Department of Endocrinology, Royal Prince Alfred Hospital, and Department of Medicine, University of Sydney, Sydney, Australia THE THYROTROPHIN RECEPTOR IN HUMAN THYROID PLASMA MEMBRANES: EFFECT OF SERUM IMMUNOGLOBULINS By Janet M. Bryson, Aet Joasoo and John R. Turtle ABSTRACT A homologous receptor assay system using human thyroid membranes and 125I-labelledhuman TSH (htsh) was used to study the effect of serum and serum fractions on the binding of [125I]hTSH to the membrane. Scatchard analysis showed a single population of binding sites for TSH. Gamma globulin fractions prepared from sera of patients with Graves disease were able to displace [125I]hTSH from the membrane to a greater extent than normal gamma globulin in 21 out of 45 cases. Increased displacement activity was seen in patients with thyroiditis, hot nodules and euthyroid eye disease but not in patients with toxic multinodular goitres. Further fractionation of the gamma globulin fraction showed that the stimulatory activity was not confined to the IgG fraction. Scatchard plots showed gamma globulin fractions decreased the number of receptor sites available for TSH binding but did not alter the affinity of the receptor for TSH. IgG fractions showed different slopes and intercepts and appeared to decrease the affinity of the receptor for TSH. LATS activity in human serum may be explained on the basis of these observations on the properties of the TSH receptor. Hyperthyroidism in Graves' disease is thought to be due to thyrotrophin-like activity of immunoglobulins such as the long acting thyroid stimulator (LATS) (Adams 8c Purves 1958), or LATS-protector (LATS-P) (Adams 8c Kennedy 1967). The failure of the McKenzie (1958) mouse bioassay to detect LATS in
the serum of all patients with Graves' disease may be due to the insensitivity of the heterologous assay system (Burke 1968). It is now thought that other immu noglobulins may be involved. Adams claimed that a human specific stimulator, LATS-protector, may be found in the serum of all patients with Graves' disease not showing LATS activity (Adams 8c Kennedy 1971). LATS and thyrotrophin (TSH) have been shown to bind to human thyroid glands (Smith 1971). LATS, like TSH, produces its effect by activation of the adenyl cyclase system, although LATS-P has been shown to have no effect on adenyl cyclase activity (Kendall-Taylor 1973). If thyroid stimulators other than TSH act via the TSH receptor site, these stimulators should be measured from their ability to displace labelled TSH from its receptor site on thyroid plasma membranes. The following studies were undertaken to evaluate human thyroid stimulators in an homologous human assay system. The effect of both normal and abnormal sera and serum fractions on the binding of [125I]hTSH to thyroid plasma membranes was observed. Similar studies have been reported recently by Manley et al. (1974a,ô) using guinea pig thyroid membranes and by Smith & Hall (1974a,b) using both guinea pig and human thyroid membranes. MATERIALS AND METHODS 1. Preparation of human thyroid plasma membranes Thyroid glands were obtained at thyroidectomy and stored in liquid air until required. Tissue from patients with Graves' disease or nodular goitre were both used. The plasma membranes were isolated using the method of Wolff Se Jones (1971) with the exception that the last stage of purification (discontinuous sucrose gradient cen trifugation) was omitted. The homogenizing medium contained 0.25 M sucrose, 1.10-3 M EGTA, 3.10-3 M Tris HC1 ph 7.4. A motor driven Teflon glass homogenizer was used. Membrane protein was determined by the method of Lowry et al. (1951) after the membrane has been dissolved in 100 pi 1 N NaOH in a boiling water bath. Membranes were stored until required in liquid air with no significant change in binding of TSH being observed after 3 months of storage. 2. Receptor assay Incubation was carried out in small plastic LP3 tubes, the incubation medium con taining 0.25 M sucrose, 1.10-3 M EGTA, 3.10-3 m Tris HC1, 2.5 /o bovine serum albumin, at final ph 5.3. To each tube was added TSH standard or test fraction, 100 pg membrane protein, 40 000 cpm [125I] htsh in a final volume of 160 pi. The tubes were incubated at 30 C for 10 min. Buffer (300 pi) was added to stop the reaction and to give a larger volume for centrifugation. The tubes were centrifuged at 10 000 g for 5 min to pellet the membrane. The supernatant was aspirated and the precipitate washed with 10 %> sucrose and centrifuged again. Radioactivity of the pellet was measured in an automatic gamma counter.
