A, amino acid sequence of the C-terminal tails of WT CXCR4 and the mutant form of CXCR4 (WHIM R334X) utilized in the current study. B, HeLa cells stably transduced with GFP-WT CXCR4 or GFP-WHIM were lysed and analyzed by SDS-PAGE and Western blotting using anti-GFP antibodies or immunoprecipitated with anti-GFP antibody and immunoblotted with anti-CXCR4 antibody. Accuracy of loading was assessed by reprobing the membrane with anti-actin antibodies. C, flow cytometric analysis of GFP expression in control (non-transduced, NT) Hela cells and HeLa cells stably transduced with GFP-CXCR4 (WT and WHIM).

A, HeLa cells stably expressing GFP-WT CXCR4 or GFP-WHIM CXCR4 were incubated at 37°C with SDF-1/CXCL12 (25 ng/ml) for 0, 5, 30 and 90 min in the presence of labeled (Alexa 568) transferrin and anti-GFP antibodies. The cells were fixed, immunostained with anti-GFP antibodies and examined by confocal microscopy. Representative images depicting the distribution of internalized CXCR4 (green), internalized transferrin (red) and colocalization of internalized CXCR4 and transferrin (yellow). The white arrows highlight the different degree of receptor internalization by the WT and the WHIM CXCR4 at time 0 and 90 min. B, HeLa cells stably expressing GFP-WT CXCR4 or GFP-WHIM CXCR4 were transfected with plasmids encoding Rab11-DsRed2, Rab5-RFP, or Rab7-RFP, incubated at 37°C overnight, fixed and mounted.

A, HeLa cells expressing GFP-CXCR4 (WT or WHIM mutant) were incubated with SDF-1/CXCL12 (25 ng/ml, 37°C, 30 min); cell lysates were immunoprecipitated with anti-GFP antibodies to immunoprecipitate GFP-CXCR4; the immunoprecipitates were separated by SDS-PAGE and immunoblotted with an antibody to Grk6. The blots were re-probed with anti-GFP antibodies to detect transduced CXCR4. B and C, Hela cells expressing GFP-CXCR4 (WT and WHIM) constructs were incubated with SDF-1/CXCL12 (25 ng/ml, 37°C, 30 min) immunoprecipitated with anti-GFP antibody to pull down transduced CXCR4, probed for Grk3, and reprobed for CXCR4 with anti-CXCR4 antibodies. Representative results are shown in B; average ratio (±SD) of Grk3 and CXCR4 band intensity in 3 separate immunoprecipitation experiments shown in C. D and E, levels of Grk6 (D) or Grk3 (E) in HeLa cells expressing GFP-WT CXCR4 after 3-day treatment with control or specific siRNAs detected by immunoblotting with antibodies to Grk3 or Grk6. The membranes were reprobed with anti-actin antibodies. F, Representative images from confocal microscopy showing internalization of cell surface GFP-CXCR4 after treatment with SDF-1/CXCR4 (25 ng/ml, 37°C, 30 min) in the presence of anti-GFP antibodies. The HeLa cells were either treated with control siRNA or depleted of Grk3 or Grk6 using specific siRNA. G, HeLa cells expressing GFP-WT CXCR4 were transfected with scrambled or siRNAs specific to Grk6, and exposed to SDF-1/CXCL12 (25 ng/ml) for the indicated time intervals. Cell lysates were separated by SDS-PAGE and analyzed by Western blotting using specific antibodies to phosphorylated pErk 1/2; membranes were re-probed with antibodies to total Erk 1/2 for loading controls.

A–C, HeLa cells stably expressing GFP-WT CXCR4 or GFP-WHIM CXCR4 constructs were exposed to 25 ng/ml SDF-1/CXCL12 for 0, 40 or 80 min at 37°C and then stained for cell surface CXCR4 using antibodies to GFP to detect the transduced CXCR4 and distinguish it from the endogenous protein; levels of cell surface GFP-CXCR4 were measured by flow cytometry. The red line depicts results from control staining with isotype control immunoglobulin and secondary antibody. Results in A and B reflect a representative experiment showing CXCR4 mean fluorescence intensities at the indicated time-points; results in C reflect the mean (±SD) levels of cell surface CXCR4 (measured by mean fluorescence intensity, MFI) at the 80 min time-point from 3 independent experiments; the results are expressed as a percentage of receptor levels (as measured by MFI) found on the surface at time 0 (0 min). D, Hela cells stably expressing GFP-WT CXCR4 or GFP-WHIM CXCR4 were exposed to SDF-1/CXCL12 (25 ng/ml) for the indicated time intervals; cell lysates were separated by SDS-PAGE and analyzed by Western blotting using specific antibodies to phosphorylated Akt and Erk 1/2; for loading controls, membranes were re-probed with antibodies to total Erk 1/2. E, HEK 293 cells were transiently transfected with WT, WHIM, or both receptors; HeLa cells were transiently transfected with both WT and WHIM receptors. Transfected cells were exposed to SDF-1/CXCL12 (25 ng/ml) for the indicated times and cell lysates analyzed by SDS-PAGE and Western blotting. F, Huvec were plated in monolayers onto 96-well plates, and were activated overnight with 2 ng/ml of TNFα. CFSE-labeled KG1a cells expressing, WT or WHIM CXCR4 were incubated at 5×10⁴ cells per well onto TNFα−preactivated Huvec monolayers in triplicate for 30 min. After removal of non-adherent cells, adherent cells were counted by reading fluorescence at ∼520 nm. The results reflect the mean +/− SD of triplicate wells in a representative experiment performed three times.

A, Non transduced (NT) Hela cells, or HeLa cells stably expressing GFP-WT CXCR4 or GFP-WHIM CXCR4 were transiently transfected with a FLAG-tagged β-Arrestin 2 construct; 48 hours after transfection, the cells were incubated at 37°C with SDF-1/CXCL12 (25 ng/ml) for 10 min; cell lysates were immunoprecipitated with anti-GFP antibodies to detect transduced CXCR4; the immunoprecipitates were separated through SDS-PAGE and analyzed by Western blotting. The results reflect immunoblotting with anti-FLAG antibodies to detect β-Arrestin 2 and reprobing with antibodies to GFP to detect the immunoprecipitated GFP-CXCR4 (representative from 5 experiments). B, The experiment was performed as in (A), except that the HeLa cells were exposed to SDF-1/CXCL12 for 5, 10 and 30 min prior to lysis. C, HeLa cells stably expressing GFP-WT CXCR4 or GFP-WHIM CXCR4 were transiently transfected with FLAG-tagged β-Arrestin 2, incubated with 25 ng/ml SDF-1/CXCL12 for 10 min or 30 min at 37°C, fixed and then stained with anti-FLAG antibody to detect β-Arrestin 2 (Barr2, red). GFP fluorescence (green) denotes the transduced CXCR4; colocalization of β-Arrestin 2 and CXCR4 is in yellow. Each of the images shown is representative of 10 fields examined. The experiment is representative of 3 independent experiments performed. D, Hela cells stably expressing GFP-WT CXCR4 or GFP-WHIM CXCR4 were either mock transfected (NT) or were transfected with a FLAG-β-Arrestin 1 construct; after 48 hours the cells were exposed to SDF-1/CXCL12 (25 ng/ml, 37°C, 10 and 30 min) and the cell lysates were immunoprecipitated with anti-GFP antibodies. Immunoprecipitates and aliquots of cell lysates used for the IP were separated onto SDS-PAGE and immunoblotted with anti-FLAG antibodies to detect β-Arrestin 1.