Table 3 Characterization of CCR2 mutants.

Cell surface expression (receptor abundance) was measured by anti–c-Myc ELISA in c-Myc–FLAG–CCR2 Flp-In T-REx 293 cells, and data are expressed as a percentage of the abundance of the WT receptor. The affinities (pKi) of MCP-1 and MCP-3 for WT and mutant CCR2 proteins were measured by 125I–MCP-1 competition binding assays with cell membrane preparations. The potency (pEC50) and efficacy (Emax) values of MCP-1 and MCP-3 for WT and mutant CCR2 proteins in ERK1/2 phosphorylation assays were measured 3 min after c-Myc–FLAG–CCR2 Flp-In T-REx 293 cells were stimulated with chemokine. pEC50 and pKi values are the negative log of EC50 and Ki values, respectively, in molar units. Emax values are relative to the positive control. Data are means ± SEM of three or four experiments, each performed in triplicate. For radioligand binding, ^P < 0.05 as compared to MCP-1 for each mutant. Analysis was by multiple t test. For ERK1/2 phosphorylation, *P < 0.05, **P < 0.01, ***P < 0.001 as compared to WT CCR2. Analysis was by one-way ANOVA with Dunnett’s multiple-comparison test.

MutationLocation#Cell surface expressionpKipERK1/2
pEC50
pERK1/2
Emax (% FBS)
MCP-1MCP-3MCP-1MCP-3MCP-1MCP-3
WT100 ± 310.82 ± 0.189.64 ± 0.19^8.01 ± 0.237.30 ± 0.2338.9 ± 335.5 ± 4.5
K34ATM1 (1.28)119 ± 1210.42 ± 0.279.70 ± 0.428.41 ± 0.247.70 ± 0.2355.5 ± 2.5***45.0 ± 2.8
Y120FTM3 (3.32)118 ± 1311.15 ± 0.189.65 ± 0.26^7.92 ± 0.327.58 ± 0.3325.4 ± 2**16.6 ± 1.6***
V187/V189AECL2108 ± 611.36 ± 0.299.85 ± 0.32^7.99 ± 0.267.28 ± 0.2330.5 ± 230.8 ± 2.3
N199A/T203ATM5 (5.35/5.39)116 ± 711.42 ± 0.2910.17 ± 0.477.66 ± 0.237.35 ± 0.3332.8 ± 220.2 ± 2.3**
R206ATM5 (5.42)112 ± 710.29 ± 0.2210.12 ± 0.338.25 ± 0.317.81 ± 0.3411.0 ± 0.8***14.8 ± 2.5***
Y259FTM6 (6.51)99 ± 610.44 ± 0.2310.20 ± 0.148.78 ± 0.36*8.57 ± 0.26**31.9 ± 1.739.3 ± 1.7
I263A/N266ATM6 (6.55/6.58)107 ± 810.79 ± 0.248.99 ± 0.17^9.46 ± 0.39**8.22 ± 0.3824.7 ± 2***36.9 ± 3.4
E270A/F272ATM6/ECL399 ± 1311.68 ± 0.3910.06 ± 0.31^7.36 ± 0.207.36 ± 0.2022.3 ± 1.3***22.1 ± 1.5**
D284ATM7 (7.32)104 ± 510.91 ± 0.169.52 ± 0.25^8.83 ± 0.40**7.80 ± 0.1834.9 ± 239.1 ± 1.9
E291ATM7 (7.39)107 ± 910.26 ± 0.249.03 ± 0.22^7.66 ± 0.407.09 ± 0.4827.9 ± 3*12.1 ± 2.2***

#Ballesteros and Weinstein numbering of TM residues is shown in parentheses (33).