Fig 2: Effects of receptor antagonists on cross-recruitment of ß-arrestin 2 to CXCR4 upon a1b-AR activation. PE: phenylephrine. (a) ß-arrestin 2 recruitment assay (PRESTO-Tango). Cells were transfected with FLAG-CXCR4-TANGO/pcDNA3 and stimulated with CXCL12 in the presence of vehicle (open circles) or AMD3100 (10 µM). N = 3 independent experiments. (b) ß-arrestin 2 recruitment assay (PRESTO-Tango). Cells were transfected with FLAG-CXCR4-TANGO/a1b-AR and stimulated with PE after pre-treatment (15 min, 37 °C) with vehicle (open circles), AMD3100 (10 µM) or phentolamine (100 µM). N = 3 independent experiments. (c) Cells were treated with various concentrations of phentolamine, as in b., and cell viability was determined by trypan blue exclusion. N = 3 independent experiments.

Fig 3: HA-CXCR4 forms heteromeric complexes with FLAG-a1b/d-AR in HEK293T cells. Typical PLA images for the detection of individual receptors (left and center) and receptor-receptor interactions (right) in HEK293T cells transfected with DNA encoding HA- and/or FLAG-tagged receptors, as indicated. PLA was performed with anti-HA and anti-FLAG. pcDNA3: control, cells were transfected with empty vector. Images show merged PLA/4',6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bars: 10 µm. Images are representative of n = 3 independent experiments.

Fig 4: Effects of peptide analogues of transmembrane domains of CXCR4 on cross-recruitment of ß-arrestin 2 to CXCR4 upon a1b-AR activation. (a) Cells were co-transfected with HA-a1b-AR and FLAG-CXCR4, treated with vehicle or peptide analogues of transmembrane domain (TM) 2 and TM4 (50 µM, 15 min, 37 °C), and used for PLA. Typical PLA images for the detection of (from top to bottom) HA-a1b-AR, FLAG-CXCR4 and interactions between both receptors are shown. Ctrl.: control, cells transfected with empty vector and stained for interactions between HA-a1b-AR and FLAG-CXCR4. Scale bars: 10 µm. (b) Quantification of PLA signals for FLAG-CXCR4-TANGO/HA-a1b-AR interactions from n = 3 independent experiments, as in a. PLA signals/cell are expressed as % of vehicle treated cells. *p < 0.05 vs. cells incubated with TM2. (c) ß-arrestin 2 recruitment assay (PRESTO-Tango). Cells were transfected with FLAG-CXCR4-TANGO/a1b-AR and stimulated with PE after pre-treatment (15 min, 37 °C) with vehicle (open circles) or the TM2 (50 µM) peptide (grey squares). N = 5 independent experiments. (d) ß-arrestin 2 recruitment assay (PRESTO-Tango). Cells were transfected with FLAG-CXCR4-TANGO/a1b-AR and stimulated with PE after pre-treatment (15 min, 37 °C) with vehicle (open circles) or the TM4 (50 µM) peptide (grey squares). N = 4 independent experiments. (e) ß-arrestin 2 recruitment assay (PRESTO-Tango). Cells were transfected as indicated and stimulated with PE after pre-treatment (15 min, 37 °C) with vehicle (open symbols) or the TM2 (50 µM) peptide (black symbols). N = 3 independent experiments.

Fig 5: a1b-AR activation within the CXCR4:a1b-AR heteromer leads to internalization of CXCR4 in HEK293T cells. (a,b) After labeling HA-CXCR4 with FITC-conjugated anti-HA at 4 °C, HEK293T cells were treated with CXCL12 (200 nM) or phenylephrine (PE, 200 µM) for 1 hour at room temperature. Images are representative of n = 3 independent experiments. Scale bar: 20 µm. HEK293T cells were transfected with HA-CXCR4/pcDNA3 (a) or with HA-CXCR4/FLAG-a1b-AR (b). (c,d) Quantification of HA-CXCR4 cell surface expression by flow cytometry with FITC-anti-HA in cells after stimulation with PE, as in a/b. Cells were transfected with HA-CXCR4/pcDNA3 (c) or HA-CXCR4/a1b-AR (d). Surface HA-CXCR4 expression is expressed as % fluorescence intensity of cells treated with vehicle. *p < 0.05 vs. vehicle. N = 4 independent experiments.