Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells.
The complementary DNA for the human TSH receptor (TSHR) translated region was amplified in the genome of stably transfected Chinese hamster ovary (CHO) cells using a dihydrofolate reductase minigene. Immunoprecipitation of TSHR in whole cells precursor-labeled with [35S]methionine and [35S]cysteine revealed an approximately 10-fold increase in TSHR expression in cells stabilized in 10,000 nM methotrexate (TSHR-10,000 cells) compared to cells with the same gene not subjected to amplification (TSHR-0 cells). Similarly, [125I]TSH cross-linking to the surface of intact CHO cells revealed a progressive increase in TSH-binding sites with dihydrofolate reductase minigene amplification, with a 12.8-fold increase in TSHR in TSHR-10,000 vs. TSHR-0 cells. Based on the known number of TSHR expressed by TSHR-0 cells, TSHR-10,000 express approximately 1.9 x 10(6) TSHR on their surface. Two ligand-TSHR complexes were evident under reducing conditions, representing the single chain 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 and 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 [125I]TSH in the presence of increasing concentrations of unlabeled TSH as well as by attempted saturation with labeled ligand. Surprisingly, in contrast to TSHR-0 cells (Kd = approximately 5 x 10(-10) M), we observed progressively lower affinities for TSH binding by TSHR-800 cells (Kd = approximately 10(-9) M) and TSHR-10,000 cells (Kd = approximately 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.
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, California 94121, USA.
G D
GD
Chazenbalk
A
A
Kakinuma
J C
JC
Jaume
S M
SM
McLachlan
B
B
Rapoport
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D007038:Hypotonic Solutions; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013972:Thyrotropin / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
D003470:Culture Media; D007038:Hypotonic Solutions; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D013972:Thyrotropin; D008715:Methionine; D000242:Cyclic AMP; D003545:Cysteine