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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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Ectopic expression of CXCR7 and CXCR4 on MDCK cells.(A) MDCK were stably transfected with empty vector (Mock), with CXCR7, CXCR4, a vector coding for a CXCR7 lacking the cytoplasmic C-terminus (ΔCXCR7), and a vector coding for a chimeric CXCR7 containing the DRYLAIV motive of CXCR4. Receptor expression was determined by FACS analysis using saturating antibody concentrations (see Methods). (B) Confocal immunofluorescence analysis of unfixed MDCK cells expressing CXCR7 (upper panels) or CXCR4 (lower panels). Cells also expressed N-ter-Lck mCherry as membrane marker (red fluorescence). Left panels: confocal images of planes cut through intracellular regions of MDCK monolayers. Right panels: x-z planes reconstructed from confocal x-y stacks. For receptor (green) detection anti-CXCR7 (11G8 R&D) or anti CXCR4 (MAB173 R&D) were used. Receptor-bound primary antibodies were revealed with goat anti mouse IgG conjugated with Alexa488 (green fluorescence).
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pone-0009175-g002: Ectopic expression of CXCR7 and CXCR4 on MDCK cells.(A) MDCK were stably transfected with empty vector (Mock), with CXCR7, CXCR4, a vector coding for a CXCR7 lacking the cytoplasmic C-terminus (ΔCXCR7), and a vector coding for a chimeric CXCR7 containing the DRYLAIV motive of CXCR4. Receptor expression was determined by FACS analysis using saturating antibody concentrations (see Methods). (B) Confocal immunofluorescence analysis of unfixed MDCK cells expressing CXCR7 (upper panels) or CXCR4 (lower panels). Cells also expressed N-ter-Lck mCherry as membrane marker (red fluorescence). Left panels: confocal images of planes cut through intracellular regions of MDCK monolayers. Right panels: x-z planes reconstructed from confocal x-y stacks. For receptor (green) detection anti-CXCR7 (11G8 R&D) or anti CXCR4 (MAB173 R&D) were used. Receptor-bound primary antibodies were revealed with goat anti mouse IgG conjugated with Alexa488 (green fluorescence).

Mentions: 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 (ΔCXCR7). 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).


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)

Ectopic expression of CXCR7 and CXCR4 on MDCK cells.(A) MDCK were stably transfected with empty vector (Mock), with CXCR7, CXCR4, a vector coding for a CXCR7 lacking the cytoplasmic C-terminus (ΔCXCR7), and a vector coding for a chimeric CXCR7 containing the DRYLAIV motive of CXCR4. Receptor expression was determined by FACS analysis using saturating antibody concentrations (see Methods). (B) Confocal immunofluorescence analysis of unfixed MDCK cells expressing CXCR7 (upper panels) or CXCR4 (lower panels). Cells also expressed N-ter-Lck mCherry as membrane marker (red fluorescence). Left panels: confocal images of planes cut through intracellular regions of MDCK monolayers. Right panels: x-z planes reconstructed from confocal x-y stacks. For receptor (green) detection anti-CXCR7 (11G8 R&D) or anti CXCR4 (MAB173 R&D) were used. Receptor-bound primary antibodies were revealed with goat anti mouse IgG conjugated with Alexa488 (green fluorescence).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820091&req=5

pone-0009175-g002: Ectopic expression of CXCR7 and CXCR4 on MDCK cells.(A) MDCK were stably transfected with empty vector (Mock), with CXCR7, CXCR4, a vector coding for a CXCR7 lacking the cytoplasmic C-terminus (ΔCXCR7), and a vector coding for a chimeric CXCR7 containing the DRYLAIV motive of CXCR4. Receptor expression was determined by FACS analysis using saturating antibody concentrations (see Methods). (B) Confocal immunofluorescence analysis of unfixed MDCK cells expressing CXCR7 (upper panels) or CXCR4 (lower panels). Cells also expressed N-ter-Lck mCherry as membrane marker (red fluorescence). Left panels: confocal images of planes cut through intracellular regions of MDCK monolayers. Right panels: x-z planes reconstructed from confocal x-y stacks. For receptor (green) detection anti-CXCR7 (11G8 R&D) or anti CXCR4 (MAB173 R&D) were used. Receptor-bound primary antibodies were revealed with goat anti mouse IgG conjugated with Alexa488 (green fluorescence).
Mentions: 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 (ΔCXCR7). 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).

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