Epitope mapping of the human TSH receptor; structure function studies.
With the aid of recombinant DNA technology (PCR/site directed mutagenesis, sequencing) the full length coding region of the human TSH receptor was manipulated to place a specific epitope peptide tag (FLAG epitope sequence) at the carboxyl end of the protein. The resulting construct was cloned into a eukaryotic expression vector and stably transfected into HeLa cells. The expression/translation of the tagged TSH receptor molecule was monitored by immune-precipitation and western blotting of protein lysates, and was found to be expressed at considerable levels using the commercially available antibodies directed towards the FLAG epitope. This analysis revealed two discrete specific bands 90-120 KDa representing, presumably, differently glycosylated forms of the receptor. TSH radio receptor assays demonstrated that the FLAG tagged TSH receptor bound TSH comparable with the wild type receptor. Furthermore TSH stimulated cAMP response in these transfected cells were comparable to the wild type receptor, thus demonstrating that the tagged receptor was functionally identical to the transfected wild type receptor. These cell lines will be of great value when analysing TSH/receptor or receptor/autoantibody interactions considering the availability of well characterized experimental anti-TSH receptor sera.
Hunt
N
N
Institute for Hormone and Fertility Research, University of Hamburg, Germany.
Ruppert
J
J
Willey
K
K
Schulte
H M
HM
D015153:Blotting, Western; D018604:Epitope Mapping; D006367:HeLa Cells; D006801:Humans; D007163:Immunosorbent Techniques; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins