Structure-Function Relationship between the Human Chemokine Receptor CXCR3 and Its Ligands*

Ian Clark-LewisDagger §, Ivan Mattioli, Jiang-Hong GongDagger , and Pius Loetscher§

From the Dagger  Biomedical Research Centre and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and the  Theodor-Kocher Institute, University of Bern, CH-3000 Bern 9, Switzerland

Received for publication, September 16, 2002, and in revised form, October 30, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

I-TAC, IP10, and Mig are interferon-gamma inducible CXC chemokines that share the same G-protein-coupled receptor CXCR3, which is preferentially expressed on Th1 lymphocytes. We have explored the structure-function relationship of the CXCR3 ligands, in particular of I-TAC, which has highest affinity for CXCR3 and is the most potent agonist. A potent antagonist for CXCR3 was obtained by NH2-terminal truncation of I-TAC. I-TAC (4-73), which lacks the first three residues, has no agonistic activity but competes for the binding of I-TAC to CXCR3-bearing cells and inhibits migration and Ca2+ changes in such cells in response to stimulation with I-TAC, IP10, and Mig. It does also not induce internalization of CXCR3, which is in support of the lack of agonistic effects. Hybrid chemokines between I-TAC and IP10 were used to identify regions responsible for the higher activity of I-TAC. I-TAC-like IP10 analogs are obtained by substituting the NH2 terminus (residues 1-8) or N-loop region (residues 12-17) of IP10 with those of I-TAC, suggesting that the differences in function of the CXCR3 ligands can be assigned to distinct regions and that these regions are interchangeable. Structure-activity studies with Mig showed that the extended basic COOH-terminal region, which is not present in I-TAC and IP10, is important for binding and activity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokines control leukocyte migration during hematopoiesis, innate and adaptive immune responses, and inflammation (1-4). They are divided into CXC, CC, C, and CX3C families and mediate their functions through binding to seven-transmembrane-domain receptors coupled to G proteins (5). The migration pattern and effector function of leukocytes are closely linked and largely defined by the expression of the chemokine receptors. The receptors often recognize more than one chemokine and, alternatively, several chemokines can bind to multiple receptors. The CXC chemokine receptors are generally ligand-specific. For instance, CXCR31 binds interferon-inducible T cell-alpha chemoattractant (I-TAC, CXCL11), interferon-inducible protein 10 (IP10, CXCL10), and monokine-induced by gamma -interferon (Mig, CXCL9); CXCR4 binds stromal cell-derived factor-1 (SDF-1, CXCL12), CXCR5 binds B cell activation chemokine-1 (CXCL13); and CXCR6 binds CXCL16 (5, 6).

I-TAC, IP10, and Mig are three non-Glu-Leu-Arg CXC chemokines that are more closely related to each other than to any other chemokine with an amino acid sequence identity of about 40% (7-9). They mainly attract activated T lymphocytes, preferentially of the Th1 phenotype, which expresses high levels of CXCR3 (10-12). The involvement in Th1 responses is also supported by the observation that the production of all three ligands by blood and tissue cells is induced by interferon-gamma , the typical Th1 cytokine (7, 13, 14). For instance, IP10 is expressed and CXCR3-positive T lymphocytes accumulate at sites of Th1-type inflammation such as multiple sclerosis (15, 16), rheumatoid arthritis (17-19), psoriasis (20, 21) and sarcoidosis (22). Recently, it has also been shown that IP10 and CXCR3 are critically involved in the development of acute allograft rejection. Cardiac allograft survival was markedly prolonged by targeting either donor-derived IP10 production or CXCR3 expression on host leukocytes as shown in studies with IP10- or CXCR3-deficient mice and with animals treated with antibodies that neutralize IP10 or CXCR3 (23, 24). In these various diseases, the expression of the CXCR3 ligands, in particular of IP10, correlates with the tissue infiltration of T lymphocytes, suggesting that these ligands play an important role in the regulation of cell recruitment to sites of inflammation.

CXCR3 is expressed on a fraction of circulating blood T cells, B cells, and NK cells (18). Blood T cells positive for CXCR3 are mostly CD45RO+ memory cells, which express high levels of beta 1 integrins (18), and the CXCR3+/CD4+ T cell subset is enriched for Th1 cells (25). T cell activation enhances CXCR3 expression and chemotactic responsiveness (26, 27). Th1 cell lines generated in vitro express higher levels of CXCR3 and migrate better to I-TAC, IP10, and Mig than Th2 cell lines (12, 17), suggesting that CXCR3 and its ligands are more active in the setting of Th1-driven inflammatory responses. In addition, CXCR3 has been reported to be expressed on plasmacytoid and myeloid dendritic cells (28, 29), leukemic B cells (30, 31), thymocyte subsets (32), and dividing microvascular endothelial cells (33, 34).

