Chemokine receptor oligomerization: A further step toward chemokine function

Immunology Letters 145 (2012) 23–29

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Review

Chemokine receptor oligomerization: A further step toward chemokine function ˜ Laura Martínez Munoz, Borja López Holgado, Carlos Martínez-A, José Miguel Rodríguez-Frade, Mario Mellado ∗ Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Darwin 3, Campus de Cantoblanco, Madrid E-28049, Spain

a r t i c l e

i n f o

a b s t r a c t

Keywords: Chemokine receptors Oligomerization GPCR

A broad array of biological responses including cell polarization, movement, immune and inflammatory responses, as well as prevention of HIV-1 infection, are triggered by the chemokines, a family of secreted and structurally related chemoattractant proteins that bind to class A-specific seven-transmembrane receptors linked to G proteins. Chemokines and their receptors should not be considered isolated entities, as they act in complex networks. Chemokines bind as oligomers, or oligomerize after binding to glycosaminoglycans on endothelial cells, and are then presented to their receptors on target cells, facilitating the generation of chemoattractant gradients. The chemokine receptors form homo- and heterodimers, as well as higher order structures at the cell surface. These structures are dynamic and are regulated by receptor expression and ligand levels. Complexity is even greater, as in addition to regulation by cytokines and decoy receptors, chemokine and receptor levels are affected by proteolytic cleavage and other protein modifications. This complex scenario should be considered when analyzing chemokine biology and the ability of their antagonists to act in vivo. Strategies based on blocking or stabilizing ligand and receptor dimers could be alternative approaches that might have broad therapeutic potential. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

Lymphocytes are therefore one of the most motile mammalian cell types, as they must be in “the right place at the right time”, making lymphocyte migration and function interdependent processes. Lymphoid organs are specialized tissues that provide the anatomic location and microenvironment in which lymphocytes achieve phenotypic and functional maturity (primary lymphoid organs) or undergo activation (secondary and tertiary lymphoid organs). Studies undertaken at the beginning of the 1990s showed that leukocyte extravasation from blood to tissues takes place in discrete steps, involving a sequence of receptor–ligand interactions, including selectin, integrins and chemokines. It was later observed that this model, defined as the multi-step paradigm, also applied for lymphocyte entry to SLO via high endothelial venules, a process also known as homing [1,4]. The activation signals that upregulate integrin adhesiveness on rolling leukocytes in a subsecond timescale are provided by chemokines. This family of small (8–12 kDa) polypeptides were originally described for their role in recruitment of innate immune cells and certain effector T cells during inflammatory processes [5]. The chemokines orchestrate physiological lymphocyte recirculation, as binding to their receptors on lymphocytes has two functional outcomes: first, certain chemokines induce integrin activation, which triggers intravascular adhesion of lymphocytes [6] and second, chemokine gradients induce directed cell migration. Chemokines thus coordinate immune responses by recruiting blood-borne cells into target tissues and by regulating

Eukaryotic cell migration is a highly coordinated process that requires the integration of extracellular signals for synchronized triggering of intracellular mechanisms that generate cell movement. Diverse physiological events, from embryonic development to wound healing, rely largely on efficient cell migration. The importance of cell migration and the contribution of various chemokines and receptors have been studied extensively, and the function of migration has been clearly established in the surveillance of the organism by cells of the immune system. For rapid and effective antigen recognition, each mature lymphocyte must sample the organism in a continuous search for its cognate antigen. To achieve this, naïve lymphocytes have evolved to migrate continuously, residing transiently in the so-called secondary lymphoid organs (SLO) strategically located throughout the body. From SLO, these cells return to the blood to continue their migration before returning to the SLO, a process known as lymphocyte recirculation [1–3]. Effector cells leave the SLO and migrate to non-lymphoid tissues, guided by local inflammatory signals elicited by pathogens [3].

∗ Corresponding author. Tel.: +34 915854852; fax: +34 913720493. E-mail address: [email protected] (M. Mellado). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2012.04.012

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homeostasis through control of the microenvironmental organization of primary and SLO [3,5,7].

2. Chemokines: key molecules that govern cell movement The chemokines have been the focus of extraordinary interest over the last 25 years [8]. They are not only linked to lymphocyte trafficking, but also participate in regulation of T cell differentiation, HIV-1 infection, angiogenesis, tumorigenesis and development [9–13]. Nearly 50 chemokines have been described, which share remarkably similar three-dimensional structures despite their limited amino acid sequence similarity [9]. The original classification of chemokines based on structural criteria (C, CC, CXC and CX3C chemokines) is being abandoned, and replaced by a functional classification by which chemokines are grouped into two main categories, homeostatic or constitutive, and inflammatory or inducible [14]. In general, constitutive chemokines are regulated during development, whereas inducible chemokine expression is regulated mainly during inflammatory processes. In addition, several viruses encode highly selective chemokine receptor ligands that function as agonists or antagonists, and might thus have a role in viral dissemination or evasion of host immune responses [15]. Chemokines act by interacting with a group of seventransmembrane receptors coupled to G proteins of the GPCR family [8]. As for the chemokines, these receptors can also be grouped into two major families, CCR and CXCR, which interact with the CC and CXC chemokines, respectively [16]. There is apparently considerable promiscuity between chemokines and their receptors, with different ligands that can associate the same receptor and distinct receptors that can bind the same ligand. Furthermore, a single cell can express several chemokine receptors, simultaneously or during different stages of its life. Based on their broad range of functions, it is easy to deduce that chemokines must be central to a variety of diseases characterized by inflammation and cell infiltration [17] such as asthma, atherosclerosis, rheumatoid arthritis, multiple sclerosis, colitis, Crohn’s disease, experimental autoimmune encephalomyelitis (EAE) or psoriasis. Finally, CXCR4 and CCR5 are the two main co-receptors for HIV-1 infection [18], and some chemokine receptors also participate in tumor metastasis [12] and transplant rejection [19]. Given the correlation between the expression of specific chemokines and the specific recruitment of cell populations in the course of certain disease processes, the chemokines and their receptors have become a major focus in research and pharmaceutical laboratories. Their medical importance has led to intense effort to obtain structural information that could aid the development and optimization of therapeutics to inhibit their functions [20–22]. Conclusions drawn from animal models unfortunately cannot always be extrapolated to man, and although there are several molecules in clinical trials to target chemokines and their receptors in inflammatory diseases, results so far are very disappointing. Blockade of a chemokine or/and a chemokine receptor binding site might be an overly simplistic approach to altering chemokine function, and a reanalysis of chemokine biology is thus justified. Expression of chemokines and their receptors is finely regulated by several factors that include cytokines, growth factors, and cell cycle status [23–25], indicating that cell context influences these responses. The complexity of this scenario is completed by the glycosaminoglycans (GAG), which present chemokines to cells [26], and by the propensity of ligands and receptors to multimerize. Antagonists and neutralizing antibodies that are active in vitro are completely inactive when tested in vivo, possibly because they cannot recognize the structure of the chemokines or of their receptors in this context.

