Allosteric ligand affinity (pKB) and binding cooperativity with NECA (logαI) for WT and mutant 3xHA-A1ARs determined from whole-cell [3H]DPCPX binding interaction studies.
Values represent the mean ± S.E.M. from indicated number of separate experiments (n) performed in duplicate.
| pKBa | logαIb (αI) | logαI + pKB | ||||
|---|---|---|---|---|---|---|
| PD81723 | VCP171 | PD81723 | VCP171 | PD81723 | VCP171 | |
| WT | 5.41 ± 0.06 (n = 30) | 5.65 ± 0.06 (n = 10) | 0.88 ± 0.05 (7.62) | 0.68 ± 0.07 (4.79) | 6.29 | 6.33 |
| F144A | 5.73 ± 0.14 (n = 7) | 5.48 ± 0.07 (n = 3) | 0.53 ± 0.15 (3.40) | 0.36 ± 0.13 (2.31) | 6.26 | 5.84 |
| G145A | 5.20 ± 0.09 (n = 6) | 5.32 ± 0.19 (n = 3) | 0.91 ± 0.11 (8.22) | 0.69 ± 0.14 (4.94) | 6.11 | 6.01 |
| W146A | 5.49 ± 0.31 (n = 3) | 5.65 ± 0.11 (n = 3) | 0.96 ± 0.27 (9.20) | 0.08 ± 0.23 (1.20)* | 6.45 | 5.73 |
| N147A | 6.15 ± 0.25 (n = 3)* | 5.48 ± 0.06 (n = 3) | 0.26 ± 0.21 (1.81)* | 0.61 ± 0.07 (4.11) | 6.41 | 6.09 |
| N148A | 5.93 ± 0.20 (n = 5)* | 5.72 ± 0.04 (n = 3) | 0.31 ± 0.08 (2.04)* | 0.36 ± 0.07 (2.29) | 6.24 | 6.08 |
| L149A | 5.77 ± 0.21 (n = 5) | 5.67 ± 0.03 (n = 3) | 0.45 ± 0.13 (2.80) | 0.44 ± 0.11 (2.79) | 6.22 | 6.11 |
| SV150AA | 5.04 ± 0.10 (n = 8) | 5.52 ± 0.19 (n = 3) | 1.16 ± 0.12 (14.35) | 0.82 ± 0.1 (6.58) | 6.20 | 6.34 |
| E153A | 5.71 ± 0.14 (n = 7) | 5.38 ± 0.11 (n = 4) | 0.58 ± 0.09 (3.76) | 0.76 ± 0.19 (5.77) | 6.29 | 6.14 |
| R154A | 5.49 ± 0.13 (n = 7) | 5.91 ± 0.08 (n = 5) | 0.75 ± 0.12 (5.57) | 0.26 ± 0.12 (1.84) | 6.24 | 6.17 |
| W156A | 5.40 ± 0.12 (n = 8) | 5.92 ± 0.13 (n = 4) | 0.95 ± 0.14 (8.87) | 0.05 ± 0.05 (1.12)* | 6.35 | 5.97 |
| N159A | 5.54 ± 0.13 (n = 6) | 5.93 ± 0.11 (n = 6) | 0.57 ± 0.1 (3.72) | 0.32 ± 0.07 (2.10) | 6.11 | 6.25 |
| G160A | 5.76 ± 0.14 (n = 7) | 5.96 ± 0.05 (n = 3) | 0.47 ± 0.11 (2.95)* | 0.39 ± 0.09 (2.47) | 6.23 | 6.35 |
| S161A | 6.18 ± 0.05 (n = 5)* | 5.63 ± 0.15 (n = 3) | −0.32 ± 0.25 (0.47)* | 0.86 ± 0.22 (7.25) | 5.86 | 6.49 |
| MGE162AAA | 5.41 ± 0.07 (n = 6) | 5.61 ± 0.02 (n = 3) | 1.04 ± 0.16 (10.99) | 0.55 ± 0.25 (3.59) | 6.45 | 6.16 |
| P165A | 5.79 ± 0.09 (n = 6) | 5.95 ± 0.12 (n = 4) | 0.77 ± 0.09 (5.85) | 0.27 ± 0.05 (1.85) | 6.56 | 6.22 |
| V166A | 5.95 ± 0.11 (n = 6)* | 5.80 ± 0.13 (n = 6) | 0.58 ± 0.09 (3.76) | 0.47 ± 0.09 (2.96) | 6.53 | 6.27 |
| I167A | 6.21 ± 0.11 (n = 6)* | 5.90 ± 0.15 (n = 6) | 0.57 ± 0.08 (3.72) | 0.72 ± 0.14 (5.29) | 6.78 | 6.62 |
| K168A | 5.86 ± 0.14 (n = 7)* | 5.49 ± 0.06 (n = 5) | 0.67 ± 0.1 (4.69) | 0.68 ± 0.07 (4.84) | 6.53 | 6.17 |
| E170A | 6.00 ± 0.11 (n = 7)* | 5.72 ± 0.11 (n = 5) | 0.46 ± 0.11 (2.89)* | 0.49 ± 0.13 (3.11) | 6.46 | 6.21 |
| E172A | 4.89 ± 0.11 (n = 5)* | 5.26 ± 0.05 (n = 6)* | 1.10 ± 0.1 (12.62) | 0.84 ± 0.04 (6.93) | 5.99 | 6.1 |
| K173A | 6.20 ± 0.11 (n = 6)* | 5.37 ± 0.09 (n = 5) | 0.32 ± 0.08 (2.08)* | 0.66 ± 0.08 (4.57) | 6.52 | 6.03 |
| V174A | 5.91 ± 0.21 (n = 4) | 5.91 ± 0.13 (n = 4) | 0.51 ± 0.18 (3.24) | 0.65 ± 0.21 (4.44) | 6.42 | 6.56 |
| I175A | 6.49 ± 0.14 (n = 4)* | 5.83 ± 0.13 (n = 3) | 0.70 ± 0.17 (5.04) | 0.24 ± 0.02 (1.74) | 7.19 | 6.07 |
| S176A | 5.78 ± 0.16 (n = 6) | 5.47 ± 0.12 (n = 6) | 0.62 ± 0.21 (4.13) | 0.80 ± 0.13 (6.27) | 6.40 | 6.27 |
| M177A | 5.73 ± 0.09 (n = 5) | 5.69 ± 0.15 (n = 6) | 0.65 ± 0.11 (4.49) | 0.84 ± 0.14 (6.92) | 6.38 | 6.53 |
↵a Negative logarithm of the equilibrium dissociation constant of PD81723 and VCP171 as estimated from eq. 1
↵b Logarithm of the binding cooperativity factor estimated from Eq. 1. Antilogarithm is shown in parentheses.
↵* P < 0.05, significantly different from WT, one-way analysis of variance, Dunnett’s post hoc test.