Production of antibodies to the human thyrotropin receptor and their use in characterising eukaryotically expressed functional receptor.
The structure of the human thyrotropin receptor expressed as a recombinant protein in eukaryotic cells was investigated by immunochemical and functional means using two types of polyclonal rabbit antisera: one raised against the large N-terminal extracellular region (residues 1-415) expressed in E. coli and the other raised against a synthetic peptide (residues 313-330). Both types of antisera gave similar results, with the former being more effective. As expected from the lack of conformation of the immunogens, the antisera worked well in immunoblotting. Less predictably, the antisera also recognised the functional receptor in its native state (detected by flow cytofluorimetry and immunoprecipitation), and inhibited the binding of thyrotropin. Thus the region 313-330 is on the outside of the receptor molecule and falls within, or close to, the binding site of thyrotropin. None of the antisera stimulated cAMP production, showing that this is a very special property, largely restricted to certain human autoantibodies. The antisera were used to immunoprecipitate radioiodinated proteins from Chinese hamster ovary cell (CHO) lines expressing recombinant receptor. The most abundant and reproducible cell-surface molecule that correlated with the presence of full-length functional receptor was a glycopolypeptide of approximately 100 kDa, of which 15 kDa is attributable to carbohydrate, in good agreement with the size predicted for the polypeptide from the cDNA sequence. Three other molecular species were also variably detected at the cell surface: 55 kDa, 180 kDa and large molecular weight material at the top of the polyacrylamide gel.(ABSTRACT TRUNCATED AT 250 WORDS)
Harfst
E
E
Department of Cellular and Molecular Sciences, St George's Hospital Medical School, London, UK.
Ross
M S
MS
Nussey
S S
SS
Johnstone
A P
AP
D000818:Animals; D000917:Antibody Formation; D001667:Binding, Competitive; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D011233:Precipitin Tests; D011817:Rabbits; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology
D018076:DNA, Complementary; D007106:Immune Sera; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins