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(Journal of Leukocyte Biology. 2001;69:977-985.)
© 2001 by Society for Leukocyte Biology

A chimeric MIP-1{alpha}/RANTES protein demonstrates the use of different regions of the RANTES protein to bind and activate its receptors

Cédric Blanpain*, Raphaële Buser{dagger}, Christine A. Power{dagger}, Michael Edgerton{dagger}, Catherine Buchanan{dagger}, Matthias Mack{ddagger}, Graham Simmons§, Paul R. Clapham§, Marc Parmentier* and Amanda E. I. Proudfoot{dagger}

* IRIBHN Université Libre de Bruxelles, Campus Erasme, Bruxelles, Belgium;
{dagger} Serono Pharmaceutical Research Institute, Geneva, Switzerland;
{ddagger} Medizinische Poliklinik, Ludwig-Maximilians-Univerity of Munich, Munich, Germany; and
§ The Wohl Virion Centre, Department of Molecular Pathology, The Windeyer Institute for Medical Sciences, University College Medical School, London, UK

Correspondence: Amanda E. I. Proudfoot, Serono Pharmaceutical Research Institute, 14, Chemin des Aulx, 1228 Plan les Ouates, Geneva, Switzerland. E-mail: amanda.proudfoot{at}serono.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human RANTES (CCL5) and MIP-1{alpha} (CCL3) bind and activate several CC chemokine receptors. RANTES is a high-affinity ligand for CCR1 and CCR5, and it binds CCR3 with moderate affinity and CCR4 with low affinity. MIP-1{alpha} has similar binding characteristics to RANTES except that it does not bind to CCR3. Here we have generated a chimera of human MIP-1{alpha} and RANTES, called MIP/RANTES, consisting of the eight amino terminal residues of MIP-1{alpha} preceding the CC motif, and the remainder of the sequence is RANTES. The chimera is able to induce chemotaxis of human monocytes. MIP/RANTES has >100-fold reduction in binding to CCR1 and does not bind to CCR3 but retains full, functional binding to CCR5. It has equivalent affinity for CCR5 to MIP-1{alpha} and RANTES, binding with an IC50 of 1.12 nM, and is able to mobilize calcium and induce endocytosis of CCR5 in PBMC in a manner equi-potent to RANTES. It also retains the ability to inhibit R5 using HIV-1 strains. Therefore, we conclude that the amino terminus of RANTES is not involved in CCR5 binding, but it is essential for CCR1 and CCR3.

Key Words: chemokine • ligand • monocyte


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are a family of polypeptides that are responsible for the recruitment and trafficking of immune-cell populations and are therefore essential for the effective organization of the immune response [1 2 3 ]. Under normal conditions, chemokines mediate the correct homing of immune and haematopoetic cells, but they are also responsible for the inflammatory, cellular infiltrates at the site of tissue injury. On this functional basis, chemokines can be subdivided into constitutive or inflammatory chemokines, although some members of the family appear to play both roles [4 ]. About 40 chemokines have been identified so far, all of which activate seven transmembrane-spanning, G protein-coupled receptors, which distinguish them from all other cytokines. To date, 18 receptors have been characterized, and the studies of receptor-ligand pairing have shown that, at least in vitro, the chemokine system appears highly redundant with few examples of absolute specificity where a receptor binds and is activated by a single ligand [5 ]. Many receptors bind two or more ligands, and most chemokines can bind and activate more than one receptor. Chemokine receptors also play a major role in AIDS pathogenesis [6 , 7 ] and are therefore potential, therapeutic targets for the prevention of human immunodeficiency virus (HIV) infectivity [8 ]. HIV entry is mediated by the interaction of the viral-envelope glycoprotein (gp120), CD4, and a coreceptor that belongs to the chemokine-receptor family. CCR5 is believed to be the principal coreceptor for M-Tropic, HIV-1 strains (or R5 strains) that are responsible for the transmission of the disease and predominate during its asymptomatic stages. Individuals deficient in CCR5 (homozygous for the nonfunctional CCR5 {triangleup}32 variant) are highly resistant to HIV-1 infection, demonstrating the essential role played by CCR5 in HIV-1 pathogenesis [9 , 10 ].

RANTES (regulated on activation, normal T expressed and secreted; CCL5) is one of the most promiscuous, inflammatory CC chemokines. The identification of CCR1, first known as the shared RANTES/macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}; CCL3) receptor, was an early example of the lack of absolute specificity [11 ]. RANTES has been shown to act on the major eosinophil, CC chemokine receptor, CCR3, although with lower affinity than eotaxin or monocyte chemoattractant protein (MCP)-3 [12 , 13 ]. The initial characterization of CCR4 showed that it could be activated by RANTES, MIP-1{alpha}, and MCP-1 (CCL2) [14 , 15 ], although its high-affinity ligands, thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), were identified later [16 , 17 ]. RANTES, MIP-1{alpha}, MIP-1ß (CCL4), and MCP-2 (CCL8) have been shown to be high-affinity ligands for CCR5 [18 19 20 ]. RANTES also binds to two human chemokine receptors without inducing signal transduction, D6 [21 ], and the Duffy antigen, DARC [22 ], as well as to the virally encoded receptor, US28 [23 ].

