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

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.

doi:10.64898/2026.05.11.724365

bioRxiv · 10.64898/2026.05.11.724365

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

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.

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Abstract

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 ({Delta}{beta}arr1/2) 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 {Delta}GRK2/3/5/6 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 {Delta}GRK2/3/5/6 cells, and G{beta}{gamma} sequestration by {beta}ARKct-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. One-sentence summaryGRKs are essential for ACKR3 endocytosis and chemokine scavenging, whereas {beta}-arrestins and receptor phosphorylation are dispensable.

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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.. 2026-05-13. GRK-dependent ACKR3 endocytosis and chemokine scavenging is independent of receptor phosphorylation and beta-arrestin. https://doi.org/10.64898/2026.05.11.724365

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