We have recently shown that I-TAC, IP10, and Mig, besides being agonists for CXCR3, act as natural antagonists for CCR3, the receptor for eotaxin, and several other CC chemokines (35). They compete for the binding of eotaxin to CCR3 and inhibit CCR3-mediated migration and Ca2+ changes. Because CXCR3 and CCR3 are differentially expressed in Th1 and Th2 cells, the findings suggest that I-TAC, IP10, and Mig, in addition to attracting CXCR3-bearing cells, have the capacity to block migration of CCR3-bearing cells, thereby enhancing the polarization of T cell recruitment. As recently shown by NMR spectroscopy, the hydrophobic cleft, which is formed by the N-loop and 40s-loop region, may provide the basis for the ability of IP10 to bind to both CXCR3 and CCR3 (36). In this study we have explored the structure-function relationship of the CXCR3 ligands, in particular of I-TAC, which has highest affinity for CXCR3 and is the most potent agonist. We show that the NH2 terminus of I-TAC is important for the agonistic and antagonistic activity. The structural motifs responsible for the higher activity of I-TAC were determined with a series of synthesized I-TAC/IP10 hybrids. The data suggest that the differences in function of the CXCR3 ligands can be assigned to distinct regions and that these regions are interchangeable.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokine Synthesis-- All chemokines and chemokine analogs were chemically synthesized using tBoc (tertiary butyloxycarbonyl) solid-phase chemistry (37). They were purified by HPLC and analyzed by electrospray mass spectrometry. For each chemokine used, the mass determined by mass spectrometry corresponded to the expected value.

Cell Preparation and Culture-- Human peripheral blood mononuclear cells were isolated from buffy-coats of donor blood (Central Laboratory of the Swiss Red Cross, Bern, Switzerland) by centrifugation on Ficoll-Paque (38). The cells were treated with phytohemagglutinin (1 µg/ml) and expanded in the presence of IL-2 (200 units/ml) in RPMI 1640 medium supplemented with 1% glutamine, non-essential amino acids, sodium pyruvate, 50 units/ml penicillin, 50 mg/ml streptomycin, 0.05 mM beta -mercaptoethanol, and 5% human serum (Swiss Red Cross Laboratory, Bern, Switzerland) for 7-15 days. Murine pre-B 300-19 cells stably expressing CXCR3 (CXCR3-B300-19 cells) were cultured in RPMI 1640 medium containing 10% fetal calf serum (10).

Receptor Binding-- Competition binding assays were performed with CXCR3-B300-19 cells using 125I-I-TAC or 125I-IP10 labeled by the Bolton-Hunter procedure (39). Briefly, the maximal binding of labeled I-TAC and IP10 was determined by measuring binding at saturating concentrations. 5 × 106 cells were incubated with 3 nM labeled chemokine and increasing concentrations of unlabeled competitor (10-9 to 3 × 10-6 M) in 200 µl of RPMI 1640 medium containing Hepes (25 mM, pH 7.4), bovine serum albumin (10 mg/ml), and sodium azide (0.1%). The incubations were carried out for 30 min at 4 °C, and cell-associated radioactivity was separated by spinning the cells through a 2:3 mixture of diacetylphthalate and dibutylphthalate and measured by gamma counting. Nonspecific binding was determined in the presence of a 100-fold concentration of unlabeled ligand and subtracted from the total. Dissociation constants (Kd values) were determined by Scatchard analysis (40).

Functional Assays-- Ca2+ mobilization was assayed in Fura-2 (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2'-amino-5'-methylphenoxy)-ethan-N-N-N'-N'-tetraacetic acid)-loaded cells after single or sequential stimulation with chemokines or chemokine analogs by recording [Ca2+]i-related fluorescence changes (41). The rate of the change was expressed as percent of Fura-2 saturation per second. Chemotaxis was assessed in 48-well Boyden microchambers (Neuro Probe Inc., Cabin John, MD) using polyvinylpyrrolidone-free polycarbonate membranes (Poretics Corp., Livermore, CA) with 3-µm pores (38). Cell suspensions and chemokine dilutions were made in RPMI 1640 medium containing 1% pasteurized plasma protein (Swiss Red Cross Laboratory, Bern, Switzerland) and 20 mM Hepes, pH 7.4. Migration was allowed to proceed for 90 min and migrated cells were counted at a 1000× magnification in five fields per well. All determinations were performed in triplicate.

Receptor Internalization-- Chemokine-induced internalization was assayed as described (42). Briefly, CXCR3-B300-19 cells were incubated for 30 min at 37 °C with increasing concentrations of chemokines to be tested. After washing twice with phosphate-buffered saline, surface-bound ligands were removed by exposure to 50 mM glycine buffer, pH 3.0, containing 100 mM NaCl for 1 min followed by washing with phosphate-buffered saline. Receptor expression was then determined by flow cytometry using phycoerythrin-conjugated mouse monoclonal anti-CXCR3 antibody (1C6, BD Biosciences), and the relative fluorescence intensity was calculated (43).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Receptor Binding and Functional Activity of NH2-terminally Truncated I-TAC Analogs-- CXCR3-B300-19 cells were used to determine the effect of NH2-terminal truncation of I-TAC on the binding to CXCR3. The competition for binding of 125I-I-TAC by unlabeled I-TAC and I-TAC analogs is shown in Fig. 1, A and B. The results show that I-TAC (2-73), (3-74), and (4-73) retained almost full CXCR3-binding activity. The dissociation constant (Kd) for I-TAC was 3.0 ± 1.6 nM (n = 8); for I-TAC, (2-73) 4.0 ± 1.1 nM (n = 3); for I-TAC (3-73), 6.6 ± 0.9 nM (n = 3); and for I-TAC (4-73) 8.5 ± 1.1 nM (n = 4). A 10- to 100-fold lower affinity was obtained with I-TAC (5-73) (Kd = 135 ± 45 nM, n = 3), I-TAC (6-73) (Kd = 121 ± 58 nM, n = 3), I-TAC (7-73) (Kd = 267 ± 70 nM, n = 3), I-TAC (8-73) (Kd = 30 ± 16 nM, n = 3), and I-TAC (9-73) (Kd = 341 ± 90 nM, n = 3). The functional activity of the analogs was assessed by measuring chemotactic migration of PHA and IL-2 activated T lymphocytes expressing CXCR3. As shown in Fig. 1C, sequential NH2-terminal truncation leads to a loss of chemotaxis. Compared with full-length I-TAC, the activity of I-TAC (2-73) and I-TAC (3-73) was reduced by about 60-70%. Interestingly, although I-TAC (4-73) retained marked binding affinity, it was completely inactive, suggesting that it is an antagonist. In agreement with the binding data, I-TAC (5-73) to (9-73) were devoid of agonistic activity (Fig. 1C and data not shown).


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Fig. 1.   Binding and chemotactic activity of I-TAC and NH2-terminal truncated analogs. A and B, displacement of 125I-I-TAC from CXCR3-B300-19 cells by I-TAC, I-TAC (2-73), (3-73), (4-73), (5-73), (6-73), (7-73), (8-73), and (9-73). The cells were incubated with 3 nM 125I-I-TAC in the presence of increasing concentrations of unlabeled competing ligand. The values were normalized by setting the specific binding of 3 nM 125I-I-TAC to 100%. C, chemotaxis of PHA and IL-2 activated T lymphocytes in response to increasing concentrations of I-TAC and NH2-terminally truncated analogs. Shown are the average numbers of migrated cells per five high-power fields in triplicate wells. The data are representative of three independent experiments.

Inhibition of Chemotaxis and [Ca2+]i Changes by I-TAC (4-73)-- In view of the observed properties, we tested whether I-TAC (4-73) has antagonistic activity for CXCR3. The migration of PHA- and IL-2-activated T lymphocytes in response to I-TAC was inhibited by I-TAC (4-73) as shown in Fig. 2A. The inhibition of migration induced by optimal concentrations of I-TAC, IP10, and Mig was concentration-dependent and complete at 1000 nM (Fig. 2B). The inhibitory effect of I-TAC (4-73) was most pronounced for IP10 and somewhat less for I-TAC and Mig. In agreement with the chemotaxis assays, the [Ca2+]i rise induced by I-TAC in PHA and IL-2 activated T lymphocytes was decreased in a concentration-dependent manner by pretreatment with I-TAC (4-73) (Fig. 2C). I-TAC (4-73) did not induce [Ca2+]i changes, even at high concentrations, confirming that it is devoid of agonistic activity. Likewise, [Ca2+]i changes induced by IP10 and Mig were inhibited in a concentration-dependent manner by I-TAC (4-73) (Fig. 2D). The same results were obtained when the effect of I-TAC (4-73) was assessed with CXCR3-B300-19 cells (data not shown). These data demonstrate that I-TAC (4-73) significantly inhibits CXCR3-mediated responses.


