CXCR3 and Heparin Binding Sites of the Chemokine IP-10 (CXCL10)

CXCR3 and Heparin Binding Sites of IP-10

1

CXCR3 and Heparin Binding Sites of the Chemokine IP-10

(CXCL10)

Gabriele S.V. Campanella∗, Elizabeth M.J. Lee∗, Jieti Sun∗, Andrew D. Luster∗§

Running title: CXCR3 and Heparin Binding Sites of IP-10

∗Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and

Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, 02114

§Address correspondence to Andrew D. Luster, Massachusetts General Hospital, Bldg 149, 13th St,

Charlestown, Massachusetts, 02129. Phone: 617-726-5710; Fax: 617-726-5651; E-mail:

[email protected]

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SUMMARY

The chemokine IP-10 (interferon-inducible protein of 10 kD, CXCL10) binds the G protein-coupled

receptor CXCR3, which is found mainly on activated T cells and NK cells, and plays an important role in

Th1-type inflammatory diseases. IP-10 also binds to glycosaminoglycans (GAGs), an interaction thought

to be important for its sequestration on endothelial and other cells. In this study, we performed an

extensive mutational analysis to identify the CXCR3 and heparin binding sites of murine IP-10. The

mutants were characterized for heparin binding, CXCR3 binding, and the ability to induce chemotaxis,

Ca2+-flux and CXCR3 internalization. Double mutations neutralizing adjacent basic residues at the C-

terminus did not lead to a significant reduction in heparin binding, indicating that the main heparin

binding site of IP-10 is not along the C-terminal α helix. Alanine exchange of R22 had the largest effect

on heparin binding, with residues R20, I24, K26, K46 and K47 further contributing to heparin binding. A

charge change mutation of R22 resulted in further reduction in heparin binding. The N-terminal residue

R8, preceding the first cysteine, was critical for CXCR3 signaling. Mutations of charged and uncharged

residues in the loop regions of residues 20-24 and 46-47, which caused reduced heparin binding, also

resulted in reduced CXCR3 binding and signaling. CXCR3 expressing GAG-deficient CHO cells

revealed that GAG binding was not required for efficient receptor binding and signaling, which suggests

that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.


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INTRODUCTION

IP-10 (Interferon-inducible protein, 10 KDa) belongs to the superfamily of chemokines (chemoattractant

cytokines), that are involved in the activation and recruitment of leukocytes, as well as non-hematopoetic

cells (1). Chemokines are 8-10 kDa proteins that have been subdivided based on the position of the first

two cysteine residues into four subfamilies. IP-10 is a member of the CXC subfamily and was first

identified as a gene markedly induced by interferon γ (2) and has since been shown to be a potent

chemoattractant of activated T cells (3). It is expressed constitutively at low levels in thymic, splenic and

lymph node stroma (4), but its expression can be highly induced by interferon α, β and γ, and LPS in a

variety of cell types, including endothelial cells, keratinocytes, fibroblasts, mesangial cells, astrocytes,

monocytes and neutrophils (5). It has been demonstrated to be highly expressed in many Th1-type

inflammatory diseases, including skin diseases (6-8), atherosclerosis (9), multiple sclerosis (10,11),

allograft rejection (12,13), and others. Studies with inhibitory antibodies and IP-10-deficient mice have

revealed that IP-10 plays an important role in the recruitment of effector T cells into inflammatory tissues

(14-17). IP-10 has also been shown to have angiostatic (18-21) and anti-tumor activity (22,23).

Chemokines activate leukocytes by binding to 7 transmembrane, G protein-coupled receptors. IP-10

binds to the CXC chemokine receptor 3 (CXCR3), which it shares with two other ligands, interferon-

inducible T cell-α chemoattractant (I-TAC/CXCL11) and monokine-induced by γ-interferon

(Mig/CXCL9). IP-10, like many chemokines, also binds to cell surface glycosaminoglycans (GAGs)

(21,24). While our understanding of the biological activities of the CXCR3 ligands has increased,

relatively little is know about the importance of their interaction with GAGs. It has been postulated that

GAGs on the cell bearing the 7 transmembrane receptor facilitate chemokine binding to its high affinity

receptor (25), while GAGs on endothelial cells and in the extracellular matrix might be important for

retaining chemokines close to their site of secretion (26). Furthermore, it has been suggested that GAGs

might also be involved in the signaling of chemokines, as has been recently shown for RANTES (CCL5)


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(27) and PF4 (platelet factor 4, CXCL4) (28). GAGs have also been shown to be important for the anti-

HIV effect of SDF-1α (stromal cell-derived factor-1α, CXCL12) (29) and RANTES (30,31). In addition,

GAGs might also be important for chemokine activity on non-hematopoetic cells, such as endothelial

cells.

In this study, we performed an extensive mutational study to locate the CXCR3 and heparin binding sites

of murine IP-10 to begin to dissect the role of CXCR3 and GAG binding in IP-10’s biological activity.

We found that the N-terminal residue R8 as well as residues in the loop regions 22-26 and 46-47 are

important for CXCR3 binding. Interestingly, these same loop regions are also the major heparin binding

site, suggesting that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.


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EXPERIMENTAL PROCEDURES

Materials: Recombinant murine IP-10 was obtained from Peprotech (Rocky Hill, NJ), [125I]-IP-10

(human) was purchased from NEN (Boston, MA). Endotoxin testing reagents were from Charlesriver

Laboratories, MA. BCA reagents were from Pierce, IL. All other materials were of biological grade and

purchased either from Sigma or Fisher.

Cell culture and transfection: 300-19 cells were maintained in complete RPMI, 10% FCS. 300-19 cells

transfected with human CXCR3 (300-19/hCXCR3) were a gift from B.Moser. CHO K1 cells and CHO

cells deficient in GAGs (CHO 745) (ATCC), a mutant cell line defective in xylosyltransferase (the first

committed enzyme involved in glycosaminoglycan biosynthesis) (32,33), were grown in F12K media

supplemented with 10% FCS. The murine CXCR3 (mCXCR3) gene in pcDNA3 was transfected into

300-19 or CHO cells by electroporation and selection was performed in G418. Clones were obtained by

limiting dilutions and selected by their mCXCR3 expression and their ability to induce Ca2+-flux in

response to murine IP-10 (mIP-10), and are referred to as 300-19/mCXCR3 or CHO/mCXCR3.

