitutive activity of CXCR1 mutants (V247A and V247N) on TM6 offers insight into the agonist-binding process and the activation of GPCRs. The amino acid residue located at TM6.40 might be critical in the stabilization of the multiple GPCRs coupled to various G proteins such as Gas, Ga15, Gai, and Gt in the inactive state. The comparison of constitutively active mutations of TM6.40 in the opsin (Met257), the TSH receptor (Leu629), the muscarinic receptor (Ile447), and the histamine H1 receptor (Ile420) (Bakker et al., 2008; Han, Smith, & Sakmar, 1998; Spalding, Burstein, Henderson, Ducote, & Brann, 1998) serves as a foundation for this conclusion. Replacement of TM6.40 in several different GPCRs (Gtcoupled rhodopsin, Gas-coupled TSHR, and Gai-/Ga15-coupled CXCR1) leads to constitutive activity of the receptors coupled to specific G proteins (Gas, Gt, Gai, and Ga15), even though the particular residues are different at this site (Met257 in bovine rhodopsin, Leu629 in TSHR, Ile447 in the muscarinic receptor, and Val247 in CXCR1). The amino acid residue located at TM6.40 is therefore critical for receptor stabilization in the inactive state, which is an intrinsic feature of the receptors (independent of the G protein type). A modification of this single amino acid residue on a structural level affects GPCR–G protein interaction and receptor activation. The constitutive activation of the Ga15 and Gai signaling pathways resulted from the substitution of V247 in transmembrane helix 6 of CXCR1. Mutants V247A and V247N were stabilized in the active state without a ligand, but ligand binding caused certain responses. This includes IP accumulation, which might result from conformational changes caused by receptor binding to IL-8; this would support the concept of existence of multiple ligand-specific conformational states. The receptor in the multistate model alternates between multiple active and inactive conformations (i.e., intermediate conformation between the inactive and active states)


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(Vauquelin & Van Liefde, 2005). In addition, an increase in chemotaxis in response to IL-8 was the result of cells expressing constitutively active mutants (CAMs) V247A and V247N of CXCR1 (Han et al., 2012). The pertussis toxin completely abolishes chemotaxis mediated by CXCR1 or its mutants, which implies that the activation of Gai is required for chemotactic responses. Thus, the constitutive activation of CXCR1 may enhance our knowledge of the activation of CXCR1; provide a rapid, sensitive readout for CXCR1 signaling; and facilitate screening and/or developing novel IL-8 antagonists for the treatment of neutrophil-mediated diseases and cancers.

2.2. CXCR2 2.2.1 Clinical significance of CXCR2 Among the chemokine receptors, the closely related CXCR1 and CXCR2 receptors share a common agonist ligand IL-8. CXCR1 and CXCR2 are widely coexpressed in immune cells, including neutrophils, CD8(+) T cells, and mast cells; noncompetitive allosteric inhibitors of these receptors inhibit neutrophil recruitment in vivo, suggesting the critical role in mediating neutrophil migration to sites of acute inflammation (Chapman et al., 2009). CXCR2 participates in chronic inflammation, sepsis, lung pathology, atherosclerosis, and neuroinflammation (Hertzer, Donald, & Hines, 2013). Also, CXCR2 controls the positioning of oligodendrocyte precursors in developing spinal cords by arresting their migration (Tsai et al., 2002). In addition, CXCR2 functions in angiogenesis and wound healing and spontaneous and inflammation-driven tumorigenesis ( Jamieson et al., 2012; Vandercappellen, Van Damme, & Struyf, 2008). Expression of the CXCR2 on many different cell types, including leukocytes and related cell lines, melanoma cells, and breast cancer cells, has been reported (Youngs, Ali, Taub, & Rees, 1997). IL-8 and Gro-a, CXCR2 ligands produced by various cell types, have shown to be angiogenic and mitogenic for endothelial cells (Koch et al., 1992). In contrast, growth arrest of CXCR2 has also been shown in IMR-90 cells, human diploid fibroblasts (WI-38), and human mammary epithelial cells (HMECs) (Acosta, O’Loghlen, Banito, Raguz, & Gil, 2008). 2.2.2 Regulation of CXCR2 signaling CXCR2 is a GPCR that engages multiple pathways once activated. CXCR2 can couple to Gai2, Ga14, and Ga16 and to transducer signal cascades following stimulation with IL-8 (Balkwill, 2004). CXCR2 activation

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Xinbing Han

by binding chemokines results in the activation of NF-kB, MAPK, PI3K, and Rac, among other signaling cascades (Balkwill, 2004). The activation of Rac by CXCR2 recruits NADPH oxidases to produce a burst of reactive oxygen species, clear pathogen infections by macrophages, and mediate the induction of apoptosis in cancer cells (Zhao, Wimmer, Trieu, Discipio, & Schraufstatter, 2004). CXCR2 directs cell trafficking and positioning by binding to a repertoire of structurally and functionally related ELR-positive CXC chemokines, including CXCL1, 2, 3, 5, 6, 7, and 8 (Addison et al., 2000). Engagement of CXCR1 or CXCR2 initiates G protein heterotrimeric dissociation, which in turn induces various downstream signaling events such as intracellular calcium mobilization and actin polymerization. Both are required for chemokine gradient-directed cell migration. A recent study has shown that CXCR2 formed a complex with its downstream effector PLC-b2 via the scaffold protein Na+/H+ exchanger regulatory factor-1 in neutrophil-like cell lines and bone marrow-derived neutrophils, mediating downstream signaling events such as calcium mobilization and neutrophilic transepithelial migration (Wu et al., 2012). 2.2.3 Constitutively active CXCR2 mutation (D138V) Point mutation causing constitutive signaling of CXCR2 was first reported by Burger et al. (1999). In this study, the replacement of the highly conserved D138 of DRY motif in the second intracellular loop with the bulky hydrophobic Val resulted in constitutive activity indicated by high levels of inositol phosphate accumulation. Moreover, the D138V mutant exhibited transforming potential similar to the KSHV-GPCR in soft agar growth assays in NIH 3T3 cells and in focus formation, a morphological manifestation of transformation associated with the loss of contact inhibition that limits cell density. These results suggested that the DRY sequence plays an essential role for G protein-coupled signaling of the CXCR2. By contrast, exchanged Asp138 of the DRY sequence in the CXCR2 with the hydrophilic Gln (D138Q) showed similarities to the CXCR2 wild type in terms of inositol phosphate turnover and foci formation potential. Nevertheless, D138 mutant demonstrated an elevated and prolonged Ca2+ mobilization, compared with a transiently increased Ca2+ concentration within detected cells (Burger et al., 1999). The DRY sequence at the junction between the third transmembrane domain and the second intracellular loop of the CXCR2, which is VRY motif in this position instead of the KSHV-GPCR, is a highly conserved motif among GPCRs. The


