TABLE 2.
Phenotype characteristics of mutations in ECL1 and transition regions to TMH2 and TMH3
| Transfected construct | Cell surface expression (% of WT TSHR) | cAMP accumulation (fold over WT TSHR basal)
|
||
|---|---|---|---|---|
| Basal | Stimulated | Slope | ||
| WT TSHR | 100 | 1.0 | 11.9 ± 0.8 | 1.0 |
| pcDNA | 7.3 ± 0.5 | 0.4 ± 0.1 | 0.5 ± 0.1 | — |
| S479A | 79.2 ± 0.9 | 0.4 ± 0.1 | 11.8 ± 0.7 | — |
| E480A | 78.5 ± 2.2 | 0.5 ± 0.1 | 9.9 ± 0.4 | — |
| N483A | 89.5 ± 3.2 | 0.4 ± 0.1 | 13.0 ± 1.2 | — |
| D487A | 94.3 ± 4.5 | 0.6 ± 0.1 | 11.4 ± 2.4 | — |
| W488A | 11.0 ± 0.3 | 0.5 ± 0.1 | 0.5 ± 0.1 | — |
| W488F | 14.0 ± 0.8 | 0.6 ± 0.1 | 1.3 ± 0.2 | — |
| W488L | 12.0 ± 0.4 | 0.5 ± 0.1 | 0.6 ± 0.1 | — |
| Q489A | 51.9 ± 1.7 | 0.6 ± 0.1 | 4.0 ± 0.5 | — |
| T490A | 85.1 ± 2.4 | 2.0 ± 0.3 | 12.9 ± 2.2 | 1.9 ± 0.7 |
Seven amino acids within ECL1 of the TSHR were characterized by alanine mutations. The side chain of Trp488 was also substituted to Phe and Leu. COS-7 cells were transfected with the WT TSHR or indicated mutated TSHRs. cAMP levels were determined after stimulation with 100 mU/ml bTSH. TSHR cell surface expression was quantified on a FACS flow cytometer. Data are means ± sd of 3 independent experiments, each carried out in duplicate. The pcDNA vector was used as a control.