Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization

Aurélien Zarca (1) , Claudia Perez (1) , Jelle van den Bor (1) , Jan Paul Bebelman (1) , Joyce Heuninck (2) , Rianna de Jonker (1) , Thierry Durroux (2) , Henry Vischer (1) , Marco Siderius (1) , Martine Smit (1)

1 VU - Vrije Universiteit Amsterdam [Amsterdam]
2 IGF - Institut de Génomique Fonctionnelle

Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization.

Mots clés: ACKR3; GPCR; GRKs; bioluminescence energy transfer (BRET); chemokine receptor; homogeneous time resolved fluorescence (HTRF); internalization; protein phosphorylation; protein recruitment; β-arrestins

Domaines: Sciences du Vivant [q-bio]; Biologie cellulaire

Aurélien Zarca, Claudia Perez, Jelle van den Bor, Jan Paul Bebelman, Joyce Heuninck, et al.. Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization. Cells, 2021, 10 (3), pp.618. ⟨10.3390/cells10030618⟩. ⟨hal-03193065⟩