Allosteric and Orthosteric Sites in CC Chemokine Receptor (CCR5), a Chimeric Receptor Approach*

Background: Characterization of 7TM biology and chemistry is needed generally and within chemokine receptors. Results: A CCR5-CCR2 receptor chimera was constructed by transferring all extracellular regions of CCR2 to CCR5. CCR2 chemokine binding was maintained and so was small molecule CCR5 agonists and antagonists. Conclusion: Orthosteric and allosteric sites could be structurally separated and still act together. Significance: New basic knowledge to be used in drug development. Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.
With several hundred members divided into four main groups, 7 transmembrane-helix (7TM) 2 receptors constitute the largest group of proteins in the human genome and are the targets for the majority of current drugs (1). Their great number reflects the multitude of chemically diverse ligands, ranging from photons over small molecule ligands to peptides and proteins. The diversity of the ligands is huge. This is also the case for the ligand-binding site, which may be located within the transmembrane area and/or the extracellular domains (2). The binding site of the endogenous ligand is referred to as the orthosteric binding site. Allosteric compounds have a binding site that is topographically distinct from the orthosteric site. They have been identified for numerous receptors and have in general become increasingly acknowledged in drug development for their higher specificity, preventing overdosing and, provided they are neutral by themselves, dependence on the presence of the endogenous ligand, therefore only taking effect at the site of interest (where the endogenous ligand is located) (3).
Chemokine receptors belong to the class A subfamily of 7TM receptors. They are involved in the regulation and development of the immune system by guiding leukocyte migration and development. The chemokine system includes more than 19 receptors and 50 ligands and is characterized by promiscuity and redundancy for the majority of its members. According to the number and spacing of the first two of usually four conserved cysteines, chemokines and their receptors are divided into four groups as follow: the CC, CXC, XC, and CX 3 C groups (4). It is generally accepted that the chemokine core binds to the extracellular domain of a given receptor, whereas the N terminus may interact with residues in the transmembrane area (5).
The central role of the chemokines in immune system regulation combined with the control of angiogenesis, cancer growth and metastasis, and HIV cell entry have facilitated drug development programs and thereby the identification of numerous small molecule antagonists and agonists (6). These small molecules usually have similar pharmacophores with an elongated structure, one or two, more or less centrally located positively charged amines and flanking aromatic residues (7,8). The positively charged amines anchor to GluVII:06 3 on top of TM-VII, a residue that is conserved in 74% of all chemokine receptors compared with Ͻ1% in nonchemokine 7TM receptors (7). This residue divides the main ligand binding crevice into a minor (delimited by TM-I, -II, -III, and -VII) and a major binding pocket (delimited by TM-III, -IV, -V, -VI, and -VII) (10). The flanking aromatic moieties of the small molecules interact with residues in the major and minor binding pocket, as described, e.g. for the CCR5 antagonists aplaviroc and SCH-C (7,(11)(12)(13)(14). Interestingly, agonists also display a similar pharmacophore with a central positive charge and flanking aromatic moieties, as observed for CCR5 and CCR8 agonists (15,16). However, recently, a novel series of metal ion chelatorbased, and thus structurally different, CCR1 agonists were identified (17). These compounds also anchored to GluVII:06 and acted as allosteric enhancers of CCL3 (MIP-1␣), 4 but not of CCL5 (regulated on activation normal T cell expressed and secreted) binding (17).
7TM receptor drugs have traditionally been developed based on the structure of the endogenous ligand and consequently target the orthosteric site. It has, however, become increasingly acknowledged that 7TM receptors have allosteric binding sites with obvious advantages over the orthosteric binding site from a drug-action point of view (as discussed above). Thus, e.g. class C 7TM receptors have large N-terminal domains that contain the orthosteric site, and the receptors can therefore be modulated via a clearly separated allosteric site within the transmembrane region (18). On the contrary, many class A receptors contain the orthosteric binding site within the transmembrane region, as described for rhodopsin, adenosine, acetylcholine, and other monoamine receptors, whereas the allosteric binding sites are located in the extracellular regions (3,19). In contrast to the cognate ligands of these receptors, chemokines are large molecules and somewhat exceptional with their orthosteric binding site located entirely in the extracellular domains, and the allosteric binding sites are located deeper in the transmembrane region (5,8).
