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First published online 3 April 2007
doi: 10.1242/jcs.03437

Journal of Cell Science 120, 1559-1571 (2007)
Published by The Company of Biologists 2007
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RhoB plays an essential role in CXCR2 sorting decisions

Nicole F. Neel1,2, Lynne A. Lapierre1,3, James R. Goldenring1,3,4 and Ann Richmond1,2,*

1 Department of Veterans Affairs, Nashville, TN 37212-2637, USA
2 Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
3 Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
4 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA


Figure 1
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  		Fig. 1. RhoB is activated upon CXCL8 stimulation. Western blot analysis with anti-RhoB antibody of input lysates (Total RhoB) and GST-TRBD bound RhoB (GTP-RhoB) separated by SDS-PAGE. HEK293 cells stably expressing CXCR2 were transiently transfected with Myc-WT RhoB or Myc-Q63L RhoB mutant. Cells transfected with Myc-Q63L RhoB were stimulated with vehicle. Cells transfected with Myc-WT RhoB were stimulated with 100ng/ml EGF for 60 minutes (EGF), vehicle (Unt), or 100 ng/ml CXCL8 for 5 minutes, 30 minutes or 60 minutes. Lysates were incubated with GST-TRBD (rhotekin Rho-binding domain) to isolate GTP-bound RhoB. Data shown are representative from three separate experiments. Induction at 5-minute and 60-minute time points when normalized to total RhoB was 1.6-fold and 1.1-fold, respectively, in the experiment shown here.

 

Figure 2
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  		Fig. 2. RhoB T19N and Q63L mutants impair CXCR2-mediated chemotaxis. (A-C) Boyden chamber assay assessing chemotaxis of HEK293 cells stably expressing CXCR2 and transiently transfected with empty vector, Myc-RhoB T19N or Myc-RhoB Q63L. A twofold reduction in CXCR2-mediated chemotaxis was observed when Myc-RhoB T19N and Myc-RhoB Q63L mutants were expressed. No effect on chemotaxis was observed when Myc-RhoB WT was expressed. CXCR2-mediated chemotaxis of cells expressing Myc-RhoB T19N (A), Myc-RhoB Q63L (B) and Myc-RhoB WT (C). The graphs display numbers of cells from twenty separate fields ± s.e.m., using the 20x objective lens. + and * indicate significant differences (P<0.01 and P<0.005; Student's t-test) of vector-transfected cells versus cells transfected with Myc-RhoB T19N or Myc-RhoB Q63L. Data shown are representative from three separate experiments.

 

Figure 3
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  		Fig. 3. Expression of dominant-negative (T19N) RhoB alters trafficking of CXCR2 following 3 hours of CXCL8 stimulation. Confocal images of immunofluorescence staining of HEK293 cells stably expressing CXCR2. (A,B) Staining of CXCR2 and LAMP-1 in cells transfected with vector (A) or Myc-RhoB T19N (B) and stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 3 hours. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is LAMP-1, blue is Myc-RhoB T19N. Bars, 10 µm. (C) Quantification of colocalization of CXCR2 with LAMP-1 in cells transfected with vector, Myc-RhoB WT or Myc-RhoB T19N. Values shown are the mean ± s.e.m. *P<0.005, significant differences (Student's t-test) of vector or Myc-RhoB WT versus Myc-RhoB T19N transfected cells. (D,E) CXCR2 and Rab11a staining in cells transfected with empty vector (D) or Myc-RhoB T19N (E) stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 3 hours. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is Rab11a, blue is Myc-RhoB T19N. Bars, 10 µm. (F) Quantification of colocalization of CXCR2 with Rab11a in cells transfected with vector, Myc-RhoB WT, or Myc-RhoB T19N. Values shown are the mean ± s.e.m. *P<=0.05, significant differences (Student's t-test) of vector or cells transfected with Myc-RhoB WT versus cells transfected with Myc-RhoB T19N. Quantification of the percentage of CXCR2 colocalized with LAMP-1 or Rab11a in 20 fields was performed using the MetaMorph Imaging system (Universal Imaging). Images were processed using Photoshop (Adobe Systems). Data shown are representative from three separate experiments.

 

Figure 4
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  		Fig. 4. Expression of Myc-RhoB T19N does not impair CXCR2 recycling. 125I-CXCL8 binding in HEK293 cells stably expressing CXCR2 and transfected with empty vector or Myc-RhoB T19N. Cells were pretreated with 20 µg/ml cycloheximide for 30 minutes and placed in the presence of 20 µg/ml cycloheximide throughout the experiment. Cells were stimulated with vehicle (total binding) or 100 ng/ml CXCL8 for 30 minutes, CXCL8 was removed, and cells were allowed to recover for 0 minutes, 30 minutes or 60 minutes. Cells were incubated with 0.1 nM 125I-CXCL8 (specific activity =2200 Ci/mmol) for 2 hours, washed to remove non-specific binding, and subjected to {gamma}-counting as described in Materials and Methods. Values represent three independent experiments and are shown as percent of total binding ± s.e.m. Data shown are representative from three separate experiments.

 

Figure 5
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  		Fig. 5. Expression of Myc-RhoB T19N impairs CXCL8-induced CXCR2 degradation following 3 hours of CXCL8 stimulation. Representative western blot analysis of CXCR2 expression in cells transfected with vector or Myc-RhoB T19N in the presence of 20 µg/ml cycloheximide following stimulation with vehicle (Unt) or 100 ng/ml CXCL8 for 30 minutes, 180 minutes or 360 minutes. Actin is shown as a control to monitor for equal loading of protein. Data shown are representative from three separate experiments.