3. Preparation of gamma globulin and non-gamma globulin fractions Serum was obtained from normal volunteers and patients with proven thyrotoxicosis before treatment was commenced. Gamma globulins were precipitated from serum with 3.75 M ammonium sulphate to a final concentration of f.6 M. The supernatant from the precipitation is referred to as the non-gamma globulin fraction. Both fractions were dialysed against several changes of distilled water for 48 h, lyophilized and taken up in 0.01 M phosphate, 0.15 M saline buffer ph 7.3. 4. Preparation of IgG and non-lgg fractions The gamma globulin fraction prepared by ammonium sulphate precipitation was fractionated using a QAE Sephadex batch technique. The fraction was diluted with an equal volume of 0.0563 M ethylene diamine, 0.077 m acetic acid buffer ph 7.0 and extracted with QAE Sephadex A 50, which had been swelled in ethylene diamine buffer and dried on a Büchner funnel, by continuous mixing for 1 h. After centrifuga tion, the supernatant containing the IgG was collected and the Sephadex washed several times with ethylene diamine buffer to remove all IgG. 0.45 M acetic acid/acetate buffer ph 4.0 was used to remove non-lgg proteins from the Sephadex. The fractions were dialysed, lyophilized and taken up in phosphosaline buffer ph 7.3. The non-lgg fraction was shown by immunoelectrophoresis to contain other immunoglobulins and traces of alpha and beta proteins. 5. Protein concentrations Protein concentrations were determined using the method of Lowry et al. (1951) using bovine albumin as the standard. 6. Preparation of ['2 IJhTSH ['25I]hTSH was prepared using the chloramine-t method of Greenwood et al. (1963). Human TSH (htsh), 2.5 pg, obtained from National Institute of Health, Bethesda, Maryland, was iodinated with 0.5 mci Na 125I. The iodinated material was applied to a 1 x 15 cm Sephadex G75 column and eluted with 0.01 M phosphate, 0.15 M saline, 0.01 m EDTA buffer ph 7.8. The [i25i]htsh was used without further purification and was stored at -20 C. 7. TSH standard Standard TSH was human TSH 68/38 from Medical Research Council, Division of Biological Standards, London. RESULTS Yields of 0.6-1.0 mg membrane protein per g wet weight thyroid tissue were obtained from multinodular thyroid glands and 1.0-3.0 mg membrane protein per g wet weight thyroid tissue was obtained from thyrotoxic glands. Binding of [125I]hTSH to thyroid plasma membranes Ten to forty per cent of [125I]hTSH (depending upon purity of the label) would bind to 100 pg membrane protein, with some variation between membrane
Fig. 1. Effect of concentration of membrane protein in the incubation mixture on binding of [!25I]hTSH to the membrane. preparations. Maximum percentage binding was the same for membranes pre pared from both thyrotoxic and nodular glands. Non-specific binding of [125I]hTSH to the tubes in the absence of membrane protein was < 1 %. Binding of htsh to the membranes was proportional up to 100 pg membrane protein per tube (0.625 mg/ml) with a maximum of 40 % binding at the highest con centration of membrane protein tested (400 pg) (Fig. 1). Fig. 2. Effect of ph of incubation mixture on binding of [^IJhTSH to the membrane.
Fig. 3. Effect of time of incubation on binding of [125I] htsh to the membrane at 2 different incubation temperatures. ( - 30 C, A- 0 C). Fig. 4: Effect of varying concentrations of htsh standard on the binding of [I25I]hTSH to the membrane. Range of htsh standard was 0-500 pu per tube.
- Maximum binding of htsh to the membranes occurred at ph between 5.0 and 5.5 (Fig. 2). This optimum ph was maintained in several different buffer systems. Twenty per cent of [125I]hTSH bound instantaneously to the membrane at both 0 and 30 C. Binding was greater at 30 C with maximum binding occurring after 10 min incubation (Fig. 3). Displacement of [mi]htsh with labelled htsh standard [i25j]htsh was displaced from the membrane by added htsh over the range 20-500 pu per tube (0.125 ^U-3.125 pu/ml) (Fig. 4). Scatchard plots gave a linear plot showing a single population of binding sites with a maximum affinity constant of 2.04 x 109l/mol which was not changed by membrane protein concentrations up to 250 pg per tube (Fig. 5). Membranes prepared from both thyrotoxic and nodular glands on the same day had identical affinity constants and the same number of binding sites per pg membrane protein. Displacement of [125l]hTSH from the membrane by serum and serum fractions 1. Whole serum. Both normal and thyrotoxic serum displaced labelled htsh from the membrane. Pre-incubation of the serum with the membrane before the addition of label gave the same displacement activity as the addition Fig. 5. Scatchard plot of B:F ratio for [125I]hTSH versus the amount of htsh bound to the membrane at different membrane concentrations (A.-A 100 pg membrane protein/ tube, - 50 /eg membrane protein/tube, - 25 pg membrane protein/tube). The affinity constant of the reaction is given by the negative slope of the plot and the binding capacity of the membrane is shown by the intercept on the horizontal axis.
