Kinetics of Thyrotropin-Stimulating Hormone (TSH) and Thyroid ...

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The Journal of Clinical Endocrinology & Metabolism 88(11):5366 –5374 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2003-030664

Kinetics of Thyrotropin-Stimulating Hormone (TSH) and Thyroid-Stimulating Antibody Binding and Action on the TSH Receptor in Intact TSH Receptor-Expressing CHO Cells J. VAN SANDE, M. J. COSTA, C. MASSART, S. SWILLENS, S. COSTAGLIOLA, J. ORGIAZZI, J. E. DUMONT

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Institute of Interdisciplinary Research, Free University of Brussels, School of Medicine, B-1070 Brussels, Belgium; and Institut National de la Sante´ et de la Recherche Me´dicale, U-449 (J.O.), Centre Hospitalier Lyon Sud 69495, Pierre Benite Cedex, Lyon, France The kinetics of TSH binding and the effects of TSH and thyroid-stimulating antibody (TSAb) on cAMP accumulation have been measured in TSH receptor-expressing CHO cells (CHO-TSHR cells). The parallel kinetics of TSH binding to its receptor and of cell cAMP concentration after the addition and withdrawal of TSH show that in the case of this receptor, signal generation and concentration are at all times proportional to occupancy. In physiological ionic medium, TSAb, but not TSH, action is slowed and in some cases almost nonexistent. The kinetics of cAMP disappearance after washout of TSAb is also slower. cAMP accumulation is faster for Fabs than for the TSAb from which they derive. Analysis of the data

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HYROID-STIMULATING antibodies (TSAb) are IgG that bind to and activate the TSH receptor. Their presence in the blood causes Graves’ disease, i.e. autoimmune thyrotoxicosis. TSAb, like TSH, stimulate through the TSH receptor, adenylate cyclase, and the cAMP cascade. Consequently, thyroid hormone secretion and the growth of the gland are enhanced, resulting in thyrotoxicosis and goiter. At high concentrations and in cells overexpressing the TSH receptor, but not in normal human thyroid cells (1), TSAb also activate the phosphatidylinositol-4,5-bisphosphate phospholipase C cascade (2). Although none has yet been purified, it is now known that TSAb are multiple and bind to different epitopes of the TSH receptor (3–7). TSAb in serum have been measured by their thyroid hormone secretory effect in mice in vivo. They have also been estimated in vitro by their competition with labeled TSH for binding to TSH receptor. This assay measures TSAb as well as thyroid-blocking antibodies that cause hypothyroidism, and it does not identify all TSAbcontaining sera. More recently and accurately, TSAb have been measured by the cAMP accumulation they induce in rat thyroid cells of the FRTL-5 cell line or in CHO cells expressing exogenous human TSH receptors (8). Other antibodies stimulating receptors to hormones or neurotransmitters have been found (9, 10), but due to the rapid Abbreviations: CHO-TSHR, TSH receptor-expressing CHO cells; KRH, Krebs-Ringer-HEPES; TSAb, thyroid-stimulating antibodies, also used in the text for serum containing the antibodies.

suggest that 1) serum TSAb are oligoclonal antibodies sets, at low concentrations, with a high affinity for the TSH receptor; 2) ionic interactions are involved in the action of TSAb on the TSH receptor; and 3) TSAb activation of the TSH receptor is at least a two-step process. Among others, a possible explanation is that the full activation of the receptor requires the binding of two or more different antibody molecules on different sites of the same TSH receptor. This analysis provides a benchmark for studies of experimentally induced monoclonal antibodies activating the TSH receptor. (J Clin Endocrinol Metab 88: 5366 –5374, 2003)

desensitization and down-regulation of these receptors they do not cause a syndrome of hyperactivation. However, antibodies stimulating the angiotensin receptor may cause hypertension in preeclampsia (11). Graves’ disease, therefore, results from the combination of the activating effect of TSAb (with their long half-life in vivo) (12) and the weak desensitization (13, 14) and down-regulation of the receptor (15). Therefore, at first sight the mechanisms of Graves’ disease are well understood. However, there are at least some discrepancies with regard to this general picture, for example, the severity of toxicosis, i.e. the importance of thyroid hormone secretion is often unrelated to the size of the goiter (although both result, in principle, from the same stimulus). Moreover, if the qualitative properties of TSAb have been studied in detail and compared with those of TSH, the kinetics of their action have been little considered. Even in the case of receptor activation by TSH, the possibility that the hormone may have a lasting effect on the receptor after its release, has been considered, but not tested. Finally, most studies made use of phosphodiesterase inhibitors, desalinated media, and global measurements of cell and medium cAMP that greatly distort the kinetics of cAMP accumulation and disposal in cells (16). Such conditions, although optimized for the sensitivity of the bioassay, may have masked important biologically relevant information. The purpose of this study was to define the kinetic characteristics of TSAb action in intact cells untreated with phosphodiesterase inhibitors and incubated in physiological medium com-

