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CXCR7 functions as a scavenger for CXCL12 and CXCL11.

Naumann U, Cameroni E, Pruenster M, Mahabaleshwar H, Raz E, Zerwes HG, Rot A, Thelen M - PLoS ONE (2010)

Bottom Line: We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation.In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations.Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine, Bellinzona, Switzerland.

ABSTRACT

Background: CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues.

Methodology/principal findings: We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium.

Conclusions/significance: The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.

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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 µM (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 37°C 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 (60′ internalization), or following an additional incubation for 30 min at 37°C to allow receptor reexpression (+30′ recycling). DMSO vehicle (circles) and 2 µM 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.
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pone-0009175-g001: 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 µM (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 37°C 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 (60′ internalization), or following an additional incubation for 30 min at 37°C to allow receptor reexpression (+30′ recycling). DMSO vehicle (circles) and 2 µM 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.

Mentions: 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 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%±10% 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 recovered after removal of the chemokine and incubation at 37°C 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.


CXCR7 functions as a scavenger for CXCL12 and CXCL11.

Naumann U, Cameroni E, Pruenster M, Mahabaleshwar H, Raz E, Zerwes HG, Rot A, Thelen M - PLoS ONE (2010)

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 µM (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 37°C 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 (60′ internalization), or following an additional incubation for 30 min at 37°C to allow receptor reexpression (+30′ recycling). DMSO vehicle (circles) and 2 µM 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820091&req=5

pone-0009175-g001: 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 µM (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 37°C 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 (60′ internalization), or following an additional incubation for 30 min at 37°C to allow receptor reexpression (+30′ recycling). DMSO vehicle (circles) and 2 µM 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.
Mentions: 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 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%±10% 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 recovered after removal of the chemokine and incubation at 37°C 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.

Bottom Line: We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation.In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations.Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine, Bellinzona, Switzerland.

ABSTRACT

Background: CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues.

Methodology/principal findings: We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium.

Conclusions/significance: The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.

Show MeSH
Related in: MedlinePlus