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 blottin g procedure. Twenty sera from thyrotropin receptor autoantibodies (TrAb)-po sitive patients with a history of hyperthyroidism and 20 sera with high lev els of TSH blocking activity were analyzed. Four of 20 sera with blocking-t ype of TRAb (i.e., TSH antagonist activity) were able to recognize the matu re, fully glycosylated 120-kd form of the receptor on blots of gels run und er reducing conditions. No sera recognized the 100-kd high mannose precurso r 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 fr agments of the mature 120-kd TSHR. In the absence of reducing agent the sam e 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 re action with the TSHR was observed on Western blots with the remaining 15 se ra with TSH blocking activity, nor with 20 sera from patients with a histor y of hyperthyroidism, nor with sera from 10 healthy blood donors, 10 Hashim oto sera (negative for TRAb) and 10 systemic lupus erythematosus sera. No c lear 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 I-125-labeled TSHR. Overall, our results in dicate that the mature 120-kd form of the TSHR that is principally responsi ble 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 t he receptor's binding sites for TSH and TRAb.