At nanomolar concentrations, some obligate chemokine monomers retain full activity for triggering receptor-mediated cell responses in vitro [27,28]. Several lines of evidence nonetheless indicate that at chemokine-rich inflammation sites in vivo, these proteins might also multimerize through GAG-mediated or -independent mechanisms, which would influence their cell activation properties [29]. As several excellent reviews have been published on the links between chemokine structure and function, these topics will not be reiterated here; instead, we limit our discussion to chemokine receptor conformation. 3. Chemokine receptors: a critical element for chemokine function 3.1. Introduction There are 20 signaling chemokine receptors, as well as three non-signaling scavenger receptors, all of which govern the immune response by binding, internalizing, and degrading chemokines [30,31], as well as several receptors encoded by viruses. All of these receptors are comprised of approximately 350 amino acids, with a molecular weight of approximately 40 kDa. The extracellular domain consists of the N terminus and three extracellular loops that act in concert to bind the chemokine ligand. The intracellular region is composed of three loops and the C terminus, which collaborate to transduce the chemokine signal. Based on their amino acid sequences, chemokine receptors belong to the class A rhodopsin-like family. Although similar to other seventransmembrane receptors, the chemokine receptors share certain structural features, such as the highly conserved DRYLAIV amino acid sequence in the second intracellular loop [32]; this motif is absent in the decoy receptors, indicating its implication in signaling. 3.2. Chemokine receptor oligomerization Several studies report that certain GPCR can function as monomers in vitro [33–35]; bioinformatic, structural and functional analyses have nonetheless advanced the concept that chemokine receptors are probably expressed at the cell surface as dimers/oligomers [36–40]. The crystal structure of CXCR4 in the presence of antagonists (a small molecule or a cyclic peptide) recently revealed a homodimeric conformation with the interface located in transmembrane regions V and VI [41]. Although initially considered a cytokine habit, evidence thus supports the existence of chemokine receptor homo- and heterodimers (Table 1). Chemokine receptor dimerization was first demonstrated for CCR2, the CCL2 receptor, using various experimental approaches that included agonist monoclonal antibodies, tagged receptors, and mutant receptors [42]. Coimmunoprecipitation and Western blot studies of chemokine receptors often detect individual receptors as well as high molecular weight species that could correspond to receptor complexes. These types of strategies nonetheless yield a static view of the system that can lead to misinterpretation of results. For example, in Western blot and coimmunoprecipitation, we detected CCR2 dimers only after ligand activation, leading us to conclude that ligands had an active role in promoting receptor dimerization [42], although later experiments using imaging-based techniques clearly showed that dimers form in the absence of chemokines [37,38,40]. The difficulty in detecting these complexes by immunoprecipitation might be due to instability of the dimer conformation in the absence of ligand, which cannot resist the detergent treatment. Analysis of live and fixed receptor-expressing cells using fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques [43] greatly enhanced our understanding of

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Table 1 Chemokine receptor homo- and heterodimers. Experimental evidence supports chemokine receptor homo- and heterodimerization. Ab, antibody; BiFC, bimolecular fluorescence microscopy; BRET, bioluminescence resonance energy transfer; co-IP, co-immunoprecipitation; FLIM, fluorescence lifetime imaging microscopy; FRET, fluorescence resonance energy transfer; IS, immunological synapse; mAb, monoclonal antibody; PCA, protein fragment complementation assay; TM, transmembrane domain.

Chemokine receptor homodimerization

Receptor

Method

Cells

Comments

References

CCR2/CCR2

Co-IP

Cell line

First description of chemokine receptor dimerization

[42]

BRET BRET Co-IP Co-IP

Cell line Cell line Cell line Cell line

CCR5/CCR5

Cell line

CXCR1/CXCR1

CXCR2/CXCR2

FRET FLIM Co-IP Bio-informatic BiFC

Cell line and primary cells

Co-IP FRET Tr-FRET BRET FRET

Cell line

Co-IP

Co-IP FRET BRET FRET CXCR4/CXCR4

Co-IP BRET

Cell line

Cell line and primary cells Cell line and primary cells Cell line

Cell line and primary cells Primary cells Cell line

BRET Co-IP FRET

Cell line

BRET BRET-BiFC

Cell line

PCA

Cell line

BRET

Cell line

Crystalization CXCR7/CXCR7

Chemokine receptor heterodimerization

CCR2/CCR5

BRET

Cell line

PCA

Cell line

BRET

Cell Line

Co-IP FRET

Cell line

Co-IP

Cell line and primary cells

In silico (lipid-facing mutational analysis) Co-IP BRET BRET

CCR2/CXCR4

Co-IP FRET BRET

Cell line Cell line

Cell line Cell line

Anti-CCR5 Ab increase BRET Anti-CCR5 mAb induce dimerization CCR5 dimerization blocks HIV-1 infection CCR532 retains CCR5 in intracellular compartments Ligands stabilize preformed receptor dimer. TM1–TM4 peptides reduce FRET signals and block function

[38] [46] [81] [82] [51] [37]