This apparent redundancy is intriguing. It may simply reflect the recent amplification of chemokine and chemokine-receptor genes, as suggested by their genomic clustering and the fact that these new genes have not acquired their nonoverlapping functions yet [24 , 25 ]. Alternatively, redundancy may be maintained as a way of increasing the robustness of the chemokine system. Primary sequence similarity between chemokines does not appear to correlate with their receptor usage. For example, RANTES and MIP-1{alpha} share only 45% identity at the primary amino acid level despite their similar pharmacology. However, the three-dimensional structure of all the chemokines solved to date has shown a remarkably conserved, monomeric fold [26 , 27 ]. Another major, biochemical difference is that RANTES is highly basic with an acidic isoelectric point of 9.8 (a feature shared by most of the chemokines), whereas MIP-1{alpha} has an acidic isoelectric point of 4.7.

Numerous studies have demonstrated that the amino terminal region of CC- and CXC-chemokines is critical for the biological activity of these proteins. On the contrary, modifications of the carboxy terminal region are well-tolerated because chemokines have been shown to be fully active when expressed as C-terminal fusion constructs with large proteins, such as alkaline phosphatase resulting in a total mass of 74 kDa [28 ]. Small modifications to RANTES, such as the retention of the initiating methionine (Met-RANTES) when the recombinant protein is produced in Escherichia coli, have profound effects on its bio-activity [29 ]. Several studies have shown that amino terminal deletions of chemokines such as interleukin (IL)-8, RANTES, MCP-1, and MCP-3 create analogues that display defective signaling while retaining receptor-binding properties, thereby creating proteins that are partial agonists or effective antagonists [30 31 32 33 34 ].

These observations led to the hypothesis that chemokines may act through a multiple-site mechanism for chemokine-chemokine receptor interaction, similar to that proposed for another chemoattractant protein of a similar size, C5a [35 ]. Indeed, using chemokine-receptor chimeras, we and others [36 , 37 ] have shown that the N-terminus of CCR2b is the major determinant of ligand selectivity, but other domains are required for receptor activation. For CCR5, the second, extracellular loop (ECL2) plays the dominant role, although a contribution of the N-terminus is required [36 , 38 ]. The impaired ability of chemokines to activate their receptors upon modification of their N-terminal regions substantiated this hypothesis.

We were interested in the consequences of replacing the amino acids preceding the CC motif of the promiscuous chemokine RANTES with those belonging to another chemokine, MIP-1{alpha}, which shares some of the RANTES receptors. We demonstrate here that this chimera, MIP/RANTES, remains fully active on one of the shared receptors, CCR5. MIP/RANTES retains the ability to bind to CCR5 with high affinity, is able to mobilize calcium with full potency, is able to mediate chemotaxis through CCR5, can cause down-modulation of the receptor, and is able to inhibit infection of R5 HIV-1 strains. It also retained the low-affinity binding properties of RANTES for CCR4. However, it presented significant, altered binding affinity for CCR1, the other shared RANTES/MIP-1{alpha} receptor, and no binding could be observed on human CCR3, which binds and is activated by RANTES but not MIP-1{alpha}. Therefore, we propose that the ligand RANTES binds CCR4 and CCR5 through regions excluding the amino terminus, whereas CCR1 and CCR3 require the amino terminus of the RANTES protein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Enzymes were from New England Biolabs (Beverly, MA), and chromatographic material was from Pharmacia (Upsala, Sweden). The anti-CCR5 monoclonal antibody (mAb; clone MC-1) was prepared as described [39 ].

Recombinant chemokines
RANTES, Met-RANTES, and aminooxypentane-RANTES were produced as described previously [29 , 40 , 41 ]. TARC, MIP-1{alpha}, and eotaxin were purchased from PeproTech (London, U.K.). The MIP/RANTES chimera was made in two steps.

In the first step, the amino terminal region of RANTES was truncated by amplifying the gene with polymerase chain reaction (PCR) primers, which introduced a unique SacI site within the first cysteine codon and a terminal ochre codon at the 3' end of the gene. This truncated gene was cloned into pET23d (Novagen, Madison, WI) as a SacI/HindIII fragment. In the second step, the chimera was made by digesting the truncated RANTES pET23d construct with SacI, followed by treatment with T4 DNA polymerase and subsequent digestion with NcoI. Two oligonucleotides were then synthesized with the preferred E. coli codon use, encoding a leader sequence, KKKWPR, followed by the N-terminal amino acids of MIP-1{alpha} (ASLAADTPTA) or the same sequence with an additional Met residue (MASLAADTPTA). The resultant oligonucleotides were ligated onto the truncated RANTES gene. Ligation products were transformed into E. coli strain BL21pLysS, and plasmid DNA from the resultant clones was sequenced to verify that no errors had been introduced into the coding sequence The KKKWPR sequence was included, because it has been shown to improve expression of the chemokines in E. coli [41 ]. The MIP/RANTES chimera was purified from E. coli exclusion bodies as described previously [41 ] with the following modifications. The leader sequences were removed by enzymic digestion with Arg C for 3 h at 37°C (enzyme:substrate, 1:100, w/w) in 50 mM Tris/HCl, pH 8.0, containing 10 mM CaCl2, 5 mM ethylenediaminetetraacetate (EDTA), and 50 mM dithiothreitol (DTT) buffer to produce Met-MIP/RANTES or, alternatively, by CNBr cleavage with a 500-fold molar excess of CNBr in 70% formic acid for 16 h in the dark at room temperature. Following the CNBr treatment, the solution was diluted tenfold with H2O and lyophilized. The cleaved products were purified by cation-exchange chromatography on a HiLoad SP 26 column, previously equilibrated in 50 mM sodium acetate, pH 4.5, containing 6 M urea and eluted with a 0–2 M NaCl gradient in the same buffer. The cleaved products were dialyzed against two changes of 1% acetic acid, once against 0.1% trifluoroacetic acid (TFA), and lyophilized. The proteins were subjected to Edman degradation and electrospray mass spectroscopy for sequence verification.

Chemotaxis assays
The proteins were analyzed for their ability to induce the directional migration of freshly isolated monocytes, purified from buffy coats, using the modified micro-Boyden chamber as described previously [42 ].

Plasmids
The coding sequences CCR5, CCR1, and CCR3 were cloned into the bicistronic expression vector pEFIN3 as previously described [36 ]. The CCR4 coding sequence was cloned into pcDNA3.1 (+) zeo (Invitrogen, San Diego, CA).

Creation of stable cell lines expressing chemokine receptors and mutant chemokines
Chinese hamster ovary (CHO)-K1 cells were cultured in HAM’s F12 medium supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Paisley, UK). A plasmid encoding an apoaequorin variant targeted to mitochondria [43 ], under control of the SR{alpha} promoter [44 ], was used to generate a stable CHO-K1 cell line as described [45 ]. Constructs encoding chemokine receptors in pEFIN3 were transfected subsequently into this apoaequorin-expressing cell line using Fugene 6. Stably transfected clones were isolated following selection for 14 days with 400 µg/ml G418 (Life Technologies) and used for binding and functional studies. Cell-surface expression of the CCR5 receptor was measured by flow cytometry using the mAb MC-5, which recognizes a linear epitope in the CCR5 amino terminal domain. Cell-surface expression of CCR1 and CCR3 were characterized by a Bmax of 5.7 pmol/mg protein and 500 fmol/mg protein for RANTES and eotaxin, respectively.

Stable cell lines expressing CCR4 were generated as follows: HEK293 cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) F12 medium containing 10% FCS, 2 mM glutamine, and 100 units/ml penicillin/streptomycin (Life Technologies) were transfected at 80% confluence with the CCR4/pcDNA3.1 (+) zeo construct using the calcium phosphate-transfection kit (Life Technologies). Forty-eight hours after transfection, the cells were harvested by trypsinization and transferred into fresh medium containing 50 µg/ml zeocin (Invitrogen), After 14 days of selection with zeocin, surviving clones were assessed for CCR4 expression by their ability to bind [125I]-TARC or [125I]-MDC (Amersham, Amersham, UK) in a whole-cell binding assay as described below.

Competition-equilibrium binding assays
CHO-K1 cells expressing wild-type CCR1, CCR3, or CCR5 were harvested using Ca2+- and Mg2+-free phosphate-buffered saline (PBS) containing 1 mM EDTA, gently pelleted for 5 min at 1000 g and resuspended in binding buffer [50 mM Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.5% bovine serum albumin (BSA)]. Competition-binding assays were performed in Minisorb tubes (Nunc, Roskilde, Denmark), using 0.1 nM [125I]-MIP-1{alpha} or [125I]-RANTES (2200 Ci/mmol; N.E.N., Zarentem, Belgium) as tracer for CCR1 and CCR5 and [125I]-eotaxin for CCR3 and variable concentrations of chemokine competitors (R&D Systems, Minneapolis, MN). The number of cells used for the CCR5 binding assay was 40,000 or 1 or 5 µg of membrane proteins for CCR1 and CCR3, respectively, in a final volume of 0.1 ml. Total binding was measured in the absence of competitor, and nonspecific binding was measured with a 100-fold excess of unlabeled ligand. Samples were incubated for 90 min at 27°C; then bound tracer was separated by filtration through GF/B filters pre-soaked in 1% BSA for [125I]-MIP-1{alpha} or 0.3% polyethylenimine (Sigma) for [125I]-RANTES and [125I]-eotaxin. Filters were counted in a ß-scintillation counter.

CCR4-expressing cells cultured to 60–80% confluence were washed twice with PBS and harvested after treatment with PBS containing 1 mM EDTA, centrifuged at 1000 g for 5 min, and resuspended in 10 ml binding buffer, pH 7.2, containing 50 mM HEPES, 5 mM MgCl2, 1 mM CaCl2, and 0.5% BSA. Cells (150,000) were incubated with 100 pM [125I]-TARC and increasing concentrations of unlabeled chemokines, namely MIP-1{alpha}, RANTES, Met-RANTES, AOP-RANTES, and MIP/RANTES, in triplicate, 96-well plates. After 90-min incubation at room temperature, the unbound radioligand was washed off with three washes of 200 µl binding buffer containing 0.5 M NaCl, covered with 50 µl Scintillate, and counted with a beta scintillation counter (Wallac, Zarentem, Belgium) for 1 min per well.

Binding parameters were determined with the Prism software (GraphPad Software) using nonlinear regression applied to a one-site competition model.

Calcium mobilization
Functional responses to chemokines were analyzed by measuring the luminescence of aequorin as described [45 , 46 ]. Cells were collected from plates with Ca2+- and Mg2+-free DMEM supplemented with 5 mM EDTA, pelleted for 2 min at 1000 g, resuspended in DMEM at a density of 5 x 106 cells/ml, and incubated for 2 h in the dark in the presence of 5 µM coelenterazine H (Molecular Probes, Junction City, OR). Cells were diluted 7.5-fold before use. Agonists in a volume of 50 µl DMEM were added to 50 µl cell suspension (33,000 cells), and luminescence was measured for 30 sec in a Berthold Luminometer.

CCR5 down-modulation
The ability of MIP/RANTES to down-regulate CCR5 from the surface of peripheral blood mononuclear cells (PBMCs) compared with the CCR5 ligands RANTES, AOP-RANTES, MIP-1{alpha}, and MIP-1ß was performed as described previously using the anti-CCR5 mAb, MC-1 [39 ].

Inhibition of HIV infectivity
HIV inhibition assays were performed as described [47 ]. Briefly, 1 x 105 phytohemagglutinin (PHA)/IL-2-stimulated PBMCs were exposed to 50 µl chemokine for 30 min at 37°C. One-thousand tissue culture infectious dose (TCID)50 of the CCR5-using HIV-1 strain SL-2 [48 ] was then added in a volume of 50 µl. Following 3 h incubation at 37°C, the cells were washed three times and resuspended in RPMI 1640 (Gibco-BRL, Paisley, UK), 20% FCS, and 10% IL-2 (Roche Diagnostics, Nutley, NJ) containing the relevant chemokine. After 7 days culture at 37°C, the samples were assayed for supernatant p24.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purification and characterization of the MIP/RANTES chimera
The enzymic digestion using endoproteinase Arg C of the MKKKWPRM-MIP/RANTES construct yielded the sequence of the MIP/RANTES chimera with an additional amino terminal Met residue, as ascertained by Edman degradation and electrospray ionization (ESI)-mass spectroscopy. The mass obtained was 7941.56 Da compared with the expected mass of 7941.23 for the oxidized protein with two disulfide bonds. Sequencing the amino terminal by Edman degradation of the first 20 amino acids of the CNBr cleavage product gave the sequence ASLAADTPTAXXFAYIARPL, confirming the expected MIP/RANTES sequence (Fig. 1 ). The authenticity of the protein was confirmed further by mass spectroscopy, because the protein had a mass of 7808.09 Da compared with the expected mass of 7810.23 for the oxidized protein.



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  		Figure 1. MIP/RANTES sequence. Alignment of the primary sequences of RANTES and MIP-1{alpha} showing identical amino acids (gray shading) and the sequence of the chimeric MIP/RANTES chemokine.

