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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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Homo- and heteromerization of ChemR23 as measured by BRET.[A] HEK293T cells were transfected with a constant amount of the ChemR23-hRLuc construct and increasing amounts of the ChemR23-EYFP (•), CXCR4-EYFP (▪) or CCR7-EYFP (□) constructs. As a control, increasing amounts of GABAbR2-EYFP (⋄, dotted curve) or TSHR-EYFP (*, dotted curve) were used as BRET acceptors [B–C] HEK293T cells were transfected with a constant amount of the CCR7-hRLuc or CXCR4-hRLuc constructs and increasing amounts of CCR7-EYFP (♦), CXCR4-EYFP (•) or ChemR23-EYFP (○) constructs. As a control, increasing amounts of GABAbR2-EYFP (⋄, dotted curve) was used as BRET acceptor. The BRET signal was recorded 5 minutes after addition of coelenterazine H. All data points were performed in triplicate (error bars indicate S.E.M.).
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pone-0058075-g001: Homo- and heteromerization of ChemR23 as measured by BRET.[A] HEK293T cells were transfected with a constant amount of the ChemR23-hRLuc construct and increasing amounts of the ChemR23-EYFP (•), CXCR4-EYFP (▪) or CCR7-EYFP (□) constructs. As a control, increasing amounts of GABAbR2-EYFP (⋄, dotted curve) or TSHR-EYFP (*, dotted curve) were used as BRET acceptors [B–C] HEK293T cells were transfected with a constant amount of the CCR7-hRLuc or CXCR4-hRLuc constructs and increasing amounts of CCR7-EYFP (♦), CXCR4-EYFP (•) or ChemR23-EYFP (○) constructs. As a control, increasing amounts of GABAbR2-EYFP (⋄, dotted curve) was used as BRET acceptor. The BRET signal was recorded 5 minutes after addition of coelenterazine H. All data points were performed in triplicate (error bars indicate S.E.M.).

Mentions: We have previously shown that heteromerization of chemokine receptors is associated with negative binding cooperativity across their respective ligands [7]–[10]. In the present study, we investigated the ability of chemokine receptors to interact with ChemR23, a member of the chemoattractant receptor family [20], and tested whether negative binding cooperativity could apply as well for heteromers involving ChemR23. In a first step, we investigated the extent of ChemR23 heteromerization in HEK293T cells by using the bioluminescence resonance energy transfer (BRET) assay. An energy transfer was detected between ChemR23-hRLuc and ChemR23-EYFP as well as between ChemR23-hRLuc and CXCR4-EYFP or CCR7-EYFP. As a specificity control, the GABAbR2-EYFP and TSHR-EYFP were used, which led to a much lower energy transfer (Figure 1 A). The calculated BRET50 parameters were in the same range for homo- and heteromers but the BRETMAX values were about two-fold lower for ChemR23 heteromers as compared to ChemR23 homomers (Figure S1 A). BRET was also detected between CXCR4-hRLuc and ChemR23-EYFP and between CCR7-hRLuc and ChemR23-EYFP (Figure 1 B, C). Again, the BRETMAX values were two to height fold lower than those obtained for the respective CXCR4 and CCR7 homomers and a two-fold increase of the BRET50 values was also detected for each heteromer. Nevertheless, the energy transfer in heteromers was much higher than when GABAbR2-EYFP was used as a negative control (Figure 1 B and 1 C inlet). Not surprisingly, no BRET signal was detected between GABAbR1-Rluc and ChemR23-EYFP, CCR7-EYFP or CXCR4-EYFP (Figure S1 A). The lower BRETMAX values detected for heteromers reflect most likely a less favorable relative orientation of BRET donor and acceptor moieties. The variations of BRET50 values between homo- and heteromers were dependent upon which receptor of the pair was the BRET donor, which prevents any definitive conclusion regarding the relative propensity of receptors to interact.


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)

Homo- and heteromerization of ChemR23 as measured by BRET.[A] HEK293T cells were transfected with a constant amount of the ChemR23-hRLuc construct and increasing amounts of the ChemR23-EYFP (•), CXCR4-EYFP (▪) or CCR7-EYFP (□) constructs. As a control, increasing amounts of GABAbR2-EYFP (⋄, dotted curve) or TSHR-EYFP (*, dotted curve) were used as BRET acceptors [B–C] HEK293T cells were transfected with a constant amount of the CCR7-hRLuc or CXCR4-hRLuc constructs and increasing amounts of CCR7-EYFP (♦), CXCR4-EYFP (•) or ChemR23-EYFP (○) constructs. As a control, increasing amounts of GABAbR2-EYFP (⋄, dotted curve) was used as BRET acceptor. The BRET signal was recorded 5 minutes after addition of coelenterazine H. All data points were performed in triplicate (error bars indicate S.E.M.).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585228&req=5

pone-0058075-g001: Homo- and heteromerization of ChemR23 as measured by BRET.[A] HEK293T cells were transfected with a constant amount of the ChemR23-hRLuc construct and increasing amounts of the ChemR23-EYFP (•), CXCR4-EYFP (▪) or CCR7-EYFP (□) constructs. As a control, increasing amounts of GABAbR2-EYFP (⋄, dotted curve) or TSHR-EYFP (*, dotted curve) were used as BRET acceptors [B–C] HEK293T cells were transfected with a constant amount of the CCR7-hRLuc or CXCR4-hRLuc constructs and increasing amounts of CCR7-EYFP (♦), CXCR4-EYFP (•) or ChemR23-EYFP (○) constructs. As a control, increasing amounts of GABAbR2-EYFP (⋄, dotted curve) was used as BRET acceptor. The BRET signal was recorded 5 minutes after addition of coelenterazine H. All data points were performed in triplicate (error bars indicate S.E.M.).
Mentions: We have previously shown that heteromerization of chemokine receptors is associated with negative binding cooperativity across their respective ligands [7]–[10]. In the present study, we investigated the ability of chemokine receptors to interact with ChemR23, a member of the chemoattractant receptor family [20], and tested whether negative binding cooperativity could apply as well for heteromers involving ChemR23. In a first step, we investigated the extent of ChemR23 heteromerization in HEK293T cells by using the bioluminescence resonance energy transfer (BRET) assay. An energy transfer was detected between ChemR23-hRLuc and ChemR23-EYFP as well as between ChemR23-hRLuc and CXCR4-EYFP or CCR7-EYFP. As a specificity control, the GABAbR2-EYFP and TSHR-EYFP were used, which led to a much lower energy transfer (Figure 1 A). The calculated BRET50 parameters were in the same range for homo- and heteromers but the BRETMAX values were about two-fold lower for ChemR23 heteromers as compared to ChemR23 homomers (Figure S1 A). BRET was also detected between CXCR4-hRLuc and ChemR23-EYFP and between CCR7-hRLuc and ChemR23-EYFP (Figure 1 B, C). Again, the BRETMAX values were two to height fold lower than those obtained for the respective CXCR4 and CCR7 homomers and a two-fold increase of the BRET50 values was also detected for each heteromer. Nevertheless, the energy transfer in heteromers was much higher than when GABAbR2-EYFP was used as a negative control (Figure 1 B and 1 C inlet). Not surprisingly, no BRET signal was detected between GABAbR1-Rluc and ChemR23-EYFP, CCR7-EYFP or CXCR4-EYFP (Figure S1 A). The lower BRETMAX values detected for heteromers reflect most likely a less favorable relative orientation of BRET donor and acceptor moieties. The variations of BRET50 values between homo- and heteromers were dependent upon which receptor of the pair was the BRET donor, which prevents any definitive conclusion regarding the relative propensity of receptors to interact.

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