CXCR3 and Heparin Binding Sites of the Chemokine IP-10 (CXCL10)
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m212077200
ISSN1083-351X
AutoresGabriele Campanella, Elizabeth M.J. Lee, Jieti Sun, Andrew D. Luster,
Tópico(s)Immune Response and Inflammation
ResumoThe chemokine IP-10 (interferon-inducible protein of 10 kDa, 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 Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, 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 Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping. The chemokine IP-10 (interferon-inducible protein of 10 kDa, 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 Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, 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 Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping. interferon inducible protein of 10 kDa lipopolysaccharide glycosaminoglycan CXC chemokine receptor interferon-inducible T cell-α chemoattractant monokine induced by γ-interferon murine human platelet factor 4 regulated on activation normal T cell expressed and secreted Chinese hamster ovary K71E/R72Q-K74Q/R75E R22A K71E/R72Q-K74Q/R75E fold induction fetal calf serum wild type fluorescence-activated cell sorter interleukin Interferon-inducible protein, 10 kDa (IP-10)1 belongs to the superfamily of chemokines (chemoattractant cytokines), that are involved in the activation and recruitment of leukocytes, as well as non-hematopoietic cells (1Luster A.D. N. Engl. J. Med. 1998; 338: 436-445Crossref PubMed Scopus (3272) Google Scholar). 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 (2Luster A.D. Unkeless J.C. Ravetch J.V. Nature. 1985; 315: 672-676Crossref PubMed Scopus (761) Google Scholar) and has since been shown to be a potent chemoattractant of activated T cells (3Taub D.D. Lloyd A.R. Conlon K. Wang J.M. Ortaldo J.R. Harada A. Matsushima K. Kelvin D.J. Oppenheim J.J. J. Exp. Med. 1993; 177: 1809-1814Crossref PubMed Scopus (693) Google Scholar). It is expressed constitutively at low levels in thymic, splenic, and lymph node stroma (4Gattass C.R. King L.B. Luster A.D. Ashwell J.D. J. Exp. 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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 (14Hancock W.W. Gao W. Csizmadia V. Faia K.L. Shemmeri N. Luster A.D. J. Exp. Med. 2001; 193: 975-980Crossref PubMed Scopus (362) Google Scholar, 15Dufour J.H. Dziejman M. Liu M.T. Leung J.H. Lane T.E. Luster A.D. J. Immunol. 2002; 168: 3195-3204Crossref PubMed Scopus (879) Google Scholar, 16Khan I.A. MacLean J.A. Lee F.S. Casciotti L. DeHaan E. Schwartzman J.D. Luster A.D. Immunity. 2000; 12: 483-494Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 17Zhang Z. Kaptanoglu L. Haddad W. Ivancic D. Alnadjim Z. Hurst S. Tishler D. Luster A.D. Barrett T.A. Fryer J. J. Immunol. 2002; 168: 3205-3212Crossref PubMed Scopus (56) Google Scholar). IP-10 has also been shown to have angiostatic (18Angiolillo A.L. Sgadari C. Taub D.D. Liao F. Farber J.M. Maheshwari S. Kleinman H.K. Reaman G.H. Tosato G. J. Exp. 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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) (21Luster A.D. Greenberg S.M. Leder P. J. Exp. Med. 1995; 182: 219-231Crossref PubMed Scopus (420) Google Scholar, 24Cole K.E. Strick C.A. Paradis T.J. Ogborne K.T. Loetscher M. Gladue R.P. Lin W. Boyd J.G. Moser B. Wood D.E. Sahagan B.G. Neote K. J. Exp. Med. 1998; 187: 2009-2021Crossref PubMed Scopus (734) Google Scholar). While our understanding of the biological activities of the CXCR3 ligands has increased, relatively little is known about the importance of their interaction with GAGs. It has been postulated that GAGs on the cell bearing the 7 transmembrane receptors facilitate chemokine binding to their high affinity receptor (25Hoogewerf A.J. Kuschert G.S. Proudfoot A.E. Borlat F. Clark-Lewis I. Power C.A. Wells T.N. Biochemistry. 1997; 36: 13570-13578Crossref PubMed Scopus (438) Google Scholar), while GAGs on endothelial cells and in the extracellular matrix might be important for retaining chemokines close to their site of secretion (26Tanaka Y. Adams D.H. Hubscher S. Hirano H. Siebenlist U. Shaw S. Nature. 1993; 361: 79-82Crossref PubMed Scopus (845) Google Scholar). Furthermore, it has been suggested that GAGs might also be involved in the signaling of chemokines, as has been recently shown for RANTES (CCL5) (27Chang T.L. Gordon C.J. Roscic-Mrkic B. Power C. Proudfoot A.E. Moore J.P. Trkola A. J. Virol. 2002; 76: 2245-2254Crossref PubMed Scopus (41) Google Scholar) and PF4 (platelet factor 4, CXCL4) (28Fleischer J. Grage-Griebenow E. Kasper B. Heine H. Ernst M. Brandt E. Flad H.D. Petersen F. J. Immunol. 2002; 169: 770-777Crossref PubMed Scopus (92) Google Scholar). GAGs have also been shown to be important for the anti-HIV effect of SDF-1α (stromal cell-derived factor-1α, CXCL12) (29Valenzuela-Fernandez A. Palanche T. Amara A. Magerus A. Altmeyer R. Delaunay T. Virelizier J.L. Baleux F. Galzi J.L. Arenzana-Seisdedos F. J. Biol. Chem. 2001; 276: 26550-26558Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and RANTES (30Oravecz T. Pall M. Wang J. Roderiquez G. Ditto M. Norcross M.A. J. Immunol. 1997; 159: 4587-4592PubMed Google Scholar, 31Wagner L. Yang O.O. Garcia-Zepeda E.A. Ge Y. Kalams S.A. Walker B.D. Pasternack M.S. Luster A.D. Nature. 1998; 391: 908-911Crossref PubMed Scopus (14) Google Scholar). In addition, GAGs might also be important for chemokine activity on non-hematopoietic 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 the biological activity of IP-10. We found that the N-terminal residue Arg-8 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. Recombinant murine IP-10 was obtained from Peprotech (Rocky Hill, NJ), 125I-labeled IP-10 (human) was purchased from PerkinElmer Life Sciences. Endotoxin testing reagents were from Charles River Laboratories, Wilmington, MA. BCA reagents were from Pierce. All other materials were of biological grade and purchased either from Sigma or Fisher. 300–19 cells were maintained in complete RPMI, 107 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) (32Esko J.D. Rostand K.S. Weinke J.L. Science. 1988; 241: 1092-1096Crossref PubMed Scopus (174) Google Scholar, 33Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (489) Google Scholar), were grown in F12K media supplemented with 107 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, 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. 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 (Clontech) or the QuikChange 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 Escherichia coli strain. Cultures were grown with 40 ॖg/ml kanamycin and 34 ॖg/ml chloramphenicol at 37 °C with shaking up to anA600 of 0.5 before inducing protein expression by the addition of 0.2 mmisopropyl-औ-d-thiogalactopyranoside. Cells were harvested 4 h after induction, pelleted, and resuspended in lysis buffer containing 0.1 mm Tris-HCl buffer, pH 8.5, 1 mmdithiothreitol, 5 mm benzamidine-HCl, 0.1 mmphenylmethylsulfonyl fluoride, 16 mm MgCl2, and 20 mg/liter 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, 0.1m 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 (Amersham Biosciences). The fractions containing monomeric IP-10 were pooled. The protein was renatured by dropwise addition to 0.1m Tris-HCl buffer, pH 8.5, containing 1 mmoxidized 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 (Amersham Biosciences) equilibrated in 50 mm sodium acetate, pH 4.5, and was eluted with a linear gradient of 0–1007 2 m NaCl in 50 mmsodium acetate, pH 4.5. Fractions containing IP-10 were dialyzed against 50 mm sodium phosphate buffer, 1.7 mammonium sulfate, pH 7.0, and loaded on a 1-ml Resource (Phenyl-Sepharose) column equilibrated in the same buffer and eluted with a 1.7–0.0 m ammonium sulfate gradient. Fractions containing pure IP-10 were identified by SDS-PAGE (4–207 Tris-HCl gels, Bio-Rad), pooled, and dialyzed twice against 17 acetic acid and once against 0.17 trifluoroacetic acid prior to lyophilization. Protein concentration was determined with a combination of BCA assay (Pierce), SDS-PAGE, and enzyme-linked immunosorbent assay. Selected purified mutants were analyzed for the presence of LPS by the limulus amebocyte assay (Charles River Laboratories). To verify the correct folding of mutants with decreased in vitroactivity, near UV CD spectra were run on an Aviv 62DS CD spectrometer at 20 °C. 20 ॖg of IP-10 protein were loaded on a 1-ml Heparin HiTrap orS-Sepharose FF (cationic exchange) column (both fromAmersham Biosciences) equilibrated in 50 mm Tris, pH 7.5, on an AKTA machine (Amersham Biosciences). 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. Binding assays were performed in 96-well tissue culture plates in a total volume of 150 ॖl of binding buffer (0.57 BSA, 50 mm HEPES, pH 7.5, 5 mmMgCl2, 1 mm CaCl2). 300–19/hCXCR3 cells (4 × 105/well) or CHO/mCXCR3 cells (3 × 105/well) were incubated with 0.04 nm125I-labeled IP-10 (PerkinElmer Life Sciences, human IP-10), and increasing concentration of IP-10 mutants (5 × 10−6 to 500 nm). After 90 min at room temperature with shaking the cells were transferred to 96-well filter plates (Millipore) previously soaked in 0.37 polyethylenimine and washed four times with 200 ॖl of binding buffer supplemented with 0.5m NaCl. Radioactivity was counted after addition of scintillation fluid in a Microbeta counter (Wallac). The data were analyzed with GraFit (34Leatherbarrow R. GraFit, Version 3.0. Erithacus Software Ltd., Staines, UK1992Google Scholar). Each experiment was performed in duplicate and repeated at least twice. Chemokine dilutions in RPMI media supplemented with 17 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 5 × 105 cells/ml, and 50 ॖl of cells were added on top of the membrane (5-ॖm pore size, polycarbonate filters). The chemotaxis plate was incubated at 37 °C for 4 h 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. 