Article Figures & Data
Figures
- FIGURE 1.
Internalization of CXCR3 by PBMCs and L1.2 transfectants. A and B, Dose-dependent nature and the respective kinetics of ligand-induced CXCR3 internalization in PBMCs as determined by flow cytometry using a specific anti-CXCR3 mAb. The ligands CXCL11 (▪), CXCL10 (•) and CXCL9 (▴) are shown. C and D, The levels of CXCR3 internalization in PBMCs and L1.2 transfectants, respectively, following incubation with 50 nM CXCL11 after pretreatment in the presence or absence of 0.4 M sucrose, 50 μM monensin, 5 μg/ml filipin, and 50 μg/ml nystatin. Cell surface CXCR3 levels were measured as described and the untreated control (□) is shown. ∗∗∗, p < 0.001 compared with CXCL11 treatment alone. E, The effect of filipin pretreatment on the specific binding of 125I-CXCL11 to L1.2 CXCR3 transfectants. Data represent the mean ± SEM of at least three different experiments.
- FIGURE 2.
Neither clathrin nor arrestins are required for the CXCL11-induced internalization of CXCR3. A–D, CXCL11-induced internalization of CXCR3 in purified T lymphocytes as deduced by flow cytometry following nucleofection in the presence or absence (mock) of a plasmid containing a dominant negative GFP-tagged DIII construct that inhibits clathrin-coated pit assembly. Nucleofection was conducted 24 h before the induction of CXCR3 internalization with 50 nM CXCL11. The percentage of cells is shown in each quadrant and the experiment shown is representative of three separate experiments. E, The relative internalization of CXCR3 in WT MEF cells (□) and β-arrestin 2- or β-arrestin 3-deficient MEF cells (▪), transiently transfected with CXCR3, following incubation with 50 nM CXCL11 or buffer alone. Data represent the mean ± SEM of four different experiments. F, The relative internalization of CXCR3 in response to 50 nM CXCL11 or buffer alone in a stable L1.2 transfectant cell line following transfection 24 h previously with either plasmid pcDNA3 (mock) or pcDNA3 containing the V53D and V54D dominant negative mutants of β-arrestin 1 and β-arrestin 2. Data represent the mean ± SEM of three different experiments ∗∗, p < 0.01 and ∗∗∗, p < 0.001 compared with buffer-treated controls.
- FIGURE 3.
CXCR3 cell surface replenishment is dependent upon de novo protein synthesis. Activated PBMCs (A), L1.2 CXCR3 transfectants (B), and L1.2 CXCR6 transfectants (C) were incubated for 1 h in serum-free medium in the presence (▪) or absence (○) of 10 μg/ml cycloheximide. Following incubation with CXCL11 (PBMCs and CXCR3 transfectants) or CXCL16 (CXCR6 transfectants), cells were further incubated with cycloheximide and samples taken at the indicated time points for the determination of cell surface expression of CXCR3 by flow cytometry. Data represent the mean ± SEM of at least three independent experiments. ∗∗, p < 0.01 and ∗∗∗, p < 0.001 compared with cycloheximide-treated cells.
- FIGURE 4.
Cell surface expression of CXCR3 is dependant upon functional ER and Golgi. Activated PBMCs (A) and L1.2 CXCR3 cells (B) were incubated with 50 nM CXCL11 for 30 min, washed three times with serum-free medium, and then incubated in serum-free medium for up to 3 h in the presence or absence of actinomycin D (5 μM), brefeldin A (5 μM), and bafilomycin A1 (100 nM). Cell surface CXCR3 was assessed by flow cytometry. The extent of CXCR3 internalization following the initial 30 min incubation with CXCL11 (□) and CXCR3 cell surface level recovery (▪) of an untreated control after 3 h are presented. Data represent the mean ± SEM of at least three independent experiments. ∗∗∗, p < 0.001 compared with untreated control.
- FIGURE 5.
The effects of C-terminal mutation upon CXCR3 function. A, The amino acid identity of the C-terminal CXCR3 mutants analyzed. B, The relative expression levels of the constructs following the transient transfection of L1.2 cells, compared with cell surface staining observed with the WT CXCR3 construct. ∗, p < 0.001. C, The relative chemotactic responses to CXCL11 of the same CXCR3 mutants. D, depicts the internalization of the CXCR3 mutants in response to increasing concentrations of CXCL11. Comparisons were made with untreated transfectants in each case.∗, p < 0.001 and ∗∗∗, p < 0.01 compared with controls. E, Constitutive internalization in the absence of ligand of both WT CXCR3 and the Δ4 construct over a 6-h period following treatment with cycloheximide to inhibit de novo synthesis of receptor. All data represent the mean ± SEM of at least three experiments.
- FIGURE 6.
Internalized CXCR3 traffics to late endosomes. T lymphocytes were processed for confocal microscopy either in the absence of treatment (A–C) or following incubation with CXCL11 for 15 min (D–F) or 60 min (G–I). Following processing, T lymphocytes were stained with Abs directed against CXCR3 (green) and LAMP-1 (red) before analysis by confocal microscopy. A, D, and G show the CXCR3 signal. B, E, and H show the LAMP-1 signal. C, F, and I show the two signals overlaid. Scale bar represents 50 μm. F, Colocalization of CXCR3 and LAMP-1 signals in cells is highlighted (arrowheads). Data shown are representative of at least three independent experiments.
- FIGURE 7.
CXCR3 is degraded following constitutive or CXCL11-induced internalization. L1.2 cells transiently transfected with plasmids encoding CXCR3 were preincubated for 30 min at 37°C with 10 μg/ml cycloheximide and an aliquot was reserved (0 h time point). Incubation of the remaining cells was then allowed to proceed in the presence or absence of 50 nM CXCL11 for 3 h. Cell lysates were generated and analyzed by Western blotting using anti-HA mAb (top). Blots were subsequently stripped and reprobed with an anti-α-tubulin (aT) Ab as a loading control (bottom). A, Both constitutive (untreated) and CXCL11-induced degradation of WT CXCR3 over the 3-h time period as deduced by a loss of immunoreactivity. B, The effects of preincubation of either 40 μM MG132 or 200 μM chloroquine on CXCL11-induced degradation. C, The CXCL11-induced degradation of WT CXCR3 and C-terminal CXCR3 mutants. Data shown are from one experiment representative of three different experiments.
- FIGURE 8.
CXCR3 function in T lymphocytes is strictly controlled. A–F, A series of experiments to compare CXCR3 expression and function in freshly isolated T lymphocytes (day 0) and T lymphocytes cultured for 10 days in medium supplemented with IL-2 and Con A (day 10). A, The relative expression levels of CXCR3 on T lymphocytes as deduced by flow cytometry using an anti-CXCR3 Ab. ∗∗, p < 0.01. B–D, The relative chemotactic responses of T lymphocytes to increasing concentrations of CXCL9, CXCL10, and CXCL11. E, The relative induction of CXCR3 internalization on freshly isolated T lymphocytes as deduced by flow cytometry, following incubation with 50 nM of CXCL9, CXCL10, or CXCL11. ∗, p < 0.05 compared with controls. F, The relative levels of specific binding of 0.1 nM 125I-CXCL10 and 125I-CXCL11 by T lymphocytes. All data represent the mean ± SEM of at least three experiments.
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