PRODUCTION OF BIOACTIVE AMINO-TERMINAL DOMAIN OF THE THYROTROPIN RECEPTOR VIA INSERTION IN THE PLASMA-MEMBRANE BY A GLYCOSYLPHOSPHATIDYLINOSITOL ANCHOR

A chimeric cDNA construct encoding the extracellular amino-terminal do main (ECD) of the thyrotropin receptor fused to the signal for additio n of glycosylphosphatidylinositol from the Thy-1 gene directs efficien t expression of the ECD at the plasma membrane of transfected CHO cell s. A cell lime (GT14) expressing over 10(6) receptors/cell was isolate d, which allows direct detection, by flow cytometry, of autoantibodies from the majority of patients with Graves' disease or autoimmune idio pathic myxedema, Treatment of GT14 cells with a glycosylphosphatidylin ositol-specific phospholipase C (PI-PLC) releases a soluble 80 kDa mol ecule which neutralizes the autoantibodies from Graves patients. Where as 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 conform ational epitope of the ECD, Our results allow production of bioactive ECD of the thyrotropin receptor in high yield, with possible applicati ons in structural analyses. (C) 1998 Federation of European Biochemica l Societies.