Common and Biased Signaling Pathways of the Chemokine Receptor CCR7 Elicited by Its Ligands CCL19 and CCL21 in Leukocytes Mark A

Erschienen in: Journal of Leukocyte Biology ; 99 (2016), 6. - S. 869-882 https://dx.doi.org/10.1189/jlb.2MR0815-380R

Common and biased signaling pathways of the chemokine receptor CCR7 elicited by its ligands CCL19 and CCL21 in leukocytes Mark A. Hauser and Daniel F. Legler1 Biotechnology Institute Thurgau at the University of Konstanz, Kreuzlingen, Switzerland

ABSTRACT Introduction Chemokines are pivotal regulators of cell migration Chemokines are chemotactic cytokines that orchestrate the during continuous immune surveillance, inflammation, coordinate positioning of cells in the body, including immune homeostasis, and development. Chemokine binding to cells. Chemokines play key roles in various processes, ranging their 7-transmembrane domain, G-protein-coupled re- from immune cell development and homeostasis to initiation of ceptors causes conformational changes that elicit in- innate and adaptive immune responses and the pathophysio- tracellular signaling pathways to acquire and maintain logical recruitment of immune cells in infection and disease. an asymmetric architectural organization and a polarized distribution of signaling molecules necessary for Chemokines constitute the largest family of cytokines, consist- ; directional cell migration. Leukocytes rely on the in- ing of 40 endogenous members in humans and mice [1]. terplay of chemokine-triggered migration modules to Chemokines can be functionally divided into 2 major groups: promote amoeboid-like locomotion. One of the most those that are primarily expressed upon activation, termed important chemokine receptors for adaptive immune inflammatory chemokines, and those that are constitutively cell migration is the CC-chemokine receptor CCR7. expressed at discrete locations in the absence of apparent CCR7 and its ligands CCL19 and CCL21 control homing activating stimuli, classified as homeostatic chemokines [1]. The of T cells and dendritic cells to areas of the lymph differences between inflammatory and homeostatic chemokines nodes where T cell priming and the initiation of the are thought to have an evolutionary origin [1]. Inflammatory adaptive immune response occur. Moreover, CCR7 chemokines are clustered on chromosomes 4 and 17 and were signaling also contributes to T cell development in the rapidly evolved [1]. The reason for this is still unclear but is thymus and to lymphorganogenesis. Although the CCR7–CCL19/CCL21 axis evolved to benefit the host, probably a result of strong, selective pressure to increase their inappropriate regulation or use of these proteins numbers when early humans experienced new pathogens that can contribute or cause pathobiology of chronic in- presented a threat to human survival [1]. In contrast, flammation, tumorigenesis, and metastasis, as well homeostatic chemokines are located in mini-clusters on as autoimmune diseases. Therefore, it appears as the different chromosomes, are well conserved between species and CCR7–CCL19/CCL21 axis is tightly regulated at numer- function in a more predictable manner [1]. ous intersections. Here, we discuss the multiple regu- Chemokines elicit their biologic function by binding to their latory mechanism of CCR7 signaling and its influence corresponding chemokine receptors on the surface of its target on CCR7 function. In particular, we focus on the cells. Chemokine receptors belong to the rhodopsin-like class A functional diversity of the 2 CCR7 ligands, CCL19 and of 7-transmembrane receptors [2]. Chemokine-binding recep- CCL21, as well as on their impact on biased signaling. The understanding of the molecular determinants tors are differentially expressed on all types of immune cells of biased signaling and the multiple layers of and can be subdivided into 2 groups: the classic GPCRs, which CCR7 regulation holds the promise for potential signal through pertussis toxin-sensitive Gai types of heterotri- future therapeutic intervention. meric G-proteins (conventional chemokine receptors), and the decoy chemokine receptors, which are scavenging chemokines in a G-protein-independent manner (atypical chemokine receptors) but do not transmit signals directly resulting in cell migration [2, 3]. There are ;20 conventional chemokine 2 2 Abbreviations: / = deficient, 2D/3D = 2/3-dimensional, Ca2+ = calcium, DC receptors and 4 "founding" members of atypical chemokine = dendritic cell, DOCK2 = dedicator of cytokinesis 2, FRC = fibroblastic receptors [3]. Furthermore, conventional chemokine receptors reticular cell, GAG = glycosaminoglycan, GEF = guanidine nucleotide exchange factor, GPCR = G-protein-coupled receptor, GRK = G-protein receptor kinase, GSK3 = glycogen synthase kinase 3, GTPase = guanosine triphosphatase, HEK = human embryonic kidney, HEV = high endothelial 1. Correspondence: BITg at the University of Konstanz, Unterseestrasse 47, (continued on next page) CH-8280 Kreuzlingen, Switzerland. E-mail: [email protected]

869 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-1ufm2jquuhz529 are classed according to their cognate chemokine family. In CCR7, CCR10, and CXCR3 [8, 9]. Interestingly, biased general, chemokines only bind receptors of their own class. receptors that bind multiple chemokines with overlapping However, within a given subfamily, there is a large promiscuity, functions are more commonly involved in inflammatory meaning that many chemokines bind multiple chemokine responses, with the exception of CCR7, which clearly owns receptors, and multiple receptors bind many chemokines [4]. homeostatic functions. Overall, homeostatic chemokine recep- Chemokine-mediated functions play a crucial role in main- tors, in contrast to inflammatory chemokine receptors, show a taining homeostasis, as well as during pathophysiological more restricted ligand use of maximal 2 ligands/receptor. Even changes, and therefore, are tightly regulated. At the systems level, in the case of 2 ligands, they are not just redundant but clearly their spatiotemporal and tissue-specific expression is 1 out of appear to have distinct roles, as exemplified by CCR7 and its 2 multiple regulation levels. On the cellular level, chemokine ligands, CCL19 and CCL21. In this review, we summarize the binding, e.g., to GAGs or chemokine receptor signaling, can vary most important functions of CCR7 and subsequently, focus on between cells, depending on the distinct expression pattern of biased signaling and function elicited by its 2 natural ligands. proteins. On the molecular level, chemokines can elicit different or biased intracellular signaling pathways. FUNCTION OF THE CCR7—CCL19/ Based on the classic model of chemokine receptor activation, CCL21 AXIS ligand binding promotes stabilization of a single active conformation of the receptor that leads to the activation of associated The chemokine receptor CCR7, together with its 2 ligands, heterotrimeric G-proteins, dependent on the strength of the plays essential roles in leukocyte homing, thereby regulating imparted signal. This activation involves the exchange of bound adaptive immunity and tolerance [10, 11]. CCR7 has 2 GDP for GTP by the Ga subunit of the G-protein, resulting in the ligands that are constitutively expressed, namely, CCL19 dissociation of the heterotrimeric G-protein into Ga and Gbg (also known as EBI-1 ligand chemokine, MIP-3b, Exodus-3, subunits [5]. The triggering of G-proteins controls the pro- or CKb11) and CCL21 (secondary lymphoid-tissue chemo- duction of second-messenger molecules, such as cAMP, in- kine, chemokine with 6 cysteines, thymus-derived chemo- tracellular Ca2+, and inositol phosphates. Modulation of those tactic agent 4, Exodus-2) [12, 13]. The major difference second messengers activates downstream effectors, including between the 2 ligands is a highly charged 40 aa C-terminal PKC and Akt (PKB). Subsequently, chemokine receptor- extension of CCL21, allowing its immobilization on GAGs mediated G-protein activation is terminated by the phosphory- that is not present in CCL19. lation of the receptor on serine and threonine residues through CCR7 is up-regulated on myeloid DCs upon encountering a specific GRKs. Serine/threonine-phosphorylated receptors re- “danger signal” (derived from pathogens, inflammatory medi- cruit b-arrestins, resulting in receptor internalization to endo- ators, or tissue damage), resulting in homing to lymph nodes via somal vesicles, thereby efficiently limiting receptor susceptibility afferent lymphatics and in induction of immunity [11, 14, 15]. to extracellular ligands. Importantly, b-arrestins are also able to Some myeloid DCs, described as semimature DCs, expresses transduce signals on their own, for instance, through activation CCR7 under steady-state conditions and hence, in the absence of the ERK1/2 pathway (reviewed in ref. [6]). of a danger signal, to home to lymph nodes, thereby As most chemokine receptors can be activated by multiple contributing to peripheral tolerance [11, 16, 17]. Plasmacytoid chemokines, the recognition of substantial differences in the DCs use CCR7 for lymph node homing through HEVs rather interaction of certain chemokine receptors with particular than lymphatics [18]. Moreover, CCR7 is expressed by ligands and multiple signaling proteins has led to the important various lymphocyte subsets, including na¨ıve T cells, central concept of biased signaling, as initially described by Kenakin in memory T cells, and regulatory T cells, but also on B and NK 1995 [7]. In this concept, chemokine stimulation has a unique cells [11, 19–21]. T cells that circulate in the blood enter quality and a magnitude of receptor activation. This is lymphoid organs via HEVs in a multistep process involving explained by the fact that each ligand does not activate the rolling, activation, arrest, and transmigration [11, 22–24]. In receptor through the stabilization of a common active state but this process, CCL21, presented by GAGs on HEVs, triggers rather, that different agonists stabilize unique and distinct active T cells, together with shear forces derived from the blood flow, states of the receptor to create a signal that is biased toward resulting in integrin activation and subsequent firm adhesion particular subsets of signaling pathways. In case of extreme andarrestoftheTcellsonHEVs[23,25–29]. Additionally, signal bias, 1 ligand would exclusively activate G-protein CCR7 ligands can bind to P-selectin glycoprotein ligand 1 to signaling, whereas another ligand would solely signal through facilitate selectin-independent T cell adhesion to HEVs and b-arrestins. Experimental comparison of signaling pathway efficient extravasation into secondary lymphoid organs [30]. activation by different chemokines revealed a biased signaling Once T cells have entered the lymph node, Gai-dependent and pattern for the chemokine receptors CCR1, CCR2, CCR5, CCR7-dependent and CCR7-independent signaling regulate locomotion and fine positioning within the T cell zone, which (continued from previous page) guarantees interactions with DCs [31–33]. venule, NMR = nuclear magnetic resonance, PKB/C = protein kinase B/C, The importance of the CCR7–CCL19/CCL21 axis for homing PLA2/C = phospholipase A2/C, plt/plt = paucity of lymph node T cells, Pyk2 to secondary lymphoid organs has been demonstrated in CCR7 = proline-rich tyrosine kinase 2, ROCK = Rho-associated coiled-coil forming gene-targeted mice [34] and in plt/plt mice, a spontaneous protein kinase, S1P1 = sphingosine-1 phosphate receptor 1, TEM = transendothelial migration, Tiam1 = T cell lymphoma invasion and metastasis mutant strain that carries an autosomal recessive mutation where 1, TM = transmembrane domain, vMIP-II = viral MIP II a large gene locus encoding CCL21-Ser and CCL19 is deleted

870 [35–38]. Of note, 2 mouse CCL21 genes were identified that Receptor desensitization differ by a single amino acid, referred to as CCL21-Ser and In lymph nodes, T cell priming by DCs was found to occur in 3 CCL21-Leu [37]. The 2 CCL21 forms in mice appear functionally successive phases [45]. The stages are characterized by identical but differ in their expression pattern; CCL21-Ser is different motile behavior of the T cells. In the first stage, na¨ıve expressed in lymphoid tissue and CCL21-Leu on lymphatic T cells perform a random guided walk on stromal cells in vessels of nonlymphoid organs. The human genome includes search for cognate antigen-bearing DCs. In the second stage, 2/2 only 1 CCL21 gene [10, 11]. As Ccl19 mice have only subtle where T cells encounter cognate peptides presented by DCs, fi steady-state defects [39], CCL21 seems to be suf cient for most their migratory speed decreases in a manner dictated by the CCR7-driven processes. The phenotypes of these mice and the TCR affinity and the amount of peptide presented. In the third – biologic functions of the CCR7 CCL19/CCL21 axis, including stage, T cells regain a motile phenotype, accompanied by their role in thymic T cell development, lymphorganogenesis, profound cell proliferation. This observation led to the “stop tumorigenesis, and autoimmunity, have been reviewed in detail and go” traffic model, presented by Michael Dustin [46]. – elsewhere [10 12]. Interestingly, the lowest T cell motility pattern is observed Here, we would like to point out that lymphatic endothelial when T cells encounter cognate DCs on FRCs, the major cells in peripheral tissues seem to express CCL21 solely [36, 40], source of CCR7 ligands in lymph nodes [41, 47]. Hence, one whereas FRCs in the T cell zone of lymph nodes are the major way of delivering a chemotactic stop signal could be the source of CCL19 and CCL21 [41]. Moreover, mature DCs that complete sequestration of the receptor from the cell surface; entered secondary lymphoid organs serve as an additional source however, this is not observed for CCR7 [42, 48]. In this regard, for CCL19 [36]. Interestingly, only CCL19 but not CCL21 can it is interesting to note that only CCL19, but not CCL21, desensitize and internalize CCR7 effectively, which contributes to induces robust CCR7 internalization. Moreover, the level of fi ne tuning of T cell migration [42, 43]. Thus, CCL19 and CCL21 CCR7 surface expression was noted not to correlate directly are not redundant chemokines binding the same receptor but with the strength of the signaling outcome [49, 50]. Therefore, instead, appear to have discrete functions. sequestration of surface CCR7 does not necessarily lead to a reduction of the maximal signal stimulation. We propose that CCR7 removal would rather cause a dose-response shift so that FUNCTIONAL DIFFERENCES BETWEEN higher CCR7 ligand concentrations are required to elicit the CCL19 AND CCL21 same magnitude of signal response. Furthermore, CCL19 binding at the leading edge of the cell causes ligand Soluble versus immobilized chemokines sequestration. The subsequent CCR7 internalization (at the On the cellular level, 2 main responses are triggered by CCR7 leading edge) renders the cell progressively less sensitive to the signaling: 1) integrin activation that causes cell adhesion, chemotactic cues. The likely scenario is that CCL21, immobi- mainly to endothelial surfaces, and 2) polarization of the lized on stoma cells in lymph nodes, provides haptokinetic actomyosin cytoskeleton that causes migration along established cues for CCR7-expressing lymphocytes and DCs, guiding them chemokine gradients [44]. Whereas both CCR7 ligands are able from the parenchyma toward the T cell zone. Then, soluble to polarize the cytoskeleton and therefore, cause chemotactic CCL19 and/or soluble CCL21 presumably dominate over movement, only CCL21 is immobilized via its C terminus to adhesive CCL21-mediated migration, as observed in vitro [44], surfaces and induces inside-out activation of integrins and cell plausibly through CCR7 desensitization [43], to guide the cells adhesion to facilitate haptokinetic movement of DCs in vitro until they arrive in the T cell zone. [44]. In fact, DCs, layered on unfixed cryosections of lymph nodes, synchronically migrated directionally from the periphery toward the T cell zone [44]. Interestingly, DCs can pro- Role in costimulation, T cell survival, and teolytically truncate CCL21 by removing its heparin sulfate- dendrite extension binding domain to generate a soluble form of CCL21, which In addition to inducing directional steering of cells, CCR7 has induces DC chemotaxis but not adhesion and is therefore been described to provide costimulatory and survival cues. Again, thought to act more like CCL19 [44]. Remarkably, when there is functionally divergence between both CCR7 ligands. For immobilized and soluble chemokines were offered in an in vitro instance, it has been shown that only CCL21-mediated interstitial setting, DCs displayed a combined response and performed a T cell motility and TCR signaling are integrated during the early directionally biased haptokinetic migration. In other words, promigratory phase of T cell–DC encountering. Thereby, CCR7, DCs were proposed to swarm into T cell zones using triggered by CCL21, lowered the TCR signal threshold and acted immobilized CCL21 for adhesive random migration and soluble as a costimulatory molecule [51]. Moreover, a nonredundant, chemokines for steering [44]. Likewise, immobilized but not additive function of CCL19, in providing IL-7-dependent survival soluble CCL21 promoted robust and sustained T cell motility signals for na¨ıve T cell homeostasis, has been described [41]. 2 2 [28]. Of note, immobilized CCL21 triggered polarized re- Consequently, Ccl19 / mice displayed a gradual decrease in the distribution of integrins on T cells but required shear forces to number of transferred T cells compared with wild-type mice [41]. activate the integrin by chemokines [28]. Thus, by use of In addition, albeit CCL19 and CCL21 are indistinguishable when chemokines that are immobilized or soluble, the same CCR7 comparing their ability to modulate DC maturation and antigen receptor system is able to integrate kinetic and tactic mecha- uptake, the 2 CCR7 ligands exert antagonistic influences on the nisms for leukocyte migration. initiation of dendritic extensions of mature DCs; whereas CCL19

871 induces, CCL21 inhibits marked dendrite extensions that subtle changes of so-called microswitch domains [58, 59]. facilitate interactions with T cells [52]. Therefore, general evidence obtained from X-ray crystallogra- phy and NMR spectroscopy of different GPCRs helps to Role in T cell emigration and DC homing understand CCR7 receptor biology. Recent structural insights Interestingly, a role for CCL19, but not for CCL21, in thymic into the dynamic process of GPCR signaling allow the extension emigration of newly generated and fully differentiated T cells has of the initial biased signaling concept by the following: 1) been identified in the neonatal thymus [53]. In fact, emigration unbound receptors exist predominantly in 2 inactive confor- of mature T cells from fetal thymic organ cultures was achieved mations, 2) ligand binding results in increased conformational by CCL19 and a truncated form of CCL21 lacking the heparin- heterogeneity and the coexistence of inactive, intermediate, and binding domain but not with (full-length) CCL21 [53]. More- active states, and 3) coupling to G-proteins leads to a complete over, antibody-mediated neutralization of CCL19 but not of transition of receptors to 1 active conformation [60]. Therefore, CCL21 in newborn mice impaired thymic emigration of T cells it is likely that ligands favor 1 over the other signaling pathway by into the peripheral circulation [53]. inducing more or less conformational receptor heterogeneity. Following antigen scanning without establishment of a fully Hereby, receptor conformations differ mainly in the rearrange- matured immunologic synapse in lymph nodes, T cells make ment of TM6 [60]. their way to efferent lymphatic vessels to re-enter circulation. b The responsible receptor for lymphocyte egress from lymph G-protein versus -arrestin activation and insights nodes is the S1P1. The relative expression and sensitivity of deduced from GPCR structures CCR7 and S1P1 determine whether the T cells remain within As a result of their differential expression and nonredundant or leave secondary lymphoid organs. In this regard, it is function, it has been speculated that binding of CCL19 and interesting to note that CCL19, but not CCL21, signaling CCL21 to CCR7 induces distinct signaling pathways. Whereas 2+ through CCR7 in murine T cells and in T cell lines was equal receptor-binding affinity, G-protein activation, and Ca reported to induce the expression of S1P1 [54]. Therefore, the mobilization [61] suggest high redundancy in CCR7 signaling above-outlined scenario can be extended: a sustained CCR7 in transfected cell lines, both chemokines differ in the ability to signaling by CCL21 and CCL19 will promote continuous T cell activate certain effector proteins (Fig. 1). As biased signaling is motility, survival, and retention in lymph nodes, but upon differentiated most often into G-protein- and b-arrestin- CCL19 triggering, CCR7 signals will become refractory, dependent signaling, we will concentrate in this section on the thereby promoting S1P1 expression and T cell egress. After differential activation of G-protein and b-arrestinbyCCL19 T cells re-enter the circulation, CCR7 reacquires its normal and CCL21. Whereas both CCR7 ligands activate GRK6 in an sensitivity and capacity for patrolling to the next secondary unbiased manner, GRK3 activity is unique to CCL19 (Fig. 1), lymphoid organ. as determined in transfected cells [63]. Therefore, CCL19 In addition to recruiting lymphocytes, lymph nodes are the stimulation results in a more robust serine/threonine phos- destination of activated, (semi-)mature DCs from peripheral phorylation and a stronger recruitment of b-arrestin (5-fold 2 2 2 2 tissues. Analysis of plt/plt, Ccl19 / , and Ccr7 / mice has over CCL21, as assessed in transfected HEK293 cells [63, 71, revealed an essential role for CCR7 and CCL21, but not CCL19, 73]). The diversity of arrestin functions and the observation in regulating the homing of DCs to secondary lymphoid organs. that ligands, like CCL19, can induce more b-arrestin re- Taken together, although acting through the same receptor, cruitment than others, emerged the so-called phosphorylation CCL19 and CCL21 appear not to serve completely overlapping barcode theory. In this concept, ligands stabilize ligand- and thus, redundant functions and therefore, display functional specific GPCR conformations, thereby inducing the specific selectivity or biased signaling. recruitment of GRKs, introducing a distinct phosphorylation pattern or "barcode" in the C terminus of the GPCR [74]. LIGAND-BIASED SIGNALING—SELECTIVITY Subsequently, the barcode promotes b-arrestin binding and FOR G-PROTEIN COUPLING AND stabilizes a certain b-arrestin conformation [75] that deter- b-ARRESTIN BINDING mines whether the receptor is desensitized (but still promotes b-arrestin-specific signaling) or desensitized and internalized The concept of biased signaling [74]. According to the phosphorylation barcode theory, The functional selectivity of CCL19 and CCL21 likely originates CCL19-mediated b-arrestin recruitment induces receptor from the situation that binding of either 1 ligand to CCR7 internalization in cell lines and primary T cells, whereas preferentially activates 1 of several available cellular signaling CCL21 hardly does [42, 76]. In spite of only marginal CCL21- pathways. The concept to explain functional selectivity by biased induced internalization, CCL21 is still able to activate signaling was introduced in 1995 and by that time, has been b-arrestin-mediated ERK1/2 phosphorylation in CCR7- referred to as "agonist trafficking" [7]. This concept presumes expressing HEK293 cells [63]. Importantly, a phosphorylated that agonists or ligands have different affinities for diverse serine/threonine cluster in the C termini of the GPCR is conformational states of the receptor, which in turn, activate essential for formation of stable GPCR-arrestin complexes that specific individual effector proteins to ultimately induce survive internalization and remain after endocytosis [77]. Of different signaling pathways, resulting in functional selectivity. note, CCR7 phosphorylation on serine/threonine residues There is common consensus that the activation of class A GPCRs within the cytoplasmic loops and the C-terminal tail on serines by ligands involves the same TM movement [55–57] regulated by 356, 357, 364, and 365 have been reported using HEK293

