CXCR7 Scavenges CXCL12 Figure 3. Degradation of CXCL12 mediated by CXCR7 and CXCR4. (A) MDCK cells stably transfected with empty vector (Mock), with CXCR7, and CXCR4 were incubated with 0.25 nM [125I]-CXCL12 for 3 h at 37uC added to the apical side. Black bars, TCA resistant radioactivity recovered from the lower part of the transwell; grey bars, radioactivity which was precipitated with TCA. Data from 5 experiments performed in triplicates. (B) Degradation of CXCL12 mediated by CXCR7 is not saturated by increasing concentrations of ligand. MDCK cells stably expressing CXCR7 or CXCR4 were incubated with increasing concentrations of [125I]-CXCL12 as in (A). Total TCA resistant counts were used to calculate the amount of degraded chemokine. Representative data shown from one experiment performed in triplicates. (C) MDCK stably transfected with empty vector (Mock) or CXCR7 were exposed to 0.25 nM [125I]-CXCL11 as in B. In three independent experiments more pronounced spontaneous degradation of CXCL11 in Mock-transfected cells compared to CXCL12 degradation was observed. (D) MDCK stably transfected with empty vector (Mock), with CXCR7, and CXCR4 were exposed to 0.25 nM [125I]-CXCL12 as in A in the presence of medium (cont.), 1 mM CXCL11 or the CXCR4 inhibitors AMD3100 (10 mM) and NIBR-1816 (10 mM). (E) The DYLAIV motif is not critical for CXCR7 activity. MDCK stably transfected with empty vector (Mock), with CXCR7, CXCR4 and a vector coding for a chimeric CXCR7 containing the DRYLAIV motive of CXCR4 were incubated as in B. Data from experiments that were performed in triplicates (n = 5, except DRYLAIV n = 2). (F) MDCK stably transfected with empty vector (Mock), with CXCR7, or a vector coding for a CXCR7 lacking the cytoplasmic C terminus (DCXCR7) were exposed to [125I]-CXCL12 as in B. doi:10.1371/journal.pone.0009175.g003 [5] and of CXCR4 for the mAb 6H8 [24]. Following removal of the protease the cells were shifted to 37uC in the presence of cycloheximide. Re-emerging of the respective receptor epitopes was measured by FACS analysis. Figure 4A shows that in MDCK cells CXCR7 rapidly reappeared after shifting to 37uC reaching an expression level of 60% after one hour compared to that measured on untreated control cells. By contrast, after one hour of incubation at 37uC the immunoreactive CXCR4 was about 20% of control. In Daudi cells the reappearance after proteinase K treatment of endogenous CXCR4 and CXCR7 was compared with a typical ligand-independent cycling receptor, the transferrin receptor. After 20 min, the surface levels of the transferrin receptor were fully restored and expression of CXCR7 recovered by 50%. Conversely, during the same time CXCR4 levels reached only 20%. Together the findings suggest an intermediate cycling activity for CXCR7 which is less rapid compared with the transferrin receptor, but markedly faster than CXCR4, consistent with the more pronounced degradation of CXCL12 mediated by CXCR7. Ligand independent cycling of CXCR7 was further investigated with antibody uptake experiments. MDCK cells expressing CXCR7 or CXCR4 were incubated for 1 h at 37uC in the absence of chemokines with specific monoclonal antibodies known to block receptor function (9C4 and 12G5, respectively) [5,37]. Following removal of extracellular antibodies by stringent washing, cells were fixed and immunecomplexes detected with F(ab9)2 goat anti mouse IgG Alexa488 by confocal microscopy. In CXCR7 expressing MDCK cells a pronounced intracellular staining was observed which is associated with endosomal compartments. By contrast, CXCR4+ cells contained much less fluorescent endosomes (Figure 4C). The marked antibody uptake in CXCR7+ cells in the absence of stimulation suggest spontane- ous receptor cycling between the plasma membrane and endosomal compartments. To further verify ligand-independent PLoS ONE | www.plosone.org 6 February 2010 | Volume 5 | Issue 2 | e9175