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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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Aequorin-based functional assay in cells co-expressing ChemR23 with CCR7 or CXCR4.The functional responses of CHO-K1 cells expressing ChemR23 (▪), CXCR4 (•), CCR7 (♦), ChemR23 + CXCR4 (□) or ChemR23 + CCR7 (○) were measured using the aequorin-based calcium mobilization assay. Cells pre-loaded with coelenterazine H were stimulated with increasing concentrations of chemerin (A, C), CXCL12 (B) or CCL19 (D) and luminescence was recorded for 30 s. The results were normalized for baseline activity (0%) and the maximal response obtained with 25 µM ATP (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of three independent experiments.
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pone-0058075-g004: Aequorin-based functional assay in cells co-expressing ChemR23 with CCR7 or CXCR4.The functional responses of CHO-K1 cells expressing ChemR23 (▪), CXCR4 (•), CCR7 (♦), ChemR23 + CXCR4 (□) or ChemR23 + CCR7 (○) were measured using the aequorin-based calcium mobilization assay. Cells pre-loaded with coelenterazine H were stimulated with increasing concentrations of chemerin (A, C), CXCL12 (B) or CCL19 (D) and luminescence was recorded for 30 s. The results were normalized for baseline activity (0%) and the maximal response obtained with 25 µM ATP (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of three independent experiments.

Mentions: We then compared the functional response of cells co-expressing CXCR4 and ChemR23 or CCR7 and ChemR23 to cell lines expressing only one receptor. The concentration-action curves upon stimulation by chemerin were similar whatever ChemR23 was expressed alone or with one of the chemokine receptors (Figure 4 A and 4 C, Table 2). Similarly, concentration-action curves upon stimulation by CXCL12 or CCL19 were not altered by the presence of ChemR23 (Figure 4 C and 4 D, Table 2).


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)

Aequorin-based functional assay in cells co-expressing ChemR23 with CCR7 or CXCR4.The functional responses of CHO-K1 cells expressing ChemR23 (▪), CXCR4 (•), CCR7 (♦), ChemR23 + CXCR4 (□) or ChemR23 + CCR7 (○) were measured using the aequorin-based calcium mobilization assay. Cells pre-loaded with coelenterazine H were stimulated with increasing concentrations of chemerin (A, C), CXCL12 (B) or CCL19 (D) and luminescence was recorded for 30 s. The results were normalized for baseline activity (0%) and the maximal response obtained with 25 µM ATP (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585228&req=5

pone-0058075-g004: Aequorin-based functional assay in cells co-expressing ChemR23 with CCR7 or CXCR4.The functional responses of CHO-K1 cells expressing ChemR23 (▪), CXCR4 (•), CCR7 (♦), ChemR23 + CXCR4 (□) or ChemR23 + CCR7 (○) were measured using the aequorin-based calcium mobilization assay. Cells pre-loaded with coelenterazine H were stimulated with increasing concentrations of chemerin (A, C), CXCL12 (B) or CCL19 (D) and luminescence was recorded for 30 s. The results were normalized for baseline activity (0%) and the maximal response obtained with 25 µM ATP (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of three independent experiments.
Mentions: We then compared the functional response of cells co-expressing CXCR4 and ChemR23 or CCR7 and ChemR23 to cell lines expressing only one receptor. The concentration-action curves upon stimulation by chemerin were similar whatever ChemR23 was expressed alone or with one of the chemokine receptors (Figure 4 A and 4 C, Table 2). Similarly, concentration-action curves upon stimulation by CXCL12 or CCL19 were not altered by the presence of ChemR23 (Figure 4 C and 4 D, Table 2).

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.

Show MeSH
Related in: MedlinePlus