The WHIM-type receptor CXCR4¹⁰¹³-mediated chemotaxis is dependent upon βarr2. (A) The CXCR4¹⁰¹³ or its counterpart with the entire C-tail CXCR4^wt was stably expressed at similar levels in HEK cells, as assessed by flow cytometric analysis using the PE-conjugated anti-CXCR4 mAb 12G5. These cells were then transiently transfected using the calcium phosphate–DNA coprecipitation method either with pN1-eGFP (N1) or pβarr2-eGFP (βarr2-GFP). Efficiency of transfection was in the same range for both cell types, with 40% to 60% of transfected cells, as deduced from flow cytometric counting of cells with green fluorescence. At 48 hours after transfection, cells were treated (+) or not (−) with 100 nM CXCL12 before immunoprecipitating of receptors. Receptors and βarr2-GFP in the immunoprecipitates were detected by Western blot analysis using the anti-CXCR4 SZ1567 and anti-βarr2 polyclonal antibodies (n = 3). (B) At 48 hours after transfection, 3 × 10⁵ transfected cells in 150 μL DMEM supplemented with 20 mM HEPES and 1% BSA were added to the upper chamber of a 8-μm-pore polycarbonate Transwell culture insert, and cell migration toward the indicated CXCL12 concentrations placed in the lower chamber proceeded for 4 hours at 37°C in humidified air with 5% CO₂. The fraction of cells that migrated across the polycarbonate membrane was assessed by flow cytometry and was calculated as follows: [(number of cells migrating to the lower chamber in response to CXCL12) / (number of cells added to the upper chamber at the start of the assay)] × 100. The percentage of input cells with green fluorescence that migrated to the lower chamber was compared with that of input CXCR4^wt- and N1-expressing cells that migrated toward 0.1 nM CXCL12 (arbitrarily set at 1, and accounting for, on average, 2% of input cells). Spontaneous migrations were marginal in these experiments. The data are means plus or minus SEM (n = 3). (C) Migration of βarr2-GFP– or N1-eGFP–expressing leukocytes from a healthy donor (CTRL) or a patient with WHIM with the CXCR4¹⁰¹³ receptor was assessed as in panel B using a 5-μm-pore polycarbonate Transwell culture insert and RPMI supplemented with HEPES and BSA as a migration medium. The percentage of input cells with green fluorescence that migrated to the lower chamber was compared with that of input N1-expressing control cells that migrated toward 0.3 nM CXCL12. Leukocytes were isolated as described¹¹  and transfected using the Amaxa Nucleofector technology. (D) CXCR4^wt- or CXCR4¹⁰¹³-expressing HEK cells were transfected with siRNA targeting βarr2 or control siRNA (NT). At 48 hours after transfection, cell lysates were prepared and expression of βarr2, βarr1, or LDH was assessed by Western blot analysis as described in “Methods.” (E) Chemotaxis of siRNA-treated cells was carried out as in panel B. The data represent means plus or minus SEM (n = 2).