Reactivity of thyrotropin receptor autoantibodies with the thyrotropin receptor on western blots.
Affinity purified recombinant human thyrotropin receptor (TSHR) was run on sodium dodecyl sulfate (SDS) gels and subjected to a renaturing and blotting procedure. Twenty sera from thyrotropin receptor autoantibodies (TRAb)-positive patients with a history of hyperthyroidism and 20 sera with high levels of TSH blocking activity were analyzed. Four of 20 sera with blocking-type of TRAb (i.e., TSH antagonist activity) were able to recognize the mature, fully glycosylated 120-kd form of the receptor on blots of gels run under reducing conditions. No sera recognized the 100-kd high mannose precursor form of the TSHR. Three of the four recognized a 74-kd band and 2 of the 4 recognized a 50-kd band. These bands are probably proteolytic cleavage fragments of the mature 120-kd TSHR. In the absence of reducing agent the same 4 of 20 sera described above together with a further serum sample (i.e., 5/20 in total) reacted with the 120-kd form of the receptor. No specific reaction with the TSHR was observed on Western blots with the remaining 15 sera with TSH blocking activity, nor with 20 sera from patients with a history of hyperthyroidism, nor with sera from 10 healthy blood donors, 10 Hashimoto sera (negative for TRAb) and 10 systemic lupus erythematosus sera. No clear differences were observed in the TRAb positive sera that were reactive and nonreactive on Western blots in terms of their ability to inhibit TSH binding or to immunoprecipitate 125I-labeled TSHR. Overall, our results indicate that the mature 120-kd form of the TSHR that is principally responsible for binding TSH is also responsible for binding TRAb (when this binding can be detected). These observations together with immunoprecipitation and TSH binding inhibition studies, emphasize the close relationship between the receptor's binding sites for TSH and TRAb.
Maruyama
M
M
FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, United Kingdom.
Sanders
J
J
Oda
Y
Y
Kiddie
A
A
Furmaniak
J
J
Rees Smith
B
B
D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D015153:Blotting, Western; D006801:Humans; D006980:Hyperthyroidism / Q000276:immunology; D018828:Immunoglobulins, Thyroid-Stimulating; D007163:Immunosorbent Techniques; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D011989:Receptors, Thyrotropin / Q000097:blood* / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D013967:Thyroiditis, Autoimmune / Q000276:immunology; D013972:Thyrotropin / Q000378:metabolism
D001323:Autoantibodies; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; C094372:thyrotropin-binding inhibitory immunoglobulin; D013972:Thyrotropin