[83] CCR5 homodimers signaling differs from that of CCR5/CXCR4 heterodimers by binding of a specific adaptor protein [40] CXCL8 does not promote changes in FRET signal

CXCL8 promotes conformational changes in homodimers GluR1 co-expression impairs dimer formation, CXCR2 deletion mutants act as dominant negative receptors CXCL8 does not promote changes in FRET signal

[45]

CXCL8 promotes conformational changes in homodimers

[45]

CXCR4 antagonists and CXCL12 alter BRET signal Ligand independent dimerization gp120 increases FRET, but AMD3100 decreases gp120-induced FRET in cells expressing hCD4 Higher-order oligomerization in a ligand independent fashion; TM implicated in oligomerization CXCL12 and pharmacological agents produced changes in bioluminescence in CXCR4/CXCR4 CXCL12 modifies the conformation of preformed CXCR4 homodimers First crystal structure for a chemokine receptor CXCL12 modifies the conformation of preformed CXCR7 homodimers Inhibitors and ligands enhance CXCR7 homodimerization AMD3100 is an allosteric agonist; CXCL12 induces conformational changes Ligands and mAb stabilize preformed receptor heterodimers, blocking HIV-1 entry Homo- and heterodimers activate distinct signaling pathways Bioinformatic analysis indicating the feasibility of CCR2/CCR5 heterodimers Cross-competition in ligand binding assays Chemokines do not influence the heterodimerization status. Negative binding cooperativity Anti-CCR2 mAb stabilizes preformed heterodimers, blocking HIV-1 entry CXCR4 TM peptides do not affect BRET signal. Ligand induces conformational changes in preformed dimers

[64]

[40]

[84] [38] [63] [85]

[36]

[86]

[76] [41] [76] [86] [87]

[88,89]

[72] [90] [56] [57]

[89] [38]

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Table 1 (Continued) Receptor

CCR5/CXCR4

CXCR1/CXCR2

CXCR4/CXCR7

CCR2/CCR5/CXCR4

Method

Cells

Comments

References

BRET

Co-IP

Cell line

Co-IP BRET

Cell line

FRET

Cell line

BRET

Cell line

BiFC

Cell line

Co-IP FRET BRET FRET BRET FRET BRET

Cell line

Allosteric transinhibition by specific inhibitors in the heterodimer Ligands induce receptor dimers; blocks HIV-1 entry CCR5 interferes with CXCR4 expression, endocytosis and HIV-1 co-receptor activity CCR5/CXCR4 heterodimers are recruited to the IS. Heterodimer is essential for the costimulatory effect in T cell activation Ligands promote conformational changes in the heterodimer Specific ligands promote conformational changes Heterodimer does not interact with NHERF1, hence CCR5/CXCR4 signaling differs from that of CCR5 homodimer Receptors form homo- as well as heterodimers

[58]

Co-IP

Cell line and primary cell Cell line

PCA BRET BiFC

Cell line and primary cells Cell line Cell line Cell line Cell line

receptor complexes at the cell surface. These methods are based on detection of receptor-coupled fluorophores excited by an external light source (FRET) or by enzyme catalysis of a luminescent substrate (BRET). Several groups used this technology to show that chemokine receptors form homodimers, complexes formed by the same receptor, and heterodimers, complexes formed by two distinct receptors. Photobleaching FRET experiments showed that all CXC chemokine receptors form homo- and heterodimers ˜ et al., submitted), and that FRET efficiency varies (Martínez-Munoz depending on the receptors analyzed. As this efficiency depends on the distance between and orientation of the fluorophores, these data indicate that the conformation adopted by a given receptor is ˜ et al., influenced by its coexpression with others (Martínez-Munoz submitted). FRET or BRET efficiency can also be determined at different ratios of donor:acceptor fluorophores coupled to receptors. These methods allow generation of saturated BRET or FRET curves that permit evaluation of the half-maximal BRET/FRET signal, that is, the relative affinity of receptor molecules in the complex. Apparent affinity calculations are used to study a specific receptor complex in a variety of experimental conditions, i.e., after ligand addition, coexpression of an unlabeled receptor, or cell/membrane modification [38,44,45]. We cannot compare the association force between different receptor complexes, however, as affinity difference is only one possible cause of the variations in FRET and BRET values [43]. As some of these methods allow evaluation of the subcellular locations of these complexes, we assume that homo- and heterodimers are found not only at the cell surface even in the absence of ligands, but also in intracellular vesicles, presumably formed during receptor synthesis and maturation [46]. As for other GPCR [47], CCR5 and CXCR4 complexes have been detected in small transGolgi vesicles [48], suggesting that receptor oligomerization is a mechanism that regulates receptor expression at the cell surface. This observation concurs with other GPCR studies suggesting that early dimerization in the endoplasmic reticulum (ER), which could have a role in trafficking, is a general feature of this receptor family [49]. CXCR2 coexpression with a mutant CXCR1 that is retained

