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Consequences of ChemR23 heteromerization with the chemokine receptors CXCR4 and CCR7.

de Poorter C, Baertsoen K, Lannoy V, Parmentier M, Springael JY - PLoS ONE (2013)

Bottom Line: Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity.As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other.Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) Université Libre de Bruxelles (U.L.B.), Campus Erasme, Brussels, Belgium.

ABSTRACT
Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

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Competition binding assays on BMDCs.[A, B] FACS analysis showing the cell surface expression of mChemR23/Dez on BMDCs generated from wild-type or ChemR23−/− mice. Cells were incubated with an anti-mChemR23 antibody (open histogram) or a control isotype (filled histogram). [C, D] Competition binding assays were performed on BMDCs generated from wild-type (C) or ChemR23−/− mice (D). Purified cells were incubated with 0.2 nM 125I-CXCL12 as tracer, and CXCL12 (300 nM), chemerin (300 nM) or a monoclonal anti-CXCR4 antibody (10 µg/ml) as competitors. After one hour incubation, unbound tracer was separated by filtration and filters washed twice before counting. The data were normalized for non-specific binding (0%) and specific binding in the absence of competitor (100%). Statistical significance as compared to the 100% values was tested by two-way analysis of variance followed by Tukey's test (***, P<0.001; **, P<0.01). All data points were performed in triplicate and the displayed data are the mean of five experiments performed with three independent cell preparations (error bars indicate S.E.M.).
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pone-0058075-g006: Competition binding assays on BMDCs.[A, B] FACS analysis showing the cell surface expression of mChemR23/Dez on BMDCs generated from wild-type or ChemR23−/− mice. Cells were incubated with an anti-mChemR23 antibody (open histogram) or a control isotype (filled histogram). [C, D] Competition binding assays were performed on BMDCs generated from wild-type (C) or ChemR23−/− mice (D). Purified cells were incubated with 0.2 nM 125I-CXCL12 as tracer, and CXCL12 (300 nM), chemerin (300 nM) or a monoclonal anti-CXCR4 antibody (10 µg/ml) as competitors. After one hour incubation, unbound tracer was separated by filtration and filters washed twice before counting. The data were normalized for non-specific binding (0%) and specific binding in the absence of competitor (100%). Statistical significance as compared to the 100% values was tested by two-way analysis of variance followed by Tukey's test (***, P<0.001; **, P<0.01). All data points were performed in triplicate and the displayed data are the mean of five experiments performed with three independent cell preparations (error bars indicate S.E.M.).

Mentions: Finally, we tested whether negative binding cooperativity could be detected in cells co-expressing ChemR23 and CXCR4 endogenously, using bone marrow dendritic cells (BMDC) generated from wild-type C57/Bl6 mice. Specific 125I-CXCL12 binding was detected on BMDCs, as demonstrated by full competition with anti-CXCR4 antibodies. Binding of 125I-CXCL12 was completely inhibited by unlabelled CXCL12 but also partially by unlabelled chemerin, demonstrating cross-competition by the ChemR23 ligand (Figure 6 B). We next performed competition experiments on cells prepared from ChemR23−/− mice and showed that chemerin did not compete for 125I-CXCL12 binding, indicating that the absence of ChemR23 completely abolished the cross-inhibition by ChemR23-specific ligands (Figure 6 C). Despite expression of ChemR23 detectable by FACS at the cell surface (Figure 6 A), only weak chemerin binding was measured on wild type BMDCs (Figure S3). This weak signal is most likely due to the relatively low expression level of ChemR23 in leukocytes combined to the poor stability of our peptidic tracer in biological media. Indeed, even on CHO-K1 cells overexpressing ChemR23, the binding assay is tedious and the specific window rather small (Figure S4). We did not investigate ChemR23/CCR7 binding cooperativity on BMDCs, as ChemR23 is inactivated and downregulated in parallel to CCR7 induction during maturation of dendritic cells [23]. The presence of both ChemR23 and CCR7 is therefore transient on these cells and difficult to study.


