Fig 2: ACKR3 gene silencing reduces ACKR3 : AVPR1A and ACKR3 : CXCR4 heteromers and increases AVPR1A : CXCR4 interactions in A7r5 cells. (a,b). Representative PLA images for the detection of individual receptors (a) and receptor–receptor interactions (b) in A7r5 cells after incubation with NT or ACKR3 siRNA. Ctrl: omission of one primary antibody. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals acquired from z-stack images (n = 10; thickness 1 µm, bottom to top). (c,d) Quantification of PLA signals per cell for the detection of individual receptors (c) and receptor–receptor interactions (d) as in (a,b). n = 6 independent experiments with n = 10 images per condition and experiment. *p < 0.05 versus cells incubated with NT-siRNA.

Fig 3: β-arrestin 2 recruitment at the ACKR3 : AVPR1A heteromeric complex. (a–c) HTLA cells were co-transfected with 0.75 µg of DNA for FLAG-AVPR1A-TANGO plus 0.75 µg of pcDNA3 or HA-ACKR3. (a) Measurement of FLAG-AVPR1A-Tango expression by flow cytometry. Cells were labelled with Alexa Fluor 647 anti-FLAG. Grey area: unstained cells. Red line: cells transfected with FLAG-AVPR1A-TANGO plus pcDNA. Green line: cells transfected with FLAG-AVPR1A-TANGO/HA-ACKR3. RFU: relative fluorescence units. Data are representative of n = 3 independent experiments. (b,c) β-arrestin 2 recruitment assay (PRESTO-Tango) for AVPR1A. Cells were stimulated with aVP (b), CXCL11 (c) and CXCL12 (c). Black symbols: cells transfected with FLAG-AVPR1A-TANGO/pcDNA3; white symbols: cells transfected with FLAG-AVPR1A-TANGO/HA-ACKR3. n = 3 independent experiments. (d–g) HTLA cells were co-transfected with 0.75 µg of DNA for FLAG-ACKR3-TANGO plus 0.75 µg of pcDNA3 or HA-AVPR1A. (d) Measurement of FLAG-ACKR3-Tango expression by flow cytometry. Cells were labelled with Alexa Fluor 647 anti-FLAG. Grey area: unstained cells. Red line: cells transfected with FLAG-ACKR3-TANGO plus pcDNA3. Green line: cells transfected with FLAG-ACKR3-TANGO/HA-AVPR1A. RFU: relative fluorescence units. Data are representative of n = 3 experiments. (e–g) β-arrestin 2 recruitment assay (PRESTO-Tango) for ACKR3. Cells were stimulated with CXCL11 (e), CXCL12 (f) and aVP (g). Black circles: cells transfected with FLAG-ACKR3-TANGO/pcDNA3; white circles: cells transfected with FLAG-ACKR3-TANGO/HA-AVPR1A. n = 3 independent experiments. (h) β-arrestin 2 recruitment assay (PRESTO-Tango) with HTLA cells co-expressing FLAG-AVPR1A-Tango/HA-ACKR3. Cells were treated with vehicle or TM2/4/7 peptides (10 µM, 30 min at 37°C) and then stimulated with aVP. n = 3 independent experiments. Black circle: vehicle. Open squares: TM2. Light grey squares: TM4. Dark grey squares: TM7. n = 4 independent experiments.

Fig 4: ACKR3 gene silencing reduces ACKR3 : AVPR1A and ACKR3 : CXCR4 heteromers and increases AVPR1A : CXCR4 interactions in hVSMCs. (a,b) Representative PLA images for the detection of individual receptors (a) and receptor–receptor interactions (b) in hVSMC after incubation with NT or ACKR3 siRNA. Ctrl: omission of one primary antibody. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals acquired from z-stack images (n = 10; thickness 1 µm, bottom to top). (c,d) Quantification of PLA signals per cell for the detection of individual receptors (c) and receptor–receptor interactions (d) as in (a,b). n = 4 independent experiments with n = 10 images per condition and experiment. *p < 0.05 versus cells incubated with NT-siRNA.

Fig 5: ACKR3-derived TM peptide analogues interfere with ACKR3 : AVPR1A and ACKR3 : CXCR4 heteromerization and inhibit aVP-induced Gaq signalling in hVSMC. (a,b) Quantification of PLA signals per cell as in figure 7 for individual receptors (a) and receptor–receptor interactions (b). n = 4 independent experiments with n = 10 images per condition and experiment. *p < 0.05 versus cells incubated with vehicle. (c) IP3 production was measured in hVSMC pretreated with vehicle or TM2/4/7 peptide analogues (10 µM, 30 min at 37°C) and then stimulated with either vehicle (-) or aVP (1 µM, 5 min) (+). n = 4 independent experiments. *p < 0.05 versus vehicle-treated cells. (d) ß-arrestin 2 recruitment assay (PRESTO-Tango) for AVPR1A. HTLA cells expressing FLAG-AVPR1A-Tango were incubated with either vehicle or ACKR3 TM2/4/7 (10 µM, 30 min at 37°C) and then stimulated with aVP overnight. RLU (%): relative luminescence units in % RLU after treatment with 10 µM aVP (=100%). n = 3 independent experiments.