Allosteric Transinhibition by Specific Antagonists in CCR2/CXCR4 Heterodimers

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.Chemokine receptors constitute an attractive family of drug targets in the frame of inflammatory diseases, human immunodeficiency virus, and cancer (1, 2). However, the complexity and the functional redundancy of the chemokine system have represented significant hurdles in this development. There is therefore a need for a better comprehension of the chemokine signaling network and how molecules (agonists and antagonists) acting on specific receptors will affect the global function of the chemokine system. One of the challenges in this area is the recent demonstration that G protein-coupled receptors, including chemokine receptors, are able to form dimers, and the functional consequences of this new concept have not been fully appreciated so far.CCR2 is a member of the CC chemokine receptor family that plays an important role in the recruitment of monocytes to atherosclerotic lesions and in the formation of intimal hyperplasia after arterial injury (3). Two isoforms of CCR2 have been described that are generated by alternative splicing and differ in their C-terminal tail, but only the longer variant (CCR2b) is expressed at high level in leukocytes. CCR2 interacts with MCP-1 (CCL2) and other CC chemokines such as MCP-2 (CCL7), MCP-3 (CCL8), and MCP-4 (CCL13) (4). CCR2 was shown to form homodimers, as well as heterodimers with CCR5, its closest structural relative within the chemokine receptor family, resulting in a negative binding cooperativity of allosteric nature (5-8). CCR2 was also reported recently to form heterodimers with CXCR4, a more distantly related chemokine receptor (9). However, no pharmacological characterization of this heterodimer was performed so far. CXCR4 binds specifically SDF-1␣, an evolutionary conserved chemokine known to play a role in a number of physiological processes, such as the homing of T cell populations to sites of inflammation and the maintenance of the cellular microenvironment in bone marrow (10). SDF-1␣ and CXCR4 h...

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