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Fig. 2.   Inhibition of CXCR3-mediated chemotaxis and [Ca2+] changes by I-TAC (4-73). A, migration of PHA and IL-2 activated T lymphocytes in response to increasing concentrations of I-TAC in the presence or absence of 1,000 nM I-TAC (4-73). Shown are the average numbers ± S.D. of migrating cells per five high power fields in triplicate wells. B, migration of PHA and IL-2 activated T lymphocytes in response to 10 nM I-TAC, 30 nM IP10, or 100 nM Mig in the presence of increasing concentrations of I-TAC (4-73). The average numbers of migrating cells per five high-power fields in triplicate wells were normalized by setting to 100% the responses of control cells (in the absence of I-TAC (4-73)). Shown are mean values ± S.D. C and D, PHA- and IL-2-activated T lymphocytes loaded with Fura-2 were exposed to increasing concentrations of I-TAC (4-73) and stimulated after 60 s with 1 nM I-TAC, 10 nM IP10, or 10 nM Mig. C, [Ca2+]i-dependent fluorescence changes are shown. D, initial rates of the [Ca2+]i rise were expressed as percent of Fura-2 saturation/sec and normalized by setting to 100% the responses of control cells (no I-TAC (4-73) pretreatment). The results are representative of two to four independent experiments.

CXCR3 Internalization-- The binding of chemokines leads to a rapid receptor internalization, which is not observed on binding of antagonists (35, 44, 45). Internalization was determined in PHA- and IL-2-activated T lymphocytes and CXCR3-B300-19 cells by flow cytometry before and after ligand exposure. As shown in Fig. 3, I-TAC and I-TAC (2-73), which have agonistic activity, induced a concentration-dependent internalization. On treatment with 100 nM I-TAC and I-TAC (2-73), CXCR3 expression levels decreased to 25 and 55%, respectively. In contrast, no internalization was observed when the cells were exposed to I-TAC (4-73) and, as expected, to the inactive analog, I-TAC (8-73). Together with the functional data, these results show that I-TAC (4-73) lacks agonistic activity and acts as a pure antagonist.


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Fig. 3.   Agonist-dependent internalization of CXCR3. PHA- and IL-2-activated T lymphocytes were incubated for 30 min at 37 °C with increasing concentrations of I-TAC, I-TAC (2-73), I-TAC (4-73), or I-TAC (8-73). The relative cell surface expression of CXCR3 was determined by flow cytometry after staining with monoclonal antibody to CXCR3. The data are representative of three independent experiments.

Generation of I-TAC-like IP10-- Of the three CXCR3 ligands, I-TAC has highest receptor affinity and is the most potent agonist as shown by chemotaxis and [Ca2+]i mobilization assays (7, 46). As shown in Fig. 5, A and B, I-TAC potently and fully displaced both 125I-labeled I-TAC or IP10 with IC50 values of 5.3 ± 1.0 nM for 125I-I-TAC (n = 10) and of 2.5 ± 0.8 nM for 125I-IP10 (n = 4). In contrast, IP10 was able to fully compete with 125I-IP10 (IC50 = 5.0 ± 1.7 nM, n = 4), but it was much less effective to displace 125I-I-TAC (IC50 = 41.4 ± 16.9 nM, n = 8), indicating that I-TAC may have an additional binding site for CXCR3. To identify amino acid residues or regions responsible for this difference, we have synthesized a series of I-TAC/IP10 hybrids (Fig. 4) and assayed them for 125I-I-TAC competition binding and [Ca2+]i mobilization (Fig. 5, C and D). In I-TAC-H1 the NH2 terminus (residues 1-8) of IP10 was substituted by that of I-TAC, and conversely, in I-TAC-H3, the NH2 terminus (residues 1-8) of I-TAC was substituted by that of IP10. The importance of the N-loop region was tested with I-TAC-H2, in which six residues of the IP10 N-loop (residues 12-17) were replaced by the ones of I-TAC. As shown in Fig. 5C, all hybrid chemokines displaced labeled I-TAC with IC50 values ranging in between the ones of I-TAC and IP10. I-TAC-H3 was the most potent competitor (IC50 = 6.5 ± 1.6 nM, n = 5) followed by I-TAC-H2 (IC50 = 29.2 ± 10.2 nM, n = 8) and I-TAC-H1 (IC50 = 38.6 ± 14.8 nM, n = 8).