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Van Sande et al. • TSAb and TSH Receptor

pared with those of TSH to further define the action of TSAb and to test various mechanistic hypotheses. Materials and Methods A human TSHR-pSVL construct and a pSV2 NEO plasmid were cotransfected into CHO cells, and the transfected cells were selected by adding 400 ␮g/ml geneticin to the culture medium. Details of construction of the human TSH receptor expression vector, transfection, selection, and characterization of the CHO cells stably expressing the human TSH receptor were given previously (8). We used a JP2626 clone, which is a subline of the initial JP26 clone (8).

cAMP measurement Between 150 –250 thousand cells were seeded in 3.5-cm diameter dishes the day before the experiment in 1 ml culture medium (Ham’s F-12 medium supplemented with 1 mm sodium pyruvate, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 2.5 ␮g/ml amphotericin B, and 10% fetal calf serum). Before the experiment the culture medium was removed, the cells were rinsed with 1 ml Krebs-Ringer-HEPES (KRH) buffer (124 mm NaCl, 5 mm KCl, 1.25 mm MgSO4, 1.45 mm CaCl2, 1.25 mm KH2PO4, 25 mm HEPES buffer, 8 mm glucose, and 0.5 g/liter BSA, pH 7.4), then preincubated for 30 min in this buffer at 37 C to equilibrate the cells. The buffer was then removed and replaced with fresh KRH buffer supplemented with the agents under study for incubations of various durations depending on the aim of the experiment. At the end of the incubation, the medium was removed, and 1 ml 0.1 m HCl was added to the dishes. The acid solution was transferred to glass tubes, which were evaporated in a vacuum concentrator (Savant Instruments, Holbrook, NY). The samples were resuspended in water and diluted appropriately for cAMP measurements by RIA according to the method of Brooker et al. (17). For measurement of cAMP in the medium, medium was added to 1 ml 0.2 m HCl and treated in the same manner as the cells. Variations of this protocol have been used for desorption experiments. For incubations longer than 2 h, cAMP was also measured in the medium. The contribution of medium cAMP to measured cell cAMP after washing was approximately 5% of the medium cAMP. This value was substracted from the cell value. As some plastic culture dishes have been reported to bind and release TSH (18), medium containing TSH and serum with TSAb activity were incubated in the plastic dishes without cells. The dishes were then rinsed by the usual protocol. Incubation for an additional 2 h in fresh medium did not release any activity, as tested in new CHO-TSHR cells (not shown). Thus, the plastic dishes used do not store and release enough TSH or TSAb to influence the results of the washout experiments. Similarly, medium from a second incubation of prestimulated cells did not acquire any detectable stimulating activity from the cells and dish.

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TSAb activity Blood from patients suffering from Graves’ disease (diagnosed by standard clinical and laboratory tests) was collected at collaborating hospitals. When sera were used as TSAb, they were decomplemented at 56 C for 30 min. For the preparation of Fab of IgGs, we used sequentially the Immunopure Plus Immobilized Protein A IgG Purification Kit to isolate IgGs from which we prepared Fabs with the Immunopure Fab Preparation Kit from Pierce (Rockford, IL). Each type of TSH experiment was repeated three times or more. The TSAb experiments were performed two or more times with each different serum depending on the availability of patients’ sera. In each experiment duplicate dishes were measured with, in the case of cAMP evaluation, four separate determinations per cell extract. The duplicates were in very close agreement (⬍5% difference between dishes) and are not reported in the representative experiment shown. When the results of several experiments are pooled, the sem is shown.

