EPITOPE MAPPING OF THE HUMAN TSH RECEPTOR - STRUCTURE-FUNCTION STUDIES

With the aid of recombinant DNA technology (PCR/site directed mutagene sis, sequencing) the full length coding region of the human TSH recept or was manipulated to place a specific epitope peptide tag (FLAG epito pe sequence) at the carboxyl end of the protein. The resulting constru ct was cloned into a eukaryotic expression vector and stably transfect ed into HeLa cells. The expression/translation of the tagged TSH recep tor molecule was monitored by immune-precipitation and western blottin g of protein lysates, and was found to be expressed at considerable le vels using the commercially available antibodies directed towards the FLAG epitope. This analysis revealed two discrete specific bands 90-12 0 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. Further more TSH stimulated cAMP response in these transfected cells were comp arable to the wild type receptor, thus demonstrating that the tagged r eceptor was functionally identical to the transfected wild type recept or. These cell lines will be of great value when analysing TSH/recepto r or receptor/autoantibody interactions considering the availability o f well characterized experimental anti-TSH receptor sera.