Limits...
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.

Show MeSH

Related in: MedlinePlus

Homo- and heteromerization of ChemR23 as measured by HTRF.[A] HEK293T cells were transfected with increasing amounts of HA-ChemR23 and incubated for one hour with (Eu)-labelled anti-HA and XL665-labelled anti-HA (ratio 1∶10) and the homogenous FRET ratio measured. [B] HEK293T cells were transfected with HA-ChemR23 only or with HA-ChemR23 and Flag-ChemR23, CXCR4, CCR7 or Rho-tagged TSHR used as competitors. Cells were incubated with (Eu)-labelled anti-HA and XL665-labelled anti-HA (ratio 1∶10) and the homogenous FRET ratio measured. For each transfection, the expression level of HA-ChemR23 and competitors was measured by FACS (Figure S1 B). All data points were performed in duplicate (error bars indicate S.E.M.).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585228&req=5

pone-0058075-g002: Homo- and heteromerization of ChemR23 as measured by HTRF.[A] HEK293T cells were transfected with increasing amounts of HA-ChemR23 and incubated for one hour with (Eu)-labelled anti-HA and XL665-labelled anti-HA (ratio 1∶10) and the homogenous FRET ratio measured. [B] HEK293T cells were transfected with HA-ChemR23 only or with HA-ChemR23 and Flag-ChemR23, CXCR4, CCR7 or Rho-tagged TSHR used as competitors. Cells were incubated with (Eu)-labelled anti-HA and XL665-labelled anti-HA (ratio 1∶10) and the homogenous FRET ratio measured. For each transfection, the expression level of HA-ChemR23 and competitors was measured by FACS (Figure S1 B). All data points were performed in duplicate (error bars indicate S.E.M.).

Mentions: With the aim of exploring further the oligomerization status of ChemR23 at the plasma membrane of HEK293T cells, we relied on a Homogenous Time Resolved FRET (HTRF) assay. Cells expressing variable amounts of HA-tagged ChemR23 were incubated with a constant 1∶10 ratio of energy-donor and -acceptor antibodies and the FRET signal resulting from the proximity of antibody pairs was recorded (Figure 2 A). In our experiments, the acceptor and donor compete for the same binding site (HA-tag). As more receptors are expressed, the amount of receptor-bound donor and acceptor increases as well, thereby increasing the FRET signal [35]. However, this increasing FRET signal might reflect either specific interactions or random contacts due to receptor crowding in the plasma membrane. With the aim of investigating the specificity of receptor interactions, competition experiments were performed by co-transfecting various constructs in HA-ChemR23-expressing cells. Results showed that the FRET signal between the (Eu)-tagged and the XL668-tagged anti-HA antibodies decreased strongly when cells co-expressed Flag-tagged ChemR23 or untagged CXCR4 or CCR7, suggesting that these receptors interact with HA-ChemR23 and compete for ChemR23 homomerization (Figure 2 B). In contrast, no competition was detected when cells were cotransfected with the Rho-tagged TSH receptor used as a negative control. As an additional control, we showed that the drop of FRET signal was not due to a decrease in HA-ChemR23 level, as the amount of HA-ChemR23 at the cell surface, as measured by FACS, remained similar whether the cells expressed or not the competitors (Figure 2 B and Figure S1 B). Altogether, the HTRF results support the BRET data and indicated that ChemR23 is present at the plasma membrane as homo- and heteromers.


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 HTRF.[A] HEK293T cells were transfected with increasing amounts of HA-ChemR23 and incubated for one hour with (Eu)-labelled anti-HA and XL665-labelled anti-HA (ratio 1∶10) and the homogenous FRET ratio measured. [B] HEK293T cells were transfected with HA-ChemR23 only or with HA-ChemR23 and Flag-ChemR23, CXCR4, CCR7 or Rho-tagged TSHR used as competitors. Cells were incubated with (Eu)-labelled anti-HA and XL665-labelled anti-HA (ratio 1∶10) and the homogenous FRET ratio measured. For each transfection, the expression level of HA-ChemR23 and competitors was measured by FACS (Figure S1 B). All data points were performed in duplicate (error bars indicate S.E.M.).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0058075-g002: Homo- and heteromerization of ChemR23 as measured by HTRF.[A] HEK293T cells were transfected with increasing amounts of HA-ChemR23 and incubated for one hour with (Eu)-labelled anti-HA and XL665-labelled anti-HA (ratio 1∶10) and the homogenous FRET ratio measured. [B] HEK293T cells were transfected with HA-ChemR23 only or with HA-ChemR23 and Flag-ChemR23, CXCR4, CCR7 or Rho-tagged TSHR used as competitors. Cells were incubated with (Eu)-labelled anti-HA and XL665-labelled anti-HA (ratio 1∶10) and the homogenous FRET ratio measured. For each transfection, the expression level of HA-ChemR23 and competitors was measured by FACS (Figure S1 B). All data points were performed in duplicate (error bars indicate S.E.M.).
Mentions: With the aim of exploring further the oligomerization status of ChemR23 at the plasma membrane of HEK293T cells, we relied on a Homogenous Time Resolved FRET (HTRF) assay. Cells expressing variable amounts of HA-tagged ChemR23 were incubated with a constant 1∶10 ratio of energy-donor and -acceptor antibodies and the FRET signal resulting from the proximity of antibody pairs was recorded (Figure 2 A). In our experiments, the acceptor and donor compete for the same binding site (HA-tag). As more receptors are expressed, the amount of receptor-bound donor and acceptor increases as well, thereby increasing the FRET signal [35]. However, this increasing FRET signal might reflect either specific interactions or random contacts due to receptor crowding in the plasma membrane. With the aim of investigating the specificity of receptor interactions, competition experiments were performed by co-transfecting various constructs in HA-ChemR23-expressing cells. Results showed that the FRET signal between the (Eu)-tagged and the XL668-tagged anti-HA antibodies decreased strongly when cells co-expressed Flag-tagged ChemR23 or untagged CXCR4 or CCR7, suggesting that these receptors interact with HA-ChemR23 and compete for ChemR23 homomerization (Figure 2 B). In contrast, no competition was detected when cells were cotransfected with the Rho-tagged TSH receptor used as a negative control. As an additional control, we showed that the drop of FRET signal was not due to a decrease in HA-ChemR23 level, as the amount of HA-ChemR23 at the cell surface, as measured by FACS, remained similar whether the cells expressed or not the competitors (Figure 2 B and Figure S1 B). Altogether, the HTRF results support the BRET data and indicated that ChemR23 is present at the plasma membrane as homo- and heteromers.

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