Figure 4.

TSH binding to mTSHR with six mutations progressively converted to the wild type. Stably transfected CHO cells expressing the indicated TSHR (see legend to Fig. 3) were incubated for 3 h at room temperature in buffer containing ¹²⁵I-bTSH supplemented with the indicated concentrations of unlabeled bTSH (see Materials and Methods). To compensate for variations in the levels of TSHR expression, data are normalized to 100% (¹²⁵I-bTSH binding in the absence of stable bTSH). In the experiment shown, total ¹²⁵I-bTSH counts per minute added per well were 9351 cpm and nonspecific binding (untransfected cells in the presence of 100 mU bTSH/ml) was 1.1%. Absolute binding for TSHR with the following number of mutations at the indicated sites was: six mutations (original cDNA), 22.0%; five mutations, 23.2%; four mutations, 20.6%; three mutations, 48.8%; two mutations, 48.7%; one mutation, 48.1%; no mutations (wild type), 47.1%. Similar data were obtained in a second experiment.