CCR5 shares the chemokine ligands CCL3 and CCL5 with CCR1, and in this study we find that CCR5, like CCR1 (17), is activated by metal ion chelator-based compounds. CCR2, the receptor most similar to CCR5 despite no overlapping chemokine binding profiles (20), is however not activated by these small molecule agonists. Until now, several series of small molecule antagonists have been described for CCR5, as for instance aplaviroc and SCH-C (both CCR5-specific) and the dual CCR5/ CCR2 antagonist TAK-779 (21)(22)(23). The small molecule agonists for CCR5 (but not CCR2) identified in this study combined with the previously identified small molecule antagonists with different degrees of CCR5 selectivity over CCR2 and two sets of completely selective chemokines for CCR5 and CCR2 inspired us to study the molecular interaction of orthosteric and allosteric ligands, especially the conformational interchange in CCR5. Therefore, we constructed a "Trojan horse" chimeric receptor, CCR5-CCR2 (all) , that consisted of the CCR5 receptor scaffold but with the extracellular domains being "shaved off" and replaced by the corresponding extracellular domains from CCR2. Construction of chimeric receptors has been applied earlier (24 -26), also for the analysis of CCR5 and CCR2 (27)(28)(29)(30). However, the majority of these were built in a "centaur" way with the N-terminal part stemming from one receptor and the C-terminal part from another receptor, therefore being sequence-related but not domain-related as in our Trojan horse chimera. CCR5-CCR2 (all) recognized CCR2 but not CCR5 chemokines, and at the small molecule level, the receptor bound the CCR5-selective nonpeptide agonists and most antagonists (except for aplaviroc). Importantly, for all ligand classes, the functional properties (agonism and antagonism) were maintained indicating that despite the different origin of the extracellular (CCR2) and transmembrane and intracellular (CCR5) domains, the receptor acted as one functional unit with transmission of activity between the domains of different origin. For aplaviroc, the C-terminal part of ECL-2 (ECL-2b) was additionally needed for maintained binding, but importantly, this was sufficient for high potency action of this antagonist. All together, by using ligands with different chemical and pharmacological properties, we studied ligand binding and receptor function across heterogeneous receptor domains in the Trojan horse CCR5-CCR2 (all) chimeric receptor that was created successfully without destroying the overall conformation and function of the receptor.

EXPERIMENTAL PROCEDURES
Materials-The human chemokines CCL3, CCL5, CCL2, and CCL7 were purchased from PeproTech. TAK-779, SCH-C, and aplaviroc were kindly provided by Gary Bridger (AnorMED). The highest concentration of metal ion chelator complexes were made from 0.2 M ZnCl 2 in water, 0.2 M CuSO 4 in water, 0.4 M phenanthroline in 70% ethanol, 0.4 M bipyridine in DMSO and were supplemented with 10% DMSO, water, and 70% ethanol. Dilutions were made in water. The concentration of chelator was double the concentration of the ions, which in turn determines the concentration of the complex, to provide optimal 1:1 complexation of the ions by chelators. The human CCR5 was cloned in-house from a leukocyte cDNA library, while CCR2 cDNA was kindly provided by Tim Wells (Glaxo-SmithKline, UK). The chimeric receptors CCR5-CCR2 (all) and CCR5-CCR2 (all⌬2b) were designed in-house and purchased from GenScript. The promiscuous G protein G␣ ⌬6qi4myr (abbreviated as G qi4myr ) was kindly provided by Evi Kostenis (University of Bonn, Germany). myo-[ 3 H]Inositol (PT6-271), Bolton-Hunter reagent, and iodinated chemokines 125 I-CCL3, 125 I-CCL5, and 125 I-CCL2 were purchased from PerkinElmer Life Sciences. 125 I-CCL7 was Bolton-Hunter-labeled and HPLC-purified in-house (31). AG 1-X8 anion exchange resin was from Bio-Rad.
Site-directed Mutagenesis-Point mutations were introduced in the receptors by the polymerase chain reaction over-lap extension technique using WT CCR5, CCR2, or CCR5-CCR2 (all) as templates. All reactions were carried out using Pfu polymerase (Stratagene) under conditions recommended by the manufacturer. The mutations were cloned into the eukaryotic expression vector pcDNA3.1 ϩ and were verified by restriction endonuclease digestion and DNA sequencing (Eurofins MWG Operon, Germany).
Transfections and Tissue Culture-COS-7 cells were grown at 10% CO 2 and 37°C in Dulbecco's modified Eagle's medium with GlutaMAX (Invitrogen) adjusted with 10% fetal bovine serum, 180 units/ml penicillin, and 45 g/ml penicillin/streptomycin. Transfection of the COS-7 cells was performed by the calcium phosphate precipitation method (32).
