Cloning and functional characterization of the rabbit C-C chemokine receptor 2.
BACKGROUND
CC-family chemokine receptor 2 (CCR2) is implicated in the trafficking of blood-borne monocytes to sites of inflammation and is implicated in the pathogenesis of several inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. The major challenge in the development of small molecule chemokine receptor antagonists is the lack of cross-species activity to the receptor in the preclinical species. Rabbit models have been widely used to study the role of various inflammatory molecules in the development of inflammatory processes. Therefore, in this study, we report the cloning and characterization of rabbit CCR2. Data regarding the activity of the CCR2 antagonist will provide valuable tools to perform toxicology and efficacy studies in the rabbit model.


RESULTS
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-1alpha or MIP-1beta. 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.


CONCLUSIONS
In this study, we report the cloning of rabbit CCR2 and demonstrate that this receptor is a functional chemotactic receptor for MCP-1.
Divisions of Atherosclerosis, Cancer, Integrative Biology and Discovery Chemistry, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285, USA. Lu_Deshun@lilly.com.
Deshun
D
Lu
Xiu-juan
XJ
Yuan
Robert J
RJ
Evans
Amy T
AT
Pappas
He
H
Wang
Eric W
EW
Su
Chafiq
C
Hamdouchi
Chandrasekar
C
Venkataraman
D000595:Amino Acid Sequence; D000818:Animals; D018932:Chemokine CCL2 / Q000378:metabolism / Q000494:pharmacology; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000378:metabolism / Q000494:pharmacology; D054410:Chemokine CCL7; D054412:Chemokine CCL8; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000378:metabolism / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008168:Lung / Q000378:metabolism; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits / Q000235:genetics*; D054390:Receptors, CCR2; D019707:Receptors, Chemokine / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000378:metabolism; D020298:U937 Cells
C099677:CCL13 protein, human; C111835:CCL2 protein, human; C515450:CCL7 protein, human; C515453:CCL8 protein, human; C515334:CCR2 protein, human; C515255:CXCL12 protein, human; C491768:Ccl2 protein, mouse; C515451:Ccl7 protein, mouse; C515454:Ccl8 protein, mouse; C515335:Ccr2 protein, mouse; D018932:Chemokine CCL2; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5; D054410:Chemokine CCL7; D054412:Chemokine CCL8; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; C515256:Cxcl12 protein, mouse; D018076:DNA, Complementary; D019402:Macrophage Inflammatory Proteins; D018945:Monocyte Chemoattractant Proteins; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D011993:Recombinant Fusion Proteins