1. Role of CXCR3 carboxyl terminus and third intracellular loop in receptor-mediated migration, adhesion and internalization in response to CXCL11
by Michal Dagan-Berger, Rotem Feniger-Barish, Shani Avniel, Hanna Wald, Eithan Galun, Valentin Grabovsky, Ronen Alon, Arnon Nagler, Adit Ben-Baruch, and Amnon Peled
Blood
Volume 107(10):
May 15, 2006
©2006 by American Society of Hematology

2. Truncations and site-directed mutagenesis in the carboxyl terminus and 3i loop of CXCR3.
Truncations and site-directed mutagenesis in the carboxyl terminus and 3i loop of CXCR3. (A) Carboxyl terminus alignment of CXCR1, CXCR2, CXCR3, and CXCR4. (B) 3i loop subdomain of CXCR1, CXCR2, CXCR3, and CXCR4. (C) CXCR3 carboxyl-terminus. The carboxyl-terminal truncations of CXCR3 were generated by introducing stop codons (*) at Ser349, Leu332, or Glu L→A site-directed mutagenesis was generated as described in “Materials and methods.” (D) CXCR3 3i loop subdomain. Site-directed mutagenesis at position 245, replacing serine with alanine, was generated as described in “Materials and methods.” Serine and threonine residues that serve as potential phosphorylation sites are underlined, and the leucine motifs are in bold. The site-directed mutagenesis is in bold italics. Positions are indicated according to Feature Aligner of ExPASy, Swiss-Prot (Swiss Institute of Bioinformatics, Basel, Switzerland).
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology

3. Cell-surface expression of WT and mutated CXCR3 receptors on HEK 293 cells.
Cell-surface expression of WT and mutated CXCR3 receptors on HEK 293 cells. WT and mutant CXCR3 cell-surface expression on stable transfected HEK 293 cell clones was tested by immunostaining with anti-CXCR3 specific antibodies and analyzed by FACS (black line). Immunostaining with IgG1 isotype control antibodies is in gray line. (A) WT CXCR3. (B) 349stopΔ20 C-tail truncated. (C) 332stopΔ37 C-tail truncated. (D) L→A C-tail mutations. (E) S245 to A 3i loop mutation. (F) Double-mutant of 332stopΔ37 C-tail truncated and S245 to A 3i loop mutation. (G) HEK 293 cells. Counts indicate relative cell number. A representative histogram of at least 3 experiments performed is presented. (H) Binding of biotinylated I-TAC (mean florescence intensity [MFI]) at different concentrations (nM) to 293 cells expressing the different mutated or truncated CXCR3.
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology

4. Dose-response of migration of HEK 293 cells expressing WT or mutated CXCR3 receptors toward CXCL11.
Dose-response of migration of HEK 293 cells expressing WT or mutated CXCR3 receptors toward CXCL11. Migration was performed as described in “Materials and methods” in response to various concentrations of CXCL11 (ng/mL). (A) WT CXCR3. (B) 349stopΔ20 C-tail truncated. (C) 332stopΔ37 C-tail truncated. (D) L→A C-tail mutations. (E) S245 to A 3i loop mutation. (F) Double-mutant of 332stopΔ37 C-tail truncated and S245 to A 3i loop mutation. A representative experiment of at least 3 performed is presented. Each value represents the mean ± SD of triplicates of the representative experiment. ANOVA was used to determine the levels of difference between CXCL11 concentrations of 50, 100, 500, and 1000 ng/mL within each CXCR3 type. The differences in number of cells per high-power field (HPF) were statistically significant for all CXCR3 types except for D37/S245A. Therefore, multiple comparisons of number of cells per HPF between these concentrations in each receptor type were done by the Newman-Keuls test. For WT CXCR3, number of cells per HPF at 50 and 100 ng/mL were not statistically different. For the mutant L→A, number of cells/HPF at 50 versus 100 ng/mL and at 500 versus 1000 ng/mL were not statistically different, but these pairs of concentrations were statistically different from each other. For the mutant S245A, number of cells per HPF at 50 versus 100 ng/mL and 500 versus 100 ng/mL were statistically different. ANOVA also showed that the differences in number of cells per HPF were statistically significant for all CXCR3 types at 50 ng/mL CXCL11. Multiple comparisons of each mutant with WT CXCR3 at this concentration were performed by the Dunnett test. Except for S245A, all other mutants were different and statistically significant from the WT.
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology

5. Migration of HEK 293 cells in responses to CXCL11.
Migration of HEK 293 cells in responses to CXCL11. (A) Migration of HEK 293 cells expressing WT CXCR3 receptors toward CXCL11 is PTx dependent. HEK 293-WT CXCR3 cells were preincubated with 100 ng/mL PTx for 2 hours at 37°C and washed prior to assay. Migration was performed as described in “Materials and methods” in response to 50 ng/mL CXCL11. A representative experiment of 3 performed is presented. Each value represents the mean SD of triplicates of the representative experiment. Using ANOVA and the Newman-Keuls test, the difference was significant for PTx/CXCL11 treatment (*P < .05). (B) Migration of HEK 293-CXCR3 was dependent mainly upon the receptor C-tail. Migration of HEK 293 cells expressing the various CXCR3 mutants was performed as described in “Materials and methods” in response to 50 ng/mL CXCL11. Percentage inhibition of migration by each mutant of CXCR3 was calculated as number of migrating cells expressing mutated CXCR3 per HPF versus the number of migrating cells expressing WT CXCR3 per HPF. Each value represents the mean ± SD of inhibition calculated of 3 independent experiments. Using ANOVA, the difference of percentage inhibition between the CXCR3 mutants was significant (*P < .05). Multiple comparisons between the mutants by the Newman-Keuls test showed that only S245A and D37 were different from each other.
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology

6. Expression and function of WT CXCR3 and S245A-mutated CXCR3 in Jurkat, YTS, and RBL hematopoietic cells.
Expression and function of WT CXCR3 and S245A-mutated CXCR3 in Jurkat, YTS, and RBL hematopoietic cells. (A) Cell-surface expression of WT or the S245A mutated CXCR3 in HEK 293, Jurkat, YTS, and RBL cells (black line). Immunostaining with immunoglobulin G1 (IgG1) isotype control antibodies is shown in filled histograms. Cell-surface expression of CXCR3 or the S245A-mutated CXCR3 in cells incubated with 1 mg/mL CXCL11 for 1 hour is also shown (gray line). (B-C) The migration of parental-, CXCR3-, and CXCR3-S245A-expressing YTS (B) and Jurkat cells (C) in response to different concentrations of CXCL11 is shown. Each value represents the mean plus or minus SD of 3 independent experiments.
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology

7. Reduction of CXCR3 cell-surface expression following CXCL11 and PMA stimulation.
Reduction of CXCR3 cell-surface expression following CXCL11 and PMA stimulation. (A) Time course of the reduction of CXCR3 expression in response to CXCL11 and PMA in 293-CXCR3 cells. CXCR3-expressing HEK 293 cells were trypsinized and incubated with either 1 mg/mL CXCL11 or 100 ng/mL PMA for the indicated time points at 37°C. The cells were then washed, immunostained with anti-CXCR3–specific antibodies, and FACS analyzed. Each value represents the mean plus or minus SE of 3 to 4 independent experiments. (B) Effect of PKC inhibitors on ligand- or PMA-induced CXCR3 down-modulation on the surface of 293 cells. 293-CXCR3 cells were trypsinized and incubated in medium alone or with a PKC inhibitor (staurosporin [0.5 μM], GF [5 μM], or rottlerin [5 μM]) for 30 minutes at 37°C, and then challenged with 1μg/mL CXCL11 or 100 ng/mL PMA for 1 hour at 37°C. The cells were then washed, and surface expression of CXCR3 receptors was analyzed. Each value represents the mean ± SE of 4 independent experiments. *P < .05 by ANOVA and Dunnett test for internalization induced by PMA with staurosporin versus without staurosporin, and with GF versus without GF. (C) The CXCR3-expressing HEK 293 cells, WT CXCR3, 349stopΔ20 C-tail truncated, 332stopΔ37 C-tail truncated, and L→A C-tail mutations were trypsinized and incubated with CXCL11 (1 μg/mL) or PMA (100 ng/mL) for 1 hour at 37°C. The cells were then washed, immunostained with anti-CXCR3–specific antibodies, and FACS analyzed. Each value represents the mean ± SE of at least 3 independent experiments. *P < 05 by ANOVA and Dunnett test for D37 CXCL11- and PMA-induced internalization versus WT.
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology

8. Reduction of S245A-mutant CXCR3 receptor expression on the cell surface of HEK 293 cells in response to CXCL11 and PMA. (A) The WT CXCR3, S245 to A 3i loop mutation CXCR3-expressing HEK 293, RBL, Jurkat, and YTS cells, were incubated with CXCL11 (1 mg/mL) f...
Reduction of S245A-mutant CXCR3 receptor expression on the cell surface of HEK 293 cells in response to CXCL11 and PMA. (A) The WT CXCR3, S245 to A 3i loop mutation CXCR3-expressing HEK 293, RBL, Jurkat, and YTS cells, were incubated with CXCL11 (1 mg/mL) for 1 hour at 37°C. The cells were then washed, immunostained with anti-CXCR3–specific antibodies, and FACS analyzed. The reduction of cell-surface expression of CXCR3 is shown. (B) The WT CXCR3, S245 to A 3i loop mutation CXCR3, and the double mutant of 332stopΔ37 C-tail truncated and S245 to A 3i loop mutation expressing HEK 293 were incubated with CXCL11 (1 mg/mL) for 1 hour at 37°C. The cells were then washed, immunostained with anti-CXCR3–specific antibodies, and FACS analyzed. The reduction of HEK 293 cell-surface expression of CXCR3 is shown. *P < .05 by ANOVA and Dunnett test for CXCL11- and PMA-induced reduction in cell-surface expression of D37/S245A versus WT. Data are expressed as the means plus or minus SD of 3 independent experiments.
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology

9. Ca2+ mobilization and F-actin polymerization in response to CXCL11 in HEK 293 cells expressing WT and the S245A CXCR3.
Ca2+ mobilization and F-actin polymerization in response to CXCL11 in HEK 293 cells expressing WT and the S245A CXCR3. (A) Intracellular Ca2+ mobilization mediated by CXCR3 in HEK 293-CXCR3 cells. Fluorescence changes in HEK 293 cells expressing WT and S245A CXCR3 loaded with 10 mM Fura-3 were measured upon stimulation with 1 mg/mL CXCL11 as indicated by arrows and described in “Materials and methods.” (B) F-actin polymerization in HEK 293 cells expressing WT and S245A CXCR3 in response to 1 mg/mL CXCL11 at various time points. F-actin polymerization was monitored by FACS as described in “Materials and methods.”
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology

10. Adhesion of Jurkat cells expressing WT and the S245A CXCR3 to VCAM-1 and FN in response to CXCL11.
Adhesion of Jurkat cells expressing WT and the S245A CXCR3 to VCAM-1 and FN in response to CXCL11. (A) Denatured and intact CXCL11 induced adhesion of Jurkat cells, expressing WT or the S245A CXCR3, to immobilized recombinant human (rh) VCAM-1 are shown. Adhesion of cells is monitored over a period of 1 minute while detachment forces (dyn/cm2) continuously increased. (B) Denatured and intact CXCL11-induced adhesion of Jurkat cells, expressing WT or the S245A CXCR3, to immobilized fibronectin are shown. Adhesion of cells is monitored over a period of 1 minute while detachment forces (dyn/cm2) continuously increased. (C) CXCL11-induced adhesion of Jurkat cells, expressing WT or the S245A CXCR3, to immobilized rhVCAM-1 5 seconds after the detachment force was increased to 5 dyn/cm2.
Michal Dagan-Berger et al. Blood 2006;107:
©2006 by American Society of Hematology