GPCR kinases shape ACKR4 functions via differential C-terminal phosphorylation.

Atypical chemokine receptor 4 (ACKR4) is a scavenger receptor that regulates the availability of chemokines, including CCL19, and consequently the responsiveness of their classical G protein coupled receptors (GPCRs). In contrast to classical chemokine receptors, ACKR4 is completely biased towards βarrestins and does not couple to G proteins. Here, we show that ACKR4 in its apo state constitutively pre-associates with βarrestins and cycles between the plasma membrane and endosomal compartments. We identify distinct serine and threonine residues in the tail region of ACKR4 involved in regulating steady-state receptor trafficking and chemokine uptake, and that a C-terminal serine/threonine cluster is key for both ligand-mediated βarrestin recruitment and efficient chemokine uptake. Moreover, different serine/threonine clusters in the tail region of ACKR4 account for steady-state and chemokine-driven association of the four non-visual GPCR kinases (GRKs), which differentially phosphorylate two serine and one threonine residues. We show that GRK5/6 primarily phosphorylate ACKR4 in the absence of chemokines, and that CCL19 stimulation recruits GRK2/3 to enhance ACKR4 phosphorylation. Notably, we found that apo ACKR4 forms a ternary complex with GRK2/3 and the G protein without activating it. Finally, we show that the C-terminal serine/threonine cluster of ACKR4 and GRK2 play leading roles in βarrestin recruitment and CCL19 internalisation.