- Fig. 6. Effect of whole serum and serum fractions on the binding of [1251] htsh to the mem brane at different protein concentrations, compared with the htsh standard curve. - TSH; o-o whole serum; - gamma globulin fraction; A-A non-gamma globulin fraction; - IgG fraction; - non-lgg fraction. of label and serum at the same time. The serum cross-reacted with htsh in the range of 200-1000 pg serum protein per tube (Fig. 6). There was no difference in the displacement activity for the two groups, expressed as equivalents of htsh as read from the htsh standard curve. htsh equivalent ± sem mu/ml Normal (14) 7.02 ± 0.96 Thyrotoxic (18) 5.99 ± 0.51 2. Non-gamma globulin fractions. The non-gamma globulin fraction (i. e. ammonium sulphate supernatant) displaced [125I]hTSH from the membrane and cross-reacted in the range of 500-1000 pg protein per tube (Fig. 6). The dis placement activities for normal and thyrotoxic non-gamma globulin fractions were similar, results being expressed as equivalents of TSH displaced per mg non-gamma globulin protein. htsh equivalent ± sem p\] htsh/mg protein Normal (14) 492.6 ± 38.4 Thyrotoxic (16) 538.0 ± 47.0
- - Twenty Fig. 7. Effect of gamma globulin fractions prepared from normal, Graves' disease and toxic multinodular goitre sera on [125I]hTSH binding to the membrane. Displacement activity of gamma globulin fractions were read from the htsh standard curve, calculated as,«u equivalents of htsh per pg protein, and expressed as a percentage of the normal mean. Number of sera tested, mean ± sem are shown. 3. Gamma globulin fraction. The gamma globulin fraction cross-reacted with htsh in the range of 100-500 pg gamma globulin protein per tube (Fig. 6). Displacement activity of the gamma globulin fraction was found to be greater than that of normals in 21 out of 45 patients with Graves' disease, the mean displacement activity for the whole group being 30 % greater than that of 27 normal gamma globulin fractions (Fig. 7). Results were calculated as pu htsh displaced per mg protein and expressed as a percentage of the normal mean. Displacement activity of 20 fractions from patients with toxic multinodular goitre was the same as that of the normal controls. Increased displacement was seen in some patients with euthyroid eye disease (3/7), thyroiditis (2/3), and hot nodules (2/4). The differences in displacement activity did not correlate with the serum thyroxine levels. 4. IgG and non-lgg fractions. gamma globulin fractions, 7 of which had high displacement activity, were fractionated further. Both IgG and non-lgg fractions cross-reacted with htsh in the range of 10-100 pg protein per tube. Of the 7, 4 showed increased displacement in both the IgG and nonlgg, 2 in the IgG and 1 in the non-lgg fraction. Of the 13 with normal activity, 1 showed increased displacement in both the IgG and non-lgg, 2 in the IgG, 1 in the non-lgg while 9 had normal displacement in both fractions.
Fig. 8. Scatchard plot of B:F ratio for [125I]hTSH versus the concentration of htsh bound to the membrane in the presence and absence of gamma globulin fraction. - htsh; - htsh + 200 pg gamma globulin protein; -A htsh + 400 pg gamma globulin protein. Fig. 9. Scatchard plot of B:F ratio for [125I]hTSH versus the concentration of htsh bound to the membrane in the presence and absence of IgG fractions from both normal and Graves' disease sera. - htsh; - htsh + 100 pg normal IgG protein; D-D htsh + 50 pg normal IgG protein; A-A htsh+100 pg Graves' disease IgG protein A-A htsh + 50 pg Graves' disease IgG protein.
5. Scatchard analysis. Both normal and Graves' disease - gamma globulin and non-lgg fractions decreased the number of binding sites available for htsh binding in linear fashion, but did not alter the affinity of the receptor site for htsh (Fig. 8). IgG fractions from both normal and Graves' disease patients decreased the affinity of the receptor site for htsh but appeared to increase the number of receptor sites available for htsh binding (Fig. 9), with different IgG fractions having different slopes and intercepts. DISCUSSION Scatchard analysis of htsh binding to receptor on thyroid membranes shows a linear plot with an affinity constant of 2.04 x 109l/mol. This suggests a single population of binding sites as found by Manley et al. (1974a) using guinea pig thyroid membrane but does not agree with Smith 8c Hall (19746), who found a non-linear plot with human thyroid membranes and suggested the presence of populations of sites with different binding affinities. Membranes prepared from thyrotoxic and nodular glands had similar affinity constants and numbers of receptor sites, indicating that thyrotoxicosis is not due to a defect in the mem brane as suggested by Solomon 8c Chopra (1972). Increased inhibition of htsh binding by gamma globulin fractions was found in only 50 % of the patients with Graves' disease, suggesting that other factors besides immunoglobulins may be responsible for the pathogenesis of Graves' disease. This increased inhibition of htsh binding may not be due to LATS as Smith 8c Hall (1974a) found no correlation between results from receptor assays and the McKenzie bioassay. Differences in htsh inhibition which were seen when results were expressed per pg protein disappeared when expressed per pi serum, based on IgG levels in the serum, suggesting that IgG alone was not responsible for this inhibition. Further fractionation of the gamma globulin using QAE Sephadex confirmed this, when the non-lgg fraction was shown to have increased htsh displacement activity. The mechanism of inhibition of htsh binding by serum fractions is unclear. The gamma globulin and non-lgg fraction did not alter the affinity of the receptor site for htsh, but decreased the number of sites available for htsh binding. This agrees with the findings of Manley et al. (19746) and supports the theory that the immunoglobulins and htsh are competing for the same receptor site. However, IgG fractions alter the affinity of the receptor for htsh and alter the number of sites available for htsh binding. This suggests that IgG fraction may cause a change in membrane configuration rather than bind to a specific receptor site as suggested by Yamashita 8c Field (1972). These results suggest that LATS activity in human serum may be explained
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