IP-10 mutagenesis and purification: The mIP-10 cDNA (in Bluescript, from Joshua Farber) was cloned

into the pET9a expression vector (Novagen). To avoid the Shine-Delgarno sequence, conservative

changes to the N-terminal DNA sequence were introduced. Mutagenesis was performed in pET9a using

the Transformer site directed mutagenesis protocol (Clonetech) or the Quick-change protocol

(Stratagene). The desired mutations were confirmed by DNA sequencing.

The pET9a expression vectors containing the mutated IP-10 gene were transformed into the BL21 DE3

pLys E.coli strain. Cultures were grown with 100 µg/ml Kanamycin and 40 µg/ml Chloramphenicol at

37° C with shaking up to an OD600 of 0.5 before inducing protein expression by the addition of 0.2 mM

isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were harvested 4 h after induction, pelleted and

resuspended in lysis buffer containing 0.1 mM Tris-HCl buffer, pH 8.5, 1 mM dithiothreitol, 5 mM


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benzamidine-HCl, 0.1 mM phenylmethylsulfonyl fluoride, 16 mM MgCl2 and 20 mg/l DNase and broken

by two passages through a French press (Carver, Wabash, IN). IP-10 was purified from the inclusion

bodies which were dissolved in 6 M guanidine-HCl, 0.1 M Tris base, pH 8.5, 1 mM dithiothreitol. To

ensure complete monomerization, the solution was heated to 60° C for 30 min and separated on a

Sephacryl S200 HR HiPrep 26-60 (Pharmacia). The fractions containing monomeric IP-10 were pooled.

The protein was renatured by dropwise addition to 0.1 M Tris-HCl buffer, pH 8.5, containing 1 mM

oxidized glutathione and 0.1 mM reduced glutathione at 4° C with stirring to reach a 15-fold dilution. The

pH of the renatured protein solution was adjusted to 4.5 with acetic acid and loaded onto a 5 ml SP

HiTrap column (Pharmacia) equilibrated in 50 mM sodium acetate, pH 4.5, and was eluted with a linear

gradient of 0 – 100% 2 M NaCl in 50 mM sodium acetate, pH 4.5. Fractions containing IP-10 were

dialyzed against 50 mM sodium phosphate buffer, 1.7 M ammonium sulfate, pH 7.0, and loaded on a 1 ml

Resource (Phenyl Sepharose) column equilibrated in the same buffer and eluted with an 1.7 M - 0.0 M

ammonium sulfate gradient. Fractions containing pure IP-10 were identified by SDS-PAGE (4-20% Tris-

HCl gels, Biorad), pooled and dialyzed twice against 1% acetic acid and once against 0.1% trifluoroacetic

acid prior to lyophilisation. Protein concentration was determined with a combination of BCA assay

(Pierce, IL), SDS-PAGE and ELISA. Selected purified mutants were analyzed for the presence of LPS

by the luminus assay (Charlesriver, MA). To verify the correct folding of mutants with decreased in vitro

activity, near UV CD spectra were run on a Aviv 62DS CD spectrometer at 20° C.

Heparin Sepharose and S Sepharose Chromatography: 20 µg IP-10 protein were loaded on a 1 ml

Heparin HiTrap or S Sepharose FF (cationic exchange) column (both from Pharmacia) equilibrated in 50

mM Tris, pH 7.5 on an AKTA machine (Pharmacia). The mutants were eluted with a 20 ml gradient of 0

– 2 M NaCl in 50 mM Tris, pH 7.5, and their elution time were measured by absorbance at 214 nm. Each

experiment was repeated at least twice.

Receptor binding assay: Binding assays were performed in 96 well tissue culture plates in a total volume

of 150 µl binding buffer (0.5 % BSA, 50 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM CaCl2). 300-


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19/hCXCR3 cells (4x105/well), or CHO/mCXCR3 cells (3x105/well), were incubated with 0.04 nM [125I]-

IP-10 (NEN, human IP-10), and increasing concentration of IP-10 mutants (5x10-6 nM – 500 nM). After

90 min at room temperature with shaking the cells were transferred to 96 well filter plates (Millipore)

previously soaked in 0.3% polyethyleinimine and washed four times with 200 µl binding buffer

supplemented with 0.5 M NaCl. Radioactivity was counted after addition of scintillation fluid in a

Microbeta counter (Wallac). The data were analyzed with GraFit (34). Each experiment was performed

in duplicate and repeated at least twice.

Chemotaxis: Chemokine dilutions in RPMI media supplemented with 1% low endotoxin BSA were

added to the bottom well of a 96 well chemotaxis plate (Neuroprobe, Gaithersburg, MD). 300-

19/mCXCR3 cells were washed into the same buffer at a concentration of 5x105 cells/ml and 50 µl of

cells were added on top of the membrane (5 µm pore size, polycarbonate filters). The chemotaxis plate

was incubated at 37° C for 4 hours and transferred to 4° C for 10 min before removing the membrane.

Cells in the bottom wells were counted under a microscope and are expressed in total cell numbers. Each

experiment was performed in duplicate and repeated a minimum of two times.