Inositol Phosphate Turnover (IP Turnover)-COS-7 cells were transfected according to the procedure mentioned above. Briefly, 6 ϫ 10 6 cells were transfected with 20 g of receptor cDNA in addition to 30 g of the promiscuous chimeric G protein, G␣ qi4myr , which turns the G␣ i -coupled signal, the most common pathway for endogenous chemokine receptors, into the G␣ q pathway (phospholipase C activation measured as IP turnover) (32,33). One day after transfection, COS-7 cells (1.5 ϫ 10 5 cells/well) were incubated for 24 h with 2 Ci of myo-[ 3 H]inositol in 0.3 ml of growth medium per well. Cells were washed twice in PBS and were incubated in 0.2 ml of Hanks' balanced salt solution (Invitrogen) supplemented with 10 mM LiCl at 37°C in the presence of various concentrations of ligands. Cells were extracted by addition of 1 ml of 10 mM formic acid to each well followed by incubation on ice for 30 -60 min. The generated [ 3 H]inositol phosphates were purified on AG 1-X8 anion exchange resin. Determinations were made in duplicates. This readout has previously been used with success in other chemokine receptors (16,17). For the test of the antagonists, the agonist was given after a 10-min preincubation with the antagonists and in concentrations that lead to ϳ80% of maximal activation for the respective ligand and receptor.
Binding Experiments-6 ϫ 10 6 COS-7 cells were transfected with 40 g of receptor cDNA and transferred to culture plates 1 day after transfection. The number of cells seeded per well was determined by the apparent expression efficiency of the receptors and was aimed at obtaining 5-10% specific binding of the added radioactive ligand. Two days after transfection, cells were assayed by competition binding for 3 h at 4°C using 10 -15 pM 125 I-CCL3, 125 I-CCL5, 125 I-CCL2, or 125 I-CCL7 plus unlabeled ligand in 0.2 ml of 50 mM Hepes buffer, pH 7.4, supplemented with 1 mM CaCl 2 , 5 mM MgCl 2 , and 0.5% (w/v) bovine serum albumin. After incubation, cells were washed quickly two times in 4°C binding buffer supplemented with 0.5 M NaCl, except for 125 I-CCL7 binding to CCR2, where the washing was performed in binding buffer without NaCl. Nonspecific binding was determined in the presence of 0.1 M unlabeled CCL3, CCL5, CCL2, or CCL7, respectively. Determinations were made in duplicates.
Receptor Surface Expression by Enzyme-Linked Immunosorbent Assay (ELISA)-COS-7 cells were transiently transfected with the N-terminal FLAG-tagged receptor variants. The cells were washed once in TBS (50 mM Tris base, 150 mM NaCl, pH 7.6), fixed in 4% glutaraldehyde for 15 min following three washes in TBS, and incubated in blocking solution (2% bovine serum albumin in TBS) for 30 min at room temperature. The cells were subsequently incubated 2 h with anti-FLAG (M1) antibody (2 g/ml) in TBS supplemented with 1% bovine serum albumin and 1 mM CaCl 2 at room temperature. After three washes in TBS with 1 mM CaCl 2 , the cells were incubated for 1 h with goat anti-mouse horseradish peroxidaseconjugated antibody in the same buffer as the anti-FLAG antibody. After three washes in TBS supplemented with 1 mM CaCl 2 , the immune reactivity was revealed by the addition of horseradish peroxidase substrate according to the manufacturer's instruction.
Generation of Stably Transfected L1.2 Cells-Receptor constructs were cloned into the bicistronic vector pIRES2-ZsGreen1 (Clontech), which results in bicistronic gene expression of receptor and GFP located on one mRNA, but translated as two proteins. L1.2 cells were grown at 5% CO 2 , 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.2 mM glutamine, 180 units/ml penicillin, and 45 g/ml penicillin/ streptomycin. L1.2 cells were transfected as described by the manufacturer using Nucleofector V solutions and program X-001 of the Nucleofector-2b-Device (Lonza, Switzerland). 48 h after transfection, G418 was added at 1 mg/ml and L1.2 cells were seeded in a 96-well plate at 25,000 cells per well. After a further 17 days, pool clones from CCR2 and CCR5-CCR2 (all) were analyzed in FACS and sorted for further cultivation before the migration experiments. For CCR5, inserted into pcDNA3.1, 10 clones were picked and tested in a migration assay. The three clones with highest expression were used for the migration experiments.