 
MIP/RANTES induced monocyte chemotaxis
The activities of the two chimerae were tested initially for their ability to induce chemotaxis of freshly isolated monocytes, which we have shown to express mRNA and functional levels of the four RANTES receptors, CCR1, CCR3, CCR4, and CCR5 [42 ]. Both chimerae were able to induce migration of human monocytes with a potency similar to RANTES but with a significant reduction in efficacy (Fig. 2 ). The EC50 for RANTES in this assay is 1 nM, with a maximal activity at 10 nM. Met-MIP/RANTES and MIP/RANTES had EC50 values of 1 nM, but the maximal activity was reached only at 100 nM. The efficacy of RANTES was donor-dependent with the chemotaxis index ranging from 4 to 10 depending on the donor [42 ], but typically, the chimerae achieved 50% of the efficacy of RANTES itself. Because the Met residue at the amino terminal had no significant effect on the activity of the chimerae, in contrast with the addition of this amino acid to the RANTES protein [29 ], subsequent studies were limited to the MIP/RANTES sequence.



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  		Figure 2. Chemotaxis of human monocytes induced by MIP/RANTES. Chemotaxis of freshly isolated human monocytes induced by RANTES ({circ}), MIP/RANTES (•), and Met-MIP/RANTES ({blacksquare}) was carried out using the modified Boyden chamber as described in the text.

 
Equilibrium competition-binding studies of MIP/RANTES to RANTES receptors
MIP-1{alpha} and RANTES are high-affinity ligands for CCR1 and CCR5. We investigated the binding affinities of MIP/RANTES and its parental chemokines on recombinant CCR1 (Fig. 3 ) and CCR5 (Fig. 4 ) using [125I]-RANTES or [125I]-MIP-1{alpha} as tracers. As shown in Figure 4 , MIP/RANTES competed for [125I]-RANTES binding on CCR5 with a potency similar to that of its parental chemokines MIP-1{alpha} and RANTES. The IC50 values were 1.12, 0.38, and 1.5 nM for MIP/RANTES, RANTES, and MIP-1{alpha}, respectively (Table 1 ). The same order of potency was obtained when [125I]-MIP-1{alpha} was used as tracer, and all three ligands competed with similar efficiencies (unpublished results). In sharp contrast, MIP/RANTES showed a 200-fold loss in potency in competition for [125I]-RANTES binding to CCR1 with an IC50 of 23 nM compared with its parental chemokines MIP-1{alpha} and RANTES, which had IC50 values of 0.16 and 0.20 nM, respectively, in this assay (Fig. 3 and Table 1 ). Similar differences in binding affinities were obtained when [125I]-MIP-1{alpha} was used as a tracer (unpublished results). The ability of MIP/RANTES to bind to CCR3 was tested using [125I]-eotaxin as tracer, and the chimera was unable to compete with this radiolabeled ligand at concentrations up to 500 nM (unpublished results). RANTES is able to compete partially with [125I]-TARC, one of the high-affinity CCR4 ligands, from HEK membranes expressing CCR4, but the IC50 is approximately 300-fold lower than that for TARC itself, and total displacement is not achieved even at concentrations as high as 1 µM (Fig. 5 ). MIP-1{alpha} is even less potent, achieving only 25% displacement at 1 µM (unpublished results). We tested the ability of MIP/RANTES and two other N-terminally modified RANTES proteins, Met-RANTES and AOP-RANTES, to compete for [125I]-TARC binding, and it displayed binding characteristics very similar to RANTES (Fig. 5) . It should be noted that MIP/RANTES competed with [125I]-TARC in a manner almost identical to its parent RANTES and more effectively than MIP-1{alpha}.



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  		Figure 3. Equilibrium competition binding assay on CCR1. Competition binding curves were performed on membrane CHO-K1 cell lines expressing CCR1 using 0.06 nM [125I]-RANTES as tracer and varying dilutions of RANTES ({square}), MIP-1{alpha} ({triangleup}), and MIP/RANTES (•). Results were analyzed by the Graphpad Prism software, using a single-site model, and data were normalized for nonspecific (0%) and specific binding in the absence of competitor (100%). All points were run in triplicate (error bars: SE). Data are representative of two independent experiments.

 


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  		Figure 4. Equilibrium competition binding assay on CCR5. Competition-binding curves were performed on CHO-K1 cell lines expressing CCR5 using 0.06 nM [125I]-RANTES as tracer and varying dilutions of RANTES ({square}), MIP-1{alpha} ({triangleup}), and MIP/RANTES (•). Data were analyzed and normalized as in Figure 3 . Data are representative of two independent experiments.