Results

Kinetics of intracellular cAMP accumulation and [125I]TSH binding have been performed simultaneously in identical conditions, aside from the presence of the tracer, on the same batch of cells. Under our conditions (i.e. 0.5 mIU/ml TSH in the medium), the values obtained from 3–90 min show a close correlation between TSH binding and cAMP levels, both expressed as a percentage of the maximal value reached at 90 min (Fig. 1). In three experiments the half-maxima were obtained after 7 ⫾ 1 min for cAMP accumulation and after 5 ⫾ 1 min for labeled TSH binding (mean ⫾ sem). The kinetics of the decrease in intracellular cAMP after a 1-h stimulation with 0.5 mIU/ml TSH and the kinetics of the decrease in binding after 1-h binding with [125I]TSH plus 0.5 mIU/ml TSH were followed, after rinsing the cells, in incubations in fresh KRH from 5 min to 4 h. The results, expressed as a percentage of the initial values (0 min ⫽ 100%), show a close parallelism for the two processes (Fig. 2). In three experiments the half-maximum was reached after 12 ⫾ 2 min for cAMP and after 10 ⫾ 2 min for TSH binding (mean ⫾ sem). Thus, for both accumulation and disposal, the level of cAMP closely follows the level of TSH binding, which sug-

[125I]TSH binding assay The binding studies, especially when a close comparison between cAMP accumulation and TSH binding was the focus of the experiment, were always performed in the same medium (KRH) and at the same temperature (37 C) as the cAMP studies. Bovine [125I]TSH [50 ␮Ci/ml (1.85 MBq); 25– 40 Ci/g (925–1480 GBq/ g)] was a gift from B.R.A.H.M.S Diagnostica GmbH (Hennigsdorf, Germany). The cells were incubated at 37 C in 1 ml KRH buffer supplemented with 150,000 –250,000 cpm [125I]TSH and 0.5 mIU/ml bovine TSH (Sigma Chemie, Bornem, Belgium). Under these incubation conditions the counts per minute bound represented a few percentages of the total counts involved. Bovine TSH (100 mIU/ml), added with the tracer, was used to estimate nonspecific binding and to compute specific binding. For the measurement of bound TSH release, the cells were first incubated for 1 h with tracer and 0.5 mIU/ml TSH, rinsed twice rapidly with 1 ml KRH at 37 C, and incubated in fresh KRH buffer for various times. At the end of the incubation, the cells were rinsed with 1 ml ice-cold KRH buffer, then solubilized in 1 ml 1 m NaOH. The solutions were counted in a ␥-counter (Packard, Downers Grove, IL). For the kinetics of TSH binding, incubations for the various times with [125I]TSH plus 0.5 mIU/ml TSH or 100 mIU/ml TSH (nonspecific binding) were ended in the same way as described above.

FIG. 1. Kinetics of labeled TSH binding and intracellular cAMP accumulation measured in parallel dishes in the same representative experiment. The concentration of cold TSH used in the two series is 0.5 mIU/ml. Specific binding was evaluated by subtracting the counts per minute bound in the presence of 100 mIU/ml TSH at each time of the kinetics. The maximal intracellular cAMP value, reported as 100%, was 124 pmol/dish, and the basal level was 0.5 pmol/dish at 3 and 12 min and 0.6 pmol/dish at 60 and 90 min. Total and specific [125I]TSH binding at 90 min (100%) were 9163 and 7548 cpm/dish, respectively. The data were obtained on duplicate dishes in three experiments. The duplicates were in very close agreement (⬍5% difference). F, cAMP; E, binding.

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FIG. 2. Kinetics of labeled TSH binding washoff and intracellular cAMP decrease measured in parallel dishes in the same representative experiment after 1-h preincubation of the cells with 0.5 mIU/ml TSH supplemented with labeled TSH for the binding. F, cAMP; E, binding.