872 GPCRs, speculations on the structural features involving G-protein and b-arrestin bias can be made. The crystal structure of the b2-adrenergic receptor in complex with agonist and heterotrimeric G-protein revealed profound changes in the Ga subunit, stabilizing TM5 and TM6 of the receptor, ˚ resulting in an extensive outward movement of ;14A of TM6 compared with its unoccupied conformation [69] (Fig. 2A). Furthermore, there is a minor outward movement and an extension of the cytoplasmic end of TM5 [78]. In addition to TM5 and TM6, the crystal structure reveals the involvement of intracellular loop 2 in the interaction of the b2-adrenergic receptor and the Ga subunit [78]. Furthermore, the recently published first crystal structure of the rhodopsin-arrestin complex now provides an excellent opportunity to examine the mechanism of arrestin versus G-protein bias signaling [79]. Similar to the b2-adrenergic receptor in complex to the Ga subunit, rhodopsin bound to arrestin reveals an extensive outward movement of the cytoplasmic end of TM6 relative to its inactive conformation [79]. However, the TM6 trans- ˚ location is, with ;14A, more pronounced in the Ga-bound ˚ state compared with ;10A in the arrestin-bound state [78, 79] (Fig. 2A). Compared with active conformations of rhodopsin bound to GaCT peptides [80, 81], arrestin-bound rhodopsin shows additional subtle conformational differences in TM1, TM4, and TM7 [79]. Together with an intermediate outward movement of the cytoplasmic end of TM6, those differences in TM1, TM4, and TM7 may constitute essential elements for arrestin-biased signaling. Insights gained from GPCR crystal structures in biased signaling are extended further by dynamic studies that use site- specific[19F]-NMR labels in the b2-adrenergic receptor, which revealed that the cytoplasmic ends of TM6 and TM7 adopt 2 major conformational states [82]. In the unstimulated and inverse-agonist-bound state, b2-adrenergic receptor exists Figure 1. Common and biased signaling pathways of CCR7 elicited by predominantly in 2 inactive conformations that exchange CCL19 and CCL21. Both CCR7 ligands elicit G-protein coupling and within hundreds of microseconds [60]. Unbiased agonist activation that differ in the signal strength, as indicated by plus signs. binding primarily shifts the equilibrium toward the (G-protein-) Chemokine-induced G-protein coupling to CCR7 was measured, for active state of TM6 [82]. In contrast, b-arrestin-biased ligands instance, by bioluminescence resonance energy transfer in transfected HEK293 cells [62]. CCL19 but not CCL21 induces robust predominantly affected the conformational state of TM7 [82]. b-arrestin recruitment (as determined by fluorescence resonance Of note, unbiased ligand occupancy resulted in the coexistence energy transfer in HEK293 transfectants [63]) and CCR7 internaliza- of inactive, intermediate, and active states of the receptor [60]. tion (in transfectants and primary T cells [42]) through the recruit- Intracellular G-protein coupling mediates the complete transi- ment of GRK3 and GRK6 (as determined by fluorescence resonance tion to the active conformation [60]. Importantly, an allosteric energy transfer in HEK293 transfectants [63]). Furthermore, both coupling between the ligand-binding pocket and the different chemokines are able to activate MAPKs, whereas CCL19 was shown to TM6 conformations has been noted for the b2-adrenergic be more efficient in activating ERK1/2 and ERK5 in various cell types [54, 61, 63]. CCL19 and CCL21 are indistinguishable in promoting receptor [60]. PI3Kg stimulation (in murine T cells and DCs [64, 65]) and the What can we learn from these structural analyses for CCR7- activation of molecules involved in small GTPase signaling, such as biased signaling mediated by CCL19 and CCL21? Based on the Rac1 (in lymphocytes [66]), Cdc42 (in bone marrow-derived DCs conformational data of GPCRs described above, we propose that [67]), Dock2 (in T cells [51]), and ROCK I/II (in T cells [68]). Both binding of CCL19 and CCL21 results in the stabilization of chemokines are similar in inducing JAK activation (in lymphocytes and different conformational states of CCR7 (Fig. 2B): whereas DCs [69, 70]) and in chemokine-mediated Ca2+ mobilization (in DCs, CCL21 may shift primarily CCR7 equilibrium toward a G-protein lymphocytes, and transfectants [13, 42, 71, 72]). active state of TM6, CCL19 may result in more diverse conformations and might impact conformational changes in transfectants as a model system [61]. Stimulation with CCL19 TM7. The multiple conformations established by movement of resulted in additional phosphorylation on serine 373 and 378 the TM domains of CCR7 can be illustrated as CCR7 traveling of CCR7 [61]. By taking into account recent structural along an energy landscape, with preferred conformations information on G-protein coupling and b-arrestin binding to existing as energy wells (Fig. 2B) [83]. Ligand binding to CCR7

873 Figure 2. Molecular determinants of heterotrimeric G-protein activation versus arrestin activation. (A) Schematic representation of the orientation of TM domains of GPCR in the steady-state (left; gray), in the full active state bound to the Ga subunit (middle; green), and in the arrestin-bound state (right; red), based on solved structures of b-adrenergic and rhodopsin receptors [78, 79]. The profound outward movement of TM6 and subtle changes of TM5 and TM7, stabilized by G-protein coupling, are illustrated by coloring the TM domains in petrol. Intermediate outward movement of TM6 and TM7, resulting in arrestin interaction, is illustrated in red (right). (B) Schematic representation of putative conformational differences of CCR7 in its unoccupied state or its CCL21 (middle)- or CCL19 (right)-bound state. CCL19 might induce a major outward movement of CCR7 TM6 and robust movement of TM7, resulting in G-protein and b-arrestin recruitment and signaling. CCL21 induces a major TM6 outward movement but might have a limited effect on TM7, leading to G-protein signaling and b-arrestin interaction that is not stabilized through TM7 movement. The multiple conformations of TM domains can be seen as a receptor moving along an energy landscape, with preferred conformations existing as energy wells. CCL19 or CCL21 binding thermodynamically builds different energy landscapes for the receptor to traverse.

results in thermodynamic changes, and ligand-bound CCR7 case, not only G-proteins and arrestins would be able to traverses a new energy landscape (Fig. 2B) [84]. Therefore, resolve and translate certain receptor conformations to signaling is linked to those preferred conformations (seen as activation of distinct signaling pathways, but also, GRKs would wells in the energy landscape) that favorably interact with cellular be important for bias receptor signaling. Indeed, there are signaling proteins (Fig. 2B) [85]. examples indicating an important involvement of GRKs in bias Further important insights came from mutagenesis studies on signaling. For instance, GRK1 and GRK2 are able to promote the chemokine receptor CCR5 in transfected COS-7 cells, high-affinity binding of arrestin-1 to rhodopsin, whereas pointing to the critical interplay between TM6 and TM7 for GRK5 was much less efficient [87]. Likewise, GRK2 and GRK3 CCR5 activation [9]. For instance, insertion of a steric hindrance effectively recruit b-arrestin-2 to desensitize vasopressin re- in the center of TM7 abolished b-arrestin recruitment and biased ceptor V2, whereas GRK5 and GRK6 had much less influence

CCR5 signaling toward Gai activation [9]. The lack of G-protein on b-arrestin-2 recruitment [88]. Likewise, CCL19 and CCL21 activation, while retaining b-arrestin coupling, is the signaling result in GRK6 recruitment, whereas GRK3 binding remains signature of decoy chemokine receptors; the studying of a decoy unique to CCL19-mediated CCR7 activation in HEK293 receptor activation mechanism would be, in addition to transfectants [63]. Although GRKs have been crystallized, no structural and mutagenesis analysis, an attractive way to gain structure of GRKs in complex with a GPCR is currently further insights into the mechanisms of biased signaling. available. As such, the exact required (bias) ligand-induced However, the restoration of the mutated Asp-Arg-Tyr motif of the receptor conformation priming, distinct GRK binding is atypical chemokine receptor 3 in transfected HEK293 cells did difﬁcult to delineate. However, mutational analysis empha- not enable the receptor to signal through the G-protein pathway sizes the importance of the relative positioning of phosphor- [86]. Therefore, the existence of bias in atypical chemokine ylation clusters. For instance, the serine-cluster proximal to receptors necessitates more in-depth studies. the NPXXY motif in TM7 of the V2 vasopressin receptor is In accordance with the phosphorylation barcode theory, necessary for internalization, whereas the threonine-serine- the distinct conformations of TM6 and TM7 could control the cluster does not inﬂuence internalization [77]. Likewise, binding of arrestins, directly or indirectly, by favoring distinct CCR7, neurotensin-1 receptor, oxytocin receptor, and phosphorylation patterns added by different GRKs. In this angiotensin II type 1A receptor display serine/threonine

874 clusters downstream of the NPXXY motif [61, 89]. In G-protein subtypes in CCR7-transfected HEK293 cells [62, contrast, receptors that lack these serine/threonine-rich 106], as depicted in Fig. 1. In addition to G-protein and clusters, such as the b2-adrenergic receptor, generally recycle b-arrestin signaling, it is likely that other effectors regulate rapidly, presumably as a result of a low affinity of the GPCR- CCR7 functional selectivity by inducing distinct signaling arrestin complex [90]. Following the same line of evidence, pathways through specificprotein–protein interaction do- b-arrestin-biased ligands induced a phosphorylation pattern mains, such as PDZ domains or Src homology 2 domains, as distinct from a fully unbiased agonist [91]. Hence, we shown for other chemokine receptors [107]. propose that different CCR7 receptor conformations induced by CCL19 or CCL21 may be read and resolved by the Ga COMMON CCR7 SIGNALING PATHWAYS subunit of the heterotrimeric G-protein or by GRK3, which would translate the distinct CCR7 conformation into a The main function of CCR7 is guiding directional cell different phosphorylation pattern that would, in turn, in- migration. Therefore, CCR7-expressing cells undergo rapid struct the conformation by b-arrestins. Distinct b-arrestin shape change upon exposure to its ligands and eventually conformations determine its functional capabilities [74] and polarize. Cell polarity has been seen as prerequisite for result in G-protein activation and b-arrestin signaling (un- chemotaxis [108]. The ubiquitously expressed, small GTPases, biased through CCL21) or in G-protein activation and Rac, RhoA, and Cdc42, are generally accepted to determine b-arrestin-mediated internalization and b-arrestin signaling shape change and cell polarization [109, 110] and are elicited (b-arrestin biased through CCL19). upon CCR7 triggering (Fig. 1). Actions of small GTPases are regulated by GTPase-activating proteins and GEFs; the latter are known downstream targets of the chemokine-activated bg BIASED SIGNALING PATHWAYS INDUCED complex of G-proteins. T cell polarization induced by CCR7 BY CCL19 OR CCL21 ligands was shown to involve rapid lamellipodia formation, lamellipodia stabilization, and subsequently, the establish- Aside from G-protein-promoted generation of second mes- ment of a defineduropod[28,111].TheclassicviewonCCR7- sengers, including cAMP, Ca2+, or phosphoinositides, which mediated migration has been that the Rho-GTPase Rac1 are equally activated by CCL19 and CCL21 [61], both controls polarized actin polymerization at the leading edge by b-arrestins and GRKs are nowadays considered as a G-protein- stimulating the actin-related protein 2/3 complex, which independent signal transducer [92–94]. In particular, nucleates actin filaments [112], and that RhoA controls b-arrestins act as multifunctional scaffolds that bind to many myosin-based contraction at the uropod, which is crucial to effector proteins [95], such as protein kinases, thereby complete TEM [113]. As a result of conflicting studies, species- changing phosphorylation of numerous intracellular targets dependent differences, and redundant protein functions, a [96].Forinstance,theb-arrestin-mediated signaling mecha- holistic view explaining GTPase- and GEF-controlled CCR7 2 2 nism includes activation of small Rho-GTPases [97], also migration appears to be difficult. However, Rac / Tcells known to be involved in large dendrite extension of DCs, a (Rac1 and Rac2 double-knockout, nondetectable with Rac3 feature unique to CCL19 [52]. Following this line of evidence, expression level) display defects in CCR7-mediated adhesion it was shown that CCR7-mediated survival of DCs depends on to ICAM1 and TEM [66]. Furthermore, these lymphocytes the activation of GSK3 [98]. In turn, b-arrestin-mediated were severely compromised in their ability to undergo signaling also involves protein phosphatase 2A-mediated chemotaxis or chemokinesis in response to CCR7 stimulation dephosphorylation of Akt, which leads to the activation of [114]. Studies in human T cells suggested that Rac1 is also GSK3 [99], and therefore, again, b-arrestin signaling might required for the rapid CCL21-induced activation of LFA-1 well be involved in CCR7-dependent DC survival. Further- [115]. In contrast to Rac-defective human T cells, mouse more, G-protein-independent activation of tyrosine kinase T cells deficient in the RacGEFs, DOCK2 and Tiam1, show Pyk2 upon CCR7 triggering in DCs has been reported [100]. normal CCL21-induced adhesion to ICAM1 and TEM Although direct evidences for CCR7 are missing, Pyk2 [116–118], suggesting that the third major GEF expressed in association to b-arrestin has been demonstrated for other T cells, Vav1, may transduce CCR7-dependent signals to Rac1. chemokine receptors [101]. Finally, not only antiapoptotic These GEFs, in turn, are targeted and activated by different effects [102] and PI3K-mediated PLA2 activation [103] have upstream signals, including Src-mediated tyrosine phosphory- been attributed to b-arrestin signaling pathways, but also lation [119] and the synthesis of specific phosphoinositides or activation of ERK-dependent protein translation (reviewed in phosphatic acid [120]. ref. [6]). In turn, CCL19-specific translation of S1P1 is Moreover, CCR7-mediated human T cell polarization and mediated via ERK5-dependent translation of Krüppel-like migration were shown to require ROCK I and II [68] (Fig. 1). In factor2andup-regulationofS1P1inaHuT78Tcelllineand DCs, pharmacological inhibition of Rho kinases or myosin II did primary T cells [54]. not affect leading-edge formation but impaired trailing-edge It is becoming more and more appreciated that the contraction, resulting in a significant reduction of migratory chemokine receptor activates ensembles of signaling pathways speed toward CCL19 [121]. Furthermore, DCs lacking Vav1, a organized as integrated networks [104, 105] rather than GEF for Rac and RhoA, showed altered cytoskeletal dynamics, signaling through linear second messenger-dependent cas- impaired integrin signaling, and cell adhesion but exhibited an cades. Therefore, CCR7 has been shown to couple to different increased migratory capacity in response to CCL21 in vitro and

