Table 2.
Location | Transfected construct | Ballesteros and Weinstein numbering | Cell surface expression | cAMP accumulation | IP accumulation | |
---|---|---|---|---|---|---|
FACS% of TSHR wt | EC50 nM | TSH stimulated % of TSHR | TSH stimulated % of TSHR | |||
wt TSHR | 100 | 1.2 (0.8–1.7) | 100 | 100 | ||
TMH1 | V424I | 1.43 | 102 ± 10 | 3.0 (2.2–4.1) | 98 ± 8 | 53 ± 4 |
TMH2 | L467V | 2.57 | 85 ± 8 | 11.2 (10.4–12.1) | 105 ± 14 | 19 ± 1 |
TMH3 | V502A | 3.33 | 44 ± 4 | 4.3 (3.9–4.7) | 84 ± 10 | 13 ± 0 |
TMH4 | L552V | 4.56 | 90 ± 5 | 1.0 (0.8–1.4) | 84 ± 19 | 79 ± 11 |
TMH5 | Y582A | 5.39 | 71 ± 5 | 3.7 (2.6–5.2) | 106 ± 19 | 45 ± 17 |
TMH5 | Y582F | 5.39 | 86 ± 8 | 1.9 (1.3–2.8) | 108 ± 12 | 114 ± 18 |
TMH5 | V586I | 5.43 | 107 ± 6 | 1.1 (0.7–1.7) | 82 ± 7 | 120 ± 6 |
TMH5 | L589V | 5.46 | 100 ± 12 | 1.1 (1.0–1.2) | 90 ± 9 | 110 ± 11 |
TMH6 | Y643A | 6.54 | 99 ± 9 | 6.0 (4.5–8.0) | 93 ± 9 | 47 ± 4 |
TMH7 | L665V | 7.40 | 95 ± 11 | 2.0 (2.0–2.0) | 100 ± 10 | 71 ± 6 |
HEK 293 cells were transfected with wt TSHR and mutated TSHRs. The TSHR cell surface expression level was quantified on a FACS flow cytometer. Receptor expression was determined by the mean fluorescence intensity by comparison to wt TSHR. Stimulation was carried out with increasing concentrations of TSH. Results for cAMP accumulation were measured by RIA. Quantification of IP accumulation was done by [3H]Inositol incorporation studies. Data are given as mean ± SD of at least two independent experiments, each carried out in duplicate. EC50 values are shown as geometric mean (95% confidence limits). The pcDNA3 vector was used as a control