gests that the cAMP levels measured reflect at all times the actual occupancy of the receptors by TSH. This is also compatible with the very short half-life (⬍30 sec) of cAMP in thyroid cells (19). The kinetics of cAMP decrease after washing were similar for cells preincubated 1 and 4 h with TSH (not shown), which shows that no change in the nature of TSH interaction with its receptor takes place during that time. As Kasagi et al. showed in 1982 that the cAMP response to TSH and TSAb in human thyroid cells was increased in a low salt medium, such conditions are widely used for TSAb assays or for TSH binding studies (20). Using our protocol, to compare the kinetics of [125I]TSH dissociation in a normal KRH medium or KRH deprived of NaCl, but with 280 mm sucrose to compensate for isotonicity, we observed a slower release from the cells incubated in NaCl-deprived medium whether the cells were incubated at 37 C or at room temperature (Fig. 3). This result accounts for the known effects of this medium; increased binding and action of TSH are therefore related, as is most often the case, to a decrease in the koff (21). This observation also demonstrates that a decrease in release can be easily demonstrated in our conditions. The presence of 2–10% normal human serum in our KRH incubation medium increases cAMP in TSH-stimulated and in nonstimulated cells by a factor of 1.7–2. Moreover, 10% serum did not modify the release of bound [125I]TSH from JP2626 (not shown). When a possible effect of 2% or 10% serum on the binding of TSH was looked after, no difference in the kinetics of binding of [125I]TSH in the presence or absence of serum was seen in an incubation of 5–90 min (not shown). The kinetics of cAMP accumulation with TSH or decrease after TSH washout were also not altered by serum. The fact that even the basal value is increased in cells incubated with serum suggests an effect of normal serum downstream from receptor binding and activation. It fits the observation that TSH binding is not modified by 10% serum in the medium. When the effects of TSAb (as patient’s sera) and TSH on cAMP kinetics were compared, the same amount of serum was present in the two series of dishes. Although the kinetics of cAMP accumulation in JP2626 cells stimulated by various TSAb from different patients show different profiles compared with 0.5 mIU/ml TSH tested in each experiment, they


FIG. 3. Kinetics of TSH binding washoff in cells incubated at 37 C in normal KRH medium (F) or in medium deprived of NaCl (E) but supplemented with sucrose to maintain isotonicity. The cells had been first incubated for 1 h with the labeled TSH and 0.5 mIU/ml cold TSH or 100 mIU/ml TSH to evaluate nonspecific binding.

share the common property of being much slower in reaching their maximal effect. This was observed independently of the fact that the maximal TSAb-induced cAMP rise was higher or lower than that reached by 0.5 mIU/ml TSH. In 17 different experiments, TSH-induced cAMP accumulation reached 80% of its maximal value within 15–30 min and 90% within 60 min. The mean time for achieving 50% of the value reached at 4 h was 6 ⫾ 1 min (mean ⫾ sem). In the same experiments the cAMP levels reached in the TSAb-stimulated cells were much more scattered, from 4.6 – 47% of the maximal value at 30 min and from 20 – 80% at 1 h. The mean time for achieving 50% of the level reached at 4 h was 61 ⫾ 11 min (mean ⫾ sem). Although the onset of the cAMP rise was slower with TSAb than with TSH, the final cAMP levels could be higher if enough time was allotted for TSAb action. During the first moments an upward concave curve of cAMP accumulation was observed in all cases with TSAb, giving the general aspect of a sigmoidal curve. Figure 4 illustrates such kinetics, representative of our observations. The disappearance of intracellular cAMP was studied in cells prestimulated with TSH or TSAb for 2 h, rinsed twice with KRH medium at 37 C, and then incubated in KRH medium without agonist for periods of 5 min to 4 h. With 9 different sera in 18 experiments, the half-life of cAMP disappearance was much longer in TSAb-stimulated cells (from 1 to ⬎4 h) than in TSH (0.5 or 1 mIU/ml)-stimulated cells (⬃15 min). Two types of curves were observed after TSAb stimulation and are presented in Fig. 5. In one category of experiments, the cAMP level remained high after the withdrawal of TSAb, decreasing very slowly thereafter (Fig. 5A). In the other category we observed an early partial drop in cAMP after washing the cells, but although in TSH-prestimulated cells the cAMP concentration continued to fall dramatically, in TSAb-prestimulated cells the cAMP concentration leveled off, remaining much higher than that in TSHprestimulated cells. This was observed even for a similar cAMP concentration at the onset of the wash (Fig. 5B). Of a total of 9 sera, 5 belong to category A and 4 to category B. The effects of increasing concentrations of TSH or TSAb were studied on cAMP accumulation after 2- or 4-h incubation, a period that brings cAMP levels close to the plateau for


FIG. 4. Kinetics of intracellular cAMP accumulation in cells incubated with 0.5 mIU/ml TSH (F) or 5% serum TSAb (E). The two main types of kinetics obtained with two different sera are illustrated in A and B. The basal values were 0.2 and 0.32 pmol cAMP/dish in A and B, respectively.

