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Production of bioactive amino‐terminal domain of the thyrotropin receptor via insertion in the plasma membrane by a glycosylphosphatidylinositol anchor
Author(s) -
Costagliola Sabine,
Khoo Daphne,
Vassart Gilbert
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01177-6
Subject(s) - terminal (telecommunication) , chemistry , membrane , domain (mathematical analysis) , microbiology and biotechnology , receptor , amino terminal , biochemistry , biology , peptide sequence , computer science , gene , mathematics , mathematical analysis , telecommunications
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 10 6 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.