The Hinge Region of Human Thyroid-Stimulating Hormone (TSH) Receptor Operates as a Tunable Switch between Hormone Binding and Receptor Activation
Figure 2
Effect TSHR MAbs on TSH-TSHR interactions.
A. Membrane preparation from HEK293-hTSHR was incubated with different TSHR MAb (50 µg IgG/ml) for 1 h at room temperature followed by incubation with 125I-hTSH and the bound hormone was determined. NMIgG and TLRR1-6 IgG were used as negative and positive control respectively. B. HEK293-hTSHR cells were incubated with TSHR MAbs (50 µg IgG/ml) in the presence of hTSH (2 nM) for 15 min at 37°C, and cAMP produced was determined by RIA. In all of experiments, the basal cAMP production was determined without hTSH or antibodies. The bracketed numbers above the bars denote percentage of inhibition of hTSH binding or stimulation in the presence of antibodies and the statistical significance of the inhibition for each MAb as compared to the control is denoted by the P-value calculated from the two-tailed unpaired t-test. C. HEK293-hTSHR cells were incubated with IgGs of TSHR MAbs (125 µg/ml) in the absence of hTSH for 1 h at 37°C, and cAMP produced determined by RIA. The r values above the bars denote fold increase in cAMP production in presence of antibodies over the basal level and the p-value the statistical significance. D. HEK293-hTSHR cells were incubated with increasing concentrations of NMIgG, or MAb 311.62 IgG for 1 h at 37°. E. HEK293 cells expressing hLHR and hFSHR were incubated with NMIgG/311.62 IgG or their respective hormones. F. HEK293-hTSHR cells were incubated with NMIgG/FAB or MAb 311.62/FAB (125 µg/ml) for 1 h at 37°C. In all experiments, cAMP produced was quantified by RIA. All of the experiments presented above are representative of at least two independent experiments.