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 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.
Costagliola
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S
I.R.I.B.H.N., Faculté de Médecine, University of Brussels, and Euroscreen s.a., Belgium.
Khoo
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Vassart
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G
D000595:Amino Acid Sequence; D000818:Animals; D001327:Autoimmune Diseases / Q000097:blood / Q000276:immunology; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006111:Graves Disease / Q000097:blood / Q000276:immunology*; D006801:Humans; D007037:Hypothyroidism / Q000097:blood / Q000276:immunology*; D018828:Immunoglobulins, Thyroid-Stimulating / Q000097:blood*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011989:Receptors, Thyrotropin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D018800:Thy-1 Antigens / Q000235:genetics; D013972:Thyrotropin / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D018828:Immunoglobulins, Thyroid-Stimulating; D010446:Peptide Fragments; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D018800:Thy-1 Antigens; D013972:Thyrotropin; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase