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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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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 37°C 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 µM CXCL11 or the CXCR4 inhibitors AMD3100 (10 µM) and NIBR-1816 (10 µM). (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 (ΔCXCR7) were exposed to [125I]-CXCL12 as in B.
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pone-0009175-g003: 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 37°C 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 µM CXCL11 or the CXCR4 inhibitors AMD3100 (10 µM) and NIBR-1816 (10 µM). (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 (ΔCXCR7) were exposed to [125I]-CXCL12 as in B.

Mentions: The scavenging activity of CXCR7 was measured using transfected MDCK cells seeded on Transwell® insets and grown to confluence. Radiolabeled [125I] CXCL12 was then added to the upper compartment and the cells incubated for 3 h at 37°C. To distinguish between degraded CXCL12 and intact or only partially proteolysed chemokine trichloroacetic acid (TCA) was added to the medium collected from the upper and lower compartment as well as the cell lysate to precipitate proteins. Radioactivity that is insensitive to TCA precipitation reflects chemokine that was cleaved by the cells during the incubation time. We compared the efficiency of MDCK cells expressing similar levels of CXCR7 and CXCR4 (Figure 2A) to degrade CXCL12. Cells expressing CXCR7 secreted significantly more CXCL12-derived radioactivity into the lower compartment than their CXCR4+ counterparts (Figure 3A). With either receptor the majority of the radioactivity was insensitive to TCA treatment, indicating that CXCL12 becomes largely degraded. Similar amounts of cleaved CXCL12 were also detected in the upper compartment of the transwell. We therefore summed all TCA-sensitive radioactivity recovered from the bottom of the well, in the cell lysate and from the upper well to calculate the total degradation activity (Figures 3B–F). We could not find evidence that the small fraction of TCA-sensitive radioactivity, which was transcytosed from the apical to the basal side by CXCR7, contained biological activity.


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)

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 37°C 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 µM CXCL11 or the CXCR4 inhibitors AMD3100 (10 µM) and NIBR-1816 (10 µM). (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 (ΔCXCR7) were exposed to [125I]-CXCL12 as in B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820091&req=5

pone-0009175-g003: 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 37°C 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 µM CXCL11 or the CXCR4 inhibitors AMD3100 (10 µM) and NIBR-1816 (10 µM). (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 (ΔCXCR7) were exposed to [125I]-CXCL12 as in B.
Mentions: The scavenging activity of CXCR7 was measured using transfected MDCK cells seeded on Transwell® insets and grown to confluence. Radiolabeled [125I] CXCL12 was then added to the upper compartment and the cells incubated for 3 h at 37°C. To distinguish between degraded CXCL12 and intact or only partially proteolysed chemokine trichloroacetic acid (TCA) was added to the medium collected from the upper and lower compartment as well as the cell lysate to precipitate proteins. Radioactivity that is insensitive to TCA precipitation reflects chemokine that was cleaved by the cells during the incubation time. We compared the efficiency of MDCK cells expressing similar levels of CXCR7 and CXCR4 (Figure 2A) to degrade CXCL12. Cells expressing CXCR7 secreted significantly more CXCL12-derived radioactivity into the lower compartment than their CXCR4+ counterparts (Figure 3A). With either receptor the majority of the radioactivity was insensitive to TCA treatment, indicating that CXCL12 becomes largely degraded. Similar amounts of cleaved CXCL12 were also detected in the upper compartment of the transwell. We therefore summed all TCA-sensitive radioactivity recovered from the bottom of the well, in the cell lysate and from the upper well to calculate the total degradation activity (Figures 3B–F). We could not find evidence that the small fraction of TCA-sensitive radioactivity, which was transcytosed from the apical to the basal side by CXCR7, contained biological activity.

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