CXCL8 promotes disruption of the heterodimer Synergic response to CXCL12 Heterodimer signals as independent entity, regulating CXCR4 signaling First evidence of chemokine receptor hetero-oligomerization

[88] [91]

[73]

[44] [54] [83]

[40]

[45] [78] [76] [86] [54]

in the ER causes a substantial reduction in cell surface expression [40]. It was hypothesized that, through dimerization with CCR5, the natural genetic mutant CCR532 retains the complex in the ER and prevents transport of normal CCR5 to the cell surface, retarding progression of HIV-1 infection [50,51]. Nonetheless, no data yet indicate whether dimerization facilitates chemokine receptor expression at the membrane, as for other GPCR [52]. Atomic force microscopy has been used to show that rhodopsin, the prototypical member of the class A GPCR, forms dimers that are condensed into supramolecular structures whose topography consists of densely packed lines [53]. Experiments using transfected cells indicate that chemokine receptor homo- and heterodimers form large receptor arrays on the cell membrane, although this remains to be validated in primary cells. At the cell membrane, CXCR4 engages in higher-order oligomerization in a ligand-independent fashion, as shown by a combination of BRET and bimolecular fluorescence complementation analysis [36]; this approach also demonstrated oligomers containing CXCR4, CCR5 and CCR2 [54]. The recent characterization of the CXCR4 crystal structure also supports interactions between CXCR4 dimers in higher order complexes [41]. Bioinformatic structure analysis of a CCR5 homology model based on the rhodopsin receptor allowed us to predict interaction of hydrophobic amino acid residues in the transmembrane regions [55]; studies of CXCR4 mutants lacking various domains [36], together with the crystal structure of CXCR4 bound to small antagonists, confirmed that chemokine receptor dimerization is promoted mainly by these interactions. Many questions remain to be answered. Do all chemokine receptors interact through similar regions, or are there dimer-specific dimerization regions depending on the receptors involved? Does homo- and heterodimerization of a given receptor always involve the same residues? Which regions are involved in the interactions between chemokine receptors in larger arrays? Is there any difference between receptor dimers alone or in the presence of ligand? The answers to these questions will be of great value in the design of new drugs able to modulate chemokine responses through interference with receptor dimerization.

