Interaction of human chorionic gonadotropin (hCG) and asialo-hCG with recombinant human thyrotropin receptor.
hCG is a putative thyroid stimulator. The present studies were undertaken to examine its interaction and that of its desialylated variant asialo-hCG with recombinant human TSH (hTSH) receptor (hTSHr). To this end, we transfected a human thyroid carcinoma cell line (HTC) lacking endogenous TSHr with the full-length cDNA of the hTSHr. Unlike the wild type, the transfected cells, termed HTC-TSHr cells, were able to bind bovine TSH (bTSH) with high affinity and increase cAMP production in response to bTSH stimulation. Of the hCG forms, intact hCG displayed a weak activity to inhibit [125I] bTSH binding to HTC-TSHr cells, with 100 mg/L (2.6 x 10(-6) mol/L) producing maximally a 20% inhibition, whereas asialo-hCG achieved half-maximum binding inhibition at a concentration of 8 mg/L (2.3 x 10(-7) mol/L). The inhibitory constant (Ki) of asialo-hCG for recombinant hTSHr was calculated from saturation experiments in the presence of variable doses of bTSH and a fixed concentration of asialo-hCG to be approximately 8 x 10(-8) mol/L. The interaction of asialo-hCG with TSHr was further assessed by studies of the direct binding of the radioactively labeled hormone to both HTC and HTC-TSHr cells. [125I]Asialo-hCG binding to HTC-TSHr cells was 4.7%, compared to 1.5% in the wild-type cells lacking TSHr and was displaceable by bTSH (0.1-100 IU/L), indicating specific binding of the tracer to TSHr. Functionally, hCG (up to 100 mg/L; 2.6 x 10(-6) mol/L) proved unable to evoke any significant cAMP response over basal values in HTC-TSHr cells, as did asialo-hCG. Asialo-hCG, but not hCG, inhibited bTSH-stimulated adenylate cyclase activity in the cells in a dose-dependent manner. In conclusion, the present data show that intact hCG binds only weakly to HTC-TSHr cells and produces no significant cAMP stimulation, which is at variance with data obtained in FRTL-5 and Chinese hamster ovary-TSHr cells, but in good accord with previous findings in human thyroid membranes. Asialo-hCG, on the other hand, strongly binds to recombinant TSHr and inhibits the cAMP response to bTSH in HTC-TSHr cells, indicating that the desialylated hCG variant directly interacts with the receptor and truly is an antagonist of the hTSHr.
Hoermann
R
R
Medical Department II, Klinikum Grosshadern, University of Munich, Germany.
Broecker
M
M
Grossmann
M
M
Mann
K
K
Derwahl
M
M
D001212:Asialoglycoproteins / Q000378:metabolism*; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure; D013972:Thyrotropin / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D001212:Asialoglycoproteins; D006063:Chorionic Gonadotropin; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; C019263:asialo-human chorionic gonadotropin; D013972:Thyrotropin; D000242:Cyclic AMP