Cloning and functional characterization of the rabbit C-C chemokine receptor 2

Sequence alignment indicated that rabbit CCR2 shares 80 % identity to human CCR2b. Tissue distribution indicated that rabbit CCR2 is abundantly expressed in spleen and lung. Recombinant rabbit CCR2 expressed as stable transfectants in U-937 cells binds radiolabeled 125I-mouse JE (murine MCP-1) with a calculated Kd of 0.1 nM. In competition binding assays, binding of radiolabeled mouse JE to rabbit CCR2 is differentially competed by human MCP-1, -2, -3 and -4, but not by RANTES, MIP-1α or MIP-1β. U-937/rabbit CCR2 stable transfectants undergo chemotaxis in response to both human MCP-1 and mouse JE with potencies comparable to those reported for human CCR2b. Finally, TAK-779, a dual CCR2/CCR5 antagonist effectively inhibits the binding of 125I-mouse JE (IC50 = 2.3 nM) to rabbit CCR2 and effectively blocks CCR2-mediated chemotaxis.In this study, we report the cloning of rabbit CCR2 and demonstrate that this receptor is a functional chemotactic receptor for MCP-1.Chemokine receptors are seven-transmembrane G-protein coupled receptors that direct the migration of various immune cells to the sites of inflammation in addition to their pivotal role in maintaining immune cell homeostasis in various lymphoid compartments [1,2]. Chemokines (8–14 kDa molecular weight), the ligands of chemokine receptors, are broadly classified based on the positioning of the two conserved cysteines into the CC, CXC, C or CX3C family [3]. Binding of chemokines to their cognate receptors on immune cells triggers a signaling cascade primarily mediated by the Gαi family of G proteins leading to a variety of cell effector functions including chemotaxis, degranulation, and release of pro-inflammatory cytokines such as TNF α [4].The CC family chemokine receptor 2 (CCR2) is primarily expressed in almost all circulating monocytes. CCR2 is believed to mediate extravasation of blood monocytes to the sites of inflammation [5-7] and is implicated in the pathogenesis of several inflammatory diseases such as