only CXCR7b and CXCL12a are involved in primordial germ cell migration [20,21]. While Valentin et al. [17] propose active signaling through CXCR7 for the migration of the lateral-line primordium, Boldajipour and colleagues suggested that CXCR7b expressed in somatic cells facilitates the CXCL12/CXCR4- mediated migration of primordial germ cells by controlling the level of the chemokine in the environment, thereby contributing to the formation of a chemotactic gradient [19]. Similarly, in the case of migration of the lateral line primordium, CXCR7b expression at the back of the cell cluster was suggested to be responsible for reduction in the level of CXCL12a at that location and to contribute to the generation of a chemotactic gradient despite the uniform expression of cxcl12a RNA [16]. In the present study we unveil the mechanism of action of CXCR7 in mammalian cells. We observed that CXCR7 cycles between the plasma membrane and endosomal compartments both in the presence and the absence of ligand. In the presence of ligand CXCR7 surface expression is biphasic showing only a moderate reduction of its cell surface levels after prolonged incubation, and is accompanied by marked uptake and degrada- tion of CXCL11 and CXCL12. By contrast, CXCR4 becomes permanently down-regulated following stimulation with CXCL12 and its internalization proceeds with a minimal degradation of the chemokine. Furthermore, we show CXCR7-mediated sequestra- tion of CXCL12 by the endothelium of mouse heart valves and of human umbilical veins. Taken together, our observations support the notion and provide mechanistic insights concerning the role of CXCR7 as a specific scavenger for CXCL12. Methods Ethics Statement Human umbilical cords were obtained from the local hospital (Ospedale San Giovanni, Bellinzona) with confirmed verbal consent of the donors. The procedure was approved by the local ethics committee Comitato Etico Cantonale, CH-6501 Bellinzona. Animals were bred and treated in accordance with the Swiss Federal Veterinary Office guidelines. Experiments were approved by the ‘‘Dipartimento della Sanita´ e della Socialita´’’. Cell Culture Madin-Darby canine kidney (MDCK) and HeLa cells were grown in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. Raji B cells were cultured in RPMI 1640 medium supplemented with 10% FBS, GlutaMAXTM-I, sodium pyruvate, non-essential amino acids, penicillin and streptomycin (all Invitrogen). Human umbilical vein endothelial cells (HUVECs) prepared and grown as described [22] were kindly provided by B. Wolff and used at passages 3–4. Plasmids and Transfections A CXCR7-DRYLAIV mutant was generated by introducing site-specific point mutations at amino acids Ser(145) and Thr(147) of human CXCR7 by PCR (AccuPrimeTM Pfx SuperMix, Invitrogen) with the following primers: 59–CCGCTACCTCGCCATCGTC- TACTTCACC, 59–GGTGAAGTAGACGATGGCGAGGTA- GCGG. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N-terminus of mCherry to generate a plasma membrane marker [23]. All mutations were confirmed by DNA sequencing. Cells were transfected with pcDNA3 plasmids encoding the human wild type and mutated receptors using LipofectamineTM 2000 (Invitrogen), followed by G418 (Sigma-Aldrich) selection. Surface receptor expression levels CXCR7 Scavenges CXCL12 were determined by FACS analysis using saturating amounts of primary antibodies (15 mg/ml anti-CXCR4 (12G5) and anti- CXCR7 (9C4)) and secondary antibodies (10 mg/ml Alexa Fluor 488-F(ab9)2 goat anti-mouse IgG). Receptor Expression on Cell Surface after CXCL12 Incubation Daudi B cells were incubated in RPMI culture medium containing 0, 100 or 1000 nM CXCL12, 50 mM cycloheximide, and washed briefly with 1 M NaCl, 50 mM glycine, pH 3. When indicated 2 mM bafiloycin A1 (Sigma) in DMSO (0.5% f.c.) was added. Receptor surface levels at 37uC were monitored over time by FACS analysis after labeling with anti-CXCR4 (12G5) and anti-CXCR7 (9C4) and Alexa Fluor 488-F(ab9)2 goat anti-mouse IgG. In control experiments cells were incubated at 0uC with or without CXCL12 for 1 hour and washed with PBS or subjected to an acid wash. After acidic wash chemokines were fully removed and did not affect antibody binding. Chemokine Uptake and Degradation MDCK cells expressing human CXCR7, CXCR7-DRYLAIV, CXCR4, mock-transfected MDCK, or primary HUVECs were grown on TranswellH insets (Costar, pore size 8 mm). When the cells reached confluence the culture medium was changed to Hank’s balanced salt solution (HBSS) containing 10 mM HEPES, and 1% BSA. Radio-labeled [125I] CXCL12 (GE Healthcare, 0.25 nM) was added to the apical side of the monolayer for 3 hours at 37uC. To assess uptake and transport of ligand across the monolayer, both top and bottom contents of the Transwell as well as cell lysates were collected. Trichloracetic acid (TCA, 10% f.c.) precipitation was used to distinguish intact (precipitated) from degraded (TCA soluble) chemokine. The radioactivity of the samples was quantified using a gamma counter (Packard Instruments). Confocal Microscopy of MDCK-CXCR7 Monolayers MDCK cells expressing human CXCR7 or CXCR4 were grown on glass bottom dishes. Confluent cells were incubated for 15 min with anti-CXCR7 (11G8 R&D) or anti CXCR4 (MAB173 R&D) in growth medium containing 0.1% saponin followed by incubation with Alexa Fluor 488-F(ab9)2 goat anti-mouse IgG in the same medium. After washing the secondary antibodies dishes were immediately analyzed with a Leica TCS SP5 confocal microscope. To monitor ligand-independent antibody uptake, MDCK cells co-expressing membrane-anchored mCherry fluorescent protein and human CXCR7 or CXCR4 were grown on glass cover slips. When confluent, cells were incubated with anti-human CXCR7 (9C4) or anti-human CXCR4 (12G5) for 60 min at 37uC. Location of the primary antibody was revealed with Alexa Fluor 488-F(ab9)2 goat anti-mouse IgG after fixing for 5 min in 4% paraformaldehyde and permeabilizing the cells with 0.02% Tween20. Slides were washed with PBS-Tween20 (0.02%) and incubated for 30 min with Alexa Fluor 488-F(ab9)2 goat anti- mouse IgG, Alexa Fluor 594 phalloidin (both Invitrogen) and DAPI. Mounted slides were viewed with a Leica TCS SP5 confocal microscope. Receptor Re-Expression at the Cell Surface MDCK cell and Daudi surface molecules were cleaved by proteinase K (0.1 mg/ml in PBS) for 3 hours on ice before transfer to PBS, 2% FBS, 2 mM EDTA, 50 mM cycloheximide. Receptor surface re-expression at 37uC was monitored over time by FACS PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9175