 

Figure 6
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  		Fig. 6. Expression of GTPase-deficient RhoB (Q63L) mutant alters trafficking of CXCR2 following 30 minutes of CXCL8 stimulation. Confocal images of immunofluorescence-stained HEK293 cells stably expressing CXCR2. (A,B) CXCR2 and Rab11a staining in cells transfected with empty vector (A) or Myc-RhoB Q63L (B) stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is Rab11a, blue is Myc-RhoB Q63L. Bars, 10 µm. (C) Quantification of colocalization of CXCR2 with Rab11a in cells transfected with vector, Myc-RhoB WT or Myc-RhoB Q63L. Values shown are the mean ± s.e.m. *P<0.05, significant differences (Student's t-test) of vector or cells transfected with Myc-RhoB WT versus cells transfected with Myc-RhoB Q63L. (D,E) CXCR2 staining and EGFP-Rab7 in cells transfected with empty vector (D) and Myc-RhoB Q63L (E) and stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is EGFP-Rab7, blue is Myc-RhoB Q63L. Bars, 10 µm. (F) Quantification of colocalization of CXCR2 with EGFP-Rab7. Values shown are the mean ± s.e.m. *P<0.05, significant differences (Student's t-test) of cells transfected with Myc-RhoB Q63L versus vector-transfected cells. Quantification of the percentage of CXCR2 colocalized with Rab11a or EGFP-Rab7 in 20 fields was performed using the MetaMorph Imaging system (Universal Imaging). Images were processed using Photoshop (Adobe Systems). Data shown are representative from three separate experiments.

 

Figure 7
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  		Fig. 7. Expression of Myc-RhoB Q63L does not impair CXCR2 recycling. 125I-CXCL8 binding in HEK293 cells stably expressing CXCR2 and transfected with empty vector or Myc-RhoB Q63L. Cells were pretreated with 20 µg/ml cycloheximide for 30 minutes and placed in the presence of 20 µg/ml cycloheximide throughout the experiment. Cells were stimulated with vehicle (total binding), 100 ng/ml CXCL8 for 30 minutes, CXCL8 was removed, and cells were allowed to recover for 0 minutes, 30 minutes or 60 minutes. Cells were incubated with 0.1 nM 125I-CXCL8 (specific activity =2200 Ci/mmol) for 2 hours, washed to remove non-specific binding, and subjected to {gamma}-counting as described in Materials and Methods. Values represent three independent experiments and are shown as percent of total binding ± s.e.m. Data shown are representative from three separate experiments.

 

Figure 8
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  		Fig. 8. Expression of Myc-RhoB Q63L impairs CXCL8-induced CXCR2 degradation and does not result in colocalization with lysosomal markers. (A) Western blot analysis of CXCR2 expression in cells transfected with vector or Myc-RhoB Q63L in the presence of 20 µg/ml cycloheximide following stimulation with vehicle (Unt) or 100 ng/ml CXCL8 for 30 minutes, 180 minutes or 360 minutes. Actin is shown as a control to monitor for equal loading of protein. (B) Confocal images from three independent experiments of immunofluorescence-stained HEK293 cells stably expressing CXCR2 and transiently transfected with Myc-RhoB Q63L. Transfected cells were identified by staining with anti-Myc antibody. CXCR2 and LAMP-1 (top panels) or CD63 (bottom panels) staining in cells stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes. Overlay images are pseudocolored; red is CXCR2, green is LAMP-1 or CD63, blue is Myc-RhoB Q63L. Images were processed using Photoshop (Adobe Systems). Bars, 10 µm. Data shown are representative from three separate experiments.

 

Figure 9
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  		Fig. 9. Expression of Q63L RhoB results in colocalization of CXCR2 with Rab4 and mannose-6-phosphate receptor (MPR). Confocal images of immunofluorescence-stained HEK293 cells stably expressing CXCR2. (A,B) CXCR2 and Rab4 staining in cells transfected with empty vector (A) or Myc-RhoB Q63L (B) and stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is Rab4, blue is Myc-RhoB Q63L. Bars, 10 µm. (C) Quantification of colocalization of CXCR2 with Rab4. Values shown are the mean ± s.e.m. *P<0.0005, significant differences (Student's t-test) of cells transfected with Myc-RhoB Q63L versus vector-transfected cells. (D,E) CXCR2 and MPR staining in cells transfected with empty vector (D) or Myc-RhoB Q63L (E) and stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is MPR, blue is Myc-RhoB Q63L. Bars, 10 µm. (F) Quantification of colocalization of CXCR2 with MPR. Values shown are the mean ± s.e.m. *P<0.0005, significant differences (Student's t-test) of cells transfected with Myc-RhoB Q63L versus vector-transfected cells. Quantification of the percentage of CXCR2 colocalized with Rab4 or MPR in 20 fields was performed using the MetaMorph Imaging system (Universal Imaging). Images were processed using Photoshop (Adobe Systems). Data shown are representative from three separate experiments.

 

Figure 10
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  		Fig. 10. Schematic representation of the effects the T19N and Q63L RhoB mutants have on CXCR2 trafficking. After 30 minutes of CXCL8 stimulation CXCR2 traffics to the Rab11a perinuclear recycling compartment and recycles back to the plasma membrane. CXCR2 traffics to the lysosome and is degraded after 3 hours of CXCL8 stimulation. Expression of T19N RhoB mutant results in constitutive recycling of CXCR2 through the Rab11a perinuclear recycling compartment after 3 hours of CXCL8 stimulation (blue arrows). Expression of the Q63L RhoB mutant results in the inability of CXCR2 to enter the Rab11a perinuclear recycling compartment after 30 minutes of CXCL8 stimulation and leads to CXCR2 recycling to the plasma membrane by alternative pathways (red arrows).

 




© The Company of Biologists Ltd 2007