300–19 or CHO (wt or GAG-deficient) cells expressing mCXCR3 were resuspended in their growth media with 17 FCS at 1 × 107 cells/ml and loaded with 5.0 ॖm acetoxymethyl ester of fura-2 (Molecular Probes, Eugene, OR) for 30 min at 37 °C in the dark. Cells were washed and resuspended at 5 × 106 cells/ml in calcium flux buffer (145 mm NaCl, 4 mm KCl, 1 mmNaHPO4, 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 and 380 nm and are presented as the relative ratio of fluorescence at 340/380 nm. Fold induction (FI) of each stimulation was calculated by dividing the peak fluorescence ratio after stimulation by the peak fluorescence ratio during the 10 s before stimulation. Each experiment was repeated at least twice. Internalization of mCXCR3 was measured as previously described (35Sauty A. Colvin R.A. Wagner L. Rochat S. Spertini F. Luster A.D. J. Immunol. 2001; 167: 7084-7093Crossref PubMed Scopus (133) Google Scholar). Briefly, 300–19/mCXCR3 were resuspended in complete RPMI, 107 FCS, and various concentrations 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 phycoerythrin-conjugated secondary antibody (Caltag, Burlingame, CA) and analyzed by FACS. Residues of 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 (36Hebert C.A. Vitangcol R.V. Baker J.B. J. Biol. 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Chem. 2001; 276: 10620-10626Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 41Ottersbach K. Graham G.J. Biochem. J. 2001; 354: 447-453Crossref PubMed Scopus (15) Google Scholar), and a set of basic residues at the C terminus, where the GAG binding site of IL-8 has been located (42Kuschert G.S. Hoogewerf A.J. Proudfoot A.E. Chung C.W. Cooke R.M. Hubbard R.E. Wells T.N. Sanderson P.N. Biochemistry. 1998; 37: 11193-11201Crossref PubMed Scopus (166) Google Scholar). Based on the results of the first series of mutants, another six mutants were created. Residues of IP-10 mutated in this study are indicated with anarrow in Fig. 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 957 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 with 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 presence of endotoxin using a Limulus Amebocyte Lysate assay at a 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 superimposable with the spectrum of wt IP-10, apart from mutant E40A, which displayed a clear shift. Mutant E40A was therefore excluded from the results. The ability of the IP-10 mutants to bind heparin was analyzed by heparin affinity chromatography. Wt IP-10 eluted at a concentration of 0.96m NaCl (Fig. 2). Of the first set of 14 mutants successfully expressed, 10 eluted at salt concentrations similar to the wt protein (difference of less than 0.10m 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 Arg-22 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 with 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 with the difference in elution from the heparin column confirmed that the residue Arg-22 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 Arg-22 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 (43Mayo K.H. Ilyina E. Roongta V. Dundas M. Joseph J. Lai C.K. Maione T. Daly T.J. Biochem. J. 1995; 312: 357-365Crossref PubMed Scopus (101) Google Scholar), to glutamic acid and glutamine yielding the mutant K71E/R72Q- K74Q/R75E (named "C-t mut"). This C-terminal mutant was also combined with the R22A mutation (C-tR22A). Mutation of Arg-22 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 K74A/R75A had only a moderate effect on heparin binding, the combination of R22A with R75E still reduced the heparin binding. Similarly, K71E/R72Q-K74Q/R75E 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. In a competitive binding assay using 300–19/hCXCR3 cells, wt IP-10 competed for the binding of 40 pm125I-labeled human IP-10 with an IC50 value of 0.11 nm (Fig.3), in agreement with publishedKd values (44Cox M.A. Jenh C.H. Gonsiorek W. Fine J. Narula S.K. Zavodny P.J. Hipkin R.W. Mol. Pharmacol. 2001; 59: 707-715Crossref PubMed Scopus (109) Google Scholar). Eight of the mutants (I12A, E28A, S33A, T44A, K62A, K66A, K71A/R72A, K74A/R75A) had IC50values 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-, 4-, and 2-fold lower receptor binding affinity, respectively. K46A/K47A, which had similarly reduced heparin binding, had a much more reduced CXCR3 binding affinity. The second set of mutants (R22E, R20A/R22A, R22A/K47A, R22A/R75E), 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. All mutants were tested at concentrations of 200 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, K71A/R72A, K74A/R75A) 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, R22A/K47A, R22AR75E, C-t mut), all displayed strongly reduced ability to cause calcium mobilization even at 1000 ng/ml. 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, R22A/K47A, R22A/R75E 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. 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 687 internalization at 10 ng/ml and 927 at 1000 ng/ml (Table I). As seen for receptor binding and chemotaxis, mutant R22A was slight
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