Human Interferon-Inducible 10-kDa Protein and Human Interferon-Inducible T Cell a Chemoattractant Are Allotopic Ligands for Human CXCR3: Differential Binding to Receptor States

The human CXC chemokines IP-10 (10-kDa interferon-inducible protein), MIG (monokine induced by human interferon-g), and I-TAC (interferon-inducible T cell a chemoattractant) attract lymphocytes through activation of CXCR3. In the studies presented here, we examined interaction of these chemokines with human CXCR3 expressed in recombinant cells and human peripheral blood lymphocytes (PBL). IP-10, MIG, and I-TAC were agonists in stimulating [S]GTPgS binding in recombinant cell and PBL membranes but had no effect in the absence of hCXCR3 expression. I-IP-10 and I-I-TAC bound hCXCR3 with high affinity, although the I-I-TAC Bmax value in saturation bindings was 7to 13-fold higher than that measured with I-IP-10. Coincubation with unlabeled chemokines decreased I-IP-10 binding with a single discernible affinity. However, with I-I-TAC, competition with IP-10 or MIG was incomplete, and multiple binding affinities were evident. Moreover, in contrast to I-TAC, IP-10 and MIG binding IC50 values did not increase predictably with increased I-I-TAC concentration in competition bindings, suggesting that these chemokines are noncompetitive (i.e., allotopic) ligands. Uncoupling of hCXCR3 eliminated I-IP-10 binding but only decreased II-TAC binding 30 to 80%, indicating that unlike IP-10, I-TAC binds with high affinity to uncoupled (R) and coupled (R*) hCXCR3. To examine chemokine binding to R*, we tested the effect of anti-hCXCR3 antibody on I-TACand IP-10-stimulated [S]GTPgS binding. The antibody attenuated [S]GTPgS binding in response to IP-10 but not to I-TAC, suggesting that the two chemokines bind differently to R*. Moreover, increased occupancy of R* with a .75-fold increase in I -IP-10 concentration did not increase the I-TAC binding IC50 value, and I-TAC increased the dissociation rate of I-IP-10. From these data, we conclude that the binding of IP-10 and I-TAC to the R* state of hCXCR3 is allotopic. Chemoattractant cytokines (chemokines) stimulate leukocyte chemotaxis by activation of various G protein-coupled receptors. As such, chemokines are important mediators of inflammatory responses. Interferon-inducible 10-kDa protein (IP-10) and monokine induced by human interferon-g (MIG) are CXC chemokines originally characterized as potent chemoattractants for activated T and natural killer cells (Luster and Leder, 1993; Farber, 1997). IP-10 and MIG attract leukocytes through activation of CXCR3 expressed on these cells. Recently, a novel non-ELR (glutamate-leucinearginine) CXC chemokine, interferon-inducible T cell a chemoattractant (I-TAC), was also identified as a potent hCXCR3 agonist in human and mouse (Cole et al., 1998; Widney et al., 2000). Interestingly, there is evidence that I-TAC expression and its regulation differ from that of MIG and IP-10 (Mach et al., 1999), suggesting that these chemokines are more than just redundant ligands for hCXCR3. Cole et al. (1998) used both signaling assays and binding studies with I-I-TAC to examine I-TAC pharmacology at hCXCR3. They found that I-TAC was more potent and efficacious than IP-10 or MIG as a chemoattractant and in stimulating calcium flux and receptor desensitization. Indeed, these findings led them to propose that I-TAC is the dominant ligand for this receptor. Competition bindings with I-I-TAC, human IP-10, and human MIG generated binding profiles that seem non-Michaelean. The authors suggested that these nonclassical binding curves reflected the poor affinity for IP-10 and MIG relative to I-TAC for hCXCR3. The studies presented here elucidate I-TAC and IP-10 binding at hCXCR3 expressed in recombinant or huABBREVIATIONS: IP-10, 10-kDa interferon-inducible protein; GTPgS, guanosine-59-O-(3-thio)triphosphate; I-TAC, interferon-inducible T cell a chemoattractant; MIG, monokine induced by human interferon-g; PBL, peripheral blood lymphocytes; WGA-SPA, wheat germ agglutinin bead-scintillation proximity assay. 0026-895X/01/5904-707–715$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 4 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 622/891372 Mol Pharmacol 59:707–715, 2001 Printed in U.S.A. 707 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from man peripheral blood lymphocytes. Using a variety of pharmacological approaches, we demonstrate that IP-10 and ITAC have vastly different affinities for uncoupled hCXCR3 and are allotopic ligands for coupled hCXCR3. Experimental Procedures Cells and Cell Culture. The cDNA encoding human CXCR3 was generated as described previously (Jenh et al., 1999) and cloned into the mammalian expression vector pME18Sneo, a derivative of the SRa expression vector (Takebe et al., 1988). Interleukin-3-dependent mouse pro-B cells (Ba/F3) were transfected to express hCXCR3 (Ba/ F3-hCXCR3) and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 mg/ml streptomycin, 1 g/ml G418 (Life Technologies, Gaithersburg, MD), and 100 U/ml penicillin, 50 mM b-mercaptoethanol, and 2 ng/ml of recombinant mouse Interleukin-3 (BioSource International, Camarillo, CA). 