875 homed faster to lymph nodes [122]. CCL21 (and CXCL12) Finally, Stein and colleagues [69] described that CCL21 stimulation in T cells promoted ZAP70-dependent dissocia- stimulation of lymphocytes led to a rapid, Gai-independent tion of Vav1 from talin, resulting in the activation of a4b1 Jak2 phosphorylation and that the Jak inhibitor tyrphostin integrin and T cell adhesion by inside-out signaling [123]. AG490 reduced lymphocyte adhesion and migration to CCR7 Whereas in T cells, integrin-mediated adhesion is an in- ligands. This is in line with the finding that Jak3 was dispensable part of the migration process, DCs can migrate phosphorylated upon CCL19 and CCL21 stimulation and that 2 2 2 2 integrin dependently [124] or integrin independently by the Jak3 / T cells [134] and Jak3 / DCs [70] own reduced sole force of the actin network expansion [121, 124]. Finally, lymph node homing capacity. Further investigations are Cdc42 often promotes stabilization of the leading edge and required to decipher how these different CCR7 signaling directed motility [125]. In chemokine (CXCL12)-induced pathways interact or adapt for distinct environmental condi- T cell polarization, Cdc42 was activated by Rap1 and induced tions to facilitate efficient leukocyte trafficking. the PAR 3/6/PKCz complex, as well as the RacGEF Tiam1 2/2 [5, 126]. In Cdc42 DCs, exposed to uniform concentrations of PUTATIVE MOLECULAR DETERMINANTS fl CCL19, actin ow, actomyosin contraction, and cell symmetry, OF CCL19 AND CCL21 ENABLING breaking was intact, and hence, cells polarized and formed BIAS SIGNALING protrusions [67]. However, CCR7-mediated protrusions in 2 2 Cdc42 / DCs were spatially and temporarily dysregulated, Despite substantial differences in their amino acid sequence, resulting in an impaired leading-edge coordination. Conse- CCL19 and CCL21 share a conserved chemokine characteristic 2 2 quently, Cdc42 / DCs were still able to migrate on a 2D tertiary structure that includes a disordered N terminus, surface but not in a 3D environment [67]. implicated in signaling, followed by a structured core domain Besides small GTPases of the Rho family, other signaling that consists of an N-loop, a 3-stranded b-sheet, and a molecules are described to contribute to CCR7-mediated C-terminal helix (Fig. 3A) [135]. The most prominent leukocyte migration. For instance, the Gbg subunit-mediated difference between the 2 CCR7 ligands is the C-terminal Ca2+ response, through PLC activation that cleaves phosphati- extension of CCL21, responsible for its immobilization to GAGs. dylinositol 4.5 bisphosphate to diacylglycerol and inositol 1,4,5 This unique architecture of CCL21 is such that it must be able triphosphate, has been shown to be necessary for CCR7-mediated to accommodate 2 very different kinds of interactions, namely DC migration [72]. Decisively for lymphocytes, it appears that binding to extracellular matrix-associated GAGs and to its signal integration of CCR7, TCR, and integrins occurs on the cognate receptor CCR7 [135]. As CCR7 is sialylated [136], it is level of intracellular Ca2+ mobilization [127]. The emerging plausible that CCL21, presented on endothelial GAGs, is principle here is that the magnitude and duration of Ca2+ spikes initially transferred to the glycan structure of CCR7. To achieve directly influence the basal speed of T cell migration in the this, the affinities of CCL21 for CCR7 and GAGs must be finely lymph nodes. However, the exact molecular mechanism linking tuned to enable the transfer from GAG to CCR7 at the right Ca2+ mobilization with cytoskeleton remodeling and migration time. GAGs are carbohydrate structures that are attached to remains to be elucidated [127]. Nonetheless, first insights protein cores of proteoglycans on cells or simply shed into the derived from studies on immature DC migration provide extracellular matrix. Most prominently, GAGs provide a scaf- evidence that Ca2+ spikes are associated with fast-motility phases fold, enabling chemokines to be gradually immobilized on cell during scanning behavior [128]. surfaces and extracellular matrices that can be referred to as In addition, the Gbg subunit is not only responsible for PLC- haptotactic chemokine gradients [137]. The chemokine–GAG mediated Ca2+ mobilization but also known to activate PI3K, interaction is mostly mediated through polar interactions; which catalyzes the phosphoinositide conversion required for whereas carbohydrate structures are highly negatively charged, the plasma membrane recruitment and the assembly of many chemokines contain highly basic domains, typically BBXB signaling complexes, including RacGEF DOCK2 [129, 130]. and BXBXXB sequences (where B is a basic amino acid and X a Although, Gbg-triggered activation of PI3K is implicated in nonconserved amino acid). The GAG-binding domain has been controlling cell migration in general [131, 132], PI3K and its identified for a number of chemokines, including CCL2 [138], downstream effector Akt are activated upon CCR7 triggering in CCL5 [139], and CXCL12 [140]. Whereas GAG-binding DCs [100], but their contribution to CCR7-driven intranodal sequences are mostly included in chemokine core domains, T cell migration remains to be incompletely understood and CCL21 has a large C-terminal extension, comprising several somehow controversial [72, 133]. Noteworthy, both DOCK2 and basic amino acids that are implicated in GAG interaction and PI3Kg signaling pathways seem to be G-protein activation immobilization [44, 141]. dependent [5]. Furthermore, by exploiting different pharma- Aside from the specific interaction of CCL21 with GAGs, cological inhibitors, it has been shown that CCR7-guided DC CCL19 and CCL21 execute their biologic function through migration was dependent on 2 different Gai-dependent binding and activation of CCR7. On part of the ligands, early pathways: one involving p38 activation and the other involving studies reveal the importance of the chemokine N terminus in ERK1/2 and JNK phosphorylation [100] (Fig. 1). Moreover, it stabilizing the active conformation of the chemokine receptor fi fi was also described that Gai-independent CCR7 signaling [142, 143]. Speci cally, N-terminal modi cation (deletions or through RhoA-Pyk2-cofilin determined the migratory speed of alanine mutations) of the first 6 aa of CCL19 were shown to DCs [100]. How these data fit into the signaling pathways convert CCL19 from an agonist into an antagonist [142]. described above remains to be determined. Surprisingly, CCL19 and CCL21 that activate a G-protein

876 Figure 3. Molecular determinants of biased signaling encoded in the structure of CCL19 and CCL21. (A) Sequence alignment of human (h) and mouse (m) CCL19 and CCL21 and visualization of characteristic differences between the 2 chemokines. Highlighted in green are lysine/arginine to serine substitutions. Shown in yellow and red are conserved acidic residues, and highlighted in blue are lysine-to-tyrosine substitutions. (B) Comparison of NMR-resolved structure of CCL21 [Protein Data Bank (PDB) ID 2L4N; left] or CCL19 (PDB ID 2MP1; right). Both chemokines are illustrated as surface representation. Highlighted in green are basic amino acids, in red acidic amino acids, and in blue tyrosine residues. To visualize the interaction of CCL19 and CCL21 with CCR7, including important residues involved in receptor binding, the NMR structures of CCL19 and CCL21 were superimposed to the structure of the CXCR4:vMIP-II complex (PDB ID 4RWS). CXCR4 is represented as a cartoon, whereas its N terminus involved in chemokine binding is shown as a stick representation.

response with similar potency but exhibit distinct signaling proteins [144]. One possible explanation for this is that pathways share only 25% sequence identity (Fig. 3B). In fact, the glutamate6 and aspartate2 of CCL21 are able to position the N-terminal 6 residues of CCL19 (GTNDAE) and CCL21 amide and acid side-chain functional groups in a similar (SDGGAQ) share only 1 conserved alanine in their sequence, orientation, as asparagine3 and aspartate4 in CCL19. Unfortu- suggesting that distinct bias-signaling abilities are encoded in the nately, b-arrestin activation or CCR7 internalization by this speciﬁc sequence. However, swapping of the N termini of CCL19 chimeric chemokines has not been addressed. and CCL21 results in comparable CCR7-driven G-protein On the chemokine receptor side, mutagenesis and truncation activation and cell migration through the newly formed chimeric studies gave rise to a concept known as the “2-step, 2-site” model

