Fig. 1.

GRKs mediate efficient CXCL12-induced internalization and chemokine scavenging by ACKR3. (A) CXCL12-promoted, active internalization following stimulation with 100 nM chemokine at 37°C was monitored by BRET between ACKR3_RlucII and rGFP_CAAX in WT and ΔGRK2/3/5/6 HEK293A cells. (B) Chemokine uptake by WT and ΔGRK2/3/5/6 cells expressing ACKR3 was detected by quantification of the remaining CXCL12 in the media by ELISA. The extent of scavenging was determined by comparison with cells transfected with empty vector. (C) Constitutive cycling, or passive internalization, of ACKR3 in WT and ΔGRK2/3/5/6 cells was quantified by tracking the loss of “prelabeled” receptor by flow cytometry. Errors are reported as standard deviations, and values are the average of three independent experiments performed in triplicate. Individual experiments are presented as points in B and C. Statistical significance was determined by unpaired t test. *P < 0.001.