Fig 1: Chemokine stimulation selectively recruits GRK3 and GRK2 to ACKR4. (A) Scheme of chemokine-mediated GRK recruitment determined by BRET. (B) HeLa cells were co-transfected with ACKR4-EYFP and GRK5-Nluc or ß-arrestin2-Nluc and stimulated with CCL19. Chemokine-mediated GRK5 or ß-arresin2 recruitment was determined by NetBRET (left). NetBRET values before chemokine stimulation (baseline) was subtracted for corrected NetBRET values (middle). Corrected NetBRET values over time after chemokine addition was integrated and depicted as area under the curve (AUC) values (right). Chemokine addition is indicated by an arrowhead. Data from one representative experiment out of three experiments is shown. (C) Spider diagram of PBS or chemokine-mediated GRK2, GRK3, GRK4, GRK5, GRK6, ß-arrestin1, and ß-arrestin2 recruitment measured as AUC over 3 min. Chemokines were used at a concentration representing about 3-times the EC50 value of ß-arrestin2 recruitment, namely 114 nM for CCL19, 408 nM for CCL21, and 352 nM for CCL25. n = 3. (D) Dose-response curve of GRK3-Nluc recruitment to ACKR4-EYFP upon ligand stimulation. n = 4. (E) Time-resolved GRK3-Nluc recruitment to ACKR4-EYFP upon 114 nM chemokine stimulation (indicated by an arrowhead). n = 3. (F) Time-resolved recruitment of GRK3-Nluc from (E), ß-arrestin1-Nluc, or ß-arrestin2-Nluc to ACKR4-EYFP upon stimulation (indicated by an arrowhead) with 114 nM CCL19. n = 3. (G) Dose-response curve of GRK2-Nluc recruitment to ACKR4-EYFP upon ligand stimulation. n = 3. (H) Time-resolved GRK2-Nluc recruitment to ACKR4-EYFP upon 114 nM chemokine stimulation (arrowhead). n = 3. (I) GRK2 (dashed lines) and GRK3 (solid lines) recruitment to ACKR4-EYFP or ACKR4t-EYFP upon 114 nM CCL19 stimulation (arrowhead) over 30 min. n = 3. (J–L) HeLa cells transfected with ACKR4-EYFP and ß-arrestin2-Nluc were pretreated for 2 h with 30 µM of the GRK2/3 inhibitor cmpd101 or solvent (DMSO) and subsequently stimulated with either 114 nM CCL19 or 408 nM CCL21 (indicated by an arrowhead). NetBRET (J) corrected NetBRET (K) or AUC (L) are depicted. n = 3.