877 for chemokine receptor interaction [145, 146]. This model CCL19, whereas lysine31 and arginine35 in CCL19 are involves the interaction between chemokine core domain and changed to serine in CCL21 (Fig. 3). Furthermore, tyrosine12 chemokine receptor N terminus (site1). Site1 docking of the of CCL21 potentially contacts the CCR7-binding pocket, which chemokine receptor’s N terminus is then thought to reorient is absent in CCL19. Despite described differences, there are the chemokine’s N terminus (important for signaling) to also structurally conserved patches, such as the acidic region enable entering of the chemokine into the major ligand pocket aspartate54; glutamate57 in CCL21, corresponding to of the receptor (site2). Recently, this concept was substantiated aspartate54; glutamate59 in CCL19; and glutamate29 in CCL21, by solving the crystal structure of CXCR4 in complex with a similar to aspartate32 in CCL19. Thus, through comparison of chemokine vMIP-II [147], known as an antagonist for multiple the solved NMR structures of both CCR7 ligands, it is likely to chemokine receptors. Importantly, the data generally confirm resolve the determinants of natural bias signaling, encoded in the concept of the 2-step, 2-site model. However, the structure the chemokines. also reveals a more extensive interface between CXCR4 and vMIP-II than anticipated by the 2-step, 2-site model. In fact, CONCLUDING REMARKS every residue of the chemokine’s N-terminal domain and the N-loop,aswellasresiduesinthethirdb-strand, interacted with Accumulated data over the last decade have demonstrated the the receptor [147]. vMIP-II binds deeply into the ligand- importance of the CCR7–CCL19/CCL21 axis in tolerance and binding pocket of CXCR4; thereby, the chemokines occupy the immunity. By mediating migration of T cells and DCs, CCR7 and major and minor ligand-binding groove. Interactions are its 2 ligands play an essential role in orchestration of the immune mediated through hydrogen bonds among aspartate97 (TM2), system, central and peripheral tolerance, and the initiation of the aspartate262 (TM6), and glutamate288 (TM7) of CXCR4 and adaptive immune response. However, there is still a lot to learn numerous van der Waals packing interactions [147]. There- about how CCL19 and CCL21, upon binding to the same fore, one could speculate that the extensive intracellular receptor CCR7, can elicit common and ligand-biased signaling displacements of TM6 and TM7 involved in activation of (bias) pathways to cover the broad range of known CCR7 functions. receptor signaling are mediated by the hydrogen bonds of Whereas the complexity and the plethora of CCR7 functions these residues. Importantly, for CCR7, mutagenesis studies make it difficult to target the CCR7–CCL19/CCL21 axis for revealed that lysine130 (TM3), lysine137 (TM3), glutamine227 therapeutic intervention in human immune-mediated diseases, (TM5), and asparagine305 (TM7) are involved in receptor the highly complex regulatory systems, which are becoming activation but are dispensable for high-affinity ligand binding increasingly apparent, suggest that appropriate controlling of [148]. Of note, mutation of lysine137 affected CCL21-mediated these systems may have significant beneficial outcomes. Biased receptor activation without interfering with CCL19-mediated ligands could be a solution to target the CCR7–CCL19/CCL21 receptor activation [148]. Therefore, one can assume that axis; by preferentially targeting 1 over the other signaling lysine137 in CCR7 represents 1 of the determinants for CCR7- pathways, the likeliness of severe side-effects might be circum- biased signaling pathways. vented. Thus, not only the delineation of the molecular signature As the crystal structure of CXCR4, in complex with vMIP-II, of CCR7 signaling pathways but also the deciphering of the reveals large interaction surfaces, it is likely that the in- mechanism behind biased signaling, including molecular deter- formation to activate 1 over the other signaling pathway is not minants on the chemokine and on the chemokine receptor side, only stored in the N terminal first 6 aa of CCL19 and CCL21. are crucial for designing and developing potent and specific anti- Hence, the comparison of the NMR structures of CCL19 and CCR7 pharmacotherapeutics. As we understand more about the CCL21 (Fig. 3B) might provide insights into their determi- specific structural basis of ligand-signaling bias, it is plausible to nants of biased signaling pathways. One of the most obvious discover that each and every ligand stabilizes distinct, active differences is the presence of a large patch of basic residues in conformations, which facilitate different patterns of proximal CCL21, consisting of lysine20, arginine44, lysine45, and effector interactions that are not limited to G-protein and arginine46, placed near the potential interaction site of the b-arrestin interactions. Therefore, it is advantageous to imagine receptor N terminus (Fig. 3B; depicted in green). This basic the multiple conformations taken by TM domains as CCR7 patch is reduced in CCL19 to arginine45 and arginine47. As moving along an energy landscape with preferred conformations frequent post-translational modifications of chemokine re- existing as energy wells (Fig. 2B). Upon binding of CCL19 or ceptors, such as sulfation and glycosylation that add additional CCL21, CCR7 becomes another thermodynamic species, and the negative charge to the N terminus and hence, contribute to ligand-bound receptor then traverses to a new energy landscape. the initial step in chemokine–chemokine receptor interaction Therefore, functionality is linked to the favored conformations, [149–152], it is likely that a post-translationally modified N as those conformations are associated with preferential affinity terminus of CCR7 interacts with this basic patch. Furthermore, for cellular signaling proteins [85]. Because the receptor CCL19 extends arginine35 toward the receptor-binding site, conformations are interconvertible, it is plausible that a ligand which is missing in CCL21. This polar residue has the potential induces not only 1 conformation that favors G-protein coupling to reach deeply into the CCR7 ligand-binding pocket to induce but rather, mediates multiple conformations that could interact CCL19-specific changes in the active state conformations of with several effector proteins. Thus, the herein presented CCR7. Notably, several similar lysine/arginine-to-serine sub- concepts of CCR7-biased signaling could help to accelerate our stitutions are apparent by comparing CCL19 and CCL21. For understanding of this fundamental molecular pathway and the instance, lysine11 in CCL21 is substituted by serine11 in cellular responses triggered by it.

878 ACKNOWLEDGMENTS 21. Campbell, D. J., Kim, C. H., Butcher, E. C. (2003) Chemokines in the systemic organization of immunity. Immunol. Rev. 195, 58–71. The authors gratefully acknowledge support from the Swiss 22. Butcher, E. C., Picker, L. J. (1996) Lymphocyte homing and homeostasis. Science 272, 60–66. National Science Foundation (SNF 31003A_143841); Thurgauische 23. Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. (2007) Getting to Stiftung für Wissenschaft und Forschung; Swiss State Secretariat the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7, 678–689. for Education, Research and Innovation; and Thurgauische 24. Nourshargh, S., Hordijk, P. L., Sixt, M. (2010) Breaching multiple Krebsliga (to D.F.L.). M.A.H. is recipient of a stipend from the barriers: leukocyte motility through venular walls and the interstitium. Nat. Rev. Mol. Cell Biol. 11, 366–378. graduate school RTG1331. 25. Stein, J. V., Rot, A., Luo, Y., Narasimhaswamy, M., Nakano, H., Gunn, M. D., Matsuzawa, A., Quackenbush, E. J., Dorf, M. E., von Andrian, U. H. (2000) The CC chemokine thymus-derived chemotactic agent 4 DISCLOSURES (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) The authors declare no conflicts of interest. triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules. J. Exp. Med. 191, 61–76. 26. Warnock, R. A., Campbell, J. J., Dorf, M. E., Matsuzawa, A., McEvoy, REFERENCES L. M., Butcher, E. C. (2000) The role of chemokines in the 1. Zlotnik, A., Yoshie, O. (2012) The chemokine superfamily revisited. microenvironmental control of T versus B cell arrest in Peyer’s patch Immunity 36, 705–716. high endothelial venules. J. Exp. Med. 191, 77–88. 2. Griffith, J. W., Sokol, C. L., Luster, A. D. (2014) Chemokines and 27. Shamri, R., Grabovsky, V., Gauguet, J. M., Feigelson, S., Manevich, E., chemokine receptors: positioning cells for host defense and immunity. Kolanus, W., Robinson, M. K., Staunton, D. E., von Andrian, U. H., Annu. Rev. Immunol. 32, 659–702. Alon, R. (2005) Lymphocyte arrest requires instantaneous induction of 3. Bachelerie, F., Ben-Baruch, A., Burkhardt, A. M., Combadiere, C., an extended LFA-1 conformation mediated by endothelium-bound Farber, J. M., Graham, G. J., Horuk, R., Sparre-Ulrich, A. H., Locati, M., chemokines. Nat. Immunol. 6, 497–506. Luster, A. D., Mantovani, A., Matsushima, K., Murphy, P. M., Nibbs, R., 28. Woolf, E., Grigorova, I., Sagiv, A., Grabovsky, V., Feigelson, S. W., Nomiyama, H., Power, C. A., Proudfoot, A. E., Rosenkilde, M. M., Rot, Shulman, Z., Hartmann, T., Sixt, M., Cyster, J. G., Alon, R. (2007) A., Sozzani, S., Thelen, M., Yoshie, O., Zlotnik, A. (2014) International Lymph node chemokines promote sustained T lymphocyte motility Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. without triggering stable integrin adhesiveness in the absence of shear Update on the extended family of chemokine receptors and forces. Nat. Immunol. 8, 1076–1085. introducing a new nomenclature for atypical chemokine receptors. 29. Kliche, S., Worbs, T., Wang, X., Degen, J., Patzak, I., Meineke, B., Togni, Pharmacol. Rev. 66, 1–79. M., Moser, M., Reinhold, A., Kiefer, F., Freund, C., Forster,¨ R., Schraven, 4. Allen, S. J., Crown, S. E., Handel, T. M. (2007) Chemokine: receptor B. (2012) CCR7-mediated LFA-1 functions in T cells are regulated by 2 structure, interactions, and antagonism. Annu. Rev. Immunol. 25, independent ADAP/SKAP55 modules. Blood 119, 777–785. 787–820. 30. Veerman, K. M., Williams, M. J., Uchimura, K., Singer, M. S., Merzaban, 5. Thelen, M., Stein, J. V. (2008) How chemokines invite leukocytes to J. S., Naus, S., Carlow, D. A., Owen, P., Rivera-Nieves, J., Rosen, S. D., dance. Nat. Immunol. 9, 953–959. Ziltener, H. J. (2007) Interaction of the selectin ligand PSGL-1 with 6. Rajagopal, S., Rajagopal, K., Lefkowitz, R. J. (2010) Teaching old chemokines CCL21 and CCL19 facilitates efficient homing of T cells to receptors new tricks: biasing seven-transmembrane receptors. Nat. Rev. secondary lymphoid organs. Nat. Immunol. 8, 532–539. Drug Discov. 9, 373–386. 31. Worbs, T., Mempel, T. R., Bölter, J., von Andrian, U. H., Förster, R. 7. Kenakin, T. (1995) Agonist-receptor efficacy. II. Agonist trafficking of (2007) CCR7 ligands stimulate the intranodal motility of T lymphocytes receptor signals. Trends Pharmacol. Sci. 16, 232–238. in vivo. J. Exp. Med. 204, 489–495. 8. Zweemer, A. J., Toraskar, J., Heitman, L. H., IJzerman, A. P. (2014) Bias 32. Okada, T., Cyster, J. G. (2007) CC chemokine receptor 7 contributes to in chemokine receptor signalling. Trends Immunol. 35, 243–252. Gi-dependent T cell motility in the lymph node. J. Immunol. 178, 9. Steen, A., Larsen, O., Thiele, S., Rosenkilde, M. M. (2014) Biased and G 2973–2978. protein-independent signaling of chemokine receptors. Front. Immunol. 33. Huang, J. H., Cardenas-Navia,´ L. I., Caldwell, C. C., Plumb, T. J., Radu, 5, 277. C. G., Rocha, P. N., Wilder, T., Bromberg, J. S., Cronstein, B. N., 10. Comerford, I., Harata-Lee, Y., Bunting, M. D., Gregor, C., Kara, E. E., Sitkovsky, M., Dewhirst, M. W., Dustin, M. L. (2007) Requirements for McColl, S. R. (2013) A myriad of functions and complex regulation of T lymphocyte migration in explanted lymph nodes. J. Immunol. 178, the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune 7747–7755. system. Cytokine Growth Factor Rev. 24, 269–283. 34. Forster,¨ R., Schubel, A., Breitfeld, D., Kremmer, E., Renner-Müller, I., 11. Förster, R., Davalos-Misslitz, A. C., Rot, A. (2008) CCR7 and its ligands: Wolf, E., Lipp, M. (1999) CCR7 coordinates the primary immune balancing immunity and tolerance. Nat. Rev. Immunol. 8, 362–371. response by establishing functional microenvironments in secondary 12. Legler, D. F., Uetz-von Allmen, E., Hauser, M. A. (2014) CCR7: roles in lymphoid organs. Cell 99, 23–33. cancer cell dissemination, migration and metastasis formation. Int. 35. Gunn, M. D., Kyuwa, S., Tam, C., Kakiuchi, T., Matsuzawa, A., Williams, J. Biochem. Cell Biol. 54, 78–82. L. T., Nakano, H. (1999) Mice lacking expression of secondary 13. Willimann, K., Legler, D. F., Loetscher, M., Roos, R. S., Delgado, lymphoid organ chemokine have defects in lymphocyte homing and M. B., Clark-Lewis, I., Baggiolini, M., Moser, B. (1998) The dendritic cell localization. J. Exp. Med. 189, 451–460. chemokine SLC is expressed in T cell areas of lymph nodes and 36. Luther, S. A., Tang, H. L., Hyman, P. L., Farr, A. G., Cyster, J. G. (2000) mucosal lymphoid tissues and attracts activated T cells via CCR7. Eur. Coexpression of the chemokines ELC and SLC by T zone stromal cells J. Immunol. 28, 2025–2034. and deletion of the ELC gene in the plt/plt mouse. Proc. Natl. Acad. Sci. 14. Randolph, G. J., Angeli, V., Swartz, M. A. (2005) Dendritic-cell USA 97, 12694–12699. trafficking to lymph nodes through lymphatic vessels. Nat. Rev. Immunol. 37. Vassileva, G., Soto, H., Zlotnik, A., Nakano, H., Kakiuchi, T., Hedrick, 5, 617–628. J. A., Lira, S. A. (1999) The reduced expression of 6Ckine in the plt 15. Alvarez, D., Vollmann, E. H., von Andrian, U. H. (2008) Mechanisms mouse results from the deletion of one of two 6Ckine genes. J. Exp. Med. and consequences of dendritic cell migration. Immunity 29, 325–342. 190, 1183–1188. 16. Merad, M., Manz, M. G., Karsunky, H., Wagers, A., Peters, W., Charo, I., 38. Nakano, H., Gunn, M. D. (2001) Gene duplications at the Weissman, I. L., Cyster, J. G., Engleman, E. G. (2002) Langerhans cells chemokine locus on mouse chromosome 4: multiple strain-specific renew in the skin throughout life under steady-state conditions. Nat. haplotypes and the deletion of secondary lymphoid-organ Immunol. 3, 1135–1141. chemokine and EBI-1 ligand chemokine genes in the plt mutation. 17. Ohl, L., Mohaupt, M., Czeloth, N., Hintzen, G., Kiafard, Z., Zwirner, J., J. Immunol. 166, 361–369. Blankenstein, T., Henning, G., Forster,¨ R. (2004) CCR7 governs skin 39. Britschgi, M. R., Favre, S., Luther, S. A. (2010) CCL21 is sufficient to dendritic cell migration under inflammatory and steady-state mediate DC migration, maturation and function in the absence of conditions. Immunity 21, 279–288. CCL19. Eur. J. Immunol. 40, 1266–1271. 18. Colonna, M., Trinchieri, G., Liu, Y. J. (2004) Plasmacytoid dendritic 40. Weber, M., Hauschild, R., Schwarz, J., Moussion, C., de Vries, I., cells in immunity. Nat. Immunol. 5, 1219–1226. Legler, D. F., Luther, S. A., Bollenbach, T., Sixt, M. (2013) Interstitial 19. Sallusto, F., Lenig, D., Forster,¨ R., Lipp, M., Lanzavecchia, A. (1999) dendritic cell guidance by haptotactic chemokine gradients. Science Two subsets of memory T lymphocytes with distinct homing potentials 339, 328–332. and effector functions. Nature 401, 708–712. 41. Link, A., Vogt, T. K., Favre, S., Britschgi, M. R., Acha-Orbea, H., Hinz, B., 20. Müller, G., Lipp, M. (2003) Shaping up adaptive immunity: the impact Cyster, J. G., Luther, S. A. (2007) Fibroblastic reticular cells in lymph of CCR7 and CXCR5 on lymphocyte trafficking. Microcirculation 10, nodes regulate the homeostasis of naive T cells. Nat. Immunol. 8, 325–334. 1255–1265.

879 42. Otero, C., Groettrup, M., Legler, D. F. (2006) Opposite fate of 66. Shulman, Z., Shinder, V., Klein, E., Grabovsky, V., Yeger, O., Geron, E., endocytosed CCR7 and its ligands: recycling versus degradation. Montresor, A., Bolomini-Vittori, M., Feigelson, S. W., Kirchhausen, T., J. Immunol. 177, 2314–2323. Laudanna, C., Shakhar, G., Alon, R. (2009) Lymphocyte crawling and 43. Bardi, G., Lipp, M., Baggiolini, M., Loetscher, P. (2001) The T cell transendothelial migration require chemokine triggering of high- chemokine receptor CCR7 is internalized on stimulation with ELC, but affinity LFA-1 integrin. Immunity 30, 384–396. not with SLC. Eur. J. Immunol. 31, 3291–3297. 67. Lammermann,¨ T., Renkawitz, J., Wu, X., Hirsch, K., Brakebusch, C., 44. Schumann, K., Lammermann,¨ T., Bruckner, M., Legler, D. F., Polleux, Sixt, M. (2009) Cdc42-dependent leading edge coordination is essential J., Spatz, J. P., Schuler, G., Forster,¨ R., Lutz, M. B., Sorokin, L., Sixt, M. for interstitial dendritic cell migration. Blood 113, 5703–5710. (2010) Immobilized chemokine fields and soluble chemokine gradients 68. Bardi, G., Niggli, V., Loetscher, P. (2003) Rho kinase is required for cooperatively shape migration patterns of dendritic cells. Immunity 32, CCR7-mediated polarization and chemotaxis of T lymphocytes. FEBS 703–713. Lett. 542, 79–83. 45. Mempel, T. R., Henrickson, S. E., Von Andrian, U. H. (2004) T-Cell 69. Stein, J. V., Soriano, S. F., M’rini, C., Nombela-Arrieta, C., de Buitrago, priming by dendritic cells in lymph nodes occurs in three distinct G. G., Rodr´ıguez-Frade, J. M., Mellado, M., Girard, J. P., Mart´ınez-A, C. phases. Nature 427, 154–159. (2003) CCR7-mediated physiological lymphocyte homing involves 46. Dustin, M. L. (2004) Stop and go traffic to tune T cell responses. activation of a tyrosine kinase pathway. Blood 101, 38–44. Immunity 21, 305–314. 70. Rivas-Caicedo, A., Soldevila, G., Fortoul, T. I., Castell-Rodr´ıguez, A., 47. Bajénoff, M., Egen, J. G., Qi, H., Huang, A. Y., Castellino, F., Germain, Flores-Romo, L., Garc´ıa-Zepeda,E.A.(2009)Jak3isinvolvedin R. N. (2007) Highways, byways and breadcrumbs: directing lymphocyte dendritic cell maturation and CCR7-dependent migration. PLoS traffic in the lymph node. Trends Immunol. 28, 346–352. One 4, e7066. 48. Ackerknecht, M., Hauser, M. A., Legler, D. F., Stein, J. V. (2015) In vivo 71. Otero, C., Eisele, P. S., Schaeuble, K., Groettrup, M., Legler, D. F. TCR signaling in CD4(+) T cells imprints a cell-intrinsic, transient low- (2008) Distinct motifs in the chemokine receptor CCR7 regulate signal motility pattern independent of chemokine receptor expression levels, transduction, receptor trafficking and chemotaxis. J. Cell Sci. 121, or microtubular network, integrin, and protein kinase C activity. Front. 2759–2767. Immunol. 6, 297. 72. Scandella, E., Men, Y., Legler, D. F., Gillessen, S., Prikler, L., Ludewig, 49. Legler, D. F., Krause, P., Scandella, E., Singer, E., Groettrup, M. (2006) B., Groettrup, M. (2004) CCL19/CCL21-triggered signal transduction Prostaglandin E2 is generally required for human dendritic cell and migration of dendritic cells requires prostaglandin E2. Blood 103, migration and exerts its effect via EP2 and EP4 receptors. J. Immunol. 1595–1601. 176, 966–973. 73. Byers, M. A., Calloway, P. A., Shannon, L., Cunningham, H. D., Smith, 50. Schaeuble, K., Hauser, M. A., Singer, E., Groettrup, M., Legler, D. F. S., Li, F., Fassold, B. C., Vines, C. M. (2008) Arrestin 3 mediates (2011) Cross-talk between TCR and CCR7 signaling sets a temporal endocytosis of CCR7 following ligation of CCL19 but not CCL21. threshold for enhanced T lymphocyte migration. J. Immunol. 187, J. Immunol. 181, 4723–4732. 5645–5652. 74. Reiter, E., Ahn, S., Shukla, A. K., Lefkowitz, R. J. (2012) Molecular 51. Gollmer, K., Asperti-Boursin, F., Tanaka, Y., Okkenhaug, K., mechanism of b-arrestin-biased agonism at seven-transmembrane Vanhaesebroeck, B., Peterson, J. R., Fukui, Y., Donnadieu, E., Stein, J. V. receptors. Annu. Rev. Pharmacol. Toxicol. 52, 179–197. (2009) CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac- 75. Shukla, A. K., Violin, J. D., Whalen, E. J., Gesty-Palmer, D., Shenoy, S. K., dependent pathway. Blood 114, 580–588. Lefkowitz, R. J. (2008) Distinct conformational changes in beta-arrestin 52. Yanagawa, Y., Onoe,´ K. (2002) CCL19 induces rapid dendritic extension report biased agonism at seven-transmembrane receptors. Proc. Natl. of murine dendritic cells. Blood 100, 1948–1956. Acad. Sci. USA 105, 9988–9993. 53. Ueno, T., Hara, K., Willis, M. S., Malin, M. A., Höpken, U. E., Gray, 76. Comerford, I., Milasta, S., Morrow, V., Milligan, G., Nibbs, R. (2006) D. H., Matsushima, K., Lipp, M., Springer, T. A., Boyd, R. L., Yoshie, O., The chemokine receptor CCX-CKR mediates effective scavenging of Takahama, Y. (2002) Role for CCR7 ligands in the emigration of newly CCL19 in vitro. Eur. J. Immunol. 36, 1904–1916. generated T lymphocytes from the neonatal thymus. Immunity 16, 77. Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., Caron, M. G. 205–218. (1999) Association of beta-arrestin with G protein-coupled receptors 54. Shannon, L. A., McBurney, T. M., Wells, M. A., Roth, M. E., Calloway, during clathrin-mediated endocytosis dictates the profile of receptor P. A., Bill, C. A., Islam, S., Vines, C. M. (2012) CCR7/CCL19 controls resensitization. J. Biol. Chem. 274, 32248–32257. expression of EDG-1 in T cells. J. Biol. Chem. 287, 11656–11664. 78. Rasmussen, S. G., DeVree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., 55. Bouvier, M. (2013) Unraveling the structural basis of GPCR activation Kobilka, T. S., Thian, F. S., Chae, P. S., Pardon, E., Calinski, D., and inactivation. Nat. Struct. Mol. Biol. 20, 539–541. Mathiesen, J. M., Shah, S. T., Lyons, J. A., Caffrey, M., Gellman, S. H., 56. Audet, M., Bouvier, M. (2012) Restructuring G-protein-coupled Steyaert, J., Skiniotis, G., Weis, W. I., Sunahara, R. K., Kobilka, B. K. receptor activation. Cell 151, 14–23. (2011) Crystal structure of the b2 adrenergic receptor-Gs protein 57. Rosenkilde, M. M., Benned-Jensen, T., Frimurer, T. M., Schwartz, T. W. complex. Nature 477, 549–555. (2010) The minor binding pocket: a major player in 7TM receptor 79. Kang, Y., Zhou, X. E., Gao, X., He, Y., Liu, W., Ishchenko, A., Barty, A., activation. Trends Pharmacol. Sci. 31, 567–574. White, T. A., Yefanov, O., Han, G. W., Xu, Q., de Waal, P. W., Ke, J., Tan, 58. Venkatakrishnan, A. J., Deupi, X., Lebon, G., Tate, C. G., Schertler, M. H., Zhang, C., Moeller, A., West, G. M., Pascal, B. D., Van Eps, N., G. F., Babu, M. M. (2013) Molecular signatures of G-protein-coupled Caro, L. N., Vishnivetskiy, S. A., Lee, R. J., Suino-Powell, K. M., Gu, X., receptors. Nature 494, 185–194. Pal, K., Ma, J., Zhi, X., Boutet, S., Williams, G. J., Messerschmidt, M., 59. Nygaard, R., Frimurer, T. M., Holst, B., Rosenkilde, M. M., Schwartz, Gati, C., Zatsepin, N. A., Wang, D., James, D., Basu, S., Roy-Chowdhury, T. W. (2009) Ligand binding and micro-switches in 7TM receptor S., Conrad, C. E., Coe, J., Liu, H., Lisova, S., Kupitz, C., Grotjohann, I., structures. Trends Pharmacol. Sci. 30, 249–259. Fromme, R., Jiang, Y., Tan, M., Yang, H., Li, J., Wang, M., Zheng, Z., Li, 60. Manglik, A., Kim, T. H., Masureel, M., Altenbach, C., Yang, Z., Hilger, D., Howe, N., Zhao, Y., Standfuss, J., Diederichs, K., Dong, Y., Potter, D., Lerch, M. T., Kobilka, T. S., Thian, F. S., Hubbell, W. L., Prosser, C. S., Carragher, B., Caffrey, M., Jiang, H., Chapman, H. N., Spence, R. S., Kobilka, B. K. (2015) Structural insights into the dynamic process J. C., Fromme, P., Weierstall, U., Ernst, O. P., Katritch, V., Gurevich, of b2-adrenergic receptor signaling. Cell 161, 1101–1111. V. V., Griffin, P. R., Hubbell, W. L., Stevens, R. C., Cherezov, V., 61. Kohout, T. A., Nicholas, S. L., Perry, S. J., Reinhart, G., Junger, S., Melcher, K., Xu, H. E. (2015) Crystal structure of rhodopsin bound to Struthers, R. S. (2004) Differential desensitization, receptor arrestin by femtosecond X-ray laser. Nature 523, 561–567. phosphorylation, beta-arrestin recruitment, and ERK1/2 activation by 80. Standfuss,J.,Edwards,P.C.,D’Antona, A., Fransen, M., Xie, G., the two endogenous ligands for the CC chemokine receptor 7. J. Biol. Oprian, D. D., Schertler, G. F. (2011) The structural basis of agonist- Chem. 279, 23214–23222. induced activation in constitutively active rhodopsin. Nature 471, 62. Corbisier, J., Gales,` C., Huszagh, A., Parmentier, M., Springael, J. Y. 656–660. (2015) Biased signaling at chemokine receptors. J. Biol. Chem. 290, 81. Scheerer, P., Park, J. H., Hildebrand, P. W., Kim, Y. J., Krauss, N., Choe, 9542–9554. H. W., Hofmann, K. P., Ernst, O. P. (2008) Crystal structure of opsin in 63. Zidar, D. A., Violin, J. D., Whalen, E. J., Lefkowitz, R. J. (2009) Selective its G-protein-interacting conformation. Nature 455, 497–502. engagement of G protein coupled receptor kinases (GRKs) encodes 82. Liu, J. J., Horst, R., Katritch, V., Stevens, R. C., Wüthrich, K. (2012) distinct functions of biased ligands. Proc. Natl. Acad. Sci. USA 106, Biased signaling pathways in b2-adrenergic receptor characterized by 9649–9654. 19F-NMR. Science 335, 1106–1110. 64. Matheu, M. P., Deane, J. A., Parker, I., Fruman, D. A., Cahalan, M. D. 83. Frauenfelder, H., Sligar, S. G., Wolynes, P. G. (1991) The energy (2007) Class IA phosphoinositide 3-kinase modulates basal lymphocyte landscapes and motions of proteins. Science 254, 1598–1603. motility in the lymph node. J. Immunol. 179, 2261–2269. 84. Peleg, G., Ghanouni, P., Kobilka, B. K., Zare, R. N. (2001) Single- 65. Del Prete, A., Vermi, W., Dander, E., Otero, K., Barberis, L., Luini, W., molecule spectroscopy of the beta(2) adrenergic receptor: observation Bernasconi, S., Sironi, M., Santoro, A., Garlanda, C., Facchetti, F., of conformational substates in a membrane protein. Proc. Natl. Acad. Sci. Wymann, M. P., Vecchi, A., Hirsch, E., Mantovani, A., Sozzani, S. (2004) USA 98, 8469–8474. Defective dendritic cell migration and activation of adaptive immunity 85. Onaran, H. O., Costa, T. (1997) Agonist efficacy and allosteric models in PI3Kgamma-deficient mice. EMBO J. 23, 3505–3515. of receptor action. Ann. N. Y. Acad. Sci. 812, 98–115.

880 86. Hoffmann, F., Müller, W., Schütz, D., Penfold, M. E., Wong, Y. H., 109. Jaffe, A. B., Hall, A. (2005) Rho GTPases: biochemistry and biology. Schulz, S., Stumm, R. (2012) Rapid uptake and degradation of CXCL12 Annu. Rev. Cell Dev. Biol. 21, 247–269. depend on CXCR7 carboxyl-terminal serine/threonine residues. J. Biol. 110. Heasman, S. J., Ridley, A. J. (2008) Mammalian Rho GTPases: new Chem. 287, 28362–28377. insights into their functions from in vivo studies. Nat. Rev. Mol. Cell Biol. 87. Vishnivetskiy, S. A., Ostermaier, M. K., Singhal, A., Panneels, V., Homan, 9, 690–701. K. T., Glukhova, A., Sligar, S. G., Tesmer, J. J., Schertler, G. F., Standfuss, 111. Real, E., Faure, S., Donnadieu, E., Delon, J. (2007) Cutting edge: J., Gurevich, V. V. (2013) Constitutively active rhodopsin mutants atypical PKCs regulate T lymphocyte polarity and scanning behavior. causing night blindness are effectively phosphorylated by GRKs but J. Immunol. 179, 5649–5652. differ in arrestin-1 binding. Cell. Signal. 25, 2155–2162. 112. Weiner, O. D., Servant, G., Welch, M. D., Mitchison, T. J., Sedat, J. W., 88. Ren, X. R., Reiter, E., Ahn, S., Kim, J., Chen, W., Lefkowitz, R. J. (2005) Bourne, H. R. (1999) Spatial control of actin polymerization during Different G protein-coupled receptor kinases govern G protein and neutrophil chemotaxis. Nat. Cell Biol. 1, 75–81. beta-arrestin-mediated signaling of V2 vasopressin receptor. Proc. Natl. 113. Worthylake, R. A., Burridge, K. (2001) Leukocyte transendothelial Acad. Sci. USA 102, 1448–1453. migration: orchestrating the underlying molecular machinery. Curr. 89. Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., Caron, M. G. Opin. Cell Biol. 13, 569–577. (2001) Molecular determinants underlying the formation of stable 114. Faroudi, M., Hons, M., Zachacz, A., Dumont, C., Lyck, R., Stein, J. V., intracellular G protein-coupled receptor-beta-arrestin complexes after Tybulewicz, V. L. (2010) Critical roles for Rac GTPases in T-cell receptor endocytosis. J. Biol. Chem. 276, 19452–19460. migration to and within lymph nodes. Blood 116, 5536–5547. 90. Ostermaier, M. K., Schertler, G. F., Standfuss, J. (2014) Molecular 115. Bolomini-Vittori, M., Montresor, A., Giagulli, C., Staunton, D., Rossi, B., mechanism of phosphorylation-dependent arrestin activation. Curr. Martinello, M., Constantin, G., Laudanna, C. (2009) Regulation of Opin. Struct. Biol. 29, 143–151. conformer-specific activation of the integrin LFA-1 by a chemokine- 91. Nobles, K. N., Xiao, K., Ahn, S., Shukla, A. K., Lam, C. M., Rajagopal, S., triggered Rho signaling module. Nat. Immunol. 10, 185–194. Strachan, R. T., Huang, T. Y., Bressler, E. A., Hara, M. R., Shenoy, S. K., 116. Nombela-Arrieta, C., Lacalle, R. A., Montoya, M. C., Kunisaki, Y., Gygi, S. P., Lefkowitz, R. J. (2011) Distinct phosphorylation sites on the Meg´ıas, D., Marques,´ M., Carrera, A. C., Mañes, S., Fukui, Y., Mart´ınez-A, b(2)-adrenergic receptor establish a barcode that encodes differential C., Stein, J. V. (2004) Differential requirements for DOCK2 and functions of b-arrestin. Sci. Signal. 4, ra51. phosphoinositide-3-kinase gamma during T and B lymphocyte homing. 92. Lefkowitz, R. J., Shenoy, S. K. (2005) Transduction of receptor signals by Immunity 21, 429–441. beta-arrestins. Science 308, 512–517. 117. Shulman, Z., Pasvolsky, R., Woolf, E., Grabovsky, V., Feigelson, S. W., 93. Lefkowitz, R. J. (1998) G Protein-coupled receptors. III. New roles for Erez, N., Fukui, Y., Alon, R. (2006) DOCK2 regulates chemokine- receptor kinases and beta-arrestins in receptor signaling and triggered lateral lymphocyte motility but not transendothelial desensitization. J. Biol. Chem. 273, 18677–18680. migration. Blood 108, 2150–2158. 94. Reiter, E., Lefkowitz, R. J. (2006) GRKs and beta-arrestins: roles in 118. Gerard,´ A., van der Kammen, R. A., Janssen, H., Ellenbroek, S. I., receptor silencing, trafficking and signaling. Trends Endocrinol. Metab. Collard, J. G. (2009) The Rac activator Tiam1 controls efficient T-cell 17, 159–165. trafficking and route of transendothelial migration. Blood 113, 95. Xiao, K., McClatchy, D. B., Shukla, A. K., Zhao, Y., Chen, M., Shenoy, 6138–6147. S. K., Yates III, J. R., Lefkowitz, R. J. (2007) Functional specialization of 119. Tybulewicz, V. L. (2005) Vav-family proteins in T-cell signalling. Curr. beta-arrestin interactions revealed by proteomic analysis. Proc. Natl. Opin. Immunol. 17, 267–274. Acad. Sci. USA 104, 12011–12016. 120. Nishikimi, A., Fukuhara, H., Su, W., Hongu, T., Takasuga, S., Mihara, 96. Xiao, K., Sun, J., Kim, J., Rajagopal, S., Zhai, B., Villen,´ J., Haas, W., H., Cao, Q., Sanematsu, F., Kanai, M., Hasegawa, H., Tanaka, Y., Kovacs, J. J., Shukla, A. K., Hara, M. R., Hernandez, M., Lachmann, A., Shibasaki, M., Kanaho, Y., Sasaki, T., Frohman, M. A., Fukui, Y. (2009) Zhao, S., Lin, Y., Cheng, Y., Mizuno, K., Ma’ayan, A., Gygi, S. P., Sequential regulation of DOCK2 dynamics by two phospholipids during Lefkowitz, R. J. (2010) Global phosphorylation analysis of beta-arrestin- neutrophil chemotaxis. Science 324, 384–387. mediated signaling downstream of a seven transmembrane receptor 121. Lammermann,¨ T., Bader, B. L., Monkley, S. J., Worbs, T., Wedlich- (7TMR). Proc. Natl. Acad. Sci. USA 107, 15299–15304. Soldner,¨ R., Hirsch, K., Keller, M., Forster,¨ R., Critchley, D. R., Fassler,¨ 97. Barnes, W. G., Reiter, E., Violin, J. D., Ren, X. R., Milligan, G., Lefkowitz, R., Sixt, M. (2008) Rapid leukocyte migration by integrin-independent R. J. (2005) beta-Arrestin 1 and Galphaq/11 coordinately activate RhoA flowing and squeezing. Nature 453, 51–55. and stress fiber formation following receptor stimulation. J. Biol. Chem. 122. Spurrell, D. R., Luckashenak, N. A., Minney, D. C., Chaplin, A., 280, 8041–8050. Penninger, J. M., Liwski, R. S., Clements, J. L., West, K. A. (2009) Vav1 98. Escribano, C., Delgado-Mart´ın, C., Rodr´ıguez-Fernandez,´ J. L. (2009) regulates the migration and adhesion of dendritic cells. J. Immunol. 183, CCR7-dependent stimulation of survival in dendritic cells involves 310–318. inhibition of GSK3beta. J. Immunol. 183, 6282–6295. 123. Garc´ıa-Bernal, D., Parmo-Cabañas, M., Dios-Esponera, A., 99. Beaulieu, J. M., Sotnikova, T. D., Marion, S., Lefkowitz, R. J., Samaniego, R., Hernán-P de la Ossa, D., Teixidó, J. (2009) Gainetdinov, R. R., Caron, M. G. (2005) An Akt/beta-arrestin 2/PP2A Chemokine-induced Zap70 kinase-mediated dissociation of the Vav1- signaling complex mediates dopaminergic neurotransmission and talin complex activates alpha4beta1 integrin for T cell adhesion. behavior. Cell 122, 261–273. Immunity 31, 953–964. 100. Riol-Blanco, L., Sanchez-S´ anchez,´ N., Torres, A., Tejedor, A., Narumiya, 124. Renkawitz, J., Schumann, K., Weber, M., Lammermann,¨ T., Pflicke, H., S., Corb´ı, A. L., Sanchez-Mateos,´ P., Rodr´ıguez-Fernandez,´ J. L. (2005) Piel, M., Polleux, J., Spatz, J. P., Sixt, M. (2009) Adaptive force The chemokine receptor CCR7 activates in dendritic cells two signaling transmission in amoeboid cell migration. Nat. Cell Biol. 11, 1438–1443. modules that independently regulate chemotaxis and migratory speed. 125. Etienne-Manneville, S. (2004) Cdc42—the centre of polarity. J. Cell Sci. J. Immunol. 174, 4070–4080. 117, 1291–1300. 101. Cheung, R., Malik, M., Ravyn, V., Tomkowicz, B., Ptasznik, A., Collman, 126. Gerard,´ A., Mertens, A. E., van der Kammen, R. A., Collard, J. G. (2007) R. G. (2009) An arrestin-dependent multi-kinase signaling complex The Par polarity complex regulates Rap1- and chemokine-induced mediates MIP-1beta/CCL4 signaling and chemotaxis of primary human T cell polarization. J. Cell Biol. 176, 863–875. macrophages. J. Leukoc. Biol. 86, 833–845. 127. Joseph, N., Reicher, B., Barda-Saad, M. (2014) The calcium feedback 102. Ahn, S., Kim, J., Hara, M. R., Ren, X. R., Lefkowitz, R. J. (2009) beta- loop and T cell activation: how cytoskeleton networks control Arrestin-2 mediates anti-apoptotic signaling through regulation of BAD intracellular calcium flux. Biochim. Biophys. Acta 1838, 557–568. phosphorylation. J. Biol. Chem. 284, 8855–8865. 128. Solanes, P., Heuze,´ M. L., Maurin, M., Bretou, M., Lautenschlaeger, 103. Walters, R. W., Shukla, A. K., Kovacs, J. J., Violin, J. D., DeWire, S. M., F.,Maiuri,P.,Terriac,E.,Thoulouze,M.I.,Launay,P.,Piel,M., Lam, C. M., Chen, J. R., Muehlbauer, M. J., Whalen, E. J., Lefkowitz, R. J. Vargas, P., Lennon-Dumenil,´ A. M. (2015) Space exploration by (2009) beta-Arrestin1 mediates nicotinic acid-induced flushing, but not dendritic cells requires maintenance of myosin II activity by IP3 its antilipolytic effect, in mice. J. Clin. Invest. 119, 1312–1321. receptor 1. EMBO J. 34, 798–810. 104. Luttrell, L. M. (2006) Transmembrane signaling by G protein-coupled 129. Ward, S. G., Westwick, J., Harris, S. (2011) Sat-Nav for T cells: Role of receptors. Methods Mol. Biol. 332, 3–49. PI3K isoforms and lipid phosphatases in migration of T lymphocytes. 105. Jean-Alphonse, F., Hanyaloglu, A. C. (2011) Regulation of GPCR Immunol. Lett. 138, 15–18. signal networks via membrane trafficking. Mol. Cell. Endocrinol. 331, 130. Vanhaesebroeck, B., Stephens, L., Hawkins, P. (2012) PI3K signalling: 205–214. the path to discovery and understanding. Nat. Rev. Mol. Cell Biol. 13, 106. Shi, G., Partida-Sanchez,´ S., Misra, R. S., Tighe, M., Borchers, M. T., Lee, 195–203. J. J., Simon, M. I., Lund, F. E. (2007) Identification of an alternative 131. Stephens, L., Milne, L., Hawkins, P. (2008) Moving towards a better Galphaq-dependent chemokine receptor signal transduction pathway in understanding of chemotaxis. Curr. Biol. 18, R485–R494. dendritic cells and granulocytes. J. Exp. Med. 204, 2705–2718. 132. Xiong, Y., Huang, C. H., Iglesias, P. A., Devreotes, P. N. (2010) Cells 107. Patrussi, L., Baldari, C. T. (2008) Intracellular mediators of CXCR4- navigate with a local-excitation, global-inhibition-biased excitable dependent signaling in T cells. Immunol. Lett. 115, 75–82. network. Proc. Natl. Acad. Sci. USA 107, 17079–17086. 108. Russell, S. (2008) How polarity shapes the destiny of T cells. J. Cell Sci. 133. Asperti-Boursin, F., Real, E., Bismuth, G., Trautmann, A., Donnadieu, E. 121, 131–136. (2007) CCR7 ligands control basal T cell motility within lymph node

