Production of bioactive amino‐terminal domain of the thyrotropin receptor via insertion in the plasma membrane by a glycosylphosphatidylinositol anchor
Open Access
- 9 October 1998
- journal article
- Published by Wiley in FEBS Letters
- Vol. 436 (3) , 427-433
- https://doi.org/10.1016/s0014-5793(98)01177-6
Abstract
A chimeric cDNA construct encoding the extracellular amino‐terminal domain (ECD) of the thyrotropin receptor fused to the signal for addition of glycosylphosphatidylinositol from the Thy‐1 gene directs efficient expression of the ECD at the plasma membrane of transfected CHO cells. A cell line (GT14) expressing over 106 receptors/cell was isolated, which allows direct detection, by flow cytometry, of autoantibodies from the majority of patients with Graves' disease or autoimmune idiopathic myxedema. Treatment of GT14 cells with a glycosylphosphatidylinositol‐specific phospholipase C (PI‐PLC) releases a soluble 80 kDa molecule which neutralizes the autoantibodies from Graves patients. Whereas it does not bind TSH when released from the cells by PI‐PLC in free form, the soluble ECD displays clear TSH binding activity when it is released as a complex with a monoclonal antibody recognizing a conformational epitope of the ECD. Our results allow production of bioactive ECD of the thyrotropin receptor in high yield, with possible applications in structural analyses.Keywords
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