Migration-ChemoTx 96-well plates (3.2 mm diameter, 5 m pore size, and 30-l well volume (Neuroprobe)) were used for migration experiments of stably transfected L1.2 cells. In short, the lower wells were filled with buffer (Hepes-modified RPMI 1640 medium, 0.1% BSA) and ligands, and the filter was applied. 20-l cell suspension drops containing 200,000 cells/ drop were placed on top of the filter for migration into the lower wells. After a 5-h incubation at 5% CO 2 , 37°C, the drops were scraped off, and the filter was removed. The lower well solution was transferred to a white 96-well plate, and 20 l of CellTiter-Glo (Promega) solution was added. The luminescence was measured in a Wallac Envision 2104 Multilabel Reader (PerkinElmer Life Sciences). Sequence Analysis-Protein sequences of 286 class A 7TM receptors were aligned in Geneious 5.3 software using Clustal W multiple sequence alignment algorithm (34). For the alignment, BLOSUM cost matrix was employed, and gap open and gap penalty costs were 10 and 0.1, respectively. Alignment results were verified for proper overlap of highly conserved NPXXY domain specific for TM-VII of class A 7TM receptors. Gaps within TM-VII upstream from NPXXY motive were manually removed. 10 receptor sequences were excluded from the analysis due to lack of similarity in TM-VII region.
Calculations-IC 50 and EC 50 values were determined by nonlinear regression using the GraphPad-Prism4 software (GraphPad Software, San Diego).

Identification of Small Molecule Agonists for CCR5 That Act Independently of Extracellular Receptor
Regions-Because of the structural similarity and considerable overlap between endogenous ligands for CCR1 and CCR5 (both are activated by CCL3 and CCL5), we tested whether previously identified small molecule agonists for CCR1 (17) also activated CCR5. Indeed, we found that the CCR1 agonists built over a metal ion chelator complex also activated CCR5, but not CCR2, with equally high potencies as observed for CCR1 (EC 50 values from 4.5 to 23 M; Fig. 2 and Table 1) (17). The interaction of these ligands with CCR5 seemed independent of any extracellular region, as the ligands activated CCR5-CCR2 (all) with similar or even higher potencies (EC 50 values ranging from 0.86 to 7.3 M) as compared with CCR5 WT (Fig. 2C). Similar to CCR1 (17), their activity was dependent on complex formation, as neither metal ions nor chelators alone showed any activity on CCR5 (data not shown). Conclusively, although the orthosteric chemokine-binding site could be transferred from CCR2 onto a CCR5 scaffold (and is thereby linked to the extracellular domains of CCR2), the small molecule activity was retained within the transmembrane regions of CCR5 and was unaffected by the exchange of the extracellular regions with those of CCR2.
Positive Cooperative Effects of Small Molecule Agonists in CCR5 Could Not Be Transferred to CCR5-CCR2 (all) -In addition to acting as agonists, the metal ion chelators were able to enhance the binding of 125 I-CCL3 to CCR5 up to 7-fold above maximum specific binding in the absence of unlabeled ligand, thus acting allosteric in relation to CCL3 similar to the previously described action in CCR1 (17). The binding affinities of metal ion chelators as measured against 125 I-CCL3 were in the same range with their agonistic potencies (K i from 7.3 to 93 M, Fig. 3A), again as observed in CCR1 (17). However, compared with the high affinity displacement of CCL5 from CCR1 (17), a 10 -30-fold lower affinity was observed for metal ion chelators in displacing 125 I-CCL5 from CCR5 (K i from 197 to 620 M, Fig.  3B). This indicates an allosteric binding mode in CCR5 related to CCL5, in contrast to a competition situation in CCR1 with overlapping binding sites between metal ion chelators and CCL5. As only CCR2-specific chemokines bound to CCR5-CCR2 (all) , we tested whether the allosteric enhancing effect on 125 I-CCL3 binding to CCR5 could be transferred from the CCR5 transmembrane unit (binding the allosteric compounds) to the CCR2 extracellular interface. Despite the maintained agonism of the small molecules in the chimeric CCR5-CCR2 (all) (Fig. 2C and Table 1), no allosteric enhancement of 125 I-CCL2 or 125 I-CCL7 binding was observed. In contrast, a low affinity displacement with K i values from 162 to 528 M for 125 I-CCL2 and from 90 to 267 M for 125 I-CCL7 was observed (Fig. 3, C  and D), i.e. with K i values lying in the same range as observed for 125 I-CCL5 binding to CCR5 (Fig. 3B).