the TSAb used. The shape of the curves obtained looked sigmoidal, more so for the TSAb-stimulated cells than for the TSH-stimulated cells (data not shown). Experimental data have been fitted to the four-parameter logistic equation using nonlinear regression, giving an index of cooperativity near 2 for both TSH (mean ⫾ sem, 1.7 ⫾ 0.2) and TSAb (2.1 ⫾ 0.2 for nine sera). However, when phosphodiesterase activity was inhibited by high concentrations of Ro 20-1724 and isobutylmethylxantine (1 mm each), n was about 1 for both TSH (1.1 ⫾ 0.1) and TSAb (1.4 ⫾ 0.4 for four sera). As shown for TSH, the positive cooperativity of cAMP accumulation therefore probably mainly reflects the negative cooperativity of the catabolizing cAMP phosphodiesterases (22, 23). The slow kinetics of TSAb action might be related to the requirement for Igs as bivalent ligands to dimerize TSH receptors. To test the possible role of TSH receptor dimerization by TSAb in the kinetics of TSAb action, Fab of IgGs of active TSAb-containing serum were prepared. As previously demonstrated, Fabs fully account for the stimulatory effect of TSAb. The activity of TSAb is therefore fully located in the antigen-binding sites of the molecules. However, the kinetics of Fab action on cAMP accumulation were faster than those of the corresponding IgGs of four different sera; each of the experiments gave the same results with different preparations of IgGs and Fabs from the same serum. In two sera the


FIG. 5. Kinetics of intracellular cAMP decrease in cells preincubated with TSH (F) or TSAb (E) and then incubated in KRH basal medium. A and B, Two types of response for two different sera. TSH, 0.5 mIU/ml; TSAb, 5% serum. A, The basal value is 0.25 pmol cAMP/dish throughout the kinetics. B, The basal value is 0.35 pmol cAMP/dish throughout the kinetics.

FIG. 6. Kinetics of intracellular cAMP accumulation in cells incubated with TSH, purified IgGs, or purified Fabs. The TSH level was 0.5 mIU/ml (F), the level of IgGs was 0.6 mg/ml (E), and the level of Fabs was 0.3 mg/ml (f).

kinetics of action of the corresponding Fabs were similar to those of TSH (Fig. 6); in two others it was still slower. To test the hypothesis that diffusion of the receptor in the two-dimensional plane of the membrane could be the limiting factor in TSAb action, the kinetics of cAMP accumulation were tested at 37 and 24 C or 37 and 15 C. Although cAMP accumulation was decreased at 24 C for both TSH and TSAb, the relative kinetics of accumulation with both TSH and TSAb were unchanged (Fig. 7). Results were similar at 15 C.

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The decrease in cAMP accumulation after washing the cells evolved in parallel for cells preincubated with TSAb for 1 or 4 h (not shown). There is therefore no indication of a progressive irreversible change in the TSH receptor in cells stimulated with TSAb. The addition of two different TSAb sera to the cells gave more or less additive effects, but no synergism (data not shown). Two different TSAb sera, which by themselves induced almost no cAMP response, did not generate a positive effect on cAMP generation when added together (data not shown). As nonionic media have been shown to give better results for the TSAb bioassay in vitro, we compared the kinetics of TSAb action on cAMP accumulation in our cells incubated in ionic and nonionic media. Figure 8 shows that in nonionic sucrose medium the kinetics of TSAb action were much faster, approximating those of TSH. The latency of the TSAb effect disappears. No such accelerating effect was observed for TSH action. Interestingly the presence or absence of NaCl had little influence on the kinetics of cAMP disposal after a washout of TSAb (data not shown). Moreover, nonionic media also accelerated the kinetics of cAMP accumulation in human thyroid cells in primary culture for TSAb, but not for TSH (data not shown). As noted by clinical laboratories, beside its kinetic effect, low ionic medium also has an overall quantitative effect on

FIG. 7. Effect of lowering the temperature on the kinetics of intracellular cAMP accumulation in cells incubated with TSH or TSAb. F, TSH (0.5 mIU/ml); E, TSAb (5% serum); solid line, 24 C; dashed line, 37 C.

