A receptor assay using [125I]bTSH-binding to guinea-pig testis membrane was developed. Unlabelled hCG and FSH inhibited [125I]bTSH binding. In patients with Graves' disease and in untreated hyperthyroid patients, almost all long-acting thyroid stimulators and thyroid-stimulating antibodies, respectively did not inhibit [125I]bTSH binding, which on the other hand was inhibited by thyroid stimulation blocking antibodies in patients with primary hypothyroidism. When the inhibitory effect on the binding of [125I]hCG and 125I-synthetic \g=a\-subunit peptide (\g=a\26-46) of hCG to testis membrane was examined, bTSH resulted in a significant inhibition. However, all three kinds of TSH receptor antibodies had no inhibitory effect. This study demonstrated 1. interaction of \g=a\-subunitof TSH and hCG with the testicular receptor; 2. binding of thyroid stimulation-blocking antibody and lack of binding of thyroid-stimulating antibody to the testicular TSH receptor in spite of binding of these TSH receptor antibodies to the thyroidal TSH receptor, and 3. lack of binding of thyroid-stimulating antibody and thyroid stimulation-blocking antibody to the testicular gonadotropin receptor.TSH receptors have been demonstrated in mem¬ branes from extrathyroid tissues (1-4). TSH, LH, FSH and hCG consist of a common ct-subunit and a hormone specific ß-subunit. Available data sug¬ gest that the ci-subunit part of pituitary glycoprotein hormones such as FSH, LH, TSH and hCG may play an important role in recognition and binding to receptor. The binding of [12oI]bovine TSH (bTSH) to its receptor was attributed to the ct-subunit (1,4-10). On the other hand, an inhibi¬ tory effect of bTSH on [125I]hCG binding to its receptor (testis or ovary) has been demonstrated (6,11,12).Long-acting thyroid stimulator (LATS) and thy¬ roid-stimulating antibody (TS-ab) have been shown to compete with TSH for binding to TSH receptor (13-16). Thyroid stimulation-blocking antibody (TSB-ab) have also a blocking action on TSH by its direct binding to the TSH receptor (17)(18)(19). How¬ ever, it has never been clear how these thyroid stimu¬ lators (TSH, LATS, TS-ab) and TSB-ab can bind to the same binding site in the receptor.In the present study binding characteristics of TSH receptor antibody (TR-ab) such as LATS, TSab, TSB-ab to the testicular TSH receptor were examined. Materials and Methods Materials and MethodsCrude bTSH (1 IU/mg protein) was obtained from Sigma Chemical Co (St. Louis, MO). Crude FSH (HMG, 75 IU/mg protein) was obtained from Nikken Co (Tokyo, Japan). The purified hCG (9000 IU/mg protein) and crude hCG preparation (1000 IU/mg protein) were ob¬ tained from UCB-Bioproducts (Brüssel, Belgium) and Mochida Co (Tokyo), respectively. Synthetic a-subunk peptide (a 26-46, Leu-Gln-CysMet-Gly-Cys-Cys-Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-LeuArg-Ser-Lys-Lys-Thr (NH2)) of hCG was synthesized by Curare Co Ltd (Osaka, Japan) (20,21).

When rabbits were immunized with porcine thyroid plasma membrane, some cases of produced antibodies showed the blocking activity for TSH stimulated cAMP production in cultured porcine thyroid cells in spite of negative TSH binding inhibitory immunoglobulin (TBII) activity. The inhibitory effect of these thyroid stimulation blocking antibodies (TSBAbs) on cAMP increase by forskolin and GTP gamma S in porcine thyroid cells was observed in 1 and 3 rabbits, respectively. When the blocking activity of these antibodies for stimulated cAMP production by forskolin, GTP gamma S and NaF was examined in the isolated porcine thyroid membrane, no blocking activity for these 3 stimulators except in 1 case was found. The blocking activity of these antibodies could be absorbed significantly by incubation with porcine thyroid plasma membrane as antigen. The blocking activity was not observed in rabbits immunized with porcine thyroglobulin. The present experiment demonstrated that the produced antibody in rabbits against porcine thyroid membrane had the blocking activity for TSH stimulation to thyroid cells without affecting TSH binding to its receptor. These facts suggest that this type of blocking antibody may be produced against any antigen of thyroid membrane origin except the TSH receptor.