Action of Small Molecule Agonists Depends upon Acidic Residues in the Top of TM-VII-To investigate the extension and possible overlap of the binding sites for chemokines and small molecules in CCR2, CCR5, and CCR5-CCR2 (all) , we initially focused on two acidic residues in TM-VII, Asp in position VII:-02 and Glu in position VII:06. Besides being anchor for many small molecule agonists and antagonists, the chemokine receptor-conserved GluVII:06 is also the anchor point for many chemokines (7,17). AspVII:-02 is also found predominantly among chemokine receptors (ϳ55% among these) as compared with ϳ6% among nonchemokine class A 7TM receptors and   OCTOBER 28, 2011 • VOLUME 286 • NUMBER 43 has been suggested as an interaction partner for the N terminus of CCL2 with CCR2 (12). Importantly, the recently published crystal structure of CXCR4 uncovered that TM-VII is extended on the extracellular side (36) and therefore includes residues from the presumed ECL-3. A computational model of CCR5 built over the crystal structure of CXCR4 (Fig. 4, C and F) illustrated that AspVII:-02 (Asp 276 ) in fact is part of TM-VII. As seen in Fig. 4, A and B, Ala substitution of AspVII:-02 (D276A) impaired the activity of small molecule agonists as compared with CCR5 WT (2.7-13-fold decreased potencies), whereas a similar substitution of GluVII:06 (E283A) completely abolished the activity (Fig. 4, A and B, and Table 2). In CCR5-CCR2 (all) , a similar dependence was observed for small molecule agonists of these acidic residues (Fig. 4, D and E). Thus, the action of small molecule agonists completely depends on GluVII:06, whereas AspVII:-02, located two helical turns above, contributes to a lesser degree. Importantly, no difference was observed between CCR5 and CCR5-CCR2 (all) .

Different Interaction of CCL2 and CCL7 with CCR2 WT as
Compared with Chimeric Receptor-Contrary to the similar dependence of the small molecule agonists on acidic residues in CCR5 WT and in CCR5-CCR2 (all) , the pattern differed for the CCR2 chemokine agonists (CCL2 and CCL7). Thus, CCL2 was highly dependent on GluVII:06 and AspVII:-02 in CCR2 WT as Ͼ13-fold decreased potency was observed for Ala substitution of either of these residues (Fig. 5A and Table 2), as reported in earlier studies (12). For CCL7, however, GluVII:06 seemed more important (63-fold decreased potency upon Ala substitution) than AspVII:-02 (5.6-fold decreased potency) (Fig. 5B). In CCR5-CCR2 (all) , a much lower dependence was observed for both acidic residues, as Ala substitution of GluVII:06 only resulted in 3.6-fold decreased CCL7 potency and 4.4-fold decreased CCL2 potency, whereas the impact of AspVII:-02 was even smaller (1.1-3.2-fold decrease) (Fig. 5, C and D). Changes in receptor surface expression could not explain the changes in potencies, as all Ala substitutions were found to be In the right panel, a molecular model of CCR5 based on the crystal structure of CXCR4 is shown in view from the extracellular side (C) and from the side (F) with residues AspVII:-02 and GluVII:06 highlighted as sticks.

TABLE 2 Surface expression of mutations AspVII:-02Ala and GluVII:06Ala in CCR5, CCR2, and CCR5-CCR2 (all) and their effect on chemokine and metal ion chelator-mediated receptor activation
The surface expression was determined by ELISA in COS-7 cells transiently transfected with FLAG-tagged receptors and is given as percent of the respective wild type receptor. IP turnover was measured in COS-7 cells co-transfected with receptor and the promiscuous G protein G qi4myr . EC 50 values are given in log and nM/M. F mut indicates the fold-decrease for the ligand potencies at the mutants as compared with the respective WT receptor. The number of experiments is shown in parentheses; ND means not determined.    Interchange in 7TM Receptors, Implications for Future Drugs equal to or even higher expressed than the respective WT receptors as determined by ELISA (Table 2). Furthermore, homologous competition binding experiments uncovered unchanged CCR2 WT-like affinities for CCL2 and CCL7 on both Ala substitutions in CCR2 (Table 3), despite the decreases in potencies (Table 2). In contrast, the two Ala substitutions in the CCR5-CCR2 (all) background resulted in minor decreases in affinities (1.9 -4.0-fold, see Table 3), which for both ligands were similar to the observed decreases in potencies (1.1-4.4fold, see Table 2). Thus, it is concluded that the two acidic residues at the top of TMVII (Asp 284 and Glu 291 ) are of huge importance for the activation but not the binding of CCL2 and CCL7 in CCR2 WT, although this dependence for chemokinemediated receptor activation is weakened in the CCR5-CCR2 (all) chimera.
Allosteric Mechanisms for the Small Molecule Agonists in CCR5 WT and CCR5-CCR2 (all) -To further investigate the action of the small molecule agonists, their effects on chemokine potency and affinity were assessed. Although only small decreases in potencies were observed for CCL3 and CCL5 activating CCR5 WT in the presence of small molecule agonists, the potencies of CCL2 and CCL7 at CCR5-CCR2 (all) were up to 100-fold reduced (Fig. 6). On the contrary, the affinities of chemokine binding to CCR5 and CCR5-CCR2 (all) were unchanged in the presence of small molecule agonists, as observed in competition binding experiments (Table 4).
Conclusively, as ligands with overlapping binding sites (i.e. competitive ligands) are expected to primarily affect the affinity of an orthosteric ligand, these ligand co-administration experiments clearly suggest an allosteric binding mode of the small molecule agonists and thereby confirm the binding and functional data in Figs. 1-5. Retained Activity of CCR5 Nonpeptide Antagonists in the CCR5-CCR2 (all) Chimera-As a third pharmacological aspect, we investigated the effect of the domain swapping in the CCR5-CCR2 (all) chimera on the CCR5 antagonists SCH-C and aplaviroc and the dual CCR5/CCR2 antagonist TAK-779. As expected from the dual nature of TAK-779, the antagonistic property was retained in the chimera. Interestingly, the activity of SCH-C was also retained and thereby independent of the extracellular CCR2 domains. This was not observed for aplaviroc, which completely lost its antagonistic functionality in CCR5-CCR2 (all) (Fig. 7 and Table 5). However, as the CCR5binding site of aplaviroc has been suggested to include residues Ser 180 and Lys 191 in ECL-2b (14), the lack of aplaviroc activity in the CCR5-CCR2 (all) chimera could be due to the absence of these particular residues in ECL-2b of CCR2 and consequently in CCR5-CCR2 (all) .

TABLE 3 Homologous chemokine binding to CCR2, CCR5, and CCR5-CCR2 (all) with or without Ala substitutions of acidic residues in TMVII
Competition binding experiments were performed in transiently transfected COS-7 cells using 125 I-CCL3, 125 I-CCL2, and 125 I-CCL7 as radioligands. The IC 50 values are given as log and in nM, and the impact of the mutants is given as fold-decrease compared with the affinities in background receptor (WT or CCR5-CCR2 (all) ). The number of experiments (n) is given. potencies in the same range as in CCR5 WT ( Fig. 8 and Table 5). As in the CCR5-CCR2 (all) chimera, the activity of the other antagonists was retained in the CCR5-CCR2 (all⌬2b) construct (Table 5). In addition, the potencies of the various chemokine (and small molecule) agonists were unaltered in CCR5-CCR2 (all⌬2b) as compared with CCR5-CCR2 (all) . Thus, CCL2 and CCL7 could activate CCR5-CCR2 (all⌬2b) with potencies of 4.4 and 1.0 nM, respectively. Also the affinities of CCL2 and CCL7 to CCR5-CCR2 (all⌬2b) were determined to be in nanomolar range (logK d(CCL2) ϭ Ϫ9.7 Ϯ 0.43 (n ϭ 2); logK d(CCL7) ϭ Ϫ9.0 Ϯ 0. 16 (n ϭ 3)). The potencies of the metal ion chelators were ranging from 1.3 M for ZnPhe, over 3.1 M for CuPhe and 11 M for ZnBip to 29 M for CuBip. This indicates that the absence of ECL-2b from CCR2 makes no difference for proper chemokine or small molecule-based activation.

Construction of Chimeric Receptors, Trojan Horse or Centaur
Principle-A chimeric receptor was constructed to investigate the behavior of allosteric and orthosteric sites in CCR5 and CCR2. Importantly, the chimeric setup in CCR5-CCR2 (all) is domain-related (Trojan horse), rather than sequence-related (centaur), meaning that the sites of interchange between the two receptors were not placed randomly and consecutively between the N and C terminus but precisely where the TM helices turn to extracellular loops. In the case of CCR5 and CCR2, which originate from one precursor by gene duplication and therefore are highly homologous (20), similar sequences were found in these areas. This allowed us to "match" the exact position between helix and loop when constructing the chimera (Fig. 1). Thus, although CCR5-CCR2 (all) looks like CCR2 from the outside (confirmed by the high affinity binding of CCR2 chemokines CCL2 and CCL7), the chimera resembles CCR5 in its core and transduces signals in a CCR5-specific manner (confirmed by the binding and action of CCR5-specific small molecules). So far, the majority of chemokine receptor chimeras have been constructed in a centaur way (27)(28)(29)(30). However, the Trojan horse chimeras have also been made previously to study CCR1 and CCR6 ligands (chimeras with CCR3 and CCR5, respectively) (25,37). Other 7TM chimeras have focused on the intracellular domains with a local or global domain exchange, designed to characterize and eventually shift the G protein coupling and signaling pathways (38 -40) and to be used in the deorphanization of receptors with unknown coupling specificity (41).