277

importance of the DRY sequence for G protein coupling has been shown for some other chemokine receptors such as CCR5 (Gosling et al., 1997). Interestingly, introducing the point mutation that exchanges the Asp for a Val into the CXCR1 (D134V CXCR1) did not result in transforming capacity as shown in CXCR2 (D134V CXCR2) and in KSHV-GPCR. This is not attributed to the poor expression of D134V CXCR1. The similarity in the signaling of the CXCR2 mutant D138V and the KSHVGPCR supports the hypothesis that the gene of the KSHV-GPCR has been pirated from CXCR2 (Burger et al., 1999). This study indicates the transforming potential of CXCR2; a further study indicated that both KSHVGPCR and the D138V CXCR2 mutant constitutively activate JAK2– STAT3 and are capable of transforming KSHV- or CXCR2-expressing NIH 3T3 cells and human microvascular endothelial cells (HMVECs) and mediating angiogenic responses that are involved in tumor development and metastasis (Burger, Hartmann, Burger, & Schraufstatter, 2005). By contrast, Acosta et al. (2008) showed that CXCR2 activation results in growth arrest (the senescence) via the activation of the p53 pathway in IMR-90 cells, WI-38, and human mammary epithelial cells (HMECs). Hence, CXCR2 may play a distinct role in cell growth, depending on the specific cell types.

2.3. CXCR3 2.3.1 Clinical significance of CXCR3 The chemokine receptor CXCR3 is mainly expressed not only in activated T helper 1 cells but also in B cells, natural killer cells, CD8 +, and mast cells (Muehlinghaus et al., 2005). Binding of ligands to the CXCR3 stimulates cellular responses such as integrin activation, actin reorganization, and directional migration, which play a key role in the recruitment of Th1 cells and other types of lymphocytes to sites of inflammation. The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10, CXCL11, and CXCL4 are involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, transplantation rejection, atherosclerosis, and inflammatory skin diseases. The CXCR3 ligands share low sequence homology (around 40% amino acid identity) and exhibit differences in their potencies and efficacies at CXCR3. CXCL11 is the dominant CXCR3 agonist with high affinity and with more potency and efficacy than CXCL10 or CXCL9 as a chemoattractant and in stimulating calcium flux and receptor desensitization. Compared with the other three ligands, CXCL4 is a weak agonist to

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evoke cell signal response and to stimulate cell migration (Korniejewska, McKnight, Johnson, Watson, & Ward, 2011). The CXCR3 ligands are upregulated at sites of inflammation, and they attract CXCR3-bearing lymphocytes, thus contributing to the inflammatory process (Verzijl et al., 2008). In addition, CXCR3 may also be involved in the metastasis of CXCR3-expressing cancer cells (Walser et al., 2006). On the other hand, expression of CXCR3 ligands such as CXCL10 (Luster & Leder, 1993) and CXCL11 (Hensbergen et al., 2005) at tumor sites may attract CXCR3-expressing immune cells to control tumor growth and metastasis. CXCR3 has been recognized as a potential attractive drug target due to its involvement in a variety of serious disorders, including cancer and inflammatory diseases mentioned in the preceding text (Verzijl et al., 2008). 2.3.2 Structural features of CXCR3 and regulation of CXCR3 signaling Upon binding to its ligand, CXCR3 activates pertussis toxin-sensitive G proteins of the Gai class, leading to chemotaxis, calcium flux, and the activation of kinases such as p44/p42 MAPK and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways in T lymphocytes (Smit et al., 2003). Binding ligands to CXCR3 activates p44/42 ERK and Akt phosphorylation responses and elicited receptor desensitization and internalization (Sauty et al., 2001). CXCL11 is the most robust ligand of CXCR3 in terms of biochemical and functional responses. It is noteworthy that CXCL11 also binds to CXCR7; however, CXCL11 does not induce calcium signaling, or p44/42 or Akt phosphorylation, through CXCR7 G protein signaling (Proost et al., 2007). The C-terminus and the DRY sequence of CXCR3 play critical roles in chemotaxis and calcium responses to all three established CXCR3 ligands. Mutation of the DRY sequence ablates CXCL11-induced calcium mobilization, p44/42 phosphorylation, and chemotaxis (Colvin, Campanella, Sun, & Luster, 2004). The internalization of CXCR3 requires distinct domains dependent on the stimulus: mutation of the DRY sequence has no effect on CXCR3 internalization (Colvin et al., 2004). CXCL11 predominantly induces internalization via a C-terminus-independent pathway, whereas CXCL9 and CXCL10 stimulate internalization via a C-terminusdependent pathway (Colvin et al., 2004). 2.3.3 Constitutively active CXCR3 mutation (N3.35A, N3.35S, and T2.56P) The CAM CXCR3 N3.35A was characterized by Verzijl et al. (2008). CXCR3 mutants N3.35A, N3.35S, and T2.56P showed constitutive


