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J Biol Chem, Vol. 273, Issue 19, 11874-11880, May 8, 1998
From the Department of Cellular and Molecular Sciences, St.
George's Hospital Medical School,
London SW17 0RE, United Kingdom
The thyrotrophin (TSH) receptor (TSHR) is
synthesized as a single polypeptide with a predicted large
extracellular domain (ECD), a seven-transmembrane pass region and a
C-terminal intracellular tail. It is a common target for production of
autoantibodies. To investigate whether the ECD is solely responsible
for ligand interaction, we directed the expression of this domain in
isolation on the cell surface by means of a
glycosylphosphatidylinositol (GPI) anchor sequence. Immunoblotting
detected TSHR material of Mr 70,000 expressed
at high levels. In immunoprecipitation studies, the GPI-anchored ECD
was recognized by experimental and pathological antibodies. The
molecule was detected on the cell surface by flow cytofluorimetry at up
to 10-fold higher amounts than the highest expressing full-length
receptor clone. Radioligand binding studies confirmed this and showed
that the recombinant molecule bound TSH with high affinity similar to
full-length receptor; however, studies with human autoimmune sera
indicated differences in the degree of inhibition when compared with
full-length receptor. The existence of the GPI anchor was confirmed by
cleavage with a GPI-specific phospholipase C and biosynthetic labeling
with [3H]ethanolamine. TSHR material was also present
inside the cell in both soluble and membrane-bound forms. Thus, the
recombinant GPI-anchored ECD is the smallest known fragment of the TSHR
that retains high-affinity TSH binding and is expressed at high levels on the cell surface as well as internally; this approach may well be
useful for other membrane proteins.
Production of the Thyrotrophin Receptor Extracellular Domain as a
Glycosylphosphatidylinositol-anchored Membrane Protein and Its
Interaction with Thyrotrophin and Autoantibodies
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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