 

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  		Table 1. Summary of the Binding and Functional Parameters of CCR1 and CCR5

 


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  		Figure 5. Equilibrium competition binding assay on CCR4. Competition binding curves were performed on HEK/CCR4 transfectants using 0.1 nM [125I]-TARC as tracer and varying dilutions of TARC ({lozenge}). (A) RANTES ({square}) and MIP-1{alpha} ({triangleup}). (B) Met-RANTES ({triangleup}), AOP-RANTES ({circ}), and MIP/RANTES (•). Data were analyzed and normalized as in Figure 3 . Data are representative of three independent experiments.

 
Receptor-functional activation by the MIP/RANTES chimera
MIP-1{alpha}, RANTES, and MIP/RANTES were also tested for their ability to activate CCR1, CCR3, and CCR5 using an aequorin-based assay as a reporter system for intracellular-calcium release, as previously described [45 , 46 ]. As shown in Figure 6A , MIP/RANTES was not able to induce calcium release in CCR1-expressing cells unlike MIP-1{alpha} and RANTES, which were efficient in this assay (Table 1) . In accordance with the binding data, MIP/RANTES, as well as MIP-1{alpha} and RANTES, induced a robust, calcium release in the CCR5-expressing cell line (Fig. 6B) . MIP/RANTES had an EC50 value of 2.75 nM, which is intermediary between the values of RANTES (1.55 nM) and MIP-1{alpha} (6.03 nM; Table 1 ). The truncated RANTES variant (8–68)RANTES shows very weak activity in this assay, achieving only 25% of the response observed for the other three chemokines. Again, in accordance with its inability to bind to CCR3, MIP/RANTES was unable to promote a functional response in the CCR3-expressing cell line even at a concentration as high as 500 nM (unpublished results). No calcium mobilization was observed with any of the amino terminally modified RANTES proteins nor by RANTES or MIP-1{alpha} on the HEK/CCR4 transfectants (unpublished results).



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  		Figure 6. Calcium mobilization induced by RANTES, MIP-1{alpha}, and MIP/RANTES. (A) The functional response of RANTES ({square}), MIP-1{alpha} ({triangleup}), and MIP/RANTES (•) in the cell line co-expressing apoaequorin and CCR1 was tested following addition of chemokines. The luminescent signal resulting from the activation of the apoaequorin-coelenterazine complex was recorded for 30 s in a luminometer. Results were analyzed by nonlinear regression using the Graphpad Prism software. Data were normalized for basal (0%) and maximal luminescence (100%). All points were run in triplicate (error bars: SE). The displayed curves represent a typical experiment out of three performed independently. (B) The functional response of RANTES ({square}), MIP-1{alpha} ({triangleup}), (8–68)RANTES ({blacktriangleup}), and MIP/RANTES (•) in the cell line co-expressing apoaequorin and CCR5 was tested following addition of chemokines. Data were analyzed as in A.

 
The chimera MIP/RANTES induces CCR5 down-modulation
We and others [20 , 39 , 49 ] have demonstrated recently that the potency of CCR5 ligands in inhibiting HIV-1 infection correlated with their ability to promote CCR5 down-modulation. Because the amino terminally modified RANTES, AOP-RANTES, displayed significantly greater ability to promote CCR5 down-regulation and anti-viral activity, we have investigated the importance of RANTES N-terminus for CCR5 endocytosis and inhibition of HIV-1 infection. We addressed the question as to whether the chimera is able to induce the endocytotic pathway of CCR5 by inducing its down-regulation. MIP/RANTES was slightly less potent at inducing CCR5 down-modulation than its parent proteins RANTES and MIP-1{alpha} and the specific CCR5 ligand, MIP-1ß, although this difference does not appear significant (Fig. 7 ). The replacement of the RANTES amino terminal sequence with that of MIP-1{alpha} did not confer the increased activity in receptor down-regulation observed for the chemically modified RANTES analogue, AOP-RANTES.



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  		Figure 7. Down-modulation of CCR5 from the cell surface of monocytes. The down-modulation of CCR5 was carried out as described in the text and was followed by flow cytometry using the mAb, MC-1. The cells were incubated for 30 min at 37°C with the indicated concentration of chemokine, RANTES ({square}), MIP-1{alpha} ({triangleup}), MIP-1ß ({triangledown}), AOP-RANTES ({circ}), and MIP/RANTES (•).

 
Inhibition of HIV-1 infectivity via CCR5
The ability of MIP/RANTES to inhibit a CCR5-using HIV-1 strain was investigated. Figure 8 shows that infection of PBMCs by the M-tropic R5 HIV-1 strain SL-2 was blocked effectively by MIP/RANTES. Although the activity observed was at least as effective as MIP-1{alpha}, RANTES inhibition was most efficient, but the difference in potency between the chimera and its parent proteins is not significant.



