CXCR7 Scavenges CXCL12 Figure 1. CXCL12 induced internalization of CXCR7 and CXCR4. (A) FACS analysis of resting Daudi B cells expressing endogenous CXCR4 and CXCR7. (B) Summary from three independent observations showing the time course of receptor expression of Daudi B cells expressing endogenous CXCR4 (open symbols) and CXCR7 (closed symbols) incubated with 100 nM (circles) or 1 mM (triangles) CXCL12. Extracellular bound chemokine was removed by an acidic wash (see Methods) and receptor surface expression was measured at the indicated times by FACS analysis. P,0.5 for ligand- induced internalization of CXCR7 and CXCR4. (C) Daudi B cells were incubated for 1 h at 37uC with 250 nM CXCL12. Extracellular bound chemokine was removed by an acidic wash and samples were split. Surface expression of CXCR7 (closed symbols) and CXCR4 (open symbols) was measured by FACS analysis directly (609 internalization), or following an additional incubation for 30 min at 37uC to allow receptor reexpression (+309 recycling). DMSO vehicle (circles) and 2 mM Bafilomycin A1 (triangles) were present during the entire procedure and a 60 min pre-treatment of the cells. MFIs are shown as percent of DMSO control without chemokine. Data from three independent observations; * P,0.05. doi:10.1371/journal.pone.0009175.g001 The scavenging activity of CXCR7 was measured using transfected MDCK cells seeded on TranswellH insets and grown to confluence. Radiolabeled [125I] CXCL12 was then added to the upper compartment and the cells incubated for 3 h at 37uC. To distinguish between degraded CXCL12 and intact or only partially proteolysed chemokine trichloroacetic acid (TCA) was added to the medium collected from the upper and lower compartment as well as the cell lysate to precipitate proteins. Radioactivity that is insensitive to TCA precipitation reflects chemokine that was cleaved by the cells during the incubation time. We compared the efficiency of MDCK cells expressing similar levels of CXCR7 and CXCR4 (Figure 2A) to degrade CXCL12. Cells expressing CXCR7 secreted significantly more CXCL12-derived radioactivity into the lower compartment than their CXCR4+ counterparts (Figure 3A). With either receptor the majority of the radioactivity was insensitive to TCA treatment, indicating that CXCL12 becomes largely degraded. Similar amounts of cleaved CXCL12 were also detected in the upper compartment of the transwell. We therefore summed all TCA- sensitive radioactivity recovered from the bottom of the well, in the cell lysate and from the upper well to calculate the total degradation activity (Figures 3B–F). We could not find evidence that the small fraction of TCA-sensitive radioactivity, which was transcytosed from the apical to the basal side by CXCR7, contained biological activity. Over the concentration range tested (0.25 nM–25 nM CXCL12) CXCR7 mediated pronounced degradation of the chemokine as opposed to CXCR4. The difference of the two receptors in promoting the proteolysis was most evident at 25 nM CXCL12. Noteworthy, the reported binding affinity of CXCL12 for CXCR7 (Kd , 0.2–0.4 nM) is considerably higher than for CXCR4 (Kd , 2–4 nM) [5,7,32]. However, when the CXCL12 concentration in the assay was raised up to 100 times the Kd the capacity of CXCR7 to mediate degradation of the chemokine appeared not to be saturated, but rather increased linearly. By contrast, the effectiveness of CXCR4 to mediate proteolysis of CXCL12 showed a maximum at 2.5 nM and did not further augment at 25 nM (Figure 3B). The more prominent CXCR4 internalization shown in figure 1, contrasts the more efficient processing of CXCL12 by CXCR7. The apparent discrepancy is best explained with a scavenger function of CXCR7. In this model the receptor continuously cycles between the plasma membrane and an intracellular compartment, where the chemokine is delivered for degradation and CXCR7 returns to the membrane at the cell surface. In this way the receptor can efficiently clear CXCL12 from the external medium. Contrarily, CXCR4 upon ligand binding is internalized and becomes to a large extent degraded [25,26,33]. The second reported ligand for CXCR7 is CXCL11. Indeed, MDCK cells expressing CXCR7 degraded [125I] CXCL11 (Figure 3C) although with lower efficiency. Nevertheless, CXCL11 readily competed proteolysis of CXCL12 by CXCR7 (Figure 3D) and, as predicted from the known receptor specificity, CXCL11 had no effect on the moderate CXCR4 mediated CXCL12 degradation. Conversely, addition of a 100 fold excess of unlabelled CXCL12 expectedly attenuated the uptake and degradation of radiolabeled ligand of both receptors (not shown). The results underline specificity of CXCR7 to scavenge CXCL12. Two small molecule inhibitors of CXCR4, AMD3100 (10 mM) and NIBR-1816 (10 mM) [34,35] markedly reduced the degrada- tion by CXCR4, but had no effect on CXCR7 mediated scavenging of CXCL12. The data indicate further that binding and internalization of cognate chemokine by CXCR7 and CXCR4 are independent of each other. Figure 3E shows that MDCK cells expressing CXCR7 when exposed to 0.25 nM [125I]-CXCL12 degraded approx. 30% of the added chemokine. By contrast, in cells transfected with an empty plasmid (mock) only marginal background proteolysis was observed. Cells carrying CXCR4 displayed a markedly reduced capacity to degrade CXCL12 compared with CXCR7. Exchang- ing the DRY-motif with the sequence of CXCR4 had no effect on CXCR7-mediated degradation, suggesting that this motif is not critical for the activity of the receptor. By contrast, deletion of the intracellular C-terminus (DCXCR7) completely abolished CXCL12 degradation mediated by CXCR7 (Figure 3F), suggest- ing that the domain is required for trafficking of the receptor. PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9175