CXCR4^wt and CXCR4¹⁰¹³ form constitutive heterodimers. (A) Coimmunoprecipitation of CXCR4¹⁰¹³ and CXCR4^wt. HEK cells stably expressing CXCR4^wt (wt) were transfected or not with a CXCR4¹⁰¹³ receptor variant fused to GFP at its N-tail (1013^GFP). At 48 hours after transfection, cells were incubated without (left and right panels) or with (left panel) 100 nM CXCL12 for 15 minutes in PBS at 37°C and treated or not with the DSS covalent cross-linker; receptors were immunoprecipitated using the anti-CXCR4 mAb 12G5. Immunoprecipitated receptors were analyzed by Western blotting as described in Figure 1. Left and right panels are representative experiments performed with 2 different GFP-CXCR4¹⁰¹³ to CXCR4^wt ratios (R), which were calculated using a LAS-1000 CCD camera with the Image Gauge 3.4 software. Experiments with parental cells (P) are also shown. The arrowheads indicate monomeric CXCR4^wt and GFP-CXCR4¹⁰¹³ at 41 and 59 kDa, respectively, and the arrows indicate GFP-CXCR4¹⁰¹³/CXCR4^wt heterodimers at 100 kDa. (B,C) Detection of receptor dimerization using BRET titration experiments. HEK cells were transfected using the transfection reagent FuGene 6 with constant amounts of cDNA coding for CXCR4^wt-RLuc (50 ng/well in 6-well dishes) and increasing amounts (from 25 ng up to 1000 ng) of cDNA encoding for CXCR4^wt-YFP (panel B; ○), YFP alone (panel B; ◇), CXCR4¹⁰¹³-YFP (panel C; □) or GBR2-YFP (panel C; ▵). Experiments were carried out 48 hours after transfection. Total fluorescence (determined using an excitation filter at 485 nm) and luminescence were used as relative measures of total expression of the RLuc- and YFP-tagged proteins. The CXCR4^wt-RLuc receptor amounts were roughly similar in the experimental conditions tested. BRET values, expressed as percent of the maximal BRET reached (BRETmax), are plotted as a function of the ratio of YFP/RLuc fusion proteins. Plotted results are from 3 independent experiments.