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activity. D3.49V mutation in the conserved DRY motif at the cytoplasmatic end of TM3 displayed basal signaling (Verzijl et al., 2008). Previous studies on CXCR2 and KSHV-GPCR have shown that substitution of DRY with VRY in these two chemokine receptors confers constitutive activity (Burger et al., 1999; Ho, Ganeshalingam, Rosenhouse-Dantsker, Osman, & Gershengorn, 2001), whereas the same mutation for CXCR1 or CCR5 (Lagane et al., 2005) did not result in constitutive activity, suggesting that the mutation of D3.49 to Val does not seem to be a universal switch for constitutive activity in chemokine receptors. Mutation of T2.56 in the conserved TXP motif (T2.56P) resulted in a CAM for CXCR3 (Verzijl et al., 2008), CCR5, and CCR2, but not for CCR1, CCR3, CCR4, CXCR2, and CXCR4 (Alvarez Arias, Navenot, Zhang, Broach, & Peiper, 2003). Replacement of N3.35 in the N(L/F)Y motif in TM3 with residue A or S resulted in CAMs for both CXCR3 (Verzijl et al., 2008) and CXCR4 (Zhang et al., 2002). Among the three CXCR3 CAMs, CXCR3 N3.35A for further characterization showed a marked increase of PLC activation in the absence of a ligand. Although all five tested nonpeptidergic antagonists behave as noncompetitive antagonists for the PLC activation by the endogenous agonists of CXCR3 (CXCL10), four of the small-molecule antagonists (VUF10472, VUF10085, VUF5834, and VUF10132) acted as full inverse agonists at the constitutively active CXCR3 N3.35A mutant. In contrast, TAK-779 acted as a partial inverse agonist, indicating a different mode of interaction with CXCR3 CAM compared with the other structural classes of compounds (Verzijl et al., 2008). In the case of CXCR3, which is upregulated under inflammatory conditions (Murphy et al., 2000; Rabin et al., 1999), constitutive activity of chemokine receptors might become apparent under pathological conditions; therefore, the use of inverse agonists may be beneficial.

2.4. CXCR4 2.4.1 Clinical significance of CXCR4 Among chemokine receptors, CXCR4 has drawn increasing attention because of its important role not only in the recruitment of leukocytes to sites of pathology and maintenance of stem cells in a microenvironmental niche leukocyte homing but also in the development of the immune, central nervous, and cardiovascular systems and in cancer metastasis (Muller et al., 2001; Sugiyama, Kohara, Noda, & Nagasawa, 2006). CXCL12 is a chemokine that acts through CXCR4 and CXCR7. CXCR4 is expressed in hematopoietic cells and mediates the chemotaxis of CD34 stem cells, playing

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a critical role in the homing of these cells in the bone marrow microenvironment (Aiuti, Webb, Bleul, Springer, & Gutierrez-Ramos, 1997). CXCL12, which binds to both CXCR4 and CXCR7, induces the migration of peripheral blood lymphocytes, CD34+ progenitor cells, and pre- and pro-B cell lines. In addition, T-tropic (X4) strains of HIV-1 utilize CXCR4 for target cell entry, and this process is blocked by CXCL12 (Bleul et al., 1996) and receptor antagonists (Schols et al., 1997). CXCR4/CXCL12 is essential during embryogenesis, hematopoiesis, and immune system organization as demonstrated in mice lacking either the CXCL12 protein or its receptor CXCR4 (Nagasawa et al., 1996; Tachibana et al., 1998; Zou, Kottmann, Kuroda, Taniuchi, & Littman, 1998). In addition, CXCR4 actively participates in the pathological process. It serves as a coreceptor for HIV-1 cellular entry and mediates the development and metastasis of many types of tumors (Alkhatib, 2009; Zlotnik, 2006). CXCR4 has been associated with more than 20 types of cancers. Increasing evidence suggests that CXCL12/CXCR4 modulates cellular growth and survival (Ptasznik et al., 2002). The immunologic blockade of CXCR4 abrogated cell adhesion and invasion demonstrates the important role of CXCL12/CXCR4 in the metastatic process (Amine et al., 2009). Therefore, insight into mechanisms to disrupt CXCR4 function by using pharmacological approaches may open important avenues for developing therapeutic approaches for myeloid malignancies and tumors and HIV-1 infection. 2.4.2 Structural features of CXCR4 and regulation of CXCR4 signaling The CXCR4 chemokine receptor is a Gai protein-coupled receptor that triggers multiple intracellular signals in response to CXCL12. Receptor internalization depends on the phosphorylation of the C-terminus part of CXCR4. Several important domains of CXCR4 and their role in transducing cell signaling, chemotaxis, and endocytosis have been studied by Roland and colleagues (2003). They demonstrated that the ICL3 of CXCR4 is specifically involved in Gai-dependent signals such as calcium mobilization and ERK activation, but it does not trigger CXCR4 internalization in response to CXCL12 (Roland et al., 2003). Furthermore, ICL2 and ICL3, as well as the C-terminus part of CXCR4, have demonstrated their important roles in transducing ligand-mediated chemotaxis (Roland et al., 2003). The DRY sequence of CXCR4 seems less important in intracellular signaling because ICL2, in the absence and presence of the aspartic acid, arginine, and tyrosine (DRY) motif, is dispensable for Gi signaling (Roland et al., 2003). Differing from other GPCRs, the crystal structure of CXCR4 indicates that the


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location and shape of the ligand-binding sites are closer to the extracellular surface (Wu et al., 2010). CXCR4-mediated cell motility, adhesion, and invasion are dependent on actin cytoskeleton dynamics, at least partially through the activation of the small GTPases RhoA, rac1, and cdc42 (Titus, Schwartz, & Theodorescu, 2005) and the activation of their downstream effectors, Rho kinases/ROCK/ROK, leading to myosin phosphorylation. In addition, CXCR4 activates the tyrosine phosphorylation of multiple focal adhesion proteins, PI3K, and mitogenic MAPK cascade (Ganju et al., 1998; Wang, Park, & Groopman, 2000). CXCR4 mediates estrogen-independent tumorigenesis and metastasis in human breast cancer through CXCL12mediated activation of downstream signaling via ERK1/2 and p38 MAPK signaling (Rhodes et al., 2011). These pathways are essential to transduce chemotactic, survival, or proliferative/differentiative signals. Ptasznik et al. (2002) reported that CXCR4-dependent stimulation of the Src-related kinase (Lyn) is associated with the activation of PI3K. This chemokine Lyn and PI3K signaling is regulated by BCR/ABL, a fusion oncoprotein expressed only in leukemia cells (Ptasznik et al., 2002). Their results define a Src tyrosine kinases-dependent mechanism, whereby BCR/ABL dysregulates CXCR4 signaling and function such as movement and retention of stem/progenitor cells within the bone marrow microenvironment. CXCR4 signaling and trafficking can be regulated by intracellular proteins b-arrestin and nucleophosmin via interaction and ubiquitination with cytosolic domains of receptors. b-arrestin redirects signaling to alternative G proteinindependent pathways of CXCR4 through its distinct interactions with the C-terminus and other regions including the third loop of CXCR4 (Cheng et al., 2000). Some other mechanisms of downmodulation include receptor ubiquitination. After CXCL12 binding, CXCR4 undergoes downmodulation and ubiquitination of the C-terminus by E3 ubiquitin ligase, thereby promoting targeting of the receptor for degradation rather than recycling via the endosomal pathway (Marchese et al., 2003). The nucleophosmin is another mechanism to desensitize CXCR4 via interaction with the C-terminus and CL-3 of the activated CXCR4 and negatively regulated induction of GTP binding by Gai subunits after CXCR4 activation. Interestingly, constitutively active CXCR4 mutants (N119A) demonstrated higher affinity to nucleophosmin than the wild-type receptor (Zhang et al., 2007). In brief, these mechanisms of “turning off” G protein receptor signaling may provide approaches to block the function of CXCR4 in the pathophysiology of tumor metastasis and the biology of leukemic myeloblasts and in HIV-1 infection.

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CXCR4 has been previously shown to homo- and heterodimerize, constitutively and upon ligand binding, by various experimental methods. CXCR4 has been reported to dimerize with CCR2, CCR5, and CXCR7, and complexes show negative binding cooperativity with their ligands, which may have implications for drug efficacy (Levoye, Balabanian, Baleux, Bachelerie, & Lagane, 2009; Luker, Gupta, & Luker, 2009). The crystal structures of CXCR4 support the concept of CXCR4 dimerization. The crystal structures of CXCR4 revealed a consistent homodimer with an interface including helices V and VI that may be involved in regulating signaling (Wu et al., 2010).

2.4.3 Constitutively active CXCR4 mutation (N119S and N119A) The importance of transmembrane helix 3 (TM3) in CXCR4 signaling was illustrated by Zhang et al. (2002). By using the Saccharomyces cerevisiae expression system to couple CXCR4 signaling to growth in the absence of histidine, they derived a CXCR4 CAM by random mutagenesis. The amino acid substitution that conferred this phenotype involved Asn-119 of TM3. Berchiche et al. (2007) detected different conformations of activated receptors and reported that mutations of the CXCR4 sequence can alter both the basal conformation and the conformational rearrangements induced by ligand binding. The constitutively active N119S mutant and wild-type CXCR4 have different conformations in the presence of CXCL12, yet both activate G proteins (Berchiche et al., 2007). Their results provide evidence that active receptor conformations can have a degree of conformational diversity. Thus, the heterogeneous CXCR4 conformations can lead to similar G protein activation, implying flexibility of active receptors. CXCR4’s CAMs (N119A and N119S) mimic many aspects of CXCR4 after CXCL12 binding, including G protein activation, constitutive phosphorylation, and constitutive internalization (Zhang et al., 2002). CXCR4 CAMs (N119S and N119A) were internalized and chronically desensitized by constitutive phosphorylation; they were constitutively phosphorylated and present in cytosolic inclusions. This mutant variant has been proved to be particularly useful to study CXCR4-associated proteins and functions that require the chronic desensitization of receptor or require a stably active CXCR4 conformation. Indeed, the physical association of nucleophosmin with constitutively active CXCR4 was identified and further characterized (Zhang et al., 2007).


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The CXCR4 CAMs may provide a powerful tool for high-throughput screening for antagonists. However, the study on CXCR4 CAM indicated that agents that inhibit the WT receptor may increase or reduce the signaling of CAMs. Analysis of the effect of CXCR4 antagonists on CXCR4 CAM signaling revealed that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists. The decrease in binding of CAM to weak partial antagonist (AMD3100 and ALX40-4C) reflects conformational shift in CXCR4 CAMs. These findings provided the evidence to utilize GPCR CAMs in pharmaceutical screening as an efficient and powerful approach for identification of novel antagonists. Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavychain antibodies. CXCR4 nanobodies (VHH-based single-variable domains) that bind to distinct but partially overlapping sites in the extracellular loops of CXCR4 competitively inhibited the CXCR4-mediated signaling and antagonized CXCL12-induced chemotaxis and CXCR4mediated HIV-1 entry ( Jahnichen et al., 2010). Interestingly, the monovalent nanobodies acted as neutral antagonists (such as AMD3100), whereas the biparatopic nanobodies behaved as inverse agonists at the constitutively active CXCR4 N3.35A (equivalent to CXCR4 N119A) (Zhang et al., 2002). Moreover, CXCR4 nanobodies potently inhibit chemotaxis and HIV-1 entry and CD34+ stem cell immobilization, demonstrating the power of this technique for the development of nanobody-based GPCR drug candidates ( Jahnichen et al., 2010).

2.5. CXCR5 2.5.1 Clinical significance of CXCR5 CXCR5 is the receptor for the chemokine CXCL13, the chemokine important for secondary lymphoid tissue orchestration and lymphoid neogenesis. CXCL13 is constitutively expressed in secondary lymphoid tissue (Cyster et al., 2000) primarily by follicular dendritic cells in the spleen, lymph nodes, tonsils, and Peyer’s patches (Cyster et al., 2000), whereas CXCR5 is highly expressed in mature B lymphocytes and a subpopulation of follicular B helper T cells (Tfh) (Forster et al., 1996). CXCL13 and its receptor CXCR5 are essential for trafficking B cells and homing B cells to lymphoid tissues and for the embryonic development of the majority of lymph nodes and Peyer’s patches (Ansel et al., 2000). Transgenic mice deficient in CXCL13 or its receptor CXCR5 manifested severely impaired lymph node development, lacking peritoneal B lymphocytes, and deficient in circulating antibodies

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to common bacterial antigens, although these mice manifested slightly increased total circulating numbers of B lymphocytes with relatively normal humoral responses to T-dependent or blood-borne antigens (Ansel, Harris, & Cyster, 2002). The involvement of CXCL13 and CXCR5 in the maintenance of pathogenic B cells in autoimmune diseases, including rheumatoid disease, multiple sclerosis, Sj€ ogren’s syndrome, autoimmune thyroid disease, and myasthenia gravis, has been well documented (Finch, Ettinger, Karnell, Herbst, & Sleeman, 2013). Recent data suggest that anti-CXCL13 might be a promising approach to modulate pathogenic immune responses while maintaining humoral host defense (Finch et al., 2013).

