C‐C chemokine receptor 2b (CCR2b) signaling: a comparison of a dog‐human chimeric receptor with the endogenously expressed human receptor

Abstract

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 (EC50 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 IC50 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.