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  		Figure 8. Inhibition of HIV-1 infection by RANTES, MIP-1{alpha}, and MIP/RANTES. The HIV infection assay of PBMC with the R5 HIV-1 strain, SL-2, was carried out as described in the text, and inhibition by RANTES ({square}), MIP-1{alpha} ({triangleup}), and MIP/RANTES (•) was measured. Supernatant p24 was measured after 7 days.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The reason why certain chemokines activate several receptors is unclear. Selectivity in vivo is governed by several factors probably, such as temporal expression of ligands and receptors, but the molecular interaction may also play a role. No clue is given by the three-dimensional structure of all chemokines solved to date, because independent of the class to which they belong, a highly conserved, monomeric fold has been identified [27 ]. Members of all subclasses have a flexible, disordered NH2-terminal region, followed by a conserved core region composed of a loop, three anti-parallel ß strands, and a COOH-terminal {alpha} helix [26 ]. However, their dimeric topology is very different; the biological role this plays is unidentified as yet.

It has been hypothesized that activation of chemokine receptors acts through a similar mechanism to that proposed for C5a, a nonchemokine, chemoattractant protein of 70 kDa, which also acts on a 7 transmembrane, G protein-coupled receptor. The hypothesis proposes that the core or body of the protein binds to a site formed by the extracellular loops of the receptor, Site 1, and that receptor activation is triggered by the flexible region at a second site, Site 2 [35 ]. In C5a, the flexible region is the carboxy terminus, but in chemokines, it is the amino terminus. To test the current hypothesis that the chemokine core is responsible for binding to the chemokine receptor, and the N-terminus is essential for receptor activation, we have generated a chemokine chimera between MIP-1{alpha} and RANTES, which are encoded by recently duplicated genes on chromosome 17 [25 ] but are only 45% identical in primary sequence. However, they share two receptors for which they bind with high affinity, CCR1 [50 ] and CCR5 [20 ]. Both chemokines also have weak affinity for CCR4 but do not activate this receptor. RANTES, but not MIP-1{alpha}, has moderate affinity for CCR3 in our hands, although high-affinity binding of RANTES to CCR3 has been studied [13 ]. The relatively low sequence homology between MIP-1{alpha} and RANTES suggests that binding of the two chemokines to their common receptors might involve different amino acids.

We have noted previously that amino terminal modifications of RANTES, as in Met-RANTES and AOP-RANTES, result in significant differences in this protein’s activity on CCR1 but less on CCR5. Although high-affinity binding is conserved to both receptors, AOP-RANTES is only a weak, partial agonist on CCR1 but is fully active on CCR5 [42 , 51 ]. Furthermore, truncated versions of the CCR5 ligands RANTES and MIP-1ß retain high-affinity binding to this receptor [34 , 52 ]. For example, [9-68]RANTES is still active on this receptor in its ability to induce receptor down-regulation [53 ] but has reduced ability to mobilize calcium on CCR5 and is practically inactive on CCR1. Therefore, we were interested in determining if full activity would be restored to RANTES by the amino terminal of another ligand that binds to both receptors—in other words, if Site 2 is common to both ligands.

The MIP/RANTES chimera was shown to behave similarly to AOP-RANTES in that it was fully active on CCR5 but almost inactive on CCR1. Furthermore, although AOP-RANTES retains high-affinity binding to CCR1, MIP/RANTES shows two orders of magnitude loss in affinity. A repulsive role of the MIP-1{alpha} amino terminus in the binding to CCR1 has been suggested recently by the demonstration that truncated MIP-1{alpha} proteins have an increased affinity for CCR1 compared with the full-length chemokine [54 , 55 ]. This demonstrates that receptor-binding affinity of RANTES to CCR1, as well as activation, requires a correct amino terminal sequence. However, CCR5 appears to be far less sensitive to the amino terminal sequence, indicating that the predominantly important region of the chemokine for receptor binding is the core of the protein. Our results suggest that the MIP-1{alpha} N-terminal domain does not play any significant role (positive or negative) in binding to CCR5 but can substitute efficiently for the RANTES N-terminus for receptor activation because the truncated [9-68]RANTES is not fully active in all assays, whereas the chimera MIP/RANTES shows full activity in all assays tested, suggesting that a full-length protein is necessary for full activation.

