(a) TSH covalent cross-linking to intact cells reveals greater level of cell surface TSHR expression in TSHR-10,000 cells than in ECD-GPI cells. After simultaneous ¹²⁵I-TSH cross-linking (same number of cells, same amount of ¹²⁵I-TSH), cell extracts were subjected to polyacrylamide gel electrophoresis (10%) under reducing conditions (see Methods). Autoradiography (representative of three experiments) was for 16 hours. In lanes 1 and 2, equal volumes of cell extract were applied. Lanes 3 and 4 were loaded with equal amounts of radioactivity. TSH cross-linking to intact cells, unlike immunoprecipitation (b), visualizes only mature TSHR expressed on the cell surface. Two forms of TSHR are seen: single-chain uncleaved TSHR and the ligand binding A subunit in the cleaved TSHR released from the serpentine region (TSHR-10,000 cells) or from the GPI-anchored portion (ECD-GPI) by disulfide bond reduction. Note the lesser proportion of cleaved versus uncleaved receptors in the ectodomain with the GPI anchor than in the wild-type TSHR. (b) Immunoprecipitation confirms higher level of TSHR expression in TSHR-10,000 cells than in ECD-GPI cells. Similar numbers of TSHR-10,000 cells and ECD-GPI cells were precursor labeled simultaneously with the same quantity of ³⁵S-methionine and ³⁵S-cysteine (see Methods). Detergent-solubilized particulate preparations were immunoprecipitated with murine mAb A9 (epitope in the midregion of the TSHR ectodomain), the most effective mAb available to us for immunoprecipitating all forms of the TSHR. After polyacrylamide gel electrophoresis (10%), autoradiography (representative of duplicate experiments) was performed for 16 hours.