EVIDENCE FOR NEGATIVE COOPERATIVITY AMONG HUMAN THYROTROPIN RECEPTORSOVEREXPRESSED IN MAMMALIAN-CELLS

The complementary DNA for the human TSH receptor (TSHR) translated reg ion was amplified in the genome of stably transfected Chinese hamster ovary (CHO) cells using a dihydrofolate reductase minigene. Immunoprec ipitation of TSHR in whole cells precursor-labeled with [S-35]methioni ne and [S-35]cysteine revealed an approximately 10-fold increase in TS HR expression in cells stabilized in 10,000 nM methotrexate (TSHR-10,0 00 cells) compared to cells with the same gene not subjected to amplif ication (TSHR-0 cells). Similarly, [I-125]TSH cross-linking to the sur face of intact CHO cells revealed a progressive increase in TSH-bindin g sites with dihydrofolate reductase minigene amplification, with a 12 .8-fold increase in TSHR in TSHR-10,000 us. TSHR-0 cells. Based on the known number of TSHR expressed by TSHR-0 cells, TSHR-10,000 express a pproximately 1.9 X 10(6) TSHR on their surface. Two ligand-TSHR comple xes were evident under reducing conditions, representing the single ch ain holoreceptor of about 115 kDa and a dissociated A subunit of about 60 kDa. In the absence of TSH, basal cAMP levels in TSHR-10,000 cells were greater than those in TSHR-0 cells (6-fold in isotonic medium an d 18.5-fold in hypotonic medium), indicating that the unliganded TSHR has significant constitutive activity. We assessed the kinetics of TSH binding to CHO cells overexpressing the TSHR using [I-125]TSH in the presence of increasing concentrations of unlabeled TSH as well as by a ttempted saturation with labeled ligand. Surprisingly, in contrast to TSHR-0 cells (K-d = similar to 5 X 10(-10) M), we observed progressive ly lower affinities for TSH binding by TSHR-800 cells (K-d = similar t o 10(-9) M) and TSHR-10,000 cells (K-d = similar to 2 X 10(-9) M). In summary, we report a high level of expression of TSHR in CHO cells and confirm the high constitutive activity of the TSHR in the absence of ligand as well as the binding of TSH to the single subunit, uncleaved TSHR. Moreover, we found that a high level of expression is associated with apparent negative cooperativity among the TSHR in terms of their affinity for ligand.