Calcium mobilization: 300-19 or CHO (wt or GAG-deficient) cells expressing mCXCR3 were

resuspended in their growth media with 1% FCS at 1x107 cells/ml and loaded with 5.0 µM of the

acetoxymethyl ester of fura-2 (Molecular Probes, Eugene, OR) for 30 min at 37° C in the dark. Cells

were washed and resuspended at 5x106 cells/ml in Calcium flux buffer (145 mM NaCl, 4 mM KCl, 1 mM

NaHPO4, 0.8 mM MgCl2, 1.8 mM CaCl2, 25 mM Hepes and 22 mM glucose). Fluorescence readings

were measured in a continuously stirring cuvette at 37° C in a DeltaRAM (Random Access

Monochromator) fluorimeter (Photon Technology International, Monmouth Junction, NJ). The data were

recorded as excitation fluorescence intensity emitted at 510 nm in response to sequential excitation at 340

nm and 380 nm and are presented as the relative ratio of fluorescence at 340/380 nm. Fold induction (FI)


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of each stimulation was calculated by dividing the peak fluorescence ratio after stimulation by the peak

fluorescence ratio during the 10 seconds before stimulation. Each experiment was repeated at least twice.

Internalization of CXCR: Internalization of mCXCR3 was measured as previously described (35).

Briefly, 300-19/mCXCR3, were resuspended in complete RPMI, 10% FCS, and various concentration of

wt and mutant IP-10 were added and incubated for 30 min at 37° C. The cells were stained with anti-

mCXCR3 antibody or isotype control (provided by Julie DiMartino, Merck), followed by a PE-labeled,

secondary antibody (Caltag, Burlingame, CA) and analyzed by FACS.


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RESULTS

Mutagenesis and Purification of IP-10 Mutants:

Residues of murine IP-10 (mIP-10) were chosen for mutagenesis by homology to the human and murine

CXCR3 ligands IP-10, Mig, I-TAC, as well as comparison to known receptor and GAG binding sites of

other CXC chemokines (Fig. 1). The first series of mutants included single point mutations to alanine or

double mutations of adjacent basic residues to alanine. We chose a total of 16 mutants, including residues

at the N-terminus, identified for a number of chemokines to be important for signaling (36-38), residues

along the 20s and 40s loop regions, shown for some chemokines to be important for receptor as well as

GAG binding (39-41) and a set of basic residues at the C-terminus, where the GAG binding site of IL-8

has been located (42). Based on the results of the first series of mutants, another 6 mutants were created.

Residues of IP-10 mutated in this study are indicated with an arrow in Figure 1.

Of the 22 mutants, one single point mutation, R38A, could not be expressed in a number of E.coli

expression strains, and was not further pursued. All the other mutants were successfully expressed in

E.coli BL21 DE3 (pLys) and purified from inclusion bodies by gel filtration, cationic exchange and

hydrophobic interaction chromatography to at least 95% homogeneity as determined by SDS-PAGE gels.

N-terminal sequencing of the purified protein from the plasmid containing the unmutated wt IP-10 gene

revealed that the N-terminal methionine was retained. We found this IP-10 to be fully active as compared

to commercially available murine IP-10 from Peprotech, which has the initiating methionine cleaved off,

in calcium-flux, chemotaxis and competitive receptor binding assays using 300-19 cells transfected with

mCXCR3 (300-19/mCXCR3) as well as for chemotaxis and CXCR3 internalization of in vitro polarized

murine Th1 cells (data not shown). The purified product from E.coli expressing the wt DNA was

therefore called wt IP-10, and all other mutants for this study were similarly purified with the N-terminal

methionine most likely retained. To eliminate the possibility that some of the effects seen with the IP-10

proteins could be due to LPS contaminations, wt IP-10 as well as selected mutants were tested for the


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presence of endotoxin using a Limulus Amebocyte Lysate assay at concentration of 1000 ng/ml and were

found to be below the detection limit of 0.03 EU/ml. Mutants with reduced CXCR3 binding and reduced

in vitro activity were analyzed for correct folding by near UV CD spectra. The spectra of all tested

mutants were super imposable with the spectrum of wt IP-10, apart from mutant E40A, which displayed a

clear shift. Mutant E40A was therefore excluded from the results.

Heparin Binding of IP-10 Mutants

The ability of the IP-10 mutants to bind heparin was analyzed by heparin affinity chromatography. Wt

IP-10 eluted at a concentration of 0.96 M NaCl (Fig. 2). Of the first set of 15 mutants successfully

expressed, 11 eluted at salt concentrations similar to the wt protein (difference of less than 0.10 M NaCl),

including single and double mutations along the C-terminal helix. Mutations along the 20s loop region

(also called N-loop) resulted in a larger difference; in particular, mutating R22 had the largest impact of a

single mutation. Mutations of the basic residues of the 40s loop also resulted in a reduction in heparin

binding.

The specificity of heparin binding compared to electrostatic interaction was investigated by eluting the

mutants from a cationic exchange column (SP HiTrap). The difference in salt concentration needed to

elute mutants from the SP column in relation to the wt protein, compared to the difference in elution from

the heparin column, confirmed that the residue R22 had the highest specific binding to heparin. In

contrast, residues along the C-terminal helix all displayed a negative differential (i.e. had at least as much

a reduction in binding to the SP column as for the heparin column), indicating that the reduction in

heparin binding due to mutation of these residues is mainly an electrostatic effect.

Based on the previous results, we created a second set of mutants, with a charge change of residues R22

to glutamic acid, and combining mutation of R22A with R20A, K47A and R75E. In addition, the four

basic residues of the C-terminal helix were mutated together, in a fashion similar to a mutant of PF4 that

showed nearly no heparin binding affinity (43), to glutamic acid and glutamine yielding the mutant


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K71ER72Q K74QR75E (named “C-t mut”). This C-terminal mutant was also combined with the R22A

mutation (C-tR22A).

Mutation of R22 to a negatively charged glutamic acid further reduced the heparin binding affinity, as did

combination with R20A and K47A (Fig. 2). These mutants displayed the highest amount of specificity

for heparin binding, revealed by the differential in SP and heparin column elution. Although the double

mutant of K74AR75A only had a moderate effect on heparin binding, the combination of R22A with

R75E still reduced the heparin binding. Similarly, K71ER72Q K74QR75E also showed a reduction of

heparin binding of 0.29 M NaCl, and combining this mutation with R22A led to the largest decrease in

heparin binding observed in this study. However, a comparison with binding to the SP column showed

that this effect was mainly electrostatic in nature.