2.6. CXCR6 2.6.1 Clinical significance of CXCR6 CXCL16 interacts with the chemokine receptor CXCR6, also known as Bonzo. CXCR6 is expressed in several subsets of T cells (memory T cells, Th1, and Tc1), natural killer T cells, and bone marrow stromal cells, and it mediates migration in response to CXCL16 (Matloubian, David, Engel, Ryan, & Cyster, 2000). Cells that produce CXCL16 include dendritic cells found in the T-cell zones of lymphoid organs and cells found in the red pulp of the spleen (Matloubian et al., 2000). CXCR6 and its ligand CXCL16 in tumor progression have been well documented (Wang, Lu, Koch, Zhang, & Taichman, 2008). CXCR6/CXCL16 plays a critical role in NKT cell activation and in the regulation of NKT cell homeostasis (Germanov et al., 2008), and it promotes inflammation and liver fibrosis (Wehr et al., 2013). CXCL16–CXCR6 interactions mediate homing of CD8(+) T cells into the human skin and thereby contribute to psoriasis pathogenesis (Gunther, Carballido-Perrig, Kaesler, Carballido, & Biedermann, 2012) and angiogenesis in systemic sclerosis of the skin (Rabquer et al., 2011). Recently, the involvement of CXCR6 and its ligand CXCL16 in tumor progression has become evident in the setting of hepatocellular carcinoma (Gao et al., 2012) and melanoma (La Porta, 2012). 2.6.2 Structural features of CXCR6 and regulation of CXCR6 signaling Unlike most chemokines that are soluble polypeptides of 10 KDa either expressed and secreted constitutively or released upon cell activation, the CX3C chemokine ligand 1/fractalkine and the CXC chemokine ligand 16 (CXCL16) are two exceptional chemokines that are expressed as type I transmembrane adhesion molecules. Larger than other chemokines (with 254 amino acids), CXCL16 consists of a CXC chemokine domain,


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a mucin-like stalk, a transmembrane domain, and a cytoplasmic tail containing a potential tyrosine phosphorylation site that may bind SH2 (Matloubian et al., 2000). These unusual features allow CXCL16 to be expressed as a cell surface-bound molecule and a soluble chemokine (Abel et al., 2004). Surface-expressed CXCL16 is capable of binding to its receptor CXCR6 expressed in leukocytes, thereby establishing a firm cell-to-cell contact. The soluble CXCL16 is constitutively generated by proteolytic cleavage of its transmembrane variant (shedding) with disintegrin-like metalloproteinase (ADAM) 10 from fibroblasts and endothelial cells (Abel et al., 2004; Gough et al., 2004). Shedding results in the release of soluble CXCL16, which then functions as a chemoattractant for CXCR6-expressing cells such as T-cell subtypes and bone marrow plasma cells (Matloubian et al., 2000; Nakayama et al., 2003). CXCL16 induces Ca(2+) influx and chemotactic migration of CD8(+) T cells in vitro (Gunther et al., 2012). Interaction of CXCL16 and CXCR6 activates the Akt/mammalian target of rapamycin (mTOR) pathways and its downstream effectors (Wang, Lu, et al., 2008).

2.7. CXCR7 2.7.1 Clinical significance of CXCR4 CXCR7 is a recently identified chemokine receptor for chemokines CXCL11 and CXCL12, which previously had been characterized as ligands only for CXCR3 or CXCR4, respectively. CXCL12 is essential for the development of the heart, gonads, nervous system, and blood vessels (Ma, Jones, & Springer, 1999; Zou et al., 1998). CXCL12 stimulates the arrest, tethering, and rolling of CD34+ progenitor cells and leukocytes on vascular endothelium (Peled et al., 1999). CXCL12 is also involved in tumor angiogenesis and metastasis. Due to its potential role in pathological inflammation and in tumor malignancy, CXCR7 has become a potential therapeutic target for the treatment of a variety of autoimmune diseases and tumors (Sanchez-Martin, Sanchez-Mateos, & Cabanas, 2013). In the future, detailed understanding of the biochemistry and pharmacology of CXCR7 would be helpful to design a therapeutic strategy for treatment of autoimmune diseases and tumors. CXCR7 functions as a specific scavenger for CXCL12 and CXCL11 by constitutively internalizing ligands and removing chemokine ligands from the extracellular space and then transporting these molecules to lysosomes for degradation (Boldajipour et al., 2008). During this process, levels of CXCR7 remain stable, indicating that only internalized ligands but not the receptor are degraded. Scavenger receptor CXCR7-dependent

