Table 2. NanoBRET Saturation- And Competition-Binding Assays in WT and Mutant Nluc-A1Ra.
| pKid |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| cell-surface expressionb | n | KDc | n | CPA | n | BnOCPA | n | HOCPA | n | adenosine | n | |
| WT | 100 ± <0.1 | 8 | 68.6 ± 7.5 | 5 | 6.95 ± 0.02 | 3 | 6.21 ± 0.04 | 4 | 6.37 ± 0.03 | 4 | 5.90 ± 0.08 | 4 |
| F81.31A | 53 ± 12** | 3 | 90.7 ± 21.5 | 3 | 7.34 ± 0.08* | 4 | 6.37 ± 0.02 | 3 | 6.95 ± 0.07** | 3 | 6.55 ± 0.17*** | 3 |
| Q91.32A | 21 ± 2**** | 3 | 60.6 ± 5.4 | 3 | 6.41 ± 0.09** | 3 | 5.44 ± 0.06**** | 3 | 5.71 ± 0.05*** | 3 | 5.07 ± 0.11**** | 3 |
| Y121.35A | 120 ± 19 | 3 | 75.8 ± 6.0 | 3 | 6.50 ± 0.06** | 4 | 5.44 ± 0.06**** | 4 | 5.78 ± 0.10*** | 4 | 5.35 ± 0.05*** | 4 |
| I692.64A | 158 ± 7*** | 3 | 147.2 ± 13.4* | 3 | 4.94 ± 0.06**** | 3 | 4.72 ± 0.04**** | 3 | 4.34 ± 0.11**** | 3 | 4.64 ± 0.13**** | 3 |
| N702.65A | 86 ± 9 | 3 | 51.6 ± 3.7 | 3 | 6.97 ± 0.12 | 3 | 5.92 ± 0.06* | 3 | 6.05 ± 0.09 | 3 | 5.83 ± 0.05 | 3 |
| N148ECL2A | 13 ± 1**** | 3 | 70.7 ± 13.2 | 3 | 7.20 ± 0.13 | 3 | 6.15 ± 0.03 | 3 | 6.15 ± 0.05 | 4 | 5.71 ± 0.07 | 3 |
| I175ECL2A | 23 ± 3**** | 3 | 153.2 ± 27.6** | 3 | 6.30 ± 0.06**** | 3 | 5.60 ± 0.05**** | 3 | 5.66 ± 0.02**** | 4 | 5.00 ± 0.11**** | 3 |
| T2576.58A | 114 ± 19 | 4 | 128.8 ± 16.0 | 3 | 7.30 ± 0.03** | 3 | 6.77 ± 0.05**** | 4 | 6.94 ± 0.05**** | 3 | 5.85 ± 0.09 | 3 |
| L2536.54A | 160 ± 24*** | 3 | 104.1 ± 19.3 | 3 | 6.17 ± 0.06**** | 4 | 5.39 ± 0.10**** | 4 | 5.49 ± 0.13**** | 3 | 5.40 ± 0.06** | 3 |
| L2586.59A | 82 ± 2 | 3 | 118.3 ± 20.6 | 3 | 6.34 ± 0.07**** | 6 | 6.03 ± 0.05*** | 5 | 5.85 ± 0.08**** | 5 | 5.66 ± 0.08 | 5 |
| H264ECL3A | 21 ± 6**** | 3 | 84.1 ± 4.6 | 3 | 6.33 ± 0.09*** | 3 | 5.60 ± 0.01**** | 3 | 6.02 ± 0.18 | 3 | 6.21 ± 0.05 | 3 |
| K265ECL3A | 68 ± 13 | 3 | 54.7 ± 8.6 | 3 | 6.59 ± 0.13 | 3 | 6.21 ± 0.05 | 4 | 6.01 ± 0.12 | 4 | 5.58 ± 0.09 | 3 |
| S2677.32A | 90 ± 6 | 3 | 89.9 ± 11.7 | 3 | 6.66 ± 0.06* | 3 | 5.85 ± 0.01*** | 3 | 5.99 ± 0.05*** | 3 | 5.66 ± 0.08 | 3 |
| Y2717.36A | 171 ± 10**** | 3 | 94.4 ± 11.1 | 3 | 5.82 ± 0.03**** | 3 | 5.14 ± 0.07**** | 4 | 5.15 ± 0.07**** | 3 | 5.15 ± 0.05*** | 3 |
| L2586.59T | 77 ± 18 | 3 | 200.4 ± 24.6**** | 3 | 6.39 ± 0.07 | 3 | 5.69 ± 0.04*** | 4 | 5.69 ± 0.04**** | 4 | 5.45 ± 0.05 | 4 |
| L2586.59F | 131 ± 2 | 4 | 123.0 ± 17.3 | 3 | 6.40 ± 0.10 | 3 | 6.12 ± 0.05**** | 3 | 5.79 ± 0.08*** | 3 | 5.47 ± 0.04 | 3 |
| L2586.59G | 15 ± 4**** | 3 | 143.3 ± 37.5* | 3 | ||||||||
CA200645 equilibrium dissociation constant (KD) and compound affinity (pKi) at WT and mutant Nluc-A1R, as determined by NanoBRET saturation and competition ligand-binding assays, respectively. Data are expressed as mean ± SEM obtained in n separate experiments. All individual experiments were conducted in duplicate. Statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001) compared to WT was determined by one-way ANOVA with the Dunnett’s post-test.
Mean fluorescence intensity of APC (% wild-type (WT)). Cell-surface expression of WT or mutant Nluc-A1R in HEK 293 cells was determined by flow cytometry. Cells were incubated with anti-Nluc antibody followed by APC-conjugated antirabbit IgG secondary antibody and the fluorescence detector FL4 (emission λ 675/25 nm) used to detect APC fluorescence.
CA200645 equilibrium dissociation constant (KD) as determined by NanoBRET saturation binding assays.
Compound affinity (pKi) (mean ± SEM) determined through NanoBRET competition-binding assays in WT/mutant Nluc-A1R stably expressing HEK 293 cells. The resulting concentration-dependent decrease in BRET ratio at 10 min was used to calculate pKi. Compound affinity could not be determined (−) for L253G Nluc-A1R given the low cell-surface expression and reduced binding affinity of CA200645 (KD).