The Peptidomimetic CXCR4 Antagonist TC14012 Recruits β-Arrestin to CXCR7

Abstract

CXCR7 is an atypical chemokine receptor that signals through ␤-arrestin in response to agonists without detectable activation of heterotrimeric G-proteins. Its cognate chemokine ligand CXCL12 also binds CXCR4, a chemokine receptor of considerable clinical interest. Here we report that TC14012, a peptidomimetic inverse agonist of CXCR4, is an agonist on CXCR7. The potency of ␤-arrestin recruitment to CXCR7 by TC14012 is much higher than that of the previously reported CXCR4 antagonist AMD3100 and differs only by one log from that of the natural ligand CXCL12 (EC 50 350 nM for TC14012, as compared with 30 nM for CXCL12 and 140 M for AMD3100). Moreover, like CXCL12, TC14012 leads to Erk 1/2 activation in U373 glioma cells that express only CXCR7, but not CXCR4. Given that with TC14012 and AMD3100 two structurally unrelated CXCR4 antagonists turn out to be agonists on CXCR7, this likely reflects differences in the activation mechanism of the arrestin pathway by both receptors. To identify the receptor domain responsible for these opposed effects, we investigated CXCR4 and CXCR7 C terminus-swapping chimeras. Using quantitative bioluminescence resonance energy transfer, we find that the CXCR7 receptor core formed by the seven-transmembrane domains and the connecting loops determines the agonistic activity of both TC14012 and AMD3100. Moreover, we find that the CXCR7 chimera bearing the CXCR4 C-terminal constitutively associates with arrestin in the absence of ligands. Our data suggest that the CXCR4 and CXCR7 cores share ligand-binding surfaces for the binding of the synthetic ligands, indicating that CXCR4 inhibitors should be tested also on CXCR7. CXCR4 is a seven-transmembrane domain (7TMR)4 chemokine receptor of considerable clinical interest, involved in stem cell homing to bone marrow niches, cancer biology and metastasis, and HIV infection. Synthetic CXCR4 ligands are being developed, and the CXCR4 blocker AMD3100 has reached the clinic for the mobilization of hematopoietic stem cells from donor bone marrow. The natural CXCR4 ligand, the chemokine CXCL12 (also called SDF-1), has later been found to also bind the atypical chemokine receptor CXCR7, which in addition recognizes the chemokine CXCL11 (also called I-TAC) (1, 2). CXCR7 is atypical in that no classical heterotrimeric G-protein signaling pathways are observed and in that it does not induce chemotaxis of motile cells. However, CXCR7 sets off G-protein-independent signaling via the ␤-arrestin pathway, leading to activation of the Erk 1/2 kinases (3, 4). Functionally, CXCR7 has been implicated in cancer cell growth and transendothelial migration (2, 5, 6). Intriguingly, CXCR7 and CXCR4 can heteromerize, and this attenuates CXCR4 G-protein signaling (7,8). Modulation of CXCR4 has been suggested as a major role of CXCR7 (9).Given that CXCR4 and CXCR7 share a chemokine ligand, we have previously tested the effects of AMD3100 on CXCR7 and found that it induces arrestin recruitment at high doses on this receptor, contrary to its effect on CXCR4 (3). This findin...

Rapid Uptake and Degradation of CXCL12 Depend on CXCR7 Carboxyl-terminal Serine/Threonine Residues

“…Little is known about structural elements underlying the different receptor functions (16,22,40). By generating tailswap mutant CXCR4 and CXCR7 receptors, we provide evidence that replacement of the CXCR7 C terminus with the CXCR4 C terminus generates a CXCR7 chimera, which shows CXCL12-dependent internalization and CXCL12-dependent phosphorylation but lacks ligand-independent internalization.…”

“…In the case of the atypical chemokine receptor D6, constitutive phosphorylation of a cluster of serine residues in the C terminus has been shown to be involved in ␤-arrestin recruitment, receptor trafficking, and receptor stability (24). Thus, the presence of potential phosphorylation sites in the C-terminal domain of CXCR7 suggests that a similar mechanism may be involved in trafficking and MAP kinase sig-naling of CXCR7 (11,22,40). Our finding that the CXCR4 C terminus shows strong constitutive phosphorylation when fused to CXCR7 points to the possibility that the CXCR7 protein prefers a conformation that attracts G protein-coupled receptor kinases.…”

CXCR7/CXCR4 Heterodimer Constitutively Recruits β-Arrestin to Enhance Cell Migration

“…However, CXCR7 is an atypical chemokine receptor that apparently does not signal through canonical G protein-linked signaling pathways even in the presence of saturating SDF-1 concentrations (11,12,14,15). Even though the signaling pathways activated by CXCR7 are still under investigation, there is increasing evidence that CXCR7 may function as a biased receptor for selective recruitment and activation of ␤-arrestin-mediated signaling (23)(24)(25)32). In this study, we show that CXCR7 modulates CXCR4 function by self-assembling in cell membranes to form heterodimers or possibly higher order hetero-oligomers with CXCR4.…”

“…For example, a study conducted by Gravel et al (32) demonstrated that a chimeric CXCR7 bearing the C-terminal tail of CXCR4 resulted in constitutive recruitment of ␤-arrestin. Such recent studies have demonstrated the usefulness of bioluminescence resonance energy transfer, luciferase complementation, and imaging techniques to investigate the ␤-arrestin-CXCR7 interaction (20,21,23,24,32). Although analogous bioluminescence resonance energy transfer/FRET techniques can be implemented to identify the specific partner of the CXCR4⅐CXCR7 dimer pair that plays a key role in ␤-arrestin recruitment, the localization and identification of amino acids involved in contacting ␤-arrestin may require more focused techniques such as unnatural amino acid mutagenesis, bio-orthogonal labeling, photochemical cross-linking, and reconstitution in membrane nanoparticles.…”

Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways

“…β-arrestin-2 recruitment to CXCR4 was measured by an intermolecular BRET assay performed as described previously (27,38). The values were corrected to net BRET by subtracting the background BRET signal obtained in cells transfected with the Rluc construct alone.…”