Figure 1
Figure 1. Rac1 inhibition causes a decrease of CXCR4 surface signal and Rac1 associates with CXCR4. (A) Schematic representation of the Rho-like GTPase C–terminal peptides fused to a protein transduction domain as used in this study. (B) HL60 cells were incubated with the Rac1 or CDC42 inhibitory peptides and CXCR4 expression was detected by flow cytometry using the fluorescently labeled mAb 12G5 or 44717. The panels show a histogram overlay (left) and bar graphs (middle and right) representing the CXCR4 surface signal. Histogram: thin solid line: isotype control mAb, long dashed line: untreated, thick solid line: control peptide, dashed line: CDC42 C–terminal peptide, and dotted line: Rac1 C–terminal peptide. The CXCR4 surface signal in the untreated condition and control peptide treated condition completely overlap (n = 3). (C) HL60 cells were incubated with the Rac1 or CDC42 inhibitory peptides and CXCR7, CCR5, and LFA-1 surface signal were measured by flow cytometry (n = 3). (D) U937 cells were treated with 50μM NSC23766 and the CXCR4 (detected by mAbs 12G5 and 44717), and CXCR7 surface signals were measured by flow cytometry (n = 3). (E) CD34+ cells isolated from CB were treated with NSC23766 and the CXCR4 signal (detected by mAb 12G5 or 44717) was measured by flow cytometry (n = 5). (F) HEK293T cells exogenously expressing CXCR4-GFP were treated with the Rac1 (mutant) C–terminal peptides or the CDC42 C–terminal peptide and the CXCR4 surface signal (detected by mAb 12G5) was measured on the GFP-positive cells by flow cytometry (n = 3). (G) Pull-down (PD) experiment was performed using lysates from HeLa cells exogenously expressing HA-CXCR4 with beads only, a control peptide, wild-type and mutant Rac1 C–terminal peptides, the Rac1 17-32 ED peptide and the CDC42 C–terminal peptide. Association of CXCR4 was detected by immunoblotting (IB) with an HA-specific monoclonal antibody (representative example of 2 independent experiments). (H) Immunoprecipitation assays were performed in HeLa cells transfected with an empty vector (pcDNA3) or with HA-CXCR4 and cotransfected with myc-Rac1Q61L or myc-Rac1T17N. Immunoprecipitation of the receptor was performed using an HA-specific antibody or an IgG control antibody and immunoblotting was performed with an HA-specific or a myc-specific antibody (representative example of 2 independent experiments). ED indicates effector domain of Rac1; HV, hypervariable domain of Rac1; PTD, protein transduction domain; Rac1 PPP→AAA, Rac1 RKR→AAA, Rac1 C–terminal peptide mutants where the 3 prolines, or RKR sequence were replaced by alanine residues, respectively; Rac 17-32, effector domain; TCL, total cell lysates; and EV, empty vector. Bars show the median (B-C) and the mean (D-F) fluorescence intensity determined by flow cytometry and expressed as percentage ± SEM compared with untreated or to control conditions (*P < .05, **P < .01).

Rac1 inhibition causes a decrease of CXCR4 surface signal and Rac1 associates with CXCR4. (A) Schematic representation of the Rho-like GTPase C–terminal peptides fused to a protein transduction domain as used in this study. (B) HL60 cells were incubated with the Rac1 or CDC42 inhibitory peptides and CXCR4 expression was detected by flow cytometry using the fluorescently labeled mAb 12G5 or 44717. The panels show a histogram overlay (left) and bar graphs (middle and right) representing the CXCR4 surface signal. Histogram: thin solid line: isotype control mAb, long dashed line: untreated, thick solid line: control peptide, dashed line: CDC42 C–terminal peptide, and dotted line: Rac1 C–terminal peptide. The CXCR4 surface signal in the untreated condition and control peptide treated condition completely overlap (n = 3). (C) HL60 cells were incubated with the Rac1 or CDC42 inhibitory peptides and CXCR7, CCR5, and LFA-1 surface signal were measured by flow cytometry (n = 3). (D) U937 cells were treated with 50μM NSC23766 and the CXCR4 (detected by mAbs 12G5 and 44717), and CXCR7 surface signals were measured by flow cytometry (n = 3). (E) CD34+ cells isolated from CB were treated with NSC23766 and the CXCR4 signal (detected by mAb 12G5 or 44717) was measured by flow cytometry (n = 5). (F) HEK293T cells exogenously expressing CXCR4-GFP were treated with the Rac1 (mutant) C–terminal peptides or the CDC42 C–terminal peptide and the CXCR4 surface signal (detected by mAb 12G5) was measured on the GFP-positive cells by flow cytometry (n = 3). (G) Pull-down (PD) experiment was performed using lysates from HeLa cells exogenously expressing HA-CXCR4 with beads only, a control peptide, wild-type and mutant Rac1 C–terminal peptides, the Rac1 17-32 ED peptide and the CDC42 C–terminal peptide. Association of CXCR4 was detected by immunoblotting (IB) with an HA-specific monoclonal antibody (representative example of 2 independent experiments). (H) Immunoprecipitation assays were performed in HeLa cells transfected with an empty vector (pcDNA3) or with HA-CXCR4 and cotransfected with myc-Rac1Q61L or myc-Rac1T17N. Immunoprecipitation of the receptor was performed using an HA-specific antibody or an IgG control antibody and immunoblotting was performed with an HA-specific or a myc-specific antibody (representative example of 2 independent experiments). ED indicates effector domain of Rac1; HV, hypervariable domain of Rac1; PTD, protein transduction domain; Rac1 PPP→AAA, Rac1 RKR→AAA, Rac1 C–terminal peptide mutants where the 3 prolines, or RKR sequence were replaced by alanine residues, respectively; Rac 17-32, effector domain; TCL, total cell lysates; and EV, empty vector. Bars show the median (B-C) and the mean (D-F) fluorescence intensity determined by flow cytometry and expressed as percentage ± SEM compared with untreated or to control conditions (*P < .05, **P < .01).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal