β-arr2 recruitment was assessed by BRET in Flp-In T-REx 293 cells transiently transfected with plasmids encoding CCR2-RLuc8 and β-arr2–YFP. Inhibition of cAMP was measured in c-Myc–FLAG–CCR2 Flp-In T-REx 293 cells transiently transfected with a plasmid encoding a BRET-based cAMP sensor. ERK1/2 phosphorylation was measured 3 min after c-Myc–FLAG–CCR2 Flp-In T-REx 293 cells were stimulated with chemokine. 125I–MCP-1 competition binding was measured in membrane preparations of c-Myc–FLAG–CCR2 Flp-In T-REx 293 cells. pEC50 and pKi values are the negative log of EC50 and inhibition constant (Ki) values, respectively, in molar units. Emax values are reported as a percentage of the value for MCP-1. Data are means ± SEM of three or four experiments, each performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Dunnett’s multiple-comparison test. Emax is shown relative to that observed with MCP-1.
β-Arrestin recruitment | cAMP inhibition | ERK1/2 phosphorylation | 125I–MCP-1 binding | ||||
pEC50 | Emax | pEC50 | Emax | pEC50 | Emax | pKi | |
MCP-1 | 8.32 ± 0.06 | 100 ± 2 | 9.10 ± 0.21 | 100 ± 9 | 9.16 ± 0.24 | 100 ± 10 | 10.60 ± 0.08 |
MCP-2 | 7.24 ± 0.26* | 23 ± 3*** | 7.34 ± 0.14*** | 113 ± 9 | 7.58 ± 0.15*** | 119 ± 9 | 8.88 ± 0.14*** |
MCP-3 | 7.33 ± 0.15* | 56 ± 4** | 8.47 ± 0.16* | 109 ± 9 | 8.09 ± 0.19*** | 116 ± 10 | 9.50 ± 0.12*** |