Differential activity and selectivity of N-terminal modified CXCL12 chemokines at the CXCR4 and ACKR3 receptors

Abstract

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors' TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2-4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.

Keywords: ACKR3; CXCL12; CXCR4; GPCR signaling; chemokine variants; pluridimensional efficacy.

FIGURE 1.. Sequences of the first 15 N-terminal residues of WT CXCL12 and the four CXCL12 variants.

Substitution of Lysine 1 (K1) and Proline 2 (P2) in CXCL12 WT are highlighted in bold.

FIGURE 2.. CXCL12 N-terminal variants differentially bind to CXCR4 and ACKR3.

HTRF-based competition experiments performed in CXCR4-expressing (A) and ACKR3-expressing (B) HEK293 cells incubated in the presence of red-CXCL12 and the indicated concentrations of ligands. Values are mean ±SEM of four experiments, each performed in triplicate expressed as percent of the maximal binding obtained without competitor.

FIGURE 3.. Inhibition of cAMP production induced by CXCL12 variants.

cAMP levels were accessed by HTRF-based assays in ST-CXCR4 cells treated with forskolin and stimulated with increasing concentrations of the indicated ligands. HTRF ratios were plotted as a function of ligand concentrations normalized to the maximal response to 1 μM WT CXCL12, and expressed as inhibition of cAMP production (% of maximal CXCL12 response). Data represent the mean ±SEM of two experiments performed in triplicate.

FIGURE 4.. Effect of CXCL12 variants on receptor internalization.

Cells stably expressing ST-CXCR4 (A) or ST-ACKR3 (B) receptors and labeled with SNAP-Lumi4Tb fluorescent substrate were incubated in medium (PBS) or in the presence of the indicated ligands (1 μM). Results represent the mean ±SEM of two independent experiments performed in triplicate. The thresholds corresponding to the signal without ligand are shown (black dashed line).

FIGURE 5.. CXCL12 variants differentially induce β-arrestin 2 recruitment to the two receptors and receptor-dependent β-arrestin 2 activation.

HEK293T cells were transiently co-transfected with Rluc-β-arrestin 2 and CXCR4-YFP (A) or Rluc-β-arrestin 2 and ACKR3-YFP (B) and stimulated with increasing concentrations of the indicated ligands. BRET data were expressed as percent of the maximal response induced by 1 μM of WT CXCL12. Data represent the mean ±SEM of three independent experiments performed in duplicate. (C-D) Intramolecular BRET assay to monitor receptor-dependent β-arrestin 2 activation. Ligand-promoted conformational changes of β-arrestin 2 were monitored by using Rluc8-β-arrestin 2-Ypet in CXCR4 (C) or ACKR3 (D) transfected cells stimulated by the indicated ligands (1 μM). Data represent the mean ±SEM of three independent experiments performed in duplicate.

FIGURE 6.. MD simulations of ACKR3-CXCL12 complexes revealed a critical role for the V3 N-terminal residue of CXCL12.

(A) Views of the interaction sites established between ACKR3 residues (marked in red) and the V3 residue of either the WT CXCL12 (left panel) or the P2G variant (right panel). Bottom panel represents a superposition of both tertiary structures. (B) Plots of the interaction strength between the V3 residue of either CXCL12 (purple line) or the P2G variant (green line) and ACKR3 residues as a function of time. ACKR3 residues are listed on the left side of the chronogram and the horizontal axis corresponds to the time of simulation in ns. Interaction strength is represented as a function of color intensity (darker color indicates stronger interaction).

FIGURE 7.. Interactions of N-terminal CXCL12 V3, S4 and L5 residues within a binding pocket of ACKR3.

(A) Views of the interaction sites established between ACKR3 residues (yellow sphere) and the V3, S4 and L5 residues of the WT CXCL12 (marked in red). (B-C) Plots of the interactions strength established between the S4 (B) and L5 (C) residues of either WT CXCL12 (purple line) or the P2G variant (green line) and ACKR3 residues as a function of time. ACKR3 residues are listed on the left side of the chronogram and horizontal axis corresponds to the time of simulation in ns. Interaction strength is represented as a function of color intensity (darker color indicates stronger interaction).