Allosteric Transinhibition by Specific Antagonists in CCR2/CXCR4 Heterodimers

Denis Sohy,Jean-Yves Springael,Marc Parmentier

doi:10.1074/jbc.m705302200

Denis Sohy, Jean-Yves Springael + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m705302200

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Abstract

Chemokine receptors are presently used as targets for candidate drugs in the frame of inflammatory diseases and human immunodeficiency virus infection. They were shown to dimerize, but the functional relevance of dimerization in terms of drug action remains poorly understood. We reported previously the existence of negative binding cooperativity between the subunits of CCR2/CCR5 heterodimers. In the present study, we extend these observations to heterodimers formed by CCR2 and CXCR4, which are more distantly related. We also show that specific antagonists of one receptor inhibit the binding of chemokines to the other receptor as a consequence of their heterodimerization, both in recombinant cell lines and primary leukocytes. This resulted in a significant functional cross-inhibition in terms of calcium mobilization and chemotaxis. These data demonstrate that chemokine receptor antagonists regulate allosterically the functional properties of receptors on which they do not bind directly, with important implications on the effects of these potential therapeutic agents.

Highlights

Communaute Francaise de Belgique, Interuniversity Attraction Poles Programme P6-14 (Belgian State, Belgian Science Policy), European Union Grants LSHB-CT-2003-503337/GPCRs and LSHB-CT-2005-518167/ INNOCHEM, the French Agence Nationale de Recherche sur le SIDA, the Fonds de la Recherche Scientifique Medicale of Belgium, and the Fondation Medicale Reine Elisabeth
Heterodimerization of CCR2 and CXCR4 in Recombinant Cell Lines—In previous studies, we showed that CCR2 forms constitutive homodimers, as well as heterodimers with its close structural relative CCR5 [6, 7]
We investigated further the ability of CCR2 to interact with more distant members of the chemokine receptor family and whether a negative binding cooperativity could apply as well for other such heterodimers

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Phycoerythrin-labeled anti-CXCR4 (MAB173), anti-CCR2 (FAB151P) monoclonal antibodies, and recombinant chemokines were obtained from R & D Systems. Expression of Rluc fusion proteins was estimated by measuring the luminescence of the cells after incubation with 2.5 ␮M coelenterazine H (Promega). The membrane preparations were incubated in the assay buffer (50 mM Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA) with 0.2 nM 125ISDF-1␣ or 0.1 nM 125I-MCP-1 as tracers and variable concentrations of unlabeled competitors. The membrane preparations were first incubated in assay buffer (50 mM Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA) with 0.1 nM 125I-MCP-1 or 125I-SDF-1␣ in a final volume of 500 ␮l. The aliquots were collected, the bound tracer was separated by filtration through GF/B filters presoaked for 1 h in 1% BSA, and the filters were counted for 1 min in a ␥-scintillation counter.

RESULTS

Tracer Competitor

DISCUSSION