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chemokine degradation does not become saturated with increasing ligand concentrations as demonstrated in mouse heart valves and human umbilical vein endothelium (Naumann et al., 2010) through constitutive endocytosis and recycling of CXCR7 (Boldajipour et al., 2008). Thus, CXCR7 may control ligands available for signaling through CXCR3 and CXCR4 through sequestering and degrading CXCL11 and CXCL12. CXCR7 may, therefore, not only modulate the activity of CXCR3 and CXCR4 in tumor cell development and metastasis and tissue invasion but also orchestrate the migration of hematopoietic cells in the bone marrow and lymphoid organs. More recently, the importance of CXCR4/CXCR7 heterodimer in regulating CXCL12-mediated G protein signaling has been appreciated. CXCR4 and CXCR7 display a wide expression pattern in mammalian tissues. They are coexpressed in T-cell and B-cell subsets, endothelial cells, spinal ganglia, descending neurons, and human renal progenitor cells and in some tumor cells, primarily human tumors (i.e., breast, lung, and prostate), and tumor-associated endothelial cells (Mazzinghi et al., 2008). Both CXCR4 and CXCR7 can differentially respond to CXCL12. For example, CXCR7 is involved in human renal progenitor cell survival and cell adhesion to endothelium, whereas CXCR4 is involved in chemotaxis (Mazzinghi et al., 2008). CXCR7 inversely correlates with the activity of CXCR4 in B cells (Infantino, Moepps, & Thelen, 2006) and is critical for CXCR4 to mediate CXCL12-dependent integrin activation in T cells (Hartmann et al., 2008). CXCR7 modulates CXCR4-mediated migration of primordial germ cells by CXCL12 sequestration (Boldajipour et al., 2008). The physical interaction of CXCR4 and CXCR7 by forming CXCR4/CXCR7 heterodimer at least partially accounts for the regulation of CXCR4 signaling by CXCR7. CXCR7 is a promising therapeutic target for diseases including cancer, multiple sclerosis, and rheumatoid arthritis. Understanding the structural domains that regulate CXCR7 would warrant the design of more effective compounds that selectively interact with this CXCR7. In addition, due to its essential role in cancer development and progression, the recently deorphanized chemokine receptor CXCR7 has become a potential therapeutic target for the treatment of a variety of tumors (Miao et al., 2007; Wang, Shiozawa, et al., 2008). In addition, the fact that the binding of CXCR4 and CXCR7 to CXCL12 with high affinity initiates a distinct cell response suggests that a detailed understanding of the biochemistry and pharmacology of CXCR4 and CXCR7 is indispensable for the efficient therapy targeting these receptors.


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2.7.2 CXCR4/CXCR7 heterodimer and biased arrestin signaling by CXCR7 Engagement of CXCL12 to CXCR4 activates heterotrimeric Gabg proteins, which then trigger numerous downstream responses, such as calcium mobilization, integrin-mediated adhesion, gene expression, and cell proliferation. Although CXCR7 conserves most of the canonical GPCR features, constitutively interacts with Gai proteins, and undergoes CXCL12-mediated conformational changes (Levoye et al., 2009), CXCR7 cannot signal directly through G protein-linked pathways; however, it can nevertheless form a heteromeric complex with CXCR4 and thereby affects cellular signaling networks. In fact, CXCR7 is the first identified seven-transmembrane receptor biased for arrestin-dependent signaling. CXCR7 selectively activates ligand-dependent signaling through b-arrestin 2 and MAPK pathways (Rajagopal et al., 2010; Wang et al., 2011). CXCL12 engagement to CXCR7 can transmit a range of cellular responses, such as the activation of ERK and AKT pathways (Wang, Shiozawa, et al., 2008), receptor internalization (Balabanian et al., 2005; Boldajipour et al., 2008), cell survival (Burns et al., 2006; Miao et al., 2007), proliferation (Meijer, Ogink, & Roos, 2008), adhesion (Hartmann et al., 2008; Mazzinghi et al., 2008), and chemotaxis of CXCR4-negative cells (Valentin, Haas, & Gilmour, 2007). This suggests that GPCRs like CXCR7 can signal through mechanisms that function independently of G proteins. Sequestering of CXCL12 by CXCR7 and then locally modifying the chemokine concentration and specifically altering CXCR4-mediated activation of G proteins through CXCR4/CXCR7 heterodimerization at least partially account for the CXCR7-mediated cellular response mentioned in the preceding text. A further study demonstrated that CXCR4/CXCR7 heterodimer impairs CXCR4promoted Gi activation and signaling, constitutively recruits b-arrestin, and activates b-arrestin-dependent signal transduction pathways, including ERK1/2, p38 MAPK, and SAPK, leading to increased cell migration of CXCR4expressing breast cancer cells (Decaillot et al., 2011). The results support the model wherein CXCR4 monomers/homodimers would signal predominantly via G protein-dependent signaling pathways, whereas CXCR4/CXCR7 heteromers would predominantly engage b-arrestin-dependent pathways (Decaillot et al., 2011). CXCR7 internalization and chemokine scavenging are controlled by clathrin-mediated endocytosis and the cytosolic adapter protein b-arrestin 2 (Kalatskaya et al., 2009; Rajagopal et al., 2010). The interaction between the intracellular tail of CXCR7 and b-arrestin 2 has been shown to be necessary for normal localization, internalization, and CXCR7-mediated

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depletion of CXCL12 (Ray et al., 2012). The phosphorylation of CXCR7 by several kinases (e.g., GRKs) at specific sites after activation recruits b-arrestin to mediate not only receptor internalization and degradation (Malik & Marchese, 2010) but also CXCL12-mediated chemotaxis via p38 MAPK (Sun, Cheng, Ma, & Pei, 2002). In addition, a recent paper demonstrated that ubiquitination of CXCR7 also controls receptor trafficking (Canals et al., 2012). The Lys residue on the C-terminus of CXCR7 is believed to be responsible for constitutive CXCR7 ubiquitination. And receptor activation by CXCL12 results in reversible deubiquitination, leading to partial restoration of the ubiquitinated receptor levels detected in the basal state (Canals et al., 2012).

2.8. KSHV-GPCR 2.8.1 Clinical significance of KSHV-GPCR KSHV is related to all forms of KS (classic, AIDS-associated, endemic (African), and iatrogenic) (Mesri et al., 2010), and it is also the etiologic agent for two lymphoproliferative disorders (primary effusion lymphoma and multicentric Castleman’s disease) (Cesarman, 2002). KS is associated with KSHV infection of the spindle-shaped tumor (spindle) cell, which is thought to have a vascular endothelial or endothelial precursor origin. Infected KS spindle cells produce elevated levels of proinflammatory and proangiogenic secretions (cytokines, chemokines, and growth factors), which may further recruit inflammatory cells and induce endothelial angiogenesis (Mesri et al., 2010). The KS-like disease in transgenic mice by KSHV-GPCR requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines (Holst et al., 2001). The implication of chemokines in the development of KS-like lesion in transgenic mice suggested that modulation of KSHV-GPCR may represent a key step in the generation of KS in humans. There is compelling evidence supporting an essential role for KSHV-GPCR in the initiation and progression of KS as shown in distinct animal models (Montaner et al., 2003; Mutlu et al., 2007). The implication of KSHV-GPCR in KS pathogenesis suggests that strategies to block its function may represent a novel approach for the treatment of KS (Montaner et al., 2013). 2.8.2 Structural features of KSHV-GPCR and regulation of KSHV-GPCR signaling The KSHV (also known as HHV-8) encodes a GPCR commonly known as KSHV-GPCR (or ORF74). KSHV-GPCR belongs to the rhodopsin/ b-adrenergic subfamily of GPCRs and is a chemokine-like receptor


