PRODUCTION OF ANTIBODIES TO THE HUMAN THYROTROPIN RECEPTOR AND THEIR USE IN CHARACTERIZING EUKARYOTICALLY EXPRESSED FUNCTIONAL RECEPTOR

The structure of the human thyrotropin receptor expressed as a recombi nant protein in eukaryotic cells was investigated by immunochemical an d functional means using two types of polyclonal rabbit antisera: one raised against the large N-terminal extracellular region (residues 1-4 15) expressed in E. coli and the other raised against a synthetic pept ide (residues 313-330). Both types of antisera gave similar results, w ith the former being more effective. As expected from the lack of conf ormation of the immunogens, the antisera worked well in immunoblotting . Less predictably, the antisera also recognised the functional recept or in its native state (detected by flow cytofluorimetry and immunopre cipitation), and inhibited the binding of thyrotropin. Thus the region 313-330 is on the outside of the receptor molecule and falls within, or close to, the binding site of thyrotropin. None of the antisera sti mulated cAMP production, showing that this is a very special property, largely restricted to certain human autoantibodies. The antisera were used to immunoprecipitate radioiodinated proteins from Chinese hamste r ovary cell (CHO) lines expressing recombinant receptor. The most abu ndant and reproducible cell-surface molecule that correlated with the presence of full-length functional receptor was a glycopolypeptide of approximately 100 kDa, of which 15 kDa is attributable to carbohydrate , in good agreement with the size predicted for the polypeptide from t he cDNA sequence. Three other molecular species were also variably det ected at the cell surface: 55 kDa, 180 kDa and large molecular weight material at the top of the polyacrylamide gel. We attribute the 55 kDa molecules to breakdown of the intact receptor during handling in vitr o; similarly we consider that the very large material is also an in vi tro artefact, possibly due to aggregation; there is no obvious explana tion for the 180 kDa band. From studies using a glucosidase inhibitor, it could be demonstrated that glycosylation is required for the expre ssion of all of these molecules at the cell surface. Thus, our data fa vour the functional thyrotropin receptor being the single glycopolypep tide predicted from its cDNA sequence. We found no evidence that furth er processing of this polypeptide in recombinant cells is required to produce the functional receptor.