Purification and characterization of a soluble bioactive amino-terminal extracellular domain of the human thyrotropin receptor.
The amino-terminal ectodomain of the human TSH receptor has been expressed at the surface of CHO cells as a glycosylphosphatidylinositol-anchored molecule containing a 10-residue histidine tag close to its C terminus. The soluble ectodomain could be released from the cells by treatment with a glycosylphosphatidylinositol-phospholipase C and purified to apparent homogeneity by cobalt-Sepharose chromatography. Two nanomoles of material was obtained, which was suitable for analysis by mass spectrometry. This allowed the identification of four out of the six potential N-glycosylation sites as being effectively glycosylated. A proportion of the purified soluble ectodomain displayed specific binding of (125)I-labeled TSH, allowing for the first time performance of classical saturation binding experiments. Two classes of high-affinity binding sites were identified: site A, K(d) 0.014 nM; site B, K(d) 0.83 nM. The significance of site A, whose affinity is much higher than for the holoreceptor at the surface of intact cells, remains to be clarified. The purified ectodomain was capable of inhibiting efficiently the thyroid stimulating activity of immunoglobulins from patients with Graves' disease. It allowed computation of the amounts of these immunoglobulins in patient's serum, giving values up to 10 microg/mL. Contrary to all currently available assays, the soluble ectodomain of the TSH receptor purified in a functionally competent conformation allows direct studies of its interactions with TSH and autoantibodies and opens the way to structural studies.
Cornelis
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IRIBHN and Department of Medical Genetics, ULB, Brussels, Belgium.
Uttenweiler-Joseph
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Panneels
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Vassart
G
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Costagliola
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D016466:CHO Cells; D002847:Chromatography, Agarose; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006031:Glycosylation; D006111:Graves Disease / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007136:Immunoglobulins / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007093:Imidazoles; D007136:Immunoglobulins; D011989:Receptors, Thyrotropin; C029899:imidazole