Chemokine Binding Depends Solely on the Extracellular Receptor Regions-It is noticeable that the construction of our chimera actually leads to a functional receptor with maintained ligand binding (Tables 3 and 4) and action (Fig. 1). Previous Trojan horse chimeras, for instance between CCR6 and CCR5

Interchange in 7TM Receptors, Implications for Future Drugs
(CCR6-CCR5 (all) ), did not succeed in maintaining CCR5 chemokine binding (37). This could be due to the low identity between these two receptors (33%) as compared with 74% between CCR5 and CCR2 and thereby a larger degree of structural changes applied to the CCR6-CCR5 (all) chimera. Importantly, our data confirm the current understanding of chemokine binding to the extracellular receptor regions. A two-state model has been proposed for the action of CCL2 with CCR2, stating that an initial high affinity interaction of the chemokine core with the extracellular domains of the receptor (especially the N terminus) is followed by a receptor-activating interaction of the chemokine N terminus with residues in the transmembrane helices. Thus, CCL2 was shown to interact with a DYDY motif in the CCR2 N terminus (42,43), followed by interaction of the chemokine N terminus with AspVII:-02 (Asp 284 ) and GluVII:06 (Glu 291 ) in TM-VII (12). These data are indeed confirmed here by the unchanged high affinity but very low potencies of CCL2 and CCL7 in the Ala substitutions of these two acidic residues in CCR2 ( Fig. 5 and Table 3). In addition, ECL-2 has earlier been shown to be important for CCL2 activity (29), again in agreement with our finding that transfer of a single region from CCR2 to CCR5 (N terminus or any of the ECLs) is insufficient for proper CCL2 or CCL7 action (supplemental Fig.  1). In CCR5, we confirm earlier studies showing that especially ECL-2 (29) and, in the case of CCL3, also ECL-3 are highly important, as we find that their replacement with CCR2 counterparts abolished CCL3 and CCL5 activity (supplemental Fig.  1). Altogether, our chimera confirms that chemokines interact with and recognize their receptors via the extracellular domains (Fig. 9).
Identification of a Novel Class of Small Molecule Agonists for CCR5-Metal ion chelators (Zn 2ϩ or Cu 2ϩ ions in complex with bipyridine or phenanthroline, Fig. 2) have earlier been shown to be complex ago-allosteric modulators of CCR1 by acting as agonists with micromolar potencies and concomitant allosterically enhancing the binding of CCL3, although they displaced CCL5 with micromolar affinities indicating partly overlapping binding sites in the area around GluVII:06, as Ala substitution in this position resulted in huge and similar decreases in potencies for CCL5 and metal ion chelators (but not CCL3) (17). In this study, we find that also CCR5 is activated by these molecules that, as in CCR1, depend on GluVII:06 (Table 2). Like in CCR1, the small molecule agonists enhance the binding of CCL3 (act allosterically) but do not displace CCL5 with the same high affinity as observed in CCR1. This could be due to a lesser degree of overlap between small molecule agonists and CCL5-binding sites in CCR5 compared with CCR1, as clearly indicated by the lower impact of Ala substitution of GluVII:06 in CCR5 (5.1-fold decrease in potency for CCL5, Table 2) as compared with 26-fold decrease in CCL5 potency in CCR1 (17). Our test of the impact of small molecule agonists on the potency (Fig. 6) and affinity (Table 4) of the chemokine ligands confirms the allosteric mode of action as no changes in the chemokine affinities were observed in the presence of small molecule agonists, although their potencies were slightly reduced at CCR5. In conclusion, CCL5 and the metal ion chelator-based agonists do not overlap in CCR5, which in turn also confirms that CCL5 interacts differently with CCR5 compared with CCR1, as suggested previously (27,44).
Separation of Small Molecule and Chemokine-binding Sites-Interestingly, the activity of small molecule agonists was retained in CCR5-CCR2 (all) and could therefore be assigned to the transmembrane region of CCR5. However, as the loops of CCR5 and CCR2 are similar to a certain extent, it cannot be excluded that also residues in the extracellular domains contribute to the small molecule agonist site. Thus, in both receptors, ECL-2, which is linked to the top of TM-III via a conserved disulfide bridge to CysIII:01, harbors a Glu in position Cys Ϫ6 (6 residues prior to the conserved Cys in ECL-2) and an aromatic residue in position Cys ϩ4 . Furthermore, ECL-2 varies greatly in length and sequence throughout class A 7TM receptors, and with its position on top of the major binding pocket, with a lid function in some receptors, ECL-2 contributes to ligand specificity in some class A 7TM receptors (45). Indeed, we find that  OCTOBER 28, 2011 • VOLUME 286 • NUMBER 43 aplaviroc loses its antagonistic property in CCR5-CCR2 (all) but could be rescued by reintroducing ECL-2b, which is in good agreement with earlier studies (14). Interestingly, the presently available crystal structures of 7TM receptors have uncovered several different conformations of ECL-2 ranging from ␤-sheets with the receptor N terminus (rhodopsin) or ECL-1 (adenosine 2A receptor) to small ␣-helices (␤ 1 -and ␤ 2 -adrenoceptors) (46 -54). In CXCR4, an anti-parallel ␤-sheet is formed, which in one case is extended with another ␤-strand from the co-crystallized peptide antagonist (36). In addition to the small molecule agonists, the activity of the antagonist SCH-C was also retained in the chimera. Thus, it can be concluded that we successfully generated a receptor with two distinct sites that bind ligands independently of each other (Fig. 9).

