reveal an effective capacity of CXCR7 to eliminate CXCL12 from its surrounding environment. The efficiency of scavenger receptors depends on their capability to mediate the elimination of a stoichiometric multiplicity of ligands from the surrounding environment. In figure 3B we demonstrate that degradation of CXCL12 by CXCR7 is not saturated even at elevated chemokine concentrations. Heptahelical receptors have been described which bind chemokines with high affinity, but fail to couple to heterotrimetric G-proteins and to induce typical responses. In mammalians three ‘atypical’ chemokine receptors were characterized namely the Duffy antigen receptor for chemokines (DARC), D6, and CCX- CKR (CCR11). DARC has recently been shown to transcytose inflammatory chemokines from the basal to the luminal site of the venular endothelium. Since chemokines remain bound to DARC expressed in the lumen of venules the receptor supports chemokine migratory activity and leukocyte extravasation [41]. On erythro- cytes DARC is assumed to function as sink or reservoir for many inflammatory chemokines [42]. The primary function of D6 and CCX-CKR is to act as scavenger for chemokines. D6 contributes to the resolution of inflammation by internalizing and destroying inflammatory CC chemokines [43,44]. CCX-CKR effectively internalizes the homeostatic CC chemokines CCL19 and CCL21 targeting them for degradation [45]. However, CXCL12 does not bind to any of the ‘atypical’ receptors. Like the other decoy receptors for chemokines CXCR7 does not activate heterotrimeric G-proteins, a compulsory step for typical signal transduction of heptahelical receptors [8,46]. Nevertheless, internalization of this class of receptors does not require coupling to G-proteins. Recently it was reported that CXCL12 induces arrestin binding to CXCR7, indicating that the agonist can promote internalization through clathrin coated pits [38]. In line with this observation we found that deletion of the C-terminus of CXCR7 markedly reduces scavenger activity (Figure 3D and 4D). Exceptionally among chemokine receptors CXCR4 can induce the sustained activation of the PI 3-kinase/PKB and MAPK cascade [46]. This responsiveness may contribute to the proliferative and pro-survival characteristics of the CXCL12/ CXCR4 axis [47,48]. Depending on the context, however, the properties can be detrimental for the host organism und must therefore be under tight control. The prolonged signaling by CXCR4 demands the continuous presence of CXCL12 at the cell surface and is abrogated when the chemokines becomes degraded, e.g. by CD26/dipeptidyl peptidase IV [26,49,50]. Blocking of the CXCL12/CXCR4 signaling axis leads to the proliferation of primitive hematopoietic stem cells [51] while excessive signaling by CXCR4 accounts for the molecular pathoetiology of the WHIM syndrome [52] and is associated with tumor growth and metastasis formation [53]. These observations indicate that the level and availability of CXCL12 for CXCR4 is tightly regulated under normal conditions and the present data indicate that CXCR7, which is widely expressed, may critically contribute to this regulation. An alternative regulation of CXCR4 activity by CXCR7 could be potential heterodimerization of the receptors. Co-transfection of CXCR7 and CXCR4 in HEK cells indicates that the receptors can functionally heterodimerize [9,15]. However, in primary References CXCR7 Scavenges CXCL12 human CD19+ B cells, which express both receptors in circulation, co-internalization, a hall mark of heptahelical receptor dimers, was not observed, suggesting that heterodimerization in primary cells is of minor relevance [1,10]. The perinatal lethality of mice with a deletion of Cxcr7 [9,54] could be explained by the lack of CXCL12 scavenging in these animals. Platelets which carry large amounts of CXCL12 may deploy the chemokine when undergoing mechanical stress in the heart circulation. The excessive presence of the chemokine may subsequently stimulate CXCR4 which is highly expressed on the heart endothelium (MT, unpublished), leading to aberrant proliferation [55]. Such view is also in line with the expression of CXCR7 on the heart valves of adult mice. The evolutionary conserved mechanism of CXCL12 scavenging by CXCR7 further supports the importance of this activity [19]. In a previous study, when CXCR7 as receptor for CXCL12 was not yet disclosed, scavenging of CXCL12 by endothelial CXCR4 was proposed, which was partially attenuated in the presence of CXCR4 inhibitors. The authors further suggested that CXCR4 translo- cates CXCL12 to the bone marrow [56]. Whether CXCR7 can transcytose CXCL12, in analogy to the activity of DARC [41], remains to be established. The present findings, however, fully support the conclusion that the principal function of CXCR7 is to act as scavenger for CXCL12. Supporting Information Movie S1 Cont Ligand-independent cycling of CXCR7 in zebrafish embryos. In embryos expressing CXCR7-EGFP the receptor localizes at the plasma membrane (grey) and intracellular vesicles (blue dot). Time laps shows that the vesicles are in contact with the membrane internalize and recycle back to the membrane. Internalization and cycling of CXCR7-EGF in the presence (control - MO) of CXCL12. Found at: doi:10.1371/journal.pone.0009175.s001 (4.61 MB MOV) Movie S2 CXCL12 Ligand-independent cycling of CXCR7 in zebrafish embryos. In embryos expressing CXCR7-EGFP the receptor localizes at the plasma membrane (grey) and intracellular vesicles (blue dot). Time laps shows that the vesicles are in contact with the membrane internalize and recycle back to the membrane. Internalization and cycling of CXCR7-EGF in the absence of CXCL12 (CXCL12 - MO). Found at: doi:10.1371/journal.pone.0009175.s002 (3.27 MB MOV) Acknowledgments The authors would like to thank Dr. B. Wolff (Vienna) for providing HUVECs, S. Volpe (Bellinzona) for the Lck-mCherry construct, P. Bombosi and L. Mudde (Vienna) for assistance. Author Contributions Conceived and designed the experiments: UN ER AR MT. Performed the experiments: UN HM. Analyzed the data: UN MT. Contributed reagents/ materials/analysis tools: EC MP HM ER HGZ AR MT. Wrote the paper: MT. 1. Thelen M, Stein JV (2008) How chemokines invite leukocytes to dance. Nat Immunol 9: 953–959. 2. Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, et al. (1996) Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382: 635–638. 3. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393: 595–599. 4. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, et al. (1998) Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9175