0021-972X/91/7304-0710$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society

Vol. 73, No. 4 Printed in U.S.A.

The Biological Activity of Bovine and Human Thyrotropin is Differently Affected by Trypsin Treatment of Human Thyroid Cells: Thyroid-Stimulating Antibody is Related to Human Thyrotropin* DANIELA FOTI, DIEGO RUSSO, GIUSEPPE COSTANTE, AND SEBASTIANO FILETTI Cattedra di Endocrinologia e Patologia Costituzionale, Universita di Catania (D.F., D.R.), Ospedale Garibaldi, Catania, Italy; and Dipartimento di Medicina Sperimentale e Clinica, Cattedra di Endocrinologia (G.C., S.FJ, Universita di Reggio Calabria, Catanzaro, Italy

ABSTRACT. Pretreatment of cultured human thyroid cells with trypsin decreased the cAMP response to bovine TSH (bTSH) (by 50-60%). In striking contrast, in trypsin treated cells the cAMP stimulation by both human TSH (hTSH) and thyroid-stimulating antibodies (TSab) was unimpaired, indicating a similar behavior for these two stimulators. The effect of trypsin on