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Fig. 4.   Sequence alignment of CXCR3 ligands and I-TAC/IP10 hybrids. Shown are the sequences of I-TAC, I-TAC-H1, I-TAC-H2, I-TAC-H3, IP10, and Mig. For the hybrid chemokines the sequence corresponding to I-TAC are underlined. The conserved cysteine residues are shown in bold.


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Fig. 5.   Displacement of 125I-I-TAC or 125I-IP10 and induction of [Ca2+] changes in CXCR3-B300-19 cells. Cells were incubated with 3 nM 125I-I-TAC (A and C) or 125I-IP10 (B) in the presence of increasing concentrations of unlabeled competing ligand. The values were normalized by setting the specific binding of 3 nM 125I-I-TAC or 125I-IP10 to 100%. The data are representative of four independent experiments. D, CXCR3-B300-19 cells loaded with Fura-2 were exposed to increasing concentrations of I-TAC, IP10, I-TAC-H1, I-TAC-H2, and I-TAC-H3 and initial rate of the [Ca2+]i rise, expressed as percent of Fura-2 saturation/sec, is shown. The results are representative of three independent experiments.

In Ca2+ mobilization assays using CXCR3-B300-19 cells (Fig. 5D) and PHA- and IL-2-activated T lymphocytes (data not shown), I-TAC was about 15-fold more potent than IP10 with EC30 values of 2.1 ± 0.4 nM for I-TAC (n = 3) and 31.9 ± 9.7 nM for IP10 (n = 3). I-TAC was also about 1.5-fold more efficacious than IP10 to mobilize calcium. All hybrid chemokines induced [Ca2+]i rises with a potency similar to I-TAC. I-TAC-H3 was the most potent agonist (EC30 = 4.1 ± 1.5, n = 3) closely followed by I-TAC-H1 (EC30 = 5.3 ± 2.5, n = 3) and I-TAC-H2 (EC30 = 8.0 ± 4.4, n = 3). I-TAC-H2 was as efficacious as I-TAC, whereas the efficacy of I-TAC-H1 and I-TAC-H3 was similar to IP10. Together with the binding studies, these results show that the amino acid residues in the N-loop region are largely responsible for the higher activity of I-TAC compared with IP10, as shown by the data obtained with I-TAC-H2 and I-TAC-H3. They also show that the NH2 terminus contributes to the observed difference between I-TAC and IP10, because an I-TAC-like IP10 analog (I-TAC-H1) is obtained by interchanging the NH2 termini. Furthermore, I-TAC-H3 demonstrates that amino acid substitutions are allowed in NH2 terminus of I-TAC without significant loss of activity. This is in agreement with the results from single amino acid replacement analogs in the NH2 terminus of I-TAC (P2G, M3A, M3F, M3Q, M3G, F4A, R8K I-TAC) which all had high affinity for CXCR3 and wild-type activity (data not shown).

Mig Structure-function-- In contrast to I-TAC and IP10, Mig has an extended basic COOH-terminal region (Fig. 4) (7, 47). The role of this extension is unknown, but it may interact with an additional site or a glycosaminoglycan binding site. Binding competition assays show that full-length Mig (1-103) displaced 125I-labeled I-TAC with an IC50 of 200 nM, indicating that it is about 30-fold less potent than I-TAC as competitor (Fig. 6A). The binding affinity was further decreased by a factor of about 15 when the COOH-terminal region was deleted to give the shortened Mig analog 1-73 (IC50 = 3000 nM). The COOH-terminal region itself (74-103) lacked detectable activity. Consistent with the binding data, the migration of PHA and IL-2 activated T lymphocytes was significantly reduced by COOH-terminal truncation of Mig and as expected, the COOH terminus itself was inactive (Fig. 6B).


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Fig. 6.   Binding and chemotactic activity of Mig and Mig analogs. A, displacement of 125I-I-TAC from CXCR3-B300-19 cells by I-TAC, Mig (1-103), Mig (1-73), and Mig (74-73). The cells were incubated with 3 nM 125I-I-TAC in the presence of increasing concentrations of unlabeled competing ligand. The values were normalized by setting the specific binding of 3 nM 125I-I-TAC to 100%. The data are representative of three independent experiments. B, chemotaxis of PHA- and IL-2-activated T lymphocytes in response to increasing concentrations of Mig and Mig analogs. Shown are the average numbers of migrated cells per five high-power fields in triplicate wells. The data are representative of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study shows that the NH2-terminal-truncated I-TAC analog, I-TAC (4-73) is a potent antagonist for CXCR3. It competes for the binding of the labeled I-TAC to CXCR3 and inhibits chemotaxis and Ca2+ changes induced in CXCR3-positive cells by stimulation with I-TAC, IP10, and Mig. In addition, it shows that the NH2 termini of I-TAC and IP10 are interchangeable, and that IP10 can be converted into an I-TAC-like chemokine by substituting the N-loop region that follows the second cysteine with that of I-TAC.

Numerous studies of structure-activity relations have shown that chemokines have two main sites of interaction with their receptors, one in the NH2 terminus and the other in the N-loop region after the second cysteine (48-50). These findings have led to the proposal of a two-step model in which receptor binding and activation are dissociated. The receptor recognizes first the binding site located in the N-loop region ("docking domain"). This initial contact facilitates the subsequent binding and proper positioning of the flexible NH2-terminal region ("triggering domain"), which activates the receptor by presumably interacting with multiple receptor helical sites and inducing a change in the receptor conformation. Thus, the N-loop region is responsible for the receptor recognition, and the NH2-terminal region triggers the receptor. For all chemokines that have been studied, minimal truncations or substitutions within the triggering domain have led to a loss of receptor activation. Often, the modified chemokines, however, retained the capacity to bind and in this way to block the receptor (51). The first antagonists, which act on CXCR1 and CXCR2, were obtained by modifying the NH2-terminal sequence of IL-8 and other Glu-Leu-Arg chemokines (52, 53). The same approach was applied to the CC chemokines MCP-1, MCP-3, and RANTES, yielding antagonists for CCR1, CCR2, CCR3, and CCR5 (39, 54, 55). More recent studies have shown that antagonists for CXCR4 can be obtained by substitution of the first two residues of SDF-1, the most potent being SDF-1(P2G) in which Pro in position 2 is replaced by Gly (56, 57). Here, we report that the same principle is true for I-TAC, as shown by the effects obtained upon NH2-terminal truncation. The most interesting analog was I-TAC (4-73), which lacks the first three amino acids. It retained significant binding affinity, but lacked the ability to trigger CXCR3 signaling, thus qualifying it as an antagonist. It is important to note that NH2-terminal truncation does not necessarily yield analogs with antagonistic activity. The two additional CXCR3 ligands, IP10 and Mig, as well as the CC chemokine eotaxin, which is a ligand for CCR3, lose the binding capacity when only a few amino acids are removed at the NH2 terminus (data not shown). Consistent with these findings are recent reports showing that the dipeptidyl-peptidase IV (CD26) reduces the activity of I-TAC (58-60), IP10 (58), Mig (58), and eotaxin (61) by cleaving off the first two NH2-terminal residues.

Of the three CXCR3 ligands, I-TAC is the dominant agonist (7, 46). It has a higher affinity for CXCR3 and is more potent and efficacious than IP10 and Mig to induce Ca2+ changes and migration. Another interesting aspect in support of this dominance is that I-TAC competes fully with both labeled I-TAC and IP10 for the binding to CXCR3, whereas IP10 competes fully with labeled IP10 but only incompletely with labeled I-TAC. These data suggest that distinct residues or stretches of residues in I-TAC are responsible for the observed differences. Using the hybrid approach, we could indeed show that the N-loop region mainly accounts for the higher activity of I-TAC. IP10 was converted into an I-TAC-like agonist when six residues in the N-loop of IP10 were replaced by the ones of I-TAC (I-TAC-H2). These results are in agreement with previous structure-function studies on SDF-1, showing that residues in the N-loop region, the so-called RFFESH motif, can augment the specific receptor-binding characteristics (56, 57). The hybrid study also underscores the importance of the NH2 terminus of I-TAC. IP10 with the NH2 terminus of I-TAC (I-TAC-H1) had I-TAC-like activity. It was, however, somewhat less efficacious than I-TAC to induce Ca2+ changes. Although the NH2 terminus is important for activity, amino acid substitutions are well tolerated within this region, as shown by the data obtained with I-TAC carrying the NH2 terminus of IP10 (I-TAC-H3), and all of the I-TAC analogs with the single amino acid substitutions. Together, these data show that both the NH2 terminus and N-loop region contribute to the higher receptor-binding affinity and activity of I-TAC compared with IP10 and Mig and suggest that these two domains are the major functional determinants.

