Title : Purification and characterization of a soluble bioactive amino-terminal extracellular
domain of the human
thyrotropin receptor
Abstract :
- 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 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