High-affinity binding of thyrotropin to the extracellular domain of its receptor transfected in Chinese hamster ovary cells.
Thyrotropin (TSH) receptor is a cell surface receptor that shares a high degree of homology with other glycoprotein hormone receptors including lutropin-choriogonadotropin (LH/CG) and follicle-stimulating hormone (FSH) receptors. Although the extracellular domain of TSH receptor is important for ligand binding, no direct information is available on whether extracellular domain alone is sufficient for high-affinity binding. Moreover, mutations made in the second cytoplasmic loop or the cytoplasmic tail of TSH receptor were reported to reduce significantly the affinity of TSH binding. In an attempt to determine whether TSH receptor extracellular domain is sufficient for high-affinity TSH binding or whether it requires transmembrane regions, we made a construct (TSHR-EX/CMV) that encodes for only the extracellular domain plus a foreign hydrophobic tail. The TSHR-EX/CMV was transfected and stably expressed in Chinese hamster ovary (CHO) cells. The truncated receptor was anchored to the cell surface through the hydrophobic tail at the carboxyl terminus. High-affinity TSH binding was observed comparable to that of the cells transfected with full-length TSH receptor. The CHO cells transfected with TSHR-EX/CMV did not respond to TSH stimulation of adenylate cyclase, whereas the cells transfected with the full-length TSH receptor cDNA did. The data presented here show that the extracellular domain of TSH receptor is sufficient to confer high-affinity TSH binding.
Department of Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia.
Y
Y
Shi
M
M
Zou
R S
RS
Parhar
N R
NR
Farid
D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells / Q000502:physiology*; D006224:Cricetinae; D000242:Cyclic AMP / Q000032:analysis; D004247:DNA / Q000032:analysis / Q000235:genetics; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D007457:Iodine Radioisotopes; D011485:Protein Binding; D011989:Receptors, Thyrotropin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism*; D014162:Transfection
D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D004247:DNA; D000242:Cyclic AMP