Interchange in 7TM Receptors, Implications for Future Drugs
As expected from studies in CCR1 (17), the small molecule agonists anchored to GluVII:06 in CCR5-CCR2 (all) and in WT CCR5 (Table 2). CCL2 and CCL7, however, have a reduced (or even lost) dependence of both GluVII:06 and AspVII:-02 in the chimera as compared with CCR2. Thus, even though both chemokines bind to the extracellular CCR2 domains in the chi-mera, the interaction with GluVII:06 and AspVII:-02 was weakened and was not sufficient to compete for the small molecule agonists, presumably because of a slightly altered (although still high affine) binding of the chemokines in the extracellular domains (Fig. 9). Consequently, CCL2 and CCL7 are only displaced by the small molecule agonists from CCR5-CCR2 (all) with rather low affinity, but neither is their binding enhanced, indicating that the positive allosteric cross-talk from the CCR5 transmembrane region (binding the ago-allosteric molecules) with the CCR5 extracellular domains (binding CCL3), could not be established in CCR5-CCR2 (all) . Thus, positive allosteric modulation not only requires that similar active conformations are stabilized but also maintained conformational interchange and communication between domains, properties that could not be established in the chimera.
Ago-allosteric Ligands in CCR5 and CCR5-CCR2 (all) -Allosteric modulators can be neutral, i.e. do not signal on their own, or they can be agonists or antagonists for a given receptor. However, independent of the efficacy, the binding of an allosteric modulator leads to changes in the balance between differ- Antagonistic activities of TAK-779, SCH-C, and aplaviroc in WT and chimeric receptors IP turnover was measured in COS-7 cells co-transfected with receptor and the promiscuous G protein G qi4myr . The receptors CCR5 WT, CCR2 WT, CCR5-CCR2 (all) , and CCR5-CCR2 (all⌬2b) were activated with chemokines or metal ion chelators ZnBip and ZnPhe. EC 50 values are given in log and nM. The number of experiments is shown in parentheses; ND means not determined. ent receptor conformations, which in turn are likely to change the affinity and/or function of the orthosteric ligands. In CCR5, we identified the metal ion chelator complexes to be agonists and positive allosteric modulators toward CCL3 (ago-allosteric ligands). The allosteric nature is furthermore underlined by the fact that these compounds competed against 125 I-CCL5 binding to CCR5 and 125 I-CCL2 and 125 I-CCL7 binding to CCR5-CCR2 (all) with very low affinities (Fig. 3, B-D), which importantly were much lower compared with the affinities for the enhancement of 125 I-CCL3 binding (Fig. 3A) and to the potencies as agonists (Fig. 2, A and C). These low affinity displacements and the decreased potencies of CCL2 and CCL7 at CCR5-CCR2 (all) in the presence of small molecule agonists (Fig.  6B) correlate well with the different dependence of chemokines and small molecule ligands on Asp 284 and Glu 291 in CCR5-CCR2 (all) . Thus, because CCL2 and CCL7 do not compete with small molecule agonists for Asp 284 and Glu 291 , higher concentrations are needed to overcome the small molecule agonistbound state to further activate the receptor, resulting in the observed decrease in potencies in the presence of small molecule agonists. Different and equally complex kinds of allosteric behaviors have earlier been observed in e.g. the action of L-692,429 at the ghrelin receptor (55) or the actions of Org-27569, Org-27759, and Org-29647 at the CB 1 receptor (56).
We conclude that Trojan horse-based chimeric receptors constitute a powerful pharmacological tool to study allosteric phenomena in 7TM receptors. The construction and combination of receptors in the chimera can be chosen so that not only a separation of the orthosteric and allosteric binding sites is achieved but also a separation between the ligand-binding and receptor-activating domains. This also emphasizes the modulelike construction of 7TM receptors and confirms the understanding that agonists may activate certain (or different) receptors in different ways (2). Furthermore, because of their advantages such as the higher specificity and dependence on the orthosteric ligand, allosteric modulators and the understanding of their action will become increasingly important in drug development.