FIG. 8. Effect of sucrose medium on the kinetics of intracellular cAMP accumulation in cells stimulated by TSH (0.5 mIU/ml) or TSAb (5% serum). The basal value is 0.37 pmol cAMP/dish in normal medium and 0.42 pmol cAMP/dish in sucrose medium. F, TSH; E, TSAb.


the cAMP response to the majority of TSAb. Most of the sera shown to be positive in low ionic media in clinical assays have little effect in saline medium. Indeed, in a series of five random sera from Graves’ disease patients, for four sera, cell cAMP was multiplied by a factor of 5 ⫾ 1 in NaCl-containing medium, but by 41 ⫾ 5 in low ionic medium (2 h). The other serum gave a moderately higher response in sucrose medium, as did TSH (Fig. 9). Similar results on total cell and medium cAMP have been reported (24). As shown previously in FRTL5 cells, enhanced cAMP accumulation in low saline media affected both intracellular and medium cAMP and therefore reflects increased generation, rather than increased efflux from the cells (25). The extension of the incu-

FIG. 9. Effect of sucrose medium on intracellular (A) or medium (B) cAMP levels of cells stimulated for 2 h by TSH (0.5 mIU/ml) or TSAb (5% serum). Five different TSAb sera were used, one that is strongly active in NaCl medium and four that have almost no activity in normal NaCl medium. The basal value of the cells in NaCl medium is 0.44 pmol cAMP/dish. f, Normal NaCl medium; o, medium without NaCl, but with sucrose.


bation to 6 h did not increase the response to the four poorly active sera in ionic medium (data not shown). Discussion

The binding of TSH to membranes containing its receptor and its action on adenylate cyclase have been much studied in acellular preparations. The action of TSH and TSAb on intact cells has also been studied. However, in most cases binding and action have been studied in unphysiological desalinated media in which the apparent affinity of TSH for its receptor is higher (20, 26). Moreover, cAMP measurements were often carried out on cells plus media and in the presence of phosphodiesterase inhibitors to increase the yield of cAMP. The latter completely modify, in fact slow down, the kinetics of cAMP accumulation and disposal (16). There are therefore few comparable kinetic data on the binding and action of TSH and on the action of TSAb in nonpharmacologically perturbed, intact cells. In this work we have carried out such experiments to answer questions about the mechanism of action of these agonists. In hormone receptor studies, one of the common implicit assumptions is that in the absence of desensitization phenomena, the hormone-bound receptor corresponds to the activated state of the receptor. In other words, the receptor is immediately activated on binding and inactivated on release of the hormone. There is no hysteresis in the action of the hormone. This assumption predicts that the kinetics of action of the hormone in the activation phase or in the desorption phase must be similar to the kinetics of binding. On the other hand, from a physiological point of view, for hormones that modulate slow-reacting organs, such as the thyroid, a hysteretic effect of the hormone would allow an integral response to short-term fluctuations of hormone levels. It has been suggested that receptor-bound TSH could convert from a rapidly dissociable state to a slowly dissociable state with time (27, 28). However, the latter studies were carried out on slices, and we have shown that the tissue architectural characteristics of this model slows the diffusion of TSH or other agents (29). Moreover, TSH binding studies in intact thyroid cells that express few TSH receptors were marred by the background binding of TSH to plastic that may exhibit properties analogous to the binding to thyroid cells (11). Our aim was to define the relative kinetics of TSH binding and action in intact cells in a system devoid of these complications. Our data demonstrate parallel kinetics of TSH binding and cAMP accumulation and of TSH desorption and cAMP decrease in identical conditions in the CHO-TSHR cells. The assumption that activation of the receptor corresponds to its occupation is thus validated for the TSH concentration used. The cell does not integrate short TSH pulses at the cAMP level. Similar results using the TSH effect on electrical resistance of cultured thyroid cells as an end point gave a similar result; the TSH action ceases immediately on dissociation by trypsin treatment (14). The fact that TSH desorption is similar after 1 and 4 h of preincubation with TSH further bears against the existence of a persistently activated, slower TSHreleasing form of the receptor (27). The increased binding and action of TSH in desalinated sucrose media that are often used for TSH binding assays and


