analysis using antibodies recognizing the extracellular C-terminus of the transferrin receptor (OKT9), and the extracellular N- termini of CXCR4 (6H8) [24] and CXCR7 (9C4) [5] and secondary Alexa Fluor 488-F(ab9)2 goat anti-mouse IgG. mRNA Expression Constructs for Zebrafish The construct used for global expression of CXCR7-EGFP was generated by cloning the cxcr7-egfp ORF into the pSP64TS vector that contains 59 and 39 UTRs of the Xenopus globin gene. Capped sense mRNA was synthesized using the mMessageMachine kit (Ambion) according to the manufacturer’s instructions. Microinjections for Global Expression and Knockdown in Zebrafish Embryos RNA and morpholino antisense oligonucleotides were microin- jected into the yolk of one-cell stage embryos. For global expression of CXCR7 225 pg of cxcr7-egfp-globin mRNA were injected. Knockdown of both zebrafish CXCL12 versions was achieved using 0.4 pmol of oligonucleotides against cxcl12a (59–TTGAGATC- CATGTTTGCAGTGTGAA-39), and cxcl12b (59-TTGCTATC- CATGCCAAGAGCGAGTG-39) Spinning Disk Confocal Microscopy of Zebrafish Embryos Confocal fluorescence images were obtained using a Zeiss AxioImager.M1 microscope fitted with a CascadeII camera (Photometrics) and VS-Laser Control, all controlled by VisiView software (Visitron Systems). Confocal Microscopy of Chemokine Uptake in Human Umbilical Cords and Mouse Hearts Sections of 3–5 mm thickness of fresh human umbilical cords were placed in DMEM containing 1% human albumin, 200 nM fluorescent chemokine or PBS. When indicated, 10 mM AMD3100 (Sigma) or 1.5 mM CXCL11 were added to the medium. After 30 min incubation the sections were washed in DMEM and mounted in O.C.T. compound (VWR). Cryosections of 20 mm were fixed for 10 min in 4% paraformaldehyde, washed in PBST, and incubated for 1 h with anti-human CD31 (BD Biosciences) in PBST, 1% goat serum (Dako). Slides were washed with PBST and incubated for 1 h with Alexa Fluor 594-F(ab9)2 goat anti-mouse IgG (Invitrogen) and DAPI. Recombinant fluorescent chemokines were produced in the laboratory by expressing CXCL12 tagged at the C-terminus with fluorescent proteins in insect cells (E.C. and M.T., unpublished). Hearts of wild-type mice (BALB/c) were excised, and heart tips were removed to access the ventricles. After rinsing with DPBS, 2% FBS, LiqueminH (Roche) the specimens were incubated with fluorescent chemokine as described above. For confocal micros- copy of the cryosections, nuclei of fixed and permeabilized cryosections were stained with DAPI. Statistical Analysis All statistical analyses were performed using SigmaPlot software (Systat Software). Statistical comparisons between datasets were made with Student’s t-test. Results We previously reported that CXCL12-induced internalization of CXCR7 is inconsistent between primary circulating human CD19+ B cells and mouse pre 300.19 B cells stably expressing the receptor [10]. The chemokine induces CXCR7 internalization in transfected cells, but has little effect on the level of surface receptor CXCR7 Scavenges CXCL12 expression on primary B cells. Similar lack of CXCR7 down- regulation after CXCL12 treatment was observed in other cells which express endogenous CXCR7, such as monocytes, the monocytic cell line THP-1, B cell clones and tumor derived cells. We now reiterated this observation in more detail using Daudi (Figure 1A) and Raji cells which express endogenous CXCR4 and CXCR7 at the plasma membrane [10]. Receptor surface expression was determined with specific monoclonal antibodies by FACS analysis. Bound chemokine, which could prevent antibody binding, was efficiently removed by an acidic wash prior antibody application [25]. In agreement with previous reports stimulation of the cells with CXCL12 induces a concentration- dependent disappearance of CXCR4 from the cell surface (not shown) [25,26]. Figure 1B shows the maximum internalization of CXCR4 and CXCR7 which was induced by CXCL12 treatment. Approx. 50% of CXCR4 became internalized after 20 min and down-regulation increased to about 70–80% (75%610% SD) after 1 h (Figure 1B). By contrast, little overall internalization (10– 25%) of CXCR7 was detected after 60 min. However, at early time points after CXCL12 addition a transient CXCR7 down- regulation was observed which showed a maximum (up to 40%) after 10 min and recovered thereafter. The time course with a minimum at 10 min was consistently observed in Daudi and Raji cells (Figure 1B). The finding suggests that CXCR7 expression at the cell surface is tightly regulated and the receptor is rapidly replenished from intracellular stores after an initial agonist- induced internalization. Indeed CXCR7 expression fully recov- ered after removal of the chemokine and incubation at 37uC for 30 min. By contrast CXCR4 expression remained down regulated under these conditions (Figure 1C). Bafilomycin A is a well established inhibitor of vATPases, which mediates the gradual acidification of organelles going from the cell surface to lysosomes [27–29]. Treatment of Daudi cells with bafilomycin A had no effect on ligand-induced CXCR4 down regulation and CXCL12 induced CXCR7 internalization (Figure 1C). However, recycling of CXCR7 was markedly attenuated, suggesting that lowering the pH in the lumen of the endosome is required for the dissociation of CXCL12 from CXCR7 and thereby promoting subsequent receptor recycling to the plasma membrane. Receptor de novo synthesis during the course of the experiments was excluded by the presence of the translation inhibitor cycloheximide. We therefore hypothesized that CXCR7 acts as a scavenger for CXCL12. To this end Madin-Darby canine kidney (MDCK) epithelial cells were stably transfected with CXCR7, CXCR4, or CXCR7 lacking the cytoplasmic C terminus (DCXCR7). Because CXCR7 does not trigger pertussis toxin sensitive responses [8], we created also a chimeric receptor where we replaced the DRYLSIT-sequence in the second intracellular loop of CXCR7 with the corresponding DRYLAIV-sequence from CXCR4. This sequence is considered critical for functional coupling of GPCRs to Gi-proteins [8,30]. Restoration of the motif in the scavenger D6 confers weak ligand-induced signaling activity [31]. The expression of all receptors on the cell surface was confirmed by FACS analysis (Figure 2A). The chimeric CXCR7 was expressed to a similar level as its wild type counterpart (Figure 2A). Nevertheless, introduction of the canonical DRYLAIV motif into CXCR7 instead of DRYLSIT did not result in CXCL12-inducible chemotaxis, calcium mobilization, or ERK activation when expressed in either MDCK cells or 300.19 pre-B cells (not shown). Confocal microscopy of permeabilized MDCK cells revealed a marked punctuated staining arising from below the cell surface, suggesting that a large fraction of both receptors resides on intracellular compartments (Figure 2B). PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9175