CXCR3 Receptor Binding

In a competitive binding assay using 300-19/hCXCR3 cells, wt IP-10 competed for the binding of 40 pM

[125I] human IP-10 with an IC50 value of 0.11 nM (Fig. 3), in agreement with published Kd values (44).

Eight of the mutants (I12A, E28A, S33A, T44A, K62A, K66A, K71AR72A, K74AR75A) had IC50 values

comparable to the wt protein. The N-terminal mutant R5A showed a 7-fold higher value, while mutant

R8A had a 60-fold higher value. Mutant R22A, I24A and K26A, which had shown lower heparin binding

affinity, displayed 6-fold, 4-fold and 2-fold lower receptor binding affinity, respectively. K46AK47A,

which had similarly reduced heparin binding, had a much more reduced CXCR3 binding affinity. The

second set of mutants (R22E, R20AR22A, R22AK47A, R22AR75E), all with further reduced heparin

binding, had 15- to 60-fold reduced binding affinity for the receptor. Mutation of the four basic residues

of the C-terminus decreased the receptor binding 80-fold, and combination with the R22A mutation

further reduced the binding another 10-fold, resulting in a nearly 1000-fold reduction in binding affinity.

Calcium Flux of IP-10 Mutants


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All mutants were tested at concentrations of 200 ng/ml and 1000 ng/ml for their ability to induce calcium

mobilization in 300-19/mCXCR3 cells with a subsequent challenge of 200 ng/ml wt IP-10 to test the

ability of the mutant proteins to desensitize the cells (Fig. 4). The eight mutants with CXCR3 binding

and heparin binding comparable to wt IP-10 (I12A, E28A, S33A, T44A, K62A, K66A, K71AR72A,

K74AR75A) all behaved equivalently to wt IP-10 in terms of calcium-flux, chemotaxis, and

internalization (data not shown), and will therefore not be listed in the remaining figures. The ability to

cause calcium flux in 300-19/mCXCR3 cells followed a similar pattern as for receptor binding. Mutant

R8A did not induce calcium mobilization at concentrations of 200 or 1000 ng/ml (Fig. 4) or at 5000 ng/ml

(data not shown), and did not desensitize the cells to a subsequent challenge of wt IP-10. Mutants R5A

and R22A, with 5- to 6-fold reduced receptor binding affinity, triggered only a small calcium

mobilization at 200 ng/ml, but were more effective at 1000 ng/ml. Mutants with more reduced CXCR3

binding (R22E, R22AK47A, R22AR75E, C-t mut), all displayed strongly reduced ability to cause

calcium mobilization even at 1000 ng/ml.

Chemotaxis Induced by IP-10 Mutants

The ability to cause chemotaxis of 300-19/mCXCR3 cells was related to the receptor binding and calcium

mobilization data (Fig. 5), with some notable exceptions. Small reductions in receptor binding affinity

did not affect the chemotactic activity of the mutants as much as it did for calcium mobilization. Mutants

R5A, R22A and K26A, with only slightly reduced CXCR3 binding, displayed nearly wt chemotactic

activity with similar potency and efficacy. Mutants R22E, R22AK47A, R22AR75E and C-t mut, which

had reduced CXCR3 and heparin binding, exhibited both reduced potency and efficacy for chemotaxis.

Mutant C-tR22A was 6- to 7-fold less potent than wt IP-10 for chemotaxis, even at concentration of 5000

ng/ml. Mutant R8A did not cause chemotaxis up to concentrations of 5000 ng/ml.

Internalization of mCXCR3 Caused by IP-10 Mutants


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All of the IP-10 mutants were analyzed for their ability to cause CXCR3 internalization. For this, 300-

19/mCXCR3 transfected cells were incubated for 30 min at 37° C with various concentrations of IP-10,

after which time the cells were stained on ice with an anti-mCXCR3 antibody and CXCR3 expression

was analyzed by FACS. No internalization was observed with incubation at 4° C (data not shown). Wt

IP-10 caused 68% internalization at 10 ng/ml and 92% at 1000 ng/ml (Table 1). As seen for receptor

binding and chemotaxis, mutant R22A was slightly less efficient than wt IP-10 in triggering

internalization, while the mutants with further reduced CXCR3 binding displayed further reduced ability

to cause internalization. Mutant R8A was the least effective mutant, triggering less than 30%

internalization at 1000 ng/ml, while mutant C-tR22A, with 50-fold lower receptor binding affinity than

R8A, caused nearly 50% internalization at 1000 ng/ml.

GAG-Deficient CHO Cells

IP-10 mutants with significantly reduced heparin binding all displayed reduced receptor binding and

reduced agonist activity. This could be due to the fact that heparin/GAG binding is promoting receptor

binding and activation, as has been shown for other chemokines (25). Alternatively, the binding sites of

CXCR3 and GAGs could be overlapping, thereby causing the mutants with reduced GAG binding affinity

to also have reduced CXCR3 binding. To address this question, wt CHO cells as well as CHO cells

deficient of GAGs (CHO 745)(33), were stably transfected with mCXCR3. Clones of wt and GAG-

deficient CHO cells with similar CXCR3 expression levels were selected based on FACS analysis. Both

wt and GAG-deficient CHO cells transfected with mCXCR3 responded with a similar rise in intracellular

calcium concentration when stimulated with 200 ng/ml wt IP-10 (Fig. 6A). The mean FI (Fold induction)

of four independent experiments for the primary challenge was 1.45 ± 0.05 for wt CHO cells and 1.45 ±

0.08 for GAG-deficient CHO cells. In a competitive binding assay, the binding of radiolabeled human

IP-10 in the absence of competitor was similar for both wt and GAG-deficient CHO cells expressing

mCXCR3, while only low background binding to mock-transfected cells was observed (Fig. 6B). The

IC50 values for wt murine IP-10 competing for the binding of the radiolabeled hIP-10 was 0.26 ± 0.05 nM


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for wt CHO cells and 0.18 ± 0.03 nM for the GAG-deficient CHO cells, indicating that GAGs on the cell

surface of the cell bearing the receptor are not essential for receptor binding.

