Characterization of the thyrotropin binding pocket.
A panel of monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) was prepared using three different immunization strategies. The mAbs obtained (n = 138) reacted with linear epitopes covering most of the TSHR extracellular domain and with conformational epitopes. mAbs that bound to five different regions of the TSHR (amino acids [aa] 32-41, aa 36-42, aa 246-260, aa 277-296, and aa 381-385) were able to inhibit (125)I-labeled thyrotropin (TSH) binding to solubilized TSHR preparations. Fab and immunoglobulin G (IgG) preparations were similarly effective inhibitors for mAbs reactive with aa 246-260, aa 277-291 and aa 381-385 suggesting that these three regions of the TSHR are involved in TSH binding. In contrast mAbs reactive with aa 32-41 and aa 36-42 were not effective at inhibiting TSH binding when Fab preparations were used, suggesting that these N terminal regions of the TSHR were less critical for TSH binding. Our studies suggest that three distinct and discontinuous regions of the TSHR (aa 246-260 and 277-296 on the TSHR A subunit) and aa 381-385 (on the TSHR B subunit) fold together to form a complex TSH binding pocket. Alignment of the aa sequences of these three regions in TSHRs from different species indicates that they are highly conserved.
Jeffreys
Jennifer
J
FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, United Kingdom.
Depraetere
Hilde
H
Sanders
Jane
J
Oda
Yasuo
Y
Evans
Michele
M
Kiddie
Angela
A
Richards
Tonya
T
Furmaniak
Jadwiga
J
Rees Smith
Bernard
B
D000818:Animals; D021461:Animals, Outbred Strains; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D000939:Epitopes; D006801:Humans; D007114:Immunization; D007457:Iodine Radioisotopes; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013972:Thyrotropin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; D013462:Sulfur Radioisotopes; D013972:Thyrotropin