881 slices in a phosphoinositide 3-kinase-independent manner. J. Exp. Med. the C-terminal domain of CCL19. Biochem. Biophys. Res. Commun. 348, 204, 1167–1179. 1089–1093. 134. Garc´ıa-Zepeda, E. A., Licona-Limon,´ I., Jimenez-S´ olomon,´ M. F., 145. Monteclaro, F. S., Charo, I. F. (1996) The amino-terminal extracellular Soldevila, G. (2007) Janus kinase 3-deficient T lymphocytes have an domain of the MCP-1 receptor, but not the RANTES/MIP-1alpha intrinsic defect in CCR7-mediated homing to peripheral lymphoid receptor, confers chemokine selectivity. Evidence for a two-step organs. Immunology 122, 247–260. mechanism for MCP-1 receptor activation. J. Biol. Chem. 271, 135. Kufareva, I., Salanga, C. L., Handel, T. M. (2015) Chemokine and 19084–19092. chemokine receptor structure and interactions: implications for 146. Crump, M. P., Gong, J. H., Loetscher, P., Rajarathnam, K., Amara, A., therapeutic strategies. Immunol. Cell Biol. 93, 372–383. Arenzana-Seisdedos, F., Virelizier, J. L., Baggiolini, M., Sykes, B. D., 136. Su, M. L., Chang, T. M., Chiang, C. H., Chang, H. C., Hou, M. F., Li, Clark-Lewis, I. (1997) Solution structure and basis for functional activity W. S., Hung, W. C. (2014) Inhibition of chemokine (C-C motif) of stromal cell-derived factor-1; dissociation of CXCR4 activation from receptor 7 sialylation suppresses CCL19-stimulated proliferation, binding and inhibition of HIV-1. EMBO J. 16, 6996–7007. invasion and anti-anoikis. PLoS One 9, e98823. 147. Qin, L., Kufareva, I., Holden, L. G., Wang, C., Zheng, Y., Zhao, C., 137. Rot, A. (1993) Neutrophil attractant/activation protein-1 (interleukin- Fenalti, G., Wu, H., Han, G. W., Cherezov, V., Abagyan, R., Stevens, 8) induces in vitro neutrophil migration by haptotactic mechanism. Eur. R. C., Handel, T. M. (2015) Structural biology. Crystal structure of the J. Immunol. 23, 303–306. chemokine receptor CXCR4 in complex with a viral chemokine. Science 138. Lau, E. K., Paavola, C. D., Johnson, Z., Gaudry, J. P., Geretti, E., Borlat, 347, 1117–1122. F., Kungl, A. J., Proudfoot, A. E., Handel, T. M. (2004) Identification of 148. Ott, T. R., Pahuja, A., Nickolls, S. A., Alleva, D. G., Struthers, R. S. (2004) the glycosaminoglycan binding site of the CC chemokine, MCP-1: Identification of CC chemokine receptor 7 residues important for implications for structure and function in vivo. J. Biol. Chem. 279, receptor activation. J. Biol. Chem. 279, 42383–42392. 22294–22305. 149. Farzan, M., Mirzabekov, T., Kolchinsky, P., Wyatt, R., Cayabyab, M., 139. Proudfoot, A. E., Fritchley, S., Borlat, F., Shaw, J. P., Vilbois, F., Zwahlen, Gerard, N. P., Gerard, C., Sodroski, J., Choe, H. (1999) Tyrosine C., Trkola, A., Marchant, D., Clapham, P. R., Wells, T. N. (2001) The sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. Cell 96, BBXB motif of RANTES is the principal site for heparin binding and 667–676. controls receptor selectivity. J. Biol. Chem. 276, 10620–10626. 150. Farzan, M., Babcock, G. J., Vasilieva, N., Wright, P. L., Kiprilov, E., 140. Sadir, R., Baleux, F., Grosdidier, A., Imberty, A., Lortat-Jacob, H. (2001) Mirzabekov, T., Choe, H. (2002) The role of post-translational Characterization of the stromal cell-derived factor-1alpha-heparin modifications of the CXCR4 amino terminus in stromal-derived complex. J. Biol. Chem. 276, 8288–8296. factor 1 alpha association and HIV-1 entry. J. Biol. Chem. 277, 141. Hirose, J., Kawashima, H., Swope Willis, M., Springer, T. A., Hasegawa, 29484–29489. H., Yoshie, O., Miyasaka, M. (2002) Chondroitin sulfate B exerts its 151. Ludwig, A., Ehlert, J. E., Flad, H. D., Brandt, E. (2000) Identification of inhibitory effect on secondary lymphoid tissue chemokine (SLC) by distinct surface-expressed and intracellular CXC-chemokine receptor 2 binding to the C-terminus of SLC. Biochim. Biophys. Acta 1571, 219–224. glycoforms in neutrophils: N-glycosylation is essential for maintenance 142. Ott, T. R., Lio, F. M., Olshefski, D., Liu, X. J., Struthers, R. S., Ling, N. of receptor surface expression. J. Immunol. 165, 1044–1052. (2004) Determinants of high-affinity binding and receptor activation in 152. Bannert, N., Craig, S., Farzan, M., Sogah, D., Santo, N. V., Choe, H., the N-terminus of CCL-19 (MIP-3 beta). Biochemistry 43, 3670–3678. Sodroski, J. (2001) Sialylated O-glycans and sulfated tyrosines in the 143. Moser, B., Dewald, B., Barella, L., Schumacher, C., Baggiolini, M., Clark- NH2-terminal domain of CC chemokine receptor 5 contribute to high Lewis, I. (1993) Interleukin-8 antagonists generated by N-terminal affinity binding of chemokines. J. Exp. Med. 194, 1661–1673. modification. J. Biol. Chem. 268, 7125–7128. 144. Ott,T.R.,Lio,F.M.,Olshefski,D.,Liu,X.J.,Ling,N.,Struthers, R. S. (2006) The N-terminal domain of CCL21 reconstitutes high KEY WORDS: affinity binding, G protein activation, and chemotactic activity, to G-protein-coupled receptor • cell migration • signal transduction

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