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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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Effect of AMD3100 on binding and functional properties of cells expressing CXCR4 and ChemR23.[A, B] Competition binding assays were performed on cells expressing CXCR4 (•) and ChemR23 (▪) only, or cells co-expressing CXCR4 and ChemR23 (○). Cells were incubated with 0.1 nM 125I-CXCL12 (A) or 125I-[145–157]-chemerin (B), as tracers and increasing concentrations of unlabelled AMD3100 as competitor. 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 unlabelled CXCL12 or chemerin respectively, and specific binding in the absence of competitor (100%). All points were run in duplicate (error bars indicate S.E.M.). The displayed data are representative of two independent experiments. The functional responses of CHO-K1 cells expressing CXCR4 (C), ChemR23 (E) or both receptors (D, F) were measured by using the aequorin-based calcium mobilization assay. [C, D] Cells preloaded with coelenterazine were stimulated with increasing concentrations of CXCL12 (▪) or CXCL12 + AMD3100 (□). [E, F] Cells preloaded with coelenterazine were stimulated with increasing concentration of chemerin (•) or chemerin + AMD3100 (○). 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-g005: Effect of AMD3100 on binding and functional properties of cells expressing CXCR4 and ChemR23.[A, B] Competition binding assays were performed on cells expressing CXCR4 (•) and ChemR23 (▪) only, or cells co-expressing CXCR4 and ChemR23 (○). Cells were incubated with 0.1 nM 125I-CXCL12 (A) or 125I-[145–157]-chemerin (B), as tracers and increasing concentrations of unlabelled AMD3100 as competitor. 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 unlabelled CXCL12 or chemerin respectively, and specific binding in the absence of competitor (100%). All points were run in duplicate (error bars indicate S.E.M.). The displayed data are representative of two independent experiments. The functional responses of CHO-K1 cells expressing CXCR4 (C), ChemR23 (E) or both receptors (D, F) were measured by using the aequorin-based calcium mobilization assay. [C, D] Cells preloaded with coelenterazine were stimulated with increasing concentrations of CXCL12 (▪) or CXCL12 + AMD3100 (□). [E, F] Cells preloaded with coelenterazine were stimulated with increasing concentration of chemerin (•) or chemerin + AMD3100 (○). 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: In previous studies, we also showed that the CXCR4-specific antagonist AMD3100 inhibits the binding of CCR2- and CCR5-specific ligands, as the result of CXCR4 heteromerization with CCR2 and CCR5 [7]; [9]. We investigated therefore whether AMD3100 also inhibits the binding of chemerin in cells co-expressing CXCR4 and ChemR23 (Figure 5 A and 5 B). In contrast to what we reported previously for other CXCR4 heteromers, AMD3100 did not inhibit the binding of chemerin on cells co-expressing ChemR3 and CXCR4, indicating that cross-competition by AMD3100 depends on the nature of the receptor with which CXCR4 is co-expressed. We also tested the effect of the CXCR4-specific antagonist AMD3100 on the functional response of cells co-expressing CXCR4 and ChemR23, and showed, in agreement with our binding data, that AMD3100 antagonized the functional response of CXCR4 but did not inhibit the response to chemerin (Figure 5 C–F). Finally, we tested whether ChemR23 heteromerization impacts on receptor endocytosis (Figure S3). Cells co-expressing ChemR23 and CXCR4 or ChemR23 and CCR7 were stimulated with 100 nM chemerin for 90 minutes. After removal of bound chemerin by an acid wash step, the amount of cell surface receptors was estimated by FACS. Results showed that stimulation with chemerin induced a decrease of cell surface ChemR23 but not of CXCR4 or CCR7. Conversely, stimulation of CCR7 or CXCR4 with either CCL19 or CXCL12 induced internalization of the chemokine receptors but without decrease of cell surface ChemR23, suggesting thus that ChemR23 does not co-internalized with chemokine receptors.