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3.3. Chemokine receptor oligomers: dynamic structures One possible consequence of the existence of receptor arrays is allosteric modulation between chemokine receptor dimers. In transfected and primary cells coexpressing CCR2/CCR5, CCR5specific ligands prevent binding of CCR2-specific ligands, although they are unable to bind CCR2 coexpressed [56,57]; similar experiments show allosteric transmodulation for CCR2 and CXCR4 [58]. The CXCR4-specific antagonist AMD3100 blocks binding of CCR2specific ligands; conversely, the CCR2, CCR5, and CXCR3 antagonist TAK779 [59] strongly inhibits CXCL12 binding in cells that coexpress CXCR4 [58]. Results were similar when TAK779 and AMD3100 were tested in cells expressing multimeric CXCR4, CCR5 and CCR2 complexes [54]. An alternative explanation for allosteric modulation is that G protein might contact the partners of a dimeric complex in a non-symmetric manner [60], leading to a differential effect of G protein on agonist affinity for the receptors involved; this could result in negative cooperativity in agonist binding. Limitations in G protein pool availability might also explain negative ligand cooperativity; even without crosstalk between the receptors that form the complex, they could attempt to bind simultaneously to the same G protein [61]. These observations are difficult to interpret if we consider the chemokine receptors as individual entities. They nonetheless fit a model that we term the “cigar bundle” [62], in which multiple chemokine receptors are expressed at the cell surface in dynamic arrays; presence of a ligand affects not only its own specific receptor, but also other receptors in the same array. In accordance with this concept, we observed that CXCR1 and CXCR2 homo- and heterodimers are dynamically regulated both by receptor expression and by ligand binding [45]. Although FRET and BRET techniques are limited in their capacity to distinguish between conformational changes and complex disruption, FRET/BRET efficiency variations have been observed after ligand addition. CXCL12 addition increases BRET between CXCR4 homodimers [38], ligand binding to CXCR4 or CCR5 alters CXCR4/CCR5 heterodimers [44], and CXCL8 abolishes FRET signals for CXCR1/CXCR2 heterodimers while stabilizing values for the two homodimers [45]. Other laboratories nonetheless do not observe chemokine effects on receptor conformation [63]; for example, CCL2 does not promote conformational changes in CXCR4/CCR2 heterodimers [38].

4. Functional consequences of chemokine receptor dimerization Although we cannot rule out the possibility that chemokine receptor monomers can be operative, several indirect observations suggest that the dimer is the minimal functional unit of chemokine receptors. Synthetic peptides that prevent receptor dimerization reduce in vitro and in vivo function [37,39]. Some non-functional receptor mutants behave as dominant negative on wild type receptors; non-functional CCR2 and CXCR2 mutants block ligand-mediated cell migration by dimerizing with their wild type receptors [42,64]. Heterodimerization between CCR5 and the non-functional receptor DARC inhibits CCR5-mediated signaling [65]. Although there are no doubts about chemokine receptor oligomerization, evidence for the functional relevance of homoand heterodimerization is nonetheless far from conclusive (Fig. 1). The discovery that GPCR form heterodimers with distinct pharmacological properties raises fascinating possibilities regarding the pharmacological plasticity and diversity of these signaling systems. Receptor dimerization affects ligand binding affinity for some GPCR [66] and modulates signal transduction [67]; there are nonetheless few data indicating affinity change of a chemokine for a given receptor expressed alone or with other receptors. Changes in IC50 for

inactive dimers

active heterodimers couples to signaling molecules

active homodimer couples to signaling molecules

FUNCTIONAL CONSEQUENCES [40, 51] 2. Allosteric modulation

[56-59] [54] [72-74]

5. Modulation of ligand functions

[65,76-80]

Fig. 1. Functional relevance of chemokine receptor oligomerization. Chemokine receptors are organized into oligomeric structures termed arrays, which contain homo- and heterodimers, probably formed during receptor synthesis and maturation. Homo- and heterodimers are dynamic conformations regulated not only by receptor expression, but also by ligand levels. Ligand binding to the receptor complex stabilizes the active conformation and signaling is triggered; these conformational changes are also propagated to neighboring receptors. After the activated complex is internalized, the array relaxes and receptors assume the resting conformation. Although few reports analyze the functional relevance of chemokine receptor heterodimers, several studies suggest their involvement in regulation of receptor trafficking, modulation of ligand affinity, promotion of allosteric modulation, triggering of distinct signaling cascades and/or modulation of behavior of other receptors in the complex.

