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

Abstract

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.

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and β-Arrestins

“…Histamine (10 µM) induced a very rapid increase in BRET between H 1 R-Rluc8 and GRK2-mVenus or GRK3-mVenus that peaked within 2 min and subsequently decreased within 20 min to an elevated steady-state level as compared to vehicle-stimulated cells ( Figure 3 A). Hence, GRK2/3 interaction with activated H 1 R indeed precedes the recruitment of β-arrestin1/2 in time, which was also previously reported for agonist-stimulated H 4 R, atypical chemokine receptor 4 (ACKR4) oxytocin, and µ-opioid receptor [ 33 , 34 , 41 , 42 ], whereas GRK2/3 and β-arrestin1/2 were recruited with comparable kinetics to ACKR3 in response to chemokine CXCL12 stimulation [ 39 ]. On the contrary, histamine (10 µM) stimulation decreased BRET between H 1 R-Rluc8 and GRK5-mVenus or GRK6-mVenus with slower kinetics as compared to the increased GRK2/3 interaction to this receptor, but comparable rates as the recruitment of β-arrestin1/2 ( Figure 3 A).…”

“…Pretreatment with the Gα q/11 protein inhibitor UBO-QIC (1 µM) [ 31 ] for 30 min did not significantly affect β-arrestin1/2 recruitment in response to 1-h stimulation with 10 µM histamine ( Figure 2 C), while completely abolishing histamine-induced nuclear factor activated T-cells (NFAT)-driven reporter gene activation in HEK293T cells expressing H 1 R-Rluc8 ( Supplementary Figure S2 ). In contrast, 30-min pretreatment with the GRK2/3 inhibitor cmpd101 (3 µM) [ 32 ] resulted in a partial (~38–46%) but significant reduction in histamine-induced β-arrestin1/2 recruitment ( Figure 2 C), suggesting that GRK2/3-mediated phosphorylation of H 1 R might in part contribute to the interaction with β-arrestins, as previously observed for several other GPCRs [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Indeed, overexpression of GRK2 was reported to increase (180%) histamine-induced phosphorylation of H 1 R in HEK293 cells that were metabolically labeled with [ 32 P]orthophosphate, whereas GRK5 and GRK6 increased H 1 R phosphorylation by 50 and 80%, respectively [ 11 ].…”

“…To evaluate which ubiquitously expressed GRK subtypes might potentially phosphorylate the histamine-activated H 1 R and consequently promote β-arrestin1/2 coupling, a BRET assay was used to measure the interaction of H 1 R with GRK2, GRK3, GRK5, and GRK6, as previously reported for the histamine H 4 receptor (H 4 R) and the atypical chemokine receptor 3 (ACKR3) [ 33 , 39 ]. Histamine (10 µM) induced a very rapid increase in BRET between H 1 R-Rluc8 and GRK2-mVenus or GRK3-mVenus that peaked within 2 min and subsequently decreased within 20 min to an elevated steady-state level as compared to vehicle-stimulated cells ( Figure 3 A).…”

“…In line, reduced BRET between agonist-activated receptor and GRK5 and/or GRK6 has been previously observed for H 4 R, β 2 -adrenergic receptor, bile acid receptor TGR5, neurokinin-1 receptor, and protease-activated receptor 2 [ 33 , 43 , 44 , 45 , 46 ]. In contrast, stimulation of ACKR3 with CXCL12 increased the BRET between this receptor and GRK5 [ 39 ]. No agonist-induced changes in BRET signal were observed for GRK6 with ACKR3 nor with bile acid receptor TGR5, and neither for GRK5/6 with ACKR4 [ 39 , 42 , 45 ].…”

“…In contrast, stimulation of ACKR3 with CXCL12 increased the BRET between this receptor and GRK5 [ 39 ]. No agonist-induced changes in BRET signal were observed for GRK6 with ACKR3 nor with bile acid receptor TGR5, and neither for GRK5/6 with ACKR4 [ 39 , 42 , 45 ].…”