293EBNA (Epstein Barr Nuclear Ag) cells (Invitrogen, Carlsbad, CA) transfected to express hCXCR3 (293-hCXCR3; Jenh et al., 1999) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 mg/ml streptomycin and 100 U/ml penicillin, 100 mg/ml G418, and 300 mg/ml hygromycin (Roche Molecular Biochemicals, Indianapolis, IN). Human peripheral blood lymphocytes (PBL) were prepared by FicollHypaque centrifugation, depleted of monocytes, (Wahl and Smith, 1991) and stimulated for 2 days with 1 mg/ml phytohemagglutinin (Murex Diagnostics, Dartford, UK) and 100 U/ml interleukin-2 (Sigma, St. Louis, MO) in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin, 1% nonessential amino acids, and 2 mM HEPES. After stimulation, PBL were cultured in above media containing 5% conditioned media (Sigma) for up to 15 days. Cell Membrane Preparation. Ba/F3-hCXCR3 were pelleted and resuspended in lysis buffer containing 10 mM HEPES, pH 7.5, and Complete protease inhibitors (1 tablet/100 ml) (Roche Molecular Biochemicals) at a cell density of 20 3 10 cells/ml. After a 5-min incubation on ice, the cells were transferred to a 4639 cell disruption bomb (Parr Instrument, Moline, IL) and applied with 1500 psi nitrogen for 30 min on ice. Large cellular debris was removed by centrifugation at 1000g. Cell membrane in the supernatant was pelleted at 100,000g. The membrane was resuspended in lysis buffer supplemented with 10% sucrose and stored at 280°C. PBL and 293hCXCR3 membranes were prepared as described previously (Hipkin et al., 1997). Cells were pelleted by centrifugation and incubated in homogenization buffer (10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, pH 7.6) and 1 mM phenylmethylsulfonyl fluoride on ice for 30 min. The cells were then lysed with a Dounce homogenizer using a stirrer type RZR3 polytron homogenizer (Caframo, Wiarton, ON) with 10 to 20 strokes at 900 rpm. The intact cells and nuclei were removed by centrifugation at 500g for 5 min. The cell membranes in the supernatant were then pelleted by centrifugation at 10,000g for 30 min. The membranes were then resuspended in glygly buffer (20 mM glycylglycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2), aliquoted, quick frozen, and stored at 280°C. Protein concentration in membrane preparations was determined using the method of Bradford (1976). Radioligand Bindings. I-I-TAC and carrier-free I-IP-10 (specific activity, 986-2200 and 2200 Ci/mmol, respectively) were obtained from PerkinElmer Life Sciences (Boston, MA). A scintillation proximity assay was used for radioligand competition and saturation binding assays. For each assay point, 0.5 to 2 mg of membrane was preincubated for 1 h at room temperature with 300 mg of wheat germ agglutinin-coated scintillation proximity assay beads (WGA-SPA; Amersham Pharmacia Biotech, Piscataway, NJ) in SPA binding buffer (50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 125 mM NaCl, 0.002% NaN3, 1.0% bovine serum albumin). The beads and membranes were transferred to a 96-well Isoplate (Wallac, Gaithersburg, MD) and incubated at room temperature with I-IP-10 or I-I-TAC and the indicated concentrations of chemokines for 3 h. Where indicated, membranes were incubated in the absence or presence of purified mouse anti-hCXCR3 monoclonal antibody (clone 1/C6/CXCR3; PharMingen International, San Diego, CA) or isotype control antibody (mouse IgG1, k) before addition of ligands and radioligand. Ligand affinities from competition bindings were calculated from binding IC50 values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973). [S]GTPgS Binding. Cell membranes were resuspended in GTPgS binding buffer [20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, and 0.2% (w/v) bovine serum albumin (factor V, lipid free), pH 7.4] and kept on ice. Membranes in 96-well plates (1–2 mg/point, in triplicate) were incubated with 1 mM GDP, 0.3 nM guanosine 59-gS-triphosphate ([S]GTPgS, triethylammonium salt; specific activity, 1250 Ci/mmol; PerkinElmer Life Sciences) in the presence or absence of various ligands for 30 to 60 min at 30°C. The reaction was terminated by placing the plates on ice and filtering the membranes through a UniFilter GF/B filter plate (Packard Instrument Co., Meriden, CT) using a Tomtec 96-well cell harvester (Hamden, CT). The filters and membranes were washed 10 times at room temperature with 20 mM HEPES and 10 mM sodium pyrophosphate. Membranebound [S]GTPgS was measured by liquid scintillation using a TopCount NXT Microplate scintillation and luminescence counter (Packard Instrument Co.). In some experiments, [S]GTPgS bindings were performed, and membranes were preincubated for 60 min at room temperature in the absence or presence of 1C6/CXCR3 or isotype control antibody (see above) before addition of ligands and [S]GTPgS. In these experiments, the bindings were done in SPA binding buffer (as described above) containing 1 mM GDP and 0.3 nM [S]GTPgS. Membrane-bound [S]GTPgS was measured by scintillation proximity assay. Materials. Chemokines were purchased from R & D Systems Inc. (Minneapolis, MN). Nonlinear regression analysis of the data was performed using Prism 2.0b (GraphPad Software, San Diego, CA) All other reagents were of the best grade available and purchased from