Extended Data Table 2.
Mutations analysis of changes in pEC50 and Emax
| CB1 | AM11542 | AM841 | CP55,940 | |||
|---|---|---|---|---|---|---|
| pEC50 | Emax (Fold) | pEC50 | Emax (Fold) | pEC50 | Emax (Fold) | |
| WT | 8.5 ± 0.21 | 2.3 ± 0.05 | 7.9 ± 0.12 | 2.5 ± 0.04 | 8.3 ± 0.15 | 2.3 ± 0.05 |
| F177A | 7.4 ± 0.17**** | 2.0 ± 0.04 | 7.3 ± 0.20**** | 2.5 ± 0.08 | 7.2 ± 0.14**** | 2.2 ± 0.05 |
| L193A | 5.8 ± 0.06**** | 1.9 ± 0.10 | 5.4 ± 0.35**** | 2.3 ± 0.42 | 5.8 ± 0.15**** | 2.2 ± 0.12 |
| D213A | 6.4 ± 0.18**** | 1.6 ± 0.05*** | 6.4 ± 0.14**** | 1.8 ± 0.05**** | 6.4 ± 0.14**** | 1.6 ± 0.04**** |
| Y275A | 6.1 ± 0.19**** | 2.1 ± 0.12 | 5.9 ± 0.22**** | 2.3 ± 0.18 | 5.4 ± 0.95**** | 1.5 ± 0.40**** |
| Y275F | 6.8 ± 0.21**** | 2.1 ± 0.09 | 6.6 ± 0.13**** | 2.5 ± 0.09 | 6.7 ± 0.13**** | 2.3 ± 0.07 |
| F379A | 5.9 ± 0.25**** | 2.0 ± 0.15 | 5.4 ± 0.30**** | 2.6 ± 0.42 | 5.3 ± 0.40**** | 2.2 ± 0.50 |
| F379W | 7.0 ± 0.12**** | 2.0 ± 0.05 | 6.6 ± 0.20**** | 2.5 ± 0.13 | 7.0± 0.15**** | 2.2 ± 0.07 |
| S383A | <5 | 1.0 ± 0.02**** | <5 | 1.6±0.51**** | <5 | 1.3 ± 0.12**** |
| T210A | 7.5 ± 0.32** | 1.9 ± 0.07** | 7.4 ± 0.22 | 2.1 ± 0.06**** | 6.9 ± 0.32*** | 1.9 ± 0.09**** |
| E273K | 8.7 ± 0.14 | 2.5 ± 0.05* | 8.1 ± 0.12 | 2.5 ± 0.06 | 8.7 ± 0.09 | 2.5 ± 0.04 |
| T283V | 8.8 ± 0.17 | 2.5 ± 0.06* | 7.9 ± 0.16 | 2.5 ± 0.07 | 8.7 ± 0.25 | 2.7 ± 0.07** |
| R340E | 8.6 ± 0.15 | 2.5 ± 0.05* | 8.1 ± 0.17 | 2.5 ± 0.09 | 8.5 ± 0.30 | 2.5 ± 0.06 |
Inhibition of forskolin-stimulated cAMP accumulation in wild-type and mutant CB1 CHO cells. Data are mean pEC50 and Emax values ± s.e.m. from fitting concentration–response data to nonlinear regression (3 parameter) analysis; n = 3 independent experiments performed in duplicate for all mutants except F177A (n = 4), T210A (n = 4 for AM841 and CP55,940) and wild type (n = 7 for AM841 and CP55,940; n = 6 for AM11542).
Compared to wild type with agonist treatment: *P < 0.05,
P < 0.01,
P < 0.0001 as determined by two-way ANOVA without repeated measures followed by Dunnett’s post hoc test. Statistical analyses were not performed on S383A as the pEC50 values were estimated at > 5 µM due to lack of response and non-convergence of the data to nonlinear regression analysis. The last four mutations represent those appearing in the crystal structure CB1 construct.