Given the possibility that GAGs could have a contribution in receptor binding and activation that could be

counterbalanced by other factors in the GAG-deficient cells, we tested the ability of selected heparin

binding reduced IP-10 mutants to compete for binding to CXCR3 in the GAG-deficient CHO cells. As

shown in Figure 6B, the IC50 values for mutant R22E were similar for binding to the wt and GAG-

deficient CHO cells expressing murine CXCR3, with mean IC50 values for three experiments of 3.4 ± 1.4

nM and 3.3 ± 0.86 nM, respectively. The IC50 values for mutants K46AK47A and R20AR22A were also

similar for binding to wt and GAG-deficient CHO cells expressing mCXCR3 (data not shown),

suggesting that the mutants of the 20s and 40s loop regions have reduced CXCR3 binding independent

from their reduced GAG binding affinity.


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DISCUSSION

In the present study we used a mutagenesis approach to define the residues of IP-10 that mediate its

interaction with CXCR3 and heparin.

Residues R20, R22, I24 and K26 as well as K46 and K47 were found to constitute the main GAG binding

domain, with mutation of R22 resulting in the largest reduction of heparin binding affinity. In an

homology model of mIP-10 based on the NMR structure of hIP-10 (45) created by the program Swiss-

Model (46,47), these residues form part of the loop region leading to and including the first residues of

the 1st β-sheet (residues 20-26) and the spatially adjacent loop region after the 2nd β-sheet (K46, K47), and

are all along the same side of the protein (Fig. 7). Residues R20, R22, K46 and K47 line a groove that

could make up the heparin binding site (Figure 7C). The basic residues on the C-terminal helix when

mutated in single or double point mutations only had a very modest effect on heparin binding. However,

when the four basic residues K71, R72, K74, R75 were mutated together with two changes to neutral and

two changes to acidic amino acids, this resulted in a mutant with a greater reduction in heparin binding.

Combining this quadruple mutation with a mutation at R22 led to the largest decrease in heparin binding

observed in this study. However, the reduction in heparin binding due to mutations along the C-terminal

helix seems to be due to an electrostatic effect, whereas mutations along the 20s and 40s loop resulted in a

heparin specific binding reduction. It is therefore difficult to evaluate the contribution of the residues

along the C-terminal helix in GAG binding. The findings are very similar to what was shown for PF4

(43), where by NMR the main GAG binding site was found to be along residues R20, R22, H23, T25,

K46 and R49. Double and even quadruple mutations of basic residues along the C-terminal α helix of

PF4 to alanine did not affect the heparin binding very much, whereas changes of four basic residues to

two glutamines and two glutamic acids reduced the heparin binding 500-fold. Since the NMR structure

of IP-10 only defines the structure up to residue 70, we could not model residues 71-75, including the

four basic residues, and cannot with confidence predict their position in relation to the other heparin


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binding residues. However, with an estimated 3.5 – 4 residues per helix turn, it is likely that residue 71

and 75 would be in close proximity to the 20s and 40s loop. Their mutation to negatively charged

residues might therefore disturb the GAG binding site of IP-10, leading to the reduction in heparin

binding observed for the K71ER72Q K74QR75E mutation, without those residues themselves being

directly involved in heparin binding.

CXCR3 binding of the mutants was investigated with a competitive binding assay. As seen for other

chemokines, residues at the N-terminus (as seen for R5 and R8) just preceding the first cysteine are

involved, though not critical, for CXCR3 binding. The 20s loop region (R20, R22, I24) is also important

for receptor binding, as are residues in the spatially adjacent 40s loop (K46, K47) (Fig. 8). Single or

double mutations of residues along the C-terminus did not have a significant impact on CXCR3 binding.

However, the quadruple mutation K72, R74, K74, R75 with two charge changes did lead to a 100-fold

reduced binding, and combined with mutation R22A to a 1000-fold reduced binding. However, since the

double mutations of the residues along the C-terminus did not affect CXCR3 binding, it is unlikely that

these residues are themselves involved in binding to CXCR3, but a double charge change might interrupt

the nearby CXCR3 binding site. It is interesting to note, that two of the main residues identified to be

involved in CXCR3 binding, namely R8 and K46, are fully conserved in all murine and human CXCR3

ligands (see Fig. 1).

A recent study published the NMR structure of an obligate monomer mutant of human IP-10 (45). In

order to investigate the interaction with CXCR3, a peptide consisting of the N-terminal residues 22-42 of

CXCR3 was titrated into the IP-10 solution. Upon titration, changes in chemical shifts were observed for

residues V7, R8, Q17, V19, Q34, R38 and T44. Although based on a completely different technique, our

study identified residues in similar regions on IP-10 interacting with CXCR3, namely residues in the N-

terminus and 20s loop. In addition, the NMR study identifies residue R38 as involved in CXCR3

binding. In our study, mutation of R38 was performed, however, the mutant did not express in bacteria.

In addition, the NMR study also identified the 30s loop to be involved in the CXCR3 binding, based on


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the change in chemical shift seen for Q34. This residue is not conserved in murine IP-10, but in our

study, mutation of S33 did not affect CXCR3 binding. However, this could be due to the relatively small

mutation of a serine to an alanine not interrupting the receptor ligand interaction. Lastly, residue T44

showed a change in chemical shift in the NMR study, but as the authors state, this residue is actually

buried and is not located on the same face of the protein as the other residues (45). T44 was chosen for

mutation in this study as it is conserved in all murine and human CXCR3 ligands, but its mutation to

alanine did not impact CXCR3 binding or signaling. Therefore, compared to the NMR study, our

mutational study identifies some similar regions (N-terminus and 20s loop), as well as some different

regions (40s loop, but not 30s loop) on IP-10 to be involved in CXCR3 binding.