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)

Effect of AMD3100 on binding and functional properties of cells expressing CXCR4 and ChemR23.[A, B] Competition binding assays were performed on cells expressing CXCR4 (•) and ChemR23 (▪) only, or cells co-expressing CXCR4 and ChemR23 (○). Cells were incubated with 0.1 nM 125I-CXCL12 (A) or 125I-[145–157]-chemerin (B), as tracers and increasing concentrations of unlabelled AMD3100 as competitor. 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 unlabelled CXCL12 or chemerin respectively, and specific binding in the absence of competitor (100%). All points were run in duplicate (error bars indicate S.E.M.). The displayed data are representative of two independent experiments. The functional responses of CHO-K1 cells expressing CXCR4 (C), ChemR23 (E) or both receptors (D, F) were measured by using the aequorin-based calcium mobilization assay. [C, D] Cells preloaded with coelenterazine were stimulated with increasing concentrations of CXCL12 (▪) or CXCL12 + AMD3100 (□). [E, F] Cells preloaded with coelenterazine were stimulated with increasing concentration of chemerin (•) or chemerin + AMD3100 (○). 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-g005: Effect of AMD3100 on binding and functional properties of cells expressing CXCR4 and ChemR23.[A, B] Competition binding assays were performed on cells expressing CXCR4 (•) and ChemR23 (▪) only, or cells co-expressing CXCR4 and ChemR23 (○). Cells were incubated with 0.1 nM 125I-CXCL12 (A) or 125I-[145–157]-chemerin (B), as tracers and increasing concentrations of unlabelled AMD3100 as competitor. 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 unlabelled CXCL12 or chemerin respectively, and specific binding in the absence of competitor (100%). All points were run in duplicate (error bars indicate S.E.M.). The displayed data are representative of two independent experiments. The functional responses of CHO-K1 cells expressing CXCR4 (C), ChemR23 (E) or both receptors (D, F) were measured by using the aequorin-based calcium mobilization assay. [C, D] Cells preloaded with coelenterazine were stimulated with increasing concentrations of CXCL12 (▪) or CXCL12 + AMD3100 (□). [E, F] Cells preloaded with coelenterazine were stimulated with increasing concentration of chemerin (•) or chemerin + AMD3100 (○). 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: In previous studies, we also showed that the CXCR4-specific antagonist AMD3100 inhibits the binding of CCR2- and CCR5-specific ligands, as the result of CXCR4 heteromerization with CCR2 and CCR5 [7]; [9]. We investigated therefore whether AMD3100 also inhibits the binding of chemerin in cells co-expressing CXCR4 and ChemR23 (Figure 5 A and 5 B). In contrast to what we reported previously for other CXCR4 heteromers, AMD3100 did not inhibit the binding of chemerin on cells co-expressing ChemR3 and CXCR4, indicating that cross-competition by AMD3100 depends on the nature of the receptor with which CXCR4 is co-expressed. We also tested the effect of the CXCR4-specific antagonist AMD3100 on the functional response of cells co-expressing CXCR4 and ChemR23, and showed, in agreement with our binding data, that AMD3100 antagonized the functional response of CXCR4 but did not inhibit the response to chemerin (Figure 5 C–F). Finally, we tested whether ChemR23 heteromerization impacts on receptor endocytosis (Figure S3). Cells co-expressing ChemR23 and CXCR4 or ChemR23 and CCR7 were stimulated with 100 nM chemerin for 90 minutes. After removal of bound chemerin by an acid wash step, the amount of cell surface receptors was estimated by FACS. Results showed that stimulation with chemerin induced a decrease of cell surface ChemR23 but not of CXCR4 or CCR7. Conversely, stimulation of CCR7 or CXCR4 with either CCL19 or CXCL12 induced internalization of the chemokine receptors but without decrease of cell surface ChemR23, suggesting thus that ChemR23 does not co-internalized with chemokine receptors.

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