CXCL12 in cells expressing CXCR4 alone or with CCR5 (1.46 nM vs. 0.35 nM) suggest modulation of CXCL12 binding affinity for CXCR4 as a result of receptor heterodimerization. In contrast, in the same conditions, IC50 for CCL4 binding to CCR5 was unaffected by CXCR4 coexpression (2.19 nM vs. 2.21 nM) [54]. Although associated with cooperative downstream signaling pathways [68] rather than with effects on ligand binding affinity, synergism between CCR7 ligands and CXCL13 renders T cells more competent in response to migratory cues [69]. CCL5/CXCL4 complexes are involved in monocyte recruitment and retention at atheroma plaques [70], and specific in vivo and in vitro functions have been reported for the CXCL8–CXCL4, CCL22–CCL19 and CCL2–CCL8 chemokine pairs [69,71]. Recent results from several groups support the hypothesis of functional diversity between chemokine receptor homo- and heterodimers. CCR2/CCR5 heterodimers trigger specific signaling events such as G11 activation, which in turn promotes specific cell functions including increased cell adhesion [72]. CCR5/CXCR4 heterodimers are recruited to the immunological synapse, where they couple to Gq/11 proteins; this leads to T cell insensitivity to chemotactic gradients, enhanced proliferation and cytokine production, and formation of more stable conjugates [73,74]. An increasing number of reports assign a role to receptor oligomerization in chemokine function. Nonetheless, lack of specific tools for stabilizing heterodimeric complexes and the constantly changing equilibrium between receptor conformations complicate study of

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the signaling and function of each conformation, as well as its pharmacological implications. Several reports nonetheless indicate that receptor heterodimerization can modulate the behavior of individual partners. CXCR7 shares the CXCL12 ligand with CXCR4, but does not activate G␣i proteins [75,76]. CXCR7/CXCR4 heterodimers are reported in both transfectants and primary cells, where CXCR7 modulates CXCR4-mediated responses [76–78]; CXCR7/CXCR4 heterodimers should thus be considered distinct functional units with novel properties, which contribute to the functional plasticity of CXCL12. Similarly, DARC inhibits CCR5-mediated functions by forming DARC/CCR5 complexes [65]. Do these data suggest that the non-signaling receptors DARC, D6 or CXC–CKR can, in addition to their scavenging properties, modulate chemokine receptor function through dimerization? Functional interaction between chemokine receptors and other unrelated GPCR has also been described. Heterodimerization of CCR5 or CXCR4 with opioid receptors alters chemokine responses [79]. Pharmacological modulation of the CXCR2 receptor results in changes in DOR (delta opioid receptor) function; interestingly, a small molecule antagonist for CXCR2 seems to enhance signaling in response to DOR agonists, although the ligands do not compete directly for the other receptors [80]. 5. Conclusions Although much evidence supports a central role for chemokines in physiological and pathological processes and the pharmaceutical industry has made an extraordinary effort to develop chemokinebased therapeutic drugs [22], results so far are very disappointing. Blockade of a chemokine or/and a receptor binding site might be an overly simplistic approach to altering the function of many chemokines. Given the complex scenario of chemokine activity, with ligands and receptors forming complexes in dynamic equilibrium, with proteins that bind and modulate their functions, and with post-transductional modifications, one can speculate that antagonists designed in vitro might not recognize a specific motif in vivo. To better understand chemokine function and improve design of new therapeutic tools, it is time to consider factors that have not been taken into account previously. Work in recent years has provided evidence that chemokine receptors are found and might function as dimers. We must confirm the functional relevance and regulation of homo- and heterodimeric complexes in the cells, tissues, and organisms in which they are naturally expressed, since most current studies use heterologous expression systems. When these questions begin to be resolved, the formidable challenge of chemokine receptor oligomerization will offer new possibilities for the design of drug-screening and therapeutic strategies. Acknowledgements We thank people at the Chemokine signaling’s group for much of the work that contributed to this review, and to C. Bastos and C. Mark for secretarial and editorial assistance, respectively. This work was supported in part by grants from the Spanish Ministry of Science and Innovation (SAF 2008-03388), from Instituto de Salud Carlos III (ISCIII) program RETICS, RD08/0075 (RIER) (RD08/0075/0010) and the European Union (Innochem LSHB-CT-2005-518167 and FP7-integrated project Masterswitch no. 223404). References [1] Butcher EC, Picker LJ. Lymphocyte homing and homeostasis. Science 1996;272:60–6. [2] Gowans JL, Knight EJ. The Route of Re-Circulation of Lymphocytes in the Rat. Proc R Soc Lond B Biol Sci 1964;159:257–82.

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