Identification of RXR motifs preventing exaggerated transport of α2C-AR to the plasma membrane at 30°C
The underlined residues in human α2C-AR were mutated from arginine to alanine using the specific primers indicated in the Materials and Methods. Subsequently, the mutants with single cluster disruption served as a template for generating double or triple mutants. The expected mutation was confirmed in each case by sequence analysis. The plasma membrane levels were determined by [3H]RX821002 binding and the results are expressed as a percentage of α2C-AR wild-type levels determined in the same experiment. n = 8–12 in each case, from three independent transfections.
| Receptors | 37°C | 30°C |
|---|---|---|
| % | ||
| With single cluster disrupted | ||
| Wild-type | 100 | 169 ± 6.2 |
| 240RTA242 | 96 ± 4.2 | 168 ± 8.4 |
| 294RRAAR298 | 93 ± 7.3 | 175 ± 10.1 |
| 306RRA308 | 102 ± 6.7 | 166 ± 7.3 |
| 358RRAAAR364 | 107 ± 9.5 | 164 ± 8.5 |
| 454RRAAR458 | 95 ± 5.5 | 176 ± 6.6 |
| With two clusters disrupted | ||
| 240RTA242/293RRAAR297 | 92 ± 6.8 | 173 ± 8.0 |
| 240RTA242/306RRA308 | 101 ± 3.1 | 168 ± 6.5 |
| 240RTA242/358RRAAAR364 | 97 ± 5.5 | 177 ± 7.5 |
| 240RTA242/454RRAAR458 | 94 ± 6.8 | 173 ± 6.6 |
| 294RRAAR298/306RRA308 | 104 ± 7.7 | 165 ± 8.2 |
| 294RRAAR298/358RRAAAR364 | 101 ± 3.1 | 177 ± 6.2 |
| 294RRAAR298/454RRAAR458 | 103 ± 6.2 | 215 ± 7.4* |
| With three clusters disrupted | ||
| 294RRAAR298/358RRAAAR364/454RRAAR458 | 104 ± 7.8 | 221 ± 8.3* |
↵* P < 0.05 compared with the wild-type human α2C-AR receptor.