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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 in cells co-expressing ChemR23 and CCR7 or CXCR4.Competition binding assays were performed on cells expressing CXCR4 (A), CCR7 (C) or ChemR23 only (E and G), and on cells co-expressing ChemR23 and CXCR4 (B and F), or ChemR23 and CCR7 (D and H). Cells were incubated with 0.1 nM 125I-CXCL12 (A and B), 125I-CCL19 (C and D) or 125I-[145–157]-chemerin (E–H), as tracers and increasing concentrations of unlabelled chemerin (•), CCL19 (□) or CXCL12 (○) as competitors. After one hour incubation, unbound tracers were separated by filtration and filters washed twice before counting. The data were normalized for nonspecific binding (0%) in the presence of 300 nM of competitor, and specific binding in the absence of competitor (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of two independent experiments.
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pone-0058075-g003: Competition binding assays in cells co-expressing ChemR23 and CCR7 or CXCR4.Competition binding assays were performed on cells expressing CXCR4 (A), CCR7 (C) or ChemR23 only (E and G), and on cells co-expressing ChemR23 and CXCR4 (B and F), or ChemR23 and CCR7 (D and H). Cells were incubated with 0.1 nM 125I-CXCL12 (A and B), 125I-CCL19 (C and D) or 125I-[145–157]-chemerin (E–H), as tracers and increasing concentrations of unlabelled chemerin (•), CCL19 (□) or CXCL12 (○) as competitors. After one hour incubation, unbound tracers were separated by filtration and filters washed twice before counting. The data were normalized for nonspecific binding (0%) in the presence of 300 nM of competitor, and specific binding in the absence of competitor (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of two independent experiments.

Mentions: We next compared the ability of ligands of the three receptors to inhibit the binding of specific radiolabelled tracers (125I-chemerin, 125I-CXCL12 or 125I-CCL19) to cells expressing single receptors or pairs of receptors. Competition binding assays showed that unlabelled chemerin inhibits the binding of 125I-CXCL12 or 125I-CCL19 on cells co-expressing ChemR23 and CXCR4 (Figure 3 B), or ChemR23 and CCR7 (Figure 3 D), but not on cells expressing CXCR4 only (Figure 3 A) or CCR7 only (Figure 3 C). Conversely, the CXCR4- and CCR7-specific ligands, CXCL12 and CCL19 respectively, inhibited the binding of 125I-chemerin, only on cells co-expressing ChemR23 and one of the chemokine receptors (Figure 3 F and 3 H). The IC50 values calculated for heterologous competition were in the same range as those estimated for homologous competition on cells expressing single receptors (Table 1). These results show thus a negative binding cooperativity between the binding pockets of ChemR23/CXCR4 and ChemR23/CCR7, similar to what we reported previously for chemokine receptor pairs. To our knowledge, this is the first example of negative binding cooperativity involving chemokines and a structurally unrelated chemoattractant protein.


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 in cells co-expressing ChemR23 and CCR7 or CXCR4.Competition binding assays were performed on cells expressing CXCR4 (A), CCR7 (C) or ChemR23 only (E and G), and on cells co-expressing ChemR23 and CXCR4 (B and F), or ChemR23 and CCR7 (D and H). Cells were incubated with 0.1 nM 125I-CXCL12 (A and B), 125I-CCL19 (C and D) or 125I-[145–157]-chemerin (E–H), as tracers and increasing concentrations of unlabelled chemerin (•), CCL19 (□) or CXCL12 (○) as competitors. After one hour incubation, unbound tracers were separated by filtration and filters washed twice before counting. The data were normalized for nonspecific binding (0%) in the presence of 300 nM of competitor, and specific binding in the absence of competitor (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585228&req=5

pone-0058075-g003: Competition binding assays in cells co-expressing ChemR23 and CCR7 or CXCR4.Competition binding assays were performed on cells expressing CXCR4 (A), CCR7 (C) or ChemR23 only (E and G), and on cells co-expressing ChemR23 and CXCR4 (B and F), or ChemR23 and CCR7 (D and H). Cells were incubated with 0.1 nM 125I-CXCL12 (A and B), 125I-CCL19 (C and D) or 125I-[145–157]-chemerin (E–H), as tracers and increasing concentrations of unlabelled chemerin (•), CCL19 (□) or CXCL12 (○) as competitors. After one hour incubation, unbound tracers were separated by filtration and filters washed twice before counting. The data were normalized for nonspecific binding (0%) in the presence of 300 nM of competitor, and specific binding in the absence of competitor (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of two independent experiments.
Mentions: We next compared the ability of ligands of the three receptors to inhibit the binding of specific radiolabelled tracers (125I-chemerin, 125I-CXCL12 or 125I-CCL19) to cells expressing single receptors or pairs of receptors. Competition binding assays showed that unlabelled chemerin inhibits the binding of 125I-CXCL12 or 125I-CCL19 on cells co-expressing ChemR23 and CXCR4 (Figure 3 B), or ChemR23 and CCR7 (Figure 3 D), but not on cells expressing CXCR4 only (Figure 3 A) or CCR7 only (Figure 3 C). Conversely, the CXCR4- and CCR7-specific ligands, CXCL12 and CCL19 respectively, inhibited the binding of 125I-chemerin, only on cells co-expressing ChemR23 and one of the chemokine receptors (Figure 3 F and 3 H). The IC50 values calculated for heterologous competition were in the same range as those estimated for homologous competition on cells expressing single receptors (Table 1). These results show thus a negative binding cooperativity between the binding pockets of ChemR23/CXCR4 and ChemR23/CCR7, similar to what we reported previously for chemokine receptor pairs. To our knowledge, this is the first example of negative binding cooperativity involving chemokines and a structurally unrelated chemoattractant protein.

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