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homologous to human CXCR1 and CXCR2 (Arvanitakis, Geras-Raaka, Varma, Gershengorn, & Cesarman, 1997). KSHV-GPCR exhibits ligand-independent constitutive activity, but it can also be activated by some CXC and CCL, including CXCR1 ligand (CXCL8 and CXCL6), CXCR2 ligand (CXCL8, CXCL7, and CXCL1), CXCR3 ligand (CXCL10, CXCL11, and CXCL9), and CC chemokines (CCL1 and CCL5) (Couty & Gershengorn, 2004). Compared with other traditional chemokine receptors, KSHV-GPCR displays its capability of interacting with a much broader array of chemokines. For example, CXCR8 and CXCL1 function as full agonists of the receptor that further activates KSHV-GPCR; CXCL10, CXCL12, and the HHV-8-encoded CC chemokine viral monocyte inflammatory protein-II are inverse agonists of KSHV8-GPCR, which inhibits KSHV8-GPCR constitutive signaling. CXCL7 and CXCL5 are neutral antagonists for HHV-8-GPCR that do not affect constitutive signaling but would compete for binding and inhibit the effects of agonists or inverse agonists (Couty & Gershengorn, 2004). KSHV-GPCR is a chemokine-like receptor that exhibits high constitutive activity through the phosphoinositide inositol trisphosphate (InsP3)– calcium/diacylglycerol–protein kinase C cascade (Arvanitakis et al., 1997; Geras-Raaka, Varma, Ho, Clark-Lewis, & Gershengorn, 1998). This receptor couples to Ga13 and Gai/o proteins. Coupling of KSHV-GPCR to the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is dependent on Gbg subunits released from PTX-sensitive and PTX-insensitive G proteins (Couty, Geras-Raaka, Weksler, & Gershengorn, 2001; Montaner, Sodhi, Pece, Mesri, & Gutkind, 2001; Smit et al., 2002). This cascade leads to the activation of NF-kB (Couty et al., 2001; Montaner et al., 2001; Smit et al., 2002). KSHV-GPCR has also been shown to activate p44/p42 MAPK (Smit et al., 2002), JNK/SAPK, and p38 MAPK as well as the proline-rich tyrosine kinase 2. These pathways are believed to play important roles both in HHV-8 replication and in KS pathogenesis. On the one hand, it is believed that the activation of p44/p42 MAPK can lead to vascular endothelial growth factor (VEGF) expression via the activation of the transcription factor hypoxia-inducible factor la (Sodhi et al., 2000). On the other hand, NF-kB, JNK/SAPK, and p38 MAPK mediate pathways that are often involved in the activation of inflammatory cytokines leading to angiogenesis and mitogenesis (Pati et al., 2001; Schwarz & Murphy, 2001). In a recently published review, Montaner et al. (2013) summarized a complex signaling network that underlies the potent sarcomagenic potential of KSHV-GPCR. KSHV-GPCR activates AKT in an autocrine manner by

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upregulating the expression of VEGF receptor KDR2 and by increasing VEGF release from endothelial cells, thereby protecting them from apoptosis and promoting the survival of KSHV-infected endothelial cells (Bais et al., 2003). AKT activation of the TSC2/mTOR pathway is necessary and sufficient for KSHV-GPCR oncogenesis (Sodhi et al., 2006). Drugs targeting protein AKT/mTOR and downstream targets may represent mechanism-based therapies to treat patients with KS (Sodhi et al., 2000). With regard to KSHV-associated lymphoid malignancies, signaling by KSHV-GPCR in B-cell lymphomas may differ from receptor-related endothelial and lymphoproliferative lesions. KSHV-GPCR activates ERK and p38 as well as the transcription factors AP-1, NF-kB, CREB, and NFAT (Cannon, Philpott, & Cesarman, 2003). The activation of AP-1 and CREB is mediated cooperatively by the Gaq-ERK and Gai-PI3K signaling pathways, whereas NF-kB and NFAT activation by KSHV-GPCR may require Rac1 (Montaner et al., 2004) and PI3/AKT pathways (Pati et al., 2003), respectively. As a result, KSHV-GPCR might upregulate the expression of lymphocyte chemoattractants and mitogens that promote B-cell recruitment and proliferation in KSHV-induced lymphoproliferative disorders. 2.8.3 Constitutively active KSHV-GPCR Given its pivotal role in the initiation and development of KS, the structural features of KSHV-GPCR warrant further investigation, which will present the opportunity to identify KSHV-GPCR allosteric modulators or inhibitors that halt its constitutive activity and transforming potential (Montaner et al., 2013). Because the protein crystal structure of KSHV-GPCR is not available, site-directed mutagenesis was undertaken to gain insight of the domains within KSHV-GPCR that are responsible for constitutive signaling. This approach was based on observations that mutations of critical residues in GPCRs can convert them from conformations that exhibit no basal activity to conformations that are constitutively active. The constitutive activity of KSHV-GPCR is attributed to a network of residue substitutions that are highly conserved in other GPCRs. The substituted residues of KSHV-GPCR may shift its basal conformational equilibrium toward the active state and result in constitutive activity and efficient coupling to G proteins. Among studied domains, the N-amino terminus of KSHVGPCR is not required for basal signaling of the receptor but is involved in ligand binding and chemokine-regulated KSHV-GPCR signaling (Ho, Du, & Gershengorn, 1999). Two critical regions that are involved


