In order to determine the functional significance of the extracellular loop of human
thyrotropin receptor (hTSHR), two peptides composed of eight amino acids were inserted
into hTSHR by ligating synthetic oligonucleotides into + 1811 NCol site of hTSHR cDNA.
Mutant hTSHR cDNAs which encode a hydrophobic peptide (ATVLVVPM) and a hydrophilic
peptide (GTTRTVAM) between +572 Met and +573 Asp were transfected into Chinese hamster
ovary (CHO) cells to develop F-cell lines and R-cell lines, respectively. Of the resulting
cloned cell lines, F-29 and R-9 were shown to express mutant hTSHs at the protein
level by Western blotting and at the mRNA level by reverse transcription-polymerase
chain reaction (RT-PCR). We show that neither thyrotropin (TSH) nor IgGs from patients
with Graves' disease stimulated cAMP production by F-29 and R-9 cells. 125I-TSH binding study revealed that F-29 and R-9 cells do not bind TSH. Our data demonstrate
that the mutations impaired TSH-binding and incapacitated the cells from responding
to TSH. The evidence suggests that the second extracellular loop of hTSHR has an important
role in TSH and thyroid stimulating antibody (TSAb)-dependent signal transduction.
Extracellular Loop - Thyrotropin Receptor - Thyrotropin - Thyroid Stimulating Antibody
- Signal Transduction