The observation that I-TAC (4-73) bound to CXCR3 but did not induce any functional responses such as chemotaxis and Ca2+ changes and failed to induce receptor internalization suggests that it acts as a true antagonist. Internalization is an agonist-driven event because of the phosphorylation of the receptor by G-protein-coupled receptor kinases and subsequent uptake in clathrin-coated pits (62, 63). We found that the internalization correlates with the agonistic activity. The decrease in agonistic activity by sequential NH2-terminal truncation of I-TAC was closely accompanied by the decrease in receptor uptake. Consistent with these findings, I-TAC was shown to be more potent than IP10 and Mig to induce CXCR3 internalization (35, 64). The lack of inducing receptor down-regulation by antagonists obtained by the modification of the NH2 terminus of chemokines was also observed with SDF-1 (P2G), which binds to CXCR4 (65), and I-TAC/EoH1, which binds to CCR3 (35). Similarly, no internalization was induced by natural chemokines acting as antagonists including I-TAC, IP10, and Mig, which bind to CCR3 (35), eotaxin, which binds to CCR2 (44), and MCP-3, which binds to CCR5 (45). Like the chemokine-based antagonists, small-molecule antagonists were unable to down-regulate chemokine receptors, as shown for TAK-779, which binds to CCR5 (66) and for UCB35625, which binds to CCR1 and CCR3 (67). These data indicate that, as a rule, the blockade of chemokine receptors by antagonists mainly depends on receptor occupancy.

In conclusion, CXCR3 is a unique chemokine receptor that selectively binds three closely related CXC chemokines and is expressed preferentially on Th1 lymphocytes. I-TAC, IP10, and Mig are unique in that they are all induced by the typical Th1 cytokine interferon-gamma in wide variety of cells. Given these properties, it is clear that CXCR3 and its ligands play an important role in Th1-driven inflammatory and immune responses. By studying the structure-function relationship of the ligands, we have identified a novel and potent antagonist, I-TAC (4-73) for CXCR3, and the structural domains that are responsible for the higher activity of I-TAC compared with IP10 and Mig. The antagonist is a valuable tool to study the function of CXCR3 in vivo and it may have the potential for therapeutic application in inflammatory diseases.

    ACKNOWLEDGEMENTS

We thank P. Tam, T. McLeod, and P. Owen for expert technical assistance with the peptide synthesis, Julie Wang for assistance with the binding studies, and B. Moser and M. Wolf for critical reading of the manuscript. Donor blood buffy coats were provided by the Swiss Central Laboratory Blood Transfusion Service, Bern, Switzerland.

    FOOTNOTES

* This work was supported by the Protein Engineering Network Centres of Excellence (Canada) and the Arthritis Society (Canada) and by Grant 31-55996.98 from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed. Tel.: 41-31-631-4164; Fax: 41-31-631-3799; E-mail: pius.loetscher@tki.unibe.ch (P. L.); or Tel.: 1-604-822-7805; Fax: 1-604-822-7274; E-mail: ian@brc.ubc.ca (I. C.-L.).

Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M209470200

    ABBREVIATIONS

The abbreviations used are: CXCR, CXC chemokine receptor; BCA-1, B cell activation chemokine-1; CCR, CC chemokine receptor; I-TAC, interferon-inducible T cell alpha -chemoattractant; I-TAC/EoH1, I-TAC-eotaxin hybrid-1; I-TAC-H1, I-TAC-H2, and I-TAC-H3, I-TAC-IP10 hybrid 1, 2 and 3; Mig, monokine induced by gamma -interferon; IP10, interferon-inducible protein 10; MCP, monocyte chemoattractant protein; RANTES, regulated on activation normal T cell expressed and secreted; SDF-1, stromal cell-derived factor-1; IL, interleukin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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