TSAb cell bioassays are here shown to be explained at least in part by a decreased koff of TSH from its receptor. That this effect bears on the TSH receptor interaction is shown by the fact that NaCl decreases the binding of TSH itself (18). NaCl does not seem to affect events downstream of the receptor, as no enhancement of cAMP accumulation in low ion media is observed after prostaglandin or forskolin treatment (24, 26). That ionic interactions are involved in TSH binding is further supported by the increased affinity of TSH in which multiple charged amino acids replace neutral amino acids (30). With regard to TSAb action in physiological medium, the main findings of this study are the slowing down of TSAb action by NaCl and, in this physiological medium, the slow kinetics of the cAMP response to TSAb stimulation or to TSAb washout, the delay in TSAb action, and the faster action of Fab compared with their corresponding TSAb. These results cast some light on the nature of TSAb-containing sera, their effect in Graves’ disease, and their mechanism of action (see below). The stimulating activity of TSAb on the TSH receptor might result from the action of a great concentration of low affinity TSAb or of a small concentration of high affinity TSAb. The low affinity of reported stimulating monoclonal antibodies against the TSH receptor would appear to support the first hypothesis (31). However, the relevance of these monoclonal antibodies to TSAb has been questioned (32). In the absence of purified monoclonal natural TSAb it is difficult to resolve this alternative. The kinetics of receptor activation and deactivation during the association and dissociation of TSH and TSAb as measured by cAMP levels, reflect the kon and koff of these agonists. Previous results on this subject using ionic media are suggestive, but not conclusive. Using various types of thyroid cells as targets, a slower cAMP response to TSAb compared with TSH had been reported, but TSH concentrations were more active than those of the TSAb (12). Isozaki et al. (33) reported slow kinetics of activation of cAMP accumulation in pig thyrocytes reorganized in follicles for some TSAb, but no systematic comparison was made with TSH. Several groups (34 –37) reported that the plateau of cAMP accumulation in mouse thyroids, FRTL-5 cells, and human thyroid cells incubated with phosphodiesterase inhibitors was reached later for long-acting thyroid stimulator than for TSH, but Vitti et al. (38) and Rapoport et al. (26) using human thyroid cells under similar conditions did not observe it. In human thyroid membrane preparations the stimulation of adenylate cyclase was slower for TSAb than for TSH (39, 40). We show here in intact CHO-TSHR cells incubated in normal saline media and in the absence of phosphodiesterase inhibitors that the kinetics of TSAb stimulation of cAMP accumulation were much slower than in TSH-stimulated cells. Similarly, the kinetics of cAMP decrease after TSAb washoff were also much slower. Thus, the kon and, more so, the koff of TSAb are slower than for TSH. It is well known that in ligand protein interactions, variations in the Kd mostly reflect variations in the koff (21). Lower koff must indicate a higher affinity of the ligand for its receptor. These data therefore suggest that the effect of TSAb results from the action of low concentrations of TSAb with high affinity for the TSH receptor. They therefore agree with the demonstration by Rapoport’s group that the concentration of

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TSAb in serum is lower than that of antiperoxidase antibodies by a factor of at least 100 (41) and that serum TSAb are neutralized by a 10⫺9 m concentration of the extracellular part of the TSH receptor (42, 43). They support the concept that only monoclonal antibodies with high affinity for the TSH receptor might be considered representative of the real in vivo TSAb (5). The disappearance curves of cAMP after TSAb washing are biphasic in some sera with a rapid component similar to TSH and a very slow component. The relative importance of the fast and slow components of the curve varies from one TSAb sample to another. This biphasic nature of the deactivating curve contrasts with the TSH curve. It fits in well with the concept that most TSAb serum preparations contain different antibodies, i.e. are oligoclonal (3, 5, 7, 44). The different kinetics of cAMP levels after TSAb washout for different sera as well as the very different effects of NaCl on the actions of these various sera (almost complete inhibition in some cases vs. mere delaying action in some others) also show that serum TSAb are different from one patient to another. As intracellular cAMP concentrations must remain elevated for 18 h to trigger DNA synthesis and proliferation in thyroid cells in culture (44), prolonged cAMP elevations are therefore necessary to induce this proliferation in vivo. The longer duration of TSAb action on cAMP coupled to the long half-life of IgGs in the blood (12) would therefore be much more likely to satisfy this requirement than the shorter effects of short-lived TSH. Dimerization of the TSH receptor by TSAb could be necessary for activation. This would result in slower kinetics of activation and an apparent negative cooperativity of TSAb action. The fact that Fab of TSAb fully reproduce the effects of TSAb bears against this hypothesis (39, 45– 47). It is interesting in this regard that Fab from monoclonal antibodies stimulating the ␤2-adrenergic receptor have no activity and become antagonists (48). Moreover, no negative cooperativity of TSAb action was observed. In fact, the converse hypothesis, i.e. that activation would require the dissociation of inactive dimers of the TSH receptor, has been proposed on the basis of direct fluorescence resonance energy transfer experiments (49). Under normal physiological conditions (physiological ionic strength, no inhibition of phosphodiesterases) TSAb action on cell cAMP accumulation shows a latency and is delayed vs. TSH action. This is not due to the use of TSAb with lower stimulating activity (12), as we showed it for TSAb with higher activity than our standard TSH concentration. This latency is largely suppressed for the Fab of the TSAb or in low ionic medium. The effect of NaCl, which in qualitative experiments was reproduced by KCl and (NH4)2SO4 (20), suggests that charge-charge interactions are involved in the binding of TSAb, as they are in the binding of TSH, and/or in the conformational change induced in the receptor. Electrostatic interactions in the binding is shown by the requirement of low salt medium for binding assays (24, 50). A role for such interactions in the activation of the receptor independently of agonist binding is suggested by the positive effect of low ionic media on the basal constitutive activity of the receptor in the absence of TSH (51). NaCl inhibition of TSAb action could correspond as such to an


inhibition of binding and to an inhibition of the conformational change induced (46, 50). It is interesting in this regard that evolution has selected a structure of the receptor with lower activity and sensitivity to TSH in physiological conditions. On the other hand, this characteristic is sufficient to inhibit TSAb action, but not to prevent Graves’ disease. Due to the fast turnover of cAMP, the kinetics of accumulation or disappearance of cAMP presumably reflect a limiting step in the cascade from the interaction of TSAb with the receptor to the activation of adenylate cyclase. The upward concave curve of cAMP accumulation suggests at least a two-step mechanism. A limiting step might occur at the binding of TSAb to the receptor, at the conformational change of the receptor, or at any downstream step (46). Several hypotheses can be rejected. That this limiting step is not the requirement for divalent binding of the TSAb inducing a necessary receptor dimerization is shown by the fact that Fabs are as active as IgGs. Similar kinetics have been observed for adenylate cyclase activation in membrane preparations (39), which shows that they do not result from cAMP metabolism. The slow kinetics of TSAb action were not further slowed at 23 or 15 C, which bears against the hypothesis that the slower step is due to membrane diffusion of the receptor. The second step in TSAb action could be an enzymatic action of the antibody such as the generation of oxygen radicals from H2O2 (52). However, no activating effect of H2O2 has been observed on cAMP accumulation in the JP2626 cells (to be published elsewhere). A slow change in TSH receptor conformation in the activation process could be hypothesized (53). However, the time scale of the latency of TSAb action (in terms of tens of minutes) makes this hypothesis improbable. An action of TSAb resulting from the necessary binding of several different antibodies would entail a multistep process. There are several arguments supporting this hypothesis: 1) the fact that TSAb sera contain several types of antibodies (polyclonal and oligoclonal) (3, 4, 7) recognizing multiple epitopes (5, 54, 55); 2) the acceleration of the activation with Fabs, which could be caused by the removal of steric hindrance; 3) some cooperativity in the concentration-action curve (although the meaning of such curves for such an heterogeneous mixture of agonists and antagonists is open to discussion); 4) the 54 –254 peptide of the external part of the TSH receptor binds TSAb. If lengthened, the peptide no longer binds TSAb, which suggests that in the whole domain the relevant epitope is masked. This implies that the binding of the TSAb requires a first step of epitope unmasking (6). A first step in the process could therefore be the unmasking of this epitope. Thus, a necessary cooperative effect of several different antibodies could be a possible explanation for the latency in the action of the TSAb active in physiological medium. The definition of the kinetic parameters of TSAb action will provide a useful basis to test the validity of the monoclonal stimulating TSH receptor antibodies that are now developed as models of these TSAb (56 –58). Acknowledgments Received April 16, 2003. Accepted August 1, 2003.


Address all correspondence and requests for reprints to: Dr. J. Van Sande, Institute of Interdisciplinary Research, University of Brussels School of Medicine, 808 route de Lennik, B-1070 Brussels, Belgium. E-mail: [email protected]. This work was supported by the Service du Premier Ministre Affaires Scientifiques, Techniques et Culturelles SSTC, the Fonds National de la Recherche Scientifique, Fonds de la Recherche Scientifique Me´ dicale, Fonds Cance´ rologique Fortis, Ope´ ration Te´ le´ vie, Fe´ de´ ration Belge contre le Cancer, and Fondacao para a Ciencia e a Tecnologia (fellowship to M.J.C.).

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