Traore, B. S.; Casella, S.; Couvineau, P.; Semache, M.; Morone, D.; D'Agostino, G.; Thelen, S.; Breton, B.; Scarpelli Pereira, P. H.; Uguccioni, M.; Legler, D. F.; Thelen, M.; Bouvier, M.

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting beta-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 beta-arrestin interaction. However, the respective roles of beta-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 beta-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in beta-arrestin-deficient (beta-arr1/2 KO) cells. We also evaluated the contribution of GRK-mediated phosphorylation. beta-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 beta-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 GRK2/3/5/6-KO cells, indicating a scaffolding role partially independent of kinase activity. Moreover, G-beta-gamma was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain-deleted GRK2 mutant restored internalization in GRK2/3/5/6-KO cells, and G-beta-gamma sequestration by BARKct-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 beta-arrestins but requires GRKs.