Consequences of ChemR23 heteromerization with the chemokine receptors CXCR4 and CCR7.

de Poorter C, Baertsoen K, Lannoy V, Parmentier M, Springael JY - PLoS ONE (2013)

Competition binding assays on BMDCs.[A, B] FACS analysis showing the cell surface expression of mChemR23/Dez on BMDCs generated from wild-type or ChemR23−/− mice. Cells were incubated with an anti-mChemR23 antibody (open histogram) or a control isotype (filled histogram). [C, D] Competition binding assays were performed on BMDCs generated from wild-type (C) or ChemR23−/− mice (D). Purified cells were incubated with 0.2 nM 125I-CXCL12 as tracer, and CXCL12 (300 nM), chemerin (300 nM) or a monoclonal anti-CXCR4 antibody (10 µg/ml) as competitors. After one hour incubation, unbound tracer was separated by filtration and filters washed twice before counting. The data were normalized for non-specific binding (0%) and specific binding in the absence of competitor (100%). Statistical significance as compared to the 100% values was tested by two-way analysis of variance followed by Tukey's test (***, P<0.001; **, P<0.01). All data points were performed in triplicate and the displayed data are the mean of five experiments performed with three independent cell preparations (error bars indicate S.E.M.).
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pone-0058075-g006: Competition binding assays on BMDCs.[A, B] FACS analysis showing the cell surface expression of mChemR23/Dez on BMDCs generated from wild-type or ChemR23−/− mice. Cells were incubated with an anti-mChemR23 antibody (open histogram) or a control isotype (filled histogram). [C, D] Competition binding assays were performed on BMDCs generated from wild-type (C) or ChemR23−/− mice (D). Purified cells were incubated with 0.2 nM 125I-CXCL12 as tracer, and CXCL12 (300 nM), chemerin (300 nM) or a monoclonal anti-CXCR4 antibody (10 µg/ml) as competitors. After one hour incubation, unbound tracer was separated by filtration and filters washed twice before counting. The data were normalized for non-specific binding (0%) and specific binding in the absence of competitor (100%). Statistical significance as compared to the 100% values was tested by two-way analysis of variance followed by Tukey's test (***, P<0.001; **, P<0.01). All data points were performed in triplicate and the displayed data are the mean of five experiments performed with three independent cell preparations (error bars indicate S.E.M.).
Mentions: Finally, we tested whether negative binding cooperativity could be detected in cells co-expressing ChemR23 and CXCR4 endogenously, using bone marrow dendritic cells (BMDC) generated from wild-type C57/Bl6 mice. Specific 125I-CXCL12 binding was detected on BMDCs, as demonstrated by full competition with anti-CXCR4 antibodies. Binding of 125I-CXCL12 was completely inhibited by unlabelled CXCL12 but also partially by unlabelled chemerin, demonstrating cross-competition by the ChemR23 ligand (Figure 6 B). We next performed competition experiments on cells prepared from ChemR23−/− mice and showed that chemerin did not compete for 125I-CXCL12 binding, indicating that the absence of ChemR23 completely abolished the cross-inhibition by ChemR23-specific ligands (Figure 6 C). Despite expression of ChemR23 detectable by FACS at the cell surface (Figure 6 A), only weak chemerin binding was measured on wild type BMDCs (Figure S3). This weak signal is most likely due to the relatively low expression level of ChemR23 in leukocytes combined to the poor stability of our peptidic tracer in biological media. Indeed, even on CHO-K1 cells overexpressing ChemR23, the binding assay is tedious and the specific window rather small (Figure S4). We did not investigate ChemR23/CCR7 binding cooperativity on BMDCs, as ChemR23 is inactivated and downregulated in parallel to CCR7 induction during maturation of dendritic cells [23]. The presence of both ChemR23 and CCR7 is therefore transient on these cells and difficult to study.

Bottom Line: Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity.As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other.Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) Université Libre de Bruxelles (U.L.B.), Campus Erasme, Brussels, Belgium.

ABSTRACT
Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

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Related in: MedlinePlus