The residues preceding the first cysteine have been shown for a number of chemokines to be critical for

signaling. In particular, in the case of IL-8, the ELR motif is essential for signaling and mutations in this

region completely abrogate function while only reducing receptor binding (37). Similarly, for MCP-1, N-

terminal residues were found to be essential for signaling, while being less important for CCR2 binding

(38). In our study, we found residue R8 of IP-10 to be critical for signaling through CXCR3. Mutation of

this residue did not affect heparin binding and led to a 60-fold reduction of CXCR3 binding. Although

other mutants with similarly reduced CXCR3 binding were still able to signal, mutation of R8 could not

induce any significant chemotaxis or calcium mobilization even at concentrations of 5000 ng/ml. Mutant

R8A did induce a small amount of CXCR3 internalization, which has a distinct signaling pathway and is

independent of Gαi protein signaling (35). The calcium flux data revealed that mutant R8A is not an

antagonist, as it did not block wt IP-10 calcium flux.

As schematized in Figure 7, mutations along the 20s and 40s loop resulted in reduced heparin as well as

CXCR3 binding. To address the question of whether the reduced ability to bind GAGs affected the

CXCR3 binding of these mutants, wt CHO cells and a mutant CHO cells line deficient in

xylosyltransferase and therefore lacking GAGs, were transfected with mCXCR3 and used to analyzed the

IP-10 mutants. Wt IP-10 induced an equivalent amount of calcium mobilization in wt and GAG-deficient


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CHO cells expressing mCXCR3 and had similar IC50 values for both, showing that the GAGs on these

transfected cells were not essential for CXCR3 binding and signaling. However, these cells are

overexpressing CXCR3, and it could be that in biological settings with a lower CXCR3 surface

expression, GAGs help to present IP-10 to the receptor. Mutants R22E, R20AR22A and K46AK47A,

which had reduced heparin and CXCR3 binding and signaling in 300-19/mCXCR3 cells, showed the

same 30- to 60-fold reduced CXCR3 binding for both wt and GAG-deficient CHO cells. This suggests

that the 20s and 40s loop region is indeed involved in CXCR3 binding, and therefore the CXCR3 and

heparin binding sites are overlapping.

Although these experiments suggest that GAGs on cells overexpressing CXCR3 are not required for

CXCR3 binding and signaling, GAGs on endothelial cells or other non CXCR3 bearing cells could still

be important for retaining and sequestrating IP-10 close to the site of secretion. In addition, GAGs might

also be involved in biological functions of IP-10 on non-CXCR3 expressing cells. We hope to explore

this hypothesis with mutants of IP-10 that have normal heparin binding affinity but reduced CXCR3

binding affinity.

In conclusion, our study has defined the N-terminal residue R8 of IP-10 as being critical for signaling

through CXCR3, and identified residues on the 20s and 40s loop as being important for CXCR3 binding.

We also demonstrated that the GAG binding site of IP-10 is partially overlapping with the CXCR3

binding site. These mutants provide useful tools to further dissect the role of CXCR3 and GAG binding

in IP-10’s known biological activities, including recruitment and activation of T cells and NK cells and

inhibition of angiogenesis, fibrosis and tumor growth. In addition, given the validation of IP-10 and

CXCR3 in certain in vivo models of disease, such as allograft rejection (14,17,48), our study provides a

structural basis for the rational design of molecular inhibitors of this pathway.


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ACKNOWLEDGEMENTS

We thank J.P. Xiong and Amin Arnaout for help with the use of the GRASP program, Sam Lehrer

(Boston Biomedical Research Institute) for help with the CD spectra and Julie DiMartino (Merck) for

generously providing the mCXCR3 antibody. This work was supported by NIH grant RO1-CA69212 and

PO1-DK50305 to ADL.

FOOTNOTES

The abbreviations used are: IP-10: interferon inducible protein of 10 kDa, GAG: glycosaminoglycan,

CXCR: CXC chemokine receptor, m: murine, h: human, I-TAC: interferon-inducible T cell-α

chemoattractant, Mig: monokine-induced by γ-interferon, PF4: platelet factor 4, RANTES: regulated on

activation normal T cell expressed and secreted, CHO: Chinese hamster ovary, C-t mut: K71ER72Q

K74QR75E, C-tR22A: R22A K71ER72Q K74QR75E


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FIGURE LEGENDS

Figure 1: Amino acid sequence of IP-10 and selected CXC chemokines. Residues in murine (m) IP-

10 conserved in relationship to human (h) IP-10 are shown in bold letters and residues mutated in this

study are indicated with an arrow. Residues in the other chemokines identical to murine IP-10 are shown

in bold. Residues in IL-8 involved in receptor binding are double underlined (36,37,49) and residues of

IL-8 and PF4 implicated in heparin binding are single underlined (42,43).

Figure 2: Heparin Sepharose and Cationic Exchange chromatography of IP-10 mutants. 20 µg of

IP-10 mutants were loaded onto either a 1 ml Heparin HiTrap column (A) or a 1 ml SP HiTrap FF

(cationic exchange) column (B) and eluted with increasing concentration of NaCl. C) Summary of

molarity of NaCl required to elute mutants from a heparin or SP column, expressed as mean ± S.D. of 2 -

4 experiments. Results of the first set of mutants are presented in upper part of C, results of second set in

lower part of C. C-t mut: K71ER72Q K74QR75E, C-tR22A: C-t mut combined with mutation R22A .

Figure 3: Competitive CXCR3 binding assay. A) The binding of [125I]-IP-10 to 300-19/hCXCR3 cells

was competed by IP-10 mutants (wt: ▲, R8A: , R22A: ●, R22E: ○, and C-tR22A: ■). B) IC50 values

were calculated for all IP-10 mutants and are expressed as mean ± S.E. from 2-4 experiments.