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in GPCR signaling, D/ERY in the intracellular region of TM3 and NPXXY within TM7, were further investigated. In KSHV-GPCR, these conserved sequences were substituted for VRY and VPXXY instead. However, KSHV-GPCR mutants in which either of these residues was substituted by the usually conserved residue exhibited no changes in basal signaling activities (Ho et al., 2001), suggesting that these residues alone are not responsible for KSHV-GPCR constitutive signaling. The DRY motif at the intracellular end of the third transmembrane a-helix (TM3) is a highly conserved sequence of GPCRs. The DRY motif draws much attention because this highly conserved domain is replaced with VRY (3.49–3.51, V142-Y144) instead. Introducing a V142 mutation in KSHV vGPCR leads to a 70% increase in its constitutive activity, whereas a double D83A/V142D mutation makes it fivefold more active in signaling assays based on PLC activation, but it shows a lack of response to either agonist or inverse agonists (Ho et al., 2001). It has been shown that two charged residues at the interface of TM3 (Arg-143) and TM2 (Asp-83) with the adjacent cytoplasmic interhelical loops are critical for the constitutive activity of KSHV-GPCR (Ho et al., 2001). An adjacent amino acid of DRY sequence, L146 (3.53) of KSHV-GPCR, is believed to contribute to conformation equilibrium of the G protein binding (Montaner et al., 2013). In addition, mutation D142V within its DRY motif contributes to KS sarcoma development through its potent transforming and proangiogenic functions (Montaner et al., 2013). Substitutions in KSHV-GPCR such as the triplet of residues at positions 2.50, 3.39, and 7.49 have structural impact. The triplet of residues, replaced with SDV (S93, D132, and V310) in KSHV-GPCR, link transmembrane helices (TMs) II, III, and VII via a network of hydrogen bonds. This “swap” disrupts the water-mediated hydrogen bonding network and results in the activated conformation of helix VII (Montaner et al., 2013). Two other highly conserved residues, located at positions 3.35 and 7.45 that typically meditate interhelical hydrogen bonding interaction in GPCRs, are substituted by tyrosine in KSHV-GPCR (Y128 and Y306). The two bulky aromatic residues greatly modify the nature and the strength of the interhelical interaction in KSHV-GPCR. Indeed, mutations of Tyr at position 3.35 in several GPCRs render them constitutively active, including CXCR3 and CXCR4. In addition, residue 6.48, substituted with a Cys in KSHV-GPCR, may restrict the rotation of TM7 and likely stabilize it into its active state (Montaner et al., 2013)

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3. CONCLUSION Chemokine receptors, especially the two big subfamilies, CC chemokines and CXC chemokines, have drawn much attention owing to their important roles in mediating immune responses on physiological conditions and their involvement in various immune-mediated diseases and tumor growth under pathological conditions. Recent revelation of the crystal structures of CXCR1 and CXCR4 has thrown light on the structure and function of CXC chemokine receptors. However, due to technical difficulties, there is no crystal structure of constitutively active chemokine receptors available. The comparison of conformational structures of constitutively active chemokine receptors with those of resting chemokine receptors, and agonist- or antagonist-bound chemokine receptors, would provide critical information about the activation of chemokine receptors and interaction between receptors and G proteins, facilitating the design and screening of effective inverse agonists and allosteric modulators targeting chemokine receptors. Recent advances in homodimerization and heterodimerization of chemokine receptors, including CXCR1/CXCR2, CCR2/CXCR4, CCR5/CXCR4, and CXCR4/CXCR7 heterodimers, have greatly deepened our understanding of chemokine receptor function and signaling transduction. Furthermore, G protein-independent signaling and biased signaling of CXCR7 also provide new avenues toward revealing the molecular mechanism of receptor activation and receptor redundancy, providing new strategies to design novel therapeutic small molecular inhibitors targeting CXC chemokine receptors. All these advances would lead us to develop promising pharmacological interventions to combat numerous diseases, including cancer, HIV infection, COPD, atherosclerosis, rheumatoid arthritis, and multiple sclerosis.

CONFLICT OF INTEREST The author has no conflicts of interest to declare.

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Association between preeclampsia and the CXC chemokine family (Review).

Constitutively active rhodopsin and retinal disease.

Stoichiometry and geometry of the CXC chemokine receptor 4 complex with CXC ligand 12: molecular modeling and experimental validation.

Chemokine receptors in Atlantic salmon.

Biased signaling at chemokine receptors.

Role of CXC chemokine receptor type 7 in carcinogenesis and lymph node metastasis of colon cancer.

CXC chemokine receptor 4 signaling upon co-activation with stromal cell-derived factor-1α and ubiquitin.

Pharmacological characterization of AZD5069, a slowly reversible CXC chemokine receptor 2 antagonist.

Inhibition of metastasis of rhabdomyosarcoma by a novel neutralizing antibody to CXC chemokine receptor-4.

A CXC chemokine gene, CXCL12, from rock bream, Oplegnathus fasciatus: Molecular characterization and transcriptional profile.

Platelet-Derived CCL5 Regulates CXC Chemokine Formation and Neutrophil Recruitment in Acute Experimental Colitis.

CXC chemokine receptor-4 signaling limits hepatocyte proliferation after hepatic ischemia-reperfusion in mice.

Staphylococcus via an interaction with the ELR+ CXC chemokine ENA-78 is associated with BOS.

STAT3-dependent CXC chemokine formation and neutrophil migration in streptococcal M1 protein-induced acute lung inflammation.

CXC chemokine CXCL12 and its receptor CXCR4 in tree shrews (Tupaia belangeri): structure, expression and function.

Urinary mRNA levels of ELR-negative CXC chemokine ligand and extracellular matrix in diabetic nephropathy.

Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices.

Transgenic expression of constitutively active RAC1 disrupts mouse rod morphogenesis.

New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity.

CXC chemokine ligand 12a enhances chondrocyte proliferation and maturation during endochondral bone formation.

Humoral Immune Pressure Selects for HIV-1 CXC-chemokine Receptor 4-using Variants.

Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival.

Function of chemokine (CXC motif) ligand 12 in periodontal ligament fibroblasts.

The role of CXC chemokine receptor 2 in Staphylococcus aureus keratitis.