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C‐C chemokine receptor 2b (CCR2b) signaling: a comparison of a dog‐human chimeric receptor with the endogenously expressed human receptor
Author(s) -
Kotnis Smita Atul,
Sullivan Nicole,
Cheng KeYi,
Kennedy Jeffrey D,
Bingham Brendan
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb83-a
Subject(s) - chemokine receptor , receptor , chemokine , microbiology and biotechnology , ccr2 , biology , pertussis toxin , cell culture , chemokine receptor ccr5 , chemistry , pharmacology , g protein , biochemistry , genetics
C‐C chemokine receptor 2b (CCR2b) and its ligand, monocyte chemoattractant protein‐1 (MCP‐1), have been implicated in chronic pain. Antagonists of CCR2b are reported to have poor cross‐species activity. To investigate species‐specificity in CCR2b signaling, we constructed a chimeric dog/human receptor. Human CCR2b sequence at the N‐ and C‐termini (including the N‐terminal high affinity MCP‐1 binding site) was coupled with intervening dog CCR2b sequence (TM1 to TM7, including the likely low affinity binding site). We compared the pharmacology of CHOK1 cells stably expressing d/hCCR2b and THP‐1 cells, a monocytic cell line endogenously expressing hCCR2b. In calcium flux assays, both cell lines responded similarly when stimulated with hMCP‐1 (EC 50 of 1.5 to 5 nM), implying a functional equivalence between the chimeric and wild‐type receptors. Calcium flux in d/hCCR2‐CHOK1 cells was sensitive to pertussis toxin, indicating normal Gi coupling. However, a CCR2b antagonist with an IC 50 of 300 nM in THP‐1 cells was substantially less potent at inhibiting hMCP‐1 induced signaling in d/hCCR2b‐expressing cells. In addition, increasing doses of the antagonist decreased the maximal response in these cells, suggesting a non‐competitive interaction. Additional mutation studies and characterization of the effects of MCP‐1 from other species may further reveal the species‐specificity of the pharmacology of CCR2b.