Figure 4: Calcium flux of 300-19/mCXCR3 cells in response to IP-10 mutants. 300-19/mCXCR3

cells were loaded with 5.0 µM of the acetoxymethyl ester of fura-2 and fluorescence intensity emitted at

510 nm in response to sequential excitation at 340 nm and 380 nm was recorded. An overlay of the

relative ratio of fluorescence at 340/380 nm for selected mutants at 200 ng/ml (A) and 1000 ng/ml (B)

initial stimulation, with a subsequent stimulation of 200 ng/ml wt IP-10. C) The fold increase (FI) of

calcium mobilization following each stimulation was calculated as described in Materials and Methods

and the mean values from at least two independent experiments are listed. The amount of desensitization

of the cells after the primary dose of wt or mutant IP-10 is measured by a second stimulation with 200


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ng/ml wt IP-10. The FI of the second dose is listed in parenthesis, with a value of 1.0 indicating complete

desensitization of the cells.

Figure 5: Chemotaxis of 300-19/mCXCR3 cell to IP-10 mutants. Chemotaxis of 300-19/mCXCR3

cells to IP-10 mutants was performed using a Neuroprobe chamber. A) representative chemotaxis of

selected mutants (wt: ▲, R8A: , R22A: ●, R22E: ○ and C-tR22A: ■, untransfected 300-19 cells/wt IP-

10: ◊). B) Peak activities compared to wt IP-10 and the peak concentrations for selected mutants. Each

experiment was performed in duplicate and repeated at least twice for each mutant.

Figure 6: Calcium flux and IP-10 binding of CXCR3 expressing GAG-deficient CHO cells. Calcium

flux of mCXCR3 expressing CHO cells (A) and GAG-deficient CHO cells (B) induced by 200 ng/ml IP-

10, with a subsequent second challenge of 200 ng/ml IP-10, each indicated with an arrow. C) The binding

of 40 pM [125I]-IP-10 to CHO cells and GAG-deficient CHO cells transfected with mCXCR3 or mock-

transfected CHO cells was competed by IP-10 mutants. Wt IP-10/wt CHO cells: ▲, wt IP-10/GAG-

deficient CHO cells: ●, R22E/wt CHO cells: , R22E/GAG-deficient CHO cells: ○, binding to mock-

transfected CHO wt cells competed by wt IP-10: ■ .

Figure 7: Schematic diagram of mIP-10 A) Side chains of residues important for heparin and CXCR3

binding are highlighted in magenta, those important for CXCR3 binding only are in yellow, those

important for heparin binding only are in green. B) Rotated view, same colors as A. C) Electrostatic

potential surface of the same view as B. Areas on the surface shaded in blue represent a positive, in red a

negative potential. Panels A and B were generated with Swiss Viewer (46), and panel C with GRASP

(50).


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% Internalization Mutant 10 ng/ml 100 ng/ml 1000 ng/ml

wt 67.7 ± 4.6 86.2 ± 2.17 91.9 ± 0.5 R5A 53.8 ± 2.7 61.5 ± 4.0 77.8 ± 3.0 R8A 8.1 ± 6.0 20.0 ± 7.2 22.7 ± 14.7

R22A 52.5 ± 7.6 74.1 ± 4.5 85.2 ± 0.5 I24A 35.3 ± 12.1 34.9 ± 5.7 79.7 ± 3.3 K26A 49.7 ± 25.9 76.7 ± 8.4 79.3 ± 7.7

K46AK47A 62.3 ± 20.4 74.2 ± 4.7 88.6 ± 2.5 R22E 21.7 ± 15.9 52.0 ± 9.7 71.9 ± 5.5

R20AR22A 9.7 ± 5.2 25.5 ± 9.7 42.6 ± 19.2 R22AK47A 35.7 ± 5.0 59.9 ± 9.6 80.5 ± 1.7 R22AR75E 34.8 ± 16.1 53.4 ± 3.9 78.9 ± 7.2

C-t mut 23.6 ± 11.4 45.8 ± 7.3 64.0 ± 4.6 C-tR22A 34.6 ± 17.5 24.4 ± 7.2 48.4 ± 7.2

Table 1: Internalization of mCXCR3 by IP-10 mutants. 300-19/mCXCR3 cells were incubated with

varying concentrations of IP-10 mutants for 30 min at 37° C. The percentage of CXCR3 internalized by

IP-10 mutants at the different concentrations is shown. Values are the mean ± S.D. of 2-4 independent

experiments.


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↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓↓ ↓ ↓ ↓↓ ↓↓ mIP-10: IPLARTVRCN CIHIDDGPVR 21MRAIGKLEII PASLSCPRVE 41IIATMKKNDE QRCLNPESKT 61IKNLMKAFSQ KRSKRAP

hIP-10: VPLSRTVRCT CISISNQPVN 21PRSLEKLEII PASQFCPRVE 41IIATMKKKGE KRCLNPESKA 61IKNLLKAVSK EMSKRSP

mI-TAC: FLMFKQGRCL CIGPGMKAVK 21MAEIEKASVI YPSNGCDKVE 41VIVTMKAHKR QRCLDPRSKQ 61ARLIMQAIEK KNFLRRQNM

mMig: GTLVIRNARCS CISTSRGTIH 22YKSLKDLKQF APSPNCNKTE 42IIATLK.NGD QTCLDPDSAN 61VKKLMKEWEK KINQKKKQKR …

hI-TAC: FPMFKRGRCL CIGPGVKAVK 21VADIEKASIM YPSNNCDKIE 41VIITLKENKG QRCLNPKSKQ 61ARLIIKKVER KNF

hMig: TPVVRKGRCS CISTNQGTIH 21LQSLKDLKQF APSPSCEKIE 41IIATLK.NGV QTCLNPDSAD 61VKELIKKWEK QVSQ

hIL-8: SAKELRCQ CIKTYSKPFH 19PKFIKELRVI ESGPHCANTE 39IIVKL.SDGR ELCLDPKENW 58VQRVVEKFL KRAENS

hPF4: EAEEDGDLQCL CVKTTSQ.VR 21PRHITSLEVI KAGPHCPTAQ 41LIATL.KNGR KICLDLQAPL 60YKKIIKKLLE S

Figure 1 Campanella et al


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0.000.540.19 ± 0.010.540.42 ± 0.01C-tR22A

-0.060.350.38 ± 0.010.290.67 ± 0.01C-t mut

0.080.270.46 ± 0.010.350.61 ± 0.02R22AR75E

0.150.160.57 ± 0.010.310.65 ± 0.03R22AK47A

0.140.170.56 ± 0.010.310.65 ± 0.01R20R22A

0.150.190.54 ± 0.010.340.62 ± 0.02R22E

-0.040.130.60 ± 0.010.090.87 ± 0.01K74AR75A

-0.070.120.61 ± 0.010.050.91 ± 0.04K71AR72A

-0.020.070.66 ± 0.010.050.91 ± 0.02K66A

-0.050.050.68 ± 0.010.000.96 ± 0.01K62A

0.030.120.61 ± 0.010.150.81 ± 0.02K46AK47A

0.020.020.71 ± 0.010.040.92 ± 0.02T44A

-0.010.000.73 ± 0.01-0.010.97 ± 0.01S33A

0.010.020.71 ± 0.010.030.93 ± 0.04E28A

0.060.050.68 ± 0.010.110.85 ± 0.01K26A

0.000.110.62 ± 0.010.110.85 ± 0.023I24A

0.080.100.63 ± 0.010.180.78 ± 0.01R22A

0.000.000.73 ± 0.010.000.96 ± 0.02I12A

0.030.000.73 ± 0.010.030.93 ± 0.01R8A

0.010.010.72 ± 0.010.020.94 ± 0.01R5A

0.73 ± 0.010.96 ± 0.02Wt IP-10

∆∆ [NaCl] (∆[NaCl]heparin -∆[NaCl]SP)

∆[NaCl]SP([NaCl]wt –[NaCl]mutant)

Elution from SP column[NaCl] M

∆[NaCl]heparin([NaCl]wt –[NaCl]mutant)

Elution from Heparin column

[NaCl] M

A B

C

Figure 2Campanella et al

28

15.0 20.0

20

40

60

80

100mS/cm

20

40

60

80

100mS/cm R22E Wt

C-tR22A R22A R22E WtC-tR22A R22A

Elution volume Elution volume


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91.7 ± 15.9C-tR22A8.45 ± 1.97C-t mut1.61 ± 0.89R22AR75E1.43 ± 0.46R22AK47A6.02 ± 0.48R20AR22A2.40 ± 0.29R22E0.18 ± 0.03K74AR75A0.28 ± 0.11K71AR72A0.25 ± 0.12K66A0.28 ± 0.14K62A2.73 ± 0.74K46AK47A0.24 ± 0.02T44A0.10 ± 0.02S33A0.26 ± 0.12E28A0.15 ± 0.06K26A0.41 ± 0.04I24A0.64 ± 0.16R22A0.25 ± 0.04I12A6.64 ± 2.3 R8A0.73 ± 0.20R5A

0.094 ± 0.017Wt IP-10IC50 (nM) ± S.E.Mutant

A BFigure 3Campanella et al

29

10210110010-110-210-310-410-510-610-7

120

100

80

60

40

20

0

[IP-10] nM

% B

indi

ng o

f max


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1.36 (1.08)1.20 (1.21)R5A

1.0 (1.30)1.0 (1.52)C-tR22A

1.13 (1.35)1.13 (1.38)C-t mut

1.23 (1.27)1.13 (1.32)R22AR75E

1.13 (1.30)1.05 (1.38)R22AK47A

1.11 (1.34)1.08 (1.53)R20AR22A

1.29 (1.39)1.00 (1.90)R22E

1.44 (1.13)1.26 (1.43)K46AK47A

1.62 (1.03)1.55 (1.09)K26A

1.35 (1.26)1.09 (1.25)I24A

1.52 (1.21)1.18 (1.28)R22A

1.00 (1.60)1.00 (1.50)R8A

1.90 (1.03)1.60 (1.11)wt

FI: 1000 ng/ml,(FI wt 200)

FI: 200 ng/ml, (FI wt 200 ng/ml)Mutant


A B

30

1

2

3

4

5

6

7

1 51 101 Time

C-tR22A

R22E

R22A

R8A

wt

1000 ng/ml wt or 200 ng/ml wt IP-10mutant protein

1 .5

2 .5

3 .5

4 .5

5 .5

6 .5

7 .5

8 .5

1 51 101 Time

Ratio

200 ng/ml wt or 200 ng/ml wt IP-10mutant proteinC


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5000 ng/ml6-7 fold lowerC-tR22A5000 ng/ml4-6 fold lowerC-t mut1000 ng/ml2-3 fold lowerR22AR75E1000 ng/ml2-3 fold lowerR22AK47A1000 ng/ml2-3 fold lowerR20AR22A5000 ng/ml2-3 fold lowerR22E200 ng/ml2-3 fold lowerK46AK47A200 ng/mlNearly wtK26A1000 ng/mlNearly wtI24A200 ng/mlNearly wtR22ANo activityNo activityR8A200 ng/ml Nearly wtR5A

Peak concentration

Peak activity in relation to wt IP-10

Mutant


A B

31

0

50

100

150

200

250

300

0 8 40 200 1000 5000

[IP-10] ng/ml

Cel

ls

wtR8AR22AR22EC-tR22Awt 300-19/ wt IP-10


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0.7

0.8

0.9

1

1 .1

1 .2

1 .3

1 .4

1 .5

1 .6

1 21 41 61 81 101 121 141 161 181Time (sec)

Rat

io

CHO wt

0.8

0.9

1

1 .1

1 .2

1 .3

1 .4

1 .5

1 .6

1 .7

1 21 41 61 81 101 121 141 161 181Time (sec)

GAG-def.CHO

A

C

B

10210110010-110-210-310-410-5

120

100

80

60

40

20

0

[IP-10] nM

% o

f max

imum

bin

ding

32


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A B


33

C


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Gabriele S.V. Campanella, Elizabeth M.J. Lee, Jieti Sun and Andrew D. LusterCXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10)

published online February 5, 2003J. Biol. Chem.

10.1074/jbc.M212077200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

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Documents

CXCR3 and Heparin Binding Sites of the Chemokine IP-10 (CXCL10)