Table 1.
Functional analysis of CCR7 mutations.
| G protein | β-arrestin-2 | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Residue | CCL21 efficacy | CCL19 potency | CCL19 efficacy | |||||||||||
| Position | Number | Efficacy at 100 nM ± SEM | Emut | -logEC50 (M) ± SEM | FMut | Efficacy at 100 nM ± SEM | Emut | |||||||
| N-term | K50A | Dead | * | (3) | 8.5 ± 0.07 | 13 | * | (3) | 55.6 ± 2.6 | 44 | * | (5) | ||
| R54A | Dead | * | (3) | 7.1 ± 0.07 | 100 | * | (3) | Dead | * | (5) | ||||
| K57A | 99 ± 6.4 | 1 | ns | (3) | 9.2 ± 0.12 | 2 | ns | (3) | 125.1 ± 13.6 | +25 | ns | (3) | ||
| TM1 | 1.35 | L61A | 44 ± 2.9 | 56 | * | (3) | 8.9 ± 0.13 | 3 | * | (3) | 36.2 ± 3.0 | 64 | * | (3) |
| 1.39 | Y65A | 44 ± 1.5 | 56 | * | (4) | 9.0 ± 0.08 | 2 | ns | (3) | 74.8 ± 6.4 | 25 | ns | (5) | |
| TM2 | 2.53 | F107A | 76 ± 7.4 | 24 | ns | (3) | 8.9 ± 0.16 | 2 | ns | (3) | 149.1 ± 14.1 | +49 | ns | (3) |
| 2.60 | W114A | 32 ± 5.6 | 68 | * | (3) | 8.0 ± 0.09 | 13 | * | (3) | 41.7 ± 3.9 | 58 | * | (5) | |
| 2.63 | S117A | 68 ± 3.0 | 32 | ns | (3) | 9.5 ±+0.07 | 1 | ns | (3) | 139.4 ± 10.3 | +29 | ns | (5) | |
| 2.64 | A118S | 67 ± 4.6 | 33 | ns | (3) | 9.7 ± 0.07 | 2 | ns | (3) | 133.5 ± 12.4 | +33 | ns | (5) | |
| TM3 | 3.26 | K130A | 42 ± 3.5 | 58 | * | (3) | 9.0 ± 0.07 | 7 | * | (3) | 113.6 ± 7.8 | +14 | ns | (3) |
| 3.29 | F133A | 27 ± 4.5 | 73 | * | (4) | 8.7 ± 0.17 | 1 | ns | (3) | 73.6 ± 2.0 | 26 | * | (3) | |
| 3.33 | K137A | 43 ± 8.5 | 57 | * | (3) | 9.2 ± 0.29 | 3 | ns | (3) | 113.9 ± 4.4 | +14 | ns | (3) | |
| TM4 | 4.60 | E193A | 45 ± 8.6 | 55 | * | (4) | 9.6 ± 0.17 | +3 | ns | (3) | 119.7 ± 4.1 | +20 | * | (4) |
| ECL2 | E205A | 48 ± 5.3 | 52 | * | (4) | 9.2 ± 0.09 | 3 | * | (3) | 56.3 ± 4.7 | 44 | * | (3) | |
| R209A | 132 ± 8.8 | +32 | * | (5) | 8.5 ± 0.14 | 8 | * | (5) | 54.3 ± 4.9 | 46 | * | (5) | ||
| TM7 | 7.32 | N305A | 53 ± 3.6 | 47 | * | (3) | 8.6 ± 0.08 | 4 | * | (3) | 87.2 ± 2.7 | 13 | ns | (5) |
| 7.36 | Y308A | 38 ± 3.4 | 62 | * | (3) | 9.1 ± 0.15 | 2 | ns | (3) | 98.9 ± 12.1 | 1 | ns | (3) | |
Signaling values for CCR7 mutations in response to CCL21 or CCL19 analyzed in G protein signaling or β-arrestin-2 recruitment assays normalized to WT signaling. Data are presented in Figures 2, 4. Residue position refers to the location in CCR7 based on the nomenclature of Ballesteros-Weinstein (41). CCL21 G protein signaling and CCL19 β-arrestin-2 recruitment are compared to WT based on changes of efficacy at maximal agonist concentration used (100 nM), as saturation of signaling curves were not established. Emut describes the percentage activity of WT, with WT activity reaching 100%. CCL19 G protein signaling is compared to WT based on fold change of potency, and Fmut describes the fold change of signaling potency compared to WT. “ns” refers to a non-significant change of signaling curve while “*” identifies significant changes between dose-response curves analyzed by ANOVA. Data are represented as mean values (±SEM) of several independent experiments performed in duplicates and always compared to the signaling values of CCR7 wild type run in parallel within the same experiments. To compensate for inter-assay variations data have been normalized to wild type within each separate experiment before the collection of data. The n number of independent experiments for each mutation is shown in brackets to the right of Fmut or Emut, (n). Bold values indicate main findings, italic values indicate statistical significance.