GRK-dependent ACKR3 endocytosis and chemokine scavenging is independent of receptor phosphorylation and β-arrestin

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting β-arrestin recruitment. Atypical chemokine receptors (ACKRs), including ACKR3, are structurally related to classical chemokine receptors but do not activate heterotrimeric G proteins. ACKR3 signaling and trafficking have been proposed to depend on GRK5-mediated phosphorylation and β-arrestin interaction. However, the respective roles of β-arrestins, GRKs, and receptor phosphorylation in chemokine scavenging and in constitutive or ligand-induced trafficking remain debated. Using bioluminescence resonance energy transfer (BRET)–based biosensors and immunofluorescence imaging with fluorescently labeled receptors and chemokines, we examined ACKR3 interaction with β-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in β-arrestin–deficient (Δβarr1/2) cells. We also evaluated the contribution of GRK-mediated phosphorylation. β-arrestins supported agonist-independent receptor internalization but were dispensable for chemokine-induced internalization and chemokine scavenging. In contrast, GRKs were required for ligand-promoted endocytosis, with either GRK2/3 or GRK5/6 being sufficient. Mutation of ACKR3 phosphorylation sites impaired β-arrestin recruitment but did not completely block internalization and scavenging, whereas complete C-terminal truncation abolished both processes. Consistently, kinase-dead GRK2 rescued ACKR3 endocytosis in ΔGRK2/3/5/6 cells, indicating a scaffolding role partially independent of kinase activity. Moreover, Gβγ was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain–deleted GRK2 mutant restored internalization in ΔGRK2/3/5/6 cells, and Gβγ sequestration by βARKct-CAAX did not inhibit this process consistent with the notion that ACKR3 does not promote G protein activation. Thus, ligand-promoted ACKR3 internalization and chemokine scavenging occur independently of β-arrestins but requires GRKs.