Binding properties of WT rmGlu5a and mutants
Saturation binding isotherms of [3H]MPEP were performed on membrane preparations from HEK-293 cells transiently transfected with the WT and point-mutated receptors as described under Materials and Methods. With exception of the mutants P654S and P654S/S657C, which are from four independent experiments, the values for other mutants are mean ± S.D. of the KD and Bmax, calculated from three independent experiments, each performed in triplicate. The mutations that affected the [3H]MPEP binding affinity are in bold. Statistical significance was determined using the two-tailed t test.
| rmGlu5a Receptor | Position in the 7TMD | KD[3H]MPEP | KD (mutant)/KD (WT) | Bmax |
|---|---|---|---|---|
| nM | pmol/mg of protein | |||
| WT | 3.1 ± 1.3 | 52.9 ± 2.1 | ||
| R647A | 3.29 | 1.4 ± 0.1 | 0.5 * | 3.6 ± 0.1 |
| P654S | 3.36 | 123.8 ± 81 | 40.0 ** | 12.5 ± 4.6 |
| S657C | 3.39 | 2.6 ± 0,7 | 0,9 | 13.8 ± 0.3 |
| P654S/S657C | 3.36,3.39 | 27.5 ± 4.3 | 9.0 ** | 48.3 ± 3.6 |
| Y658V | 3.40 | No binding | No binding | |
| Y658F | 3.40 | 2.4 ± 0.7 | 0.8 | 36.8 ± 1.5 |
| N733A | 45.51 | 3.5 ± 1.3 | 1.1 | 41.3 ± 3.5 |
| L743V | 5.47 | 12.1 ± 3.2 | 3.9* | 23.6 ± 3.6 |
| L743A | 5.47 | 14.4 ± 3.5 | 4.7 ** | 21.7 ± 1.9 |
| T780A | 6.44 | 14.2 ± 1.2 | 4.6 *** | 20.5 ± 0.5 |
| W784F | 6.48 | 10.2 ± 1.8 | 3.3** | 32.6 ± 2.9 |
| W784A | 6.48 | No binding | No binding | |
| F787A | 6.51 | No binding | No binding | |
| V788M | 6.52 | 3.5 ± 1.1 | 1.1 | 43.1 ± 1.2 |
| Y791A | 6.55 | 15.1 ± 2.1 | 4.9 ** | 21.4 ± 0.6 |
| A809V | 7.47 | No binding | No binding |