Table 2. The effect of mutations on the pharmacology of the orthosteric antagonist [3H]spiperone and agonist dopamine.
Binding parameters were determined in [3H]spiperone saturation and competition (dopamine) binding assays. Functional parameters for dopamine were measured in an assay of ERK1/2 phosphorylation. Values are expressed as mean ± S.D. from three separate experiments.
| Radioligand binding ([3H]spiperone) | pERK1/2 (dopamine) | |||
|---|---|---|---|---|
| pKD ([3H]Spiperone) | Bmax (fmol.mg−1) | pKi (dopamine) | pEC50 | |
| WT | 10.41 ± 0.11 | 257 ± 16 | 5.10 ± 0.05 | 8.30 ± 0.09 |
| Y371.35A | 10.37 ± 0.16 | 226 ± 23 | 5.21 ± 0.07 | 8.39 ± 0.07 |
| L411.39A | 10.23 ± 0.03 | 384 ± 46 | - | 6.94 ± 0.06* |
| V912.61A | ND | ND | - | 8.60 ± 0.14 |
| L942.64A | 9.93 ± 0.04 | 285 ± 86 | - | 7.90 ± 0.05 |
| E952.65A | 10.13 ± 0.16 | 216 ± 16 | 5.66 ± 0.05* | 8.18 ± 0.07 |
| G98ECL1A | 10.07 ± 0.12 | 234 ± 60 | - | 7.85 ± 0.06 |
| K101ECL1A | 10.28 ± 0.07 | 174 ± 56 | - | 7.90 ± 0.05 |
| F1103.28A | 10.48 ± 0.06 | 171 ± 23 | - | 6.64 ± 0.08* |
| D1143.32E | 9.62 ± 0.07* | 390 ± 81 | - | ND |
| F1644.54A | 10.35 ± 0.15 | 250 ± 43 | - | 7.98 ± 0.06 |
| C1684.58A | 10.39 ± 0.16 | 276 ± 52 | - | 8.10 ± 0.07 |
| L174ECL2A | 10.64 ± 0.14 | 241 ± 49 | - | 7.01 ± 0.09* |
| N180ECL2A | 10.33 ± 0.04 | 266 ± 72 | - | 8.30 ± 0.08 |
| E181ECL2A | 10.36 ± 0.05 | 328 ± 47 | 5.31 ± 0.05 | 8.67 ± 0.10 |
| I183ECL2A | 10.31 ± 0.13 | 346 ± 23 | - | 7.73 ± 0.07 |
| I184ECL2A | 10.85 ± 0.04* | 363 ± 79 | 5.24 ± 0.04 | 7.02 ± 0.05* |
| A185ECL2S | 10.45 ± 0.09 | 199 ± 17 | - | 8.08 ± 0.05 |
| N186ECL2A | 10.19 ± 0.05 | 116 ± 24 | - | 7.18 ± 0.05* |
| S1935.42A | 11.01 ± 0.08* | 227 ± 86 | 4.21 ± 0.04* | 5.13 ± 0.03* |
| S1945.43A | 10.58 ± 0.09 | 209 ± 22 | 4.36 ± 0.04* | 6.05 ± 0.06* |
| S1975.46A | 10.43 ± 0.10 | 453 ± 109 | - | ND |
| F3906.52A | 9.75 ± 0.12* | 152 ± 14 | - | 7.04 ± 0.07* |
| H3936.55A | 10.40 ± 0.01 | 461 ± 106 | - | 5.90 ± 0.12* |
| H3936.55F | 10.50 ± 0.13 | 237 ± 24 | 4.41 ± 0.07* | 6.47 ± 0.12* |
| Y4087.35A | ND | ND | - | ND |
| S4097.36A | 10.14 ± 0.11 | 262 ± 43 | 5.49 ± 0.04* | 8.16 ± 0.06 |
| F4117.38A | 10.21 ± 0.13 | 283 ± 36 | - | 8.00 ± 0.11 |
| T4127.39A | 10.28 ± 0.11 | 255 ± 39 | 5.48 ± 0.05* | 7.15 ± 0.05* |
| Y4167.43A | ND | ND | - | ND |
*
P<0.05, significantly different from the wild-type receptor determined by a one-way ANOVA, Dunnett post-hoc test
ND, either no binding of [3H]spiperone was detected up to a concentration of 10 nM or no dopamine effect was detected