These observations could be explained by the hypothesis that the amino terminus of a chemokine ligand on CCR5 is involved in certain stabilizing interactions. Inspection of the primary sequences of CCR5 ligands reveals that there are three amino acids that are common in the MIP-1{alpha} and RANTES amino termini, D6, T7, and T9, using the MIP-1{alpha} numbering. However, in the RANTES sequence, the two Thr residues are adjacent, whereas in MIP-1{alpha}, these Thr residues are separated by a Pro, which would be expected to change their spatial arrangement significantly. MIP-1ß, the third high-affinity ligand for CCR5, also has a Thr at position 9, whereas MCP-2, shown recently to be a ligand for CCR5, has a Thr residue immediately adjacent to the CC motif, which is not separated by an amino acid as in the case of RANTES, MIP-1{alpha}, and MIP-1ß. A Pro residue in position 2 has been postulated as playing a major role in CCR5 binding. RANTES, like MIP-1ß and MCP-2, has a Pro in position 2, whereas MIP-1{alpha} has a Ser. Substitution of Pro2 in RANTES with Ala has been shown to cause a dramatic reduction in binding affinity for CCR5 [56 ]. Moreover, the nonallelic variant of MIP-1{alpha}, LD78ß, which has a Pro in position 2 rather than a Ser, has significantly enhanced binding affinity for CCR5 as well as a greater HIV-suppressive activity. However, this hypothesis is contradicted by the fact that the naturally occurring, truncated form [3-68]RANTES, in which the Pro is absent, is fully active [57 ] as well as the observations here that the MIP/RANTES chimera, which lacks a Pro at this position, is also fully active. These arguments are based on the premise that the binding site for the amino terminus is shared by all the ligands that activate CCR5. However, it is possible that each chemokine has a specific or possibly overlapping binding site for the protein core and that the site used by the N-terminus is also overlapping or distinct. Currently, we are investigating this hypothesis using mutants of CCR5 that are able to distinguish RANTES and MIP-1{alpha} binding sites.

A common binding site for CC chemokines has been revealed by mutagenesis studies that have demonstrated distinct but overlapping residues in RANTES for necessary, high-affinity binding of CCR1, 3, and 5 [56 ]. An aromatic residue in the RANTES N-loop (F12) is necessary for high-affinity binding to CCR3 and CCR5. It is interesting that the same residue in MIP-1{alpha}, MIP-1ß, and MCP-1 is also crucial for binding to CCR5 and CCR2b, respectively [52 , 58 ], suggesting that this aromatic side-chain of the N-loop of CC chemokines may represent an essential element.

CCR3, which is a functional RANTES receptor, appears to follow the same activation pattern to CCR1, where alteration of the amino terminus of the chemokine abolishes binding and activation. The importance of an integral amino terminus of RANTES has been demonstrated by the loss of activity of the RANTES analogues, Met-RANTES and AOP-RANTES [59 , 60 ], on CCR3. However, the low-affinity binding of RANTES and MIP-1{alpha} to CCR4 appears to be mediated by the core of the protein, with little influence of the amino terminus, as is seen for their high-affinity binding to CCR5. The MIP/RANTES chimra shows the same properties in CCR4 binding as that of the parent RANTES protein and the amino terminally modified proteins Met-RANTES and AOP-RANTES. The biological relevance of the binding of these two ligands to CCR4 is not clear, because receptor activation was observed in oocytes over-expressing the receptor [14 ] but is not observed in vitro in most recombinant cell lines. Unexpectedly, the response to MIP-1{alpha}, but not to RANTES, in in vitro chemotaxis was lost in mouse splenocytes and thymocytes, in which the CCR4 gene has been deleted, but not in neutrophils [61 ], suggesting that cell background may be an important determinant of ligand specificity.

Altogether, our analysis of the MIP/RANTES chimera indicates that the simplistic view that chemokines have two functionally independent sites, the core being responsible for high-affinity binding and the N-terminus for receptor activation, does not necessarily hold true. Activation of CCR1 and CCR3 by RANTES seems to follow the two-site model because the core and correct N-terminus are required. Conversely, the core of RANTES seems sufficient to achieve high-affinity binding and even activation of CCR5, although activity is enhanced by the presence of an N-terminal domain.

Although the chemokine system appears to be highly redundant, as best exemplified perhaps by the specificity characteristics of RANTES, which activates several receptors, we show here that subtle differences appear to exist for receptor activation by this chemokine. Although this observation certainly does not explain the reason for the redundancy, we believe that specificity mechanisms exist in vivo that may be influenced by differential mechanisms of receptor activation. These differential-activation patterns may trigger different signal-transduction pathways. This is shown by the highly divergent effects induced by RANTES on CCR1 and CCR5 with respect to receptor trafficking [60 ].


   ACKNOWLEDGEMENTS
 
C. Blanpain is aspirant of the Fonds National de la Recherche Scientifique (FNRS) of Belgium. We thank F. Borlat for excellent technical assistance, R. E. Offord and B. Dufour for synthesis of AOP-RANTES, P. O. Regamey for mass spectrometry, E. Magnenat for Edman degradation, and T. N. C. Wells for critical reading of the manuscript.

Received October 17, 2000; accepted December 20, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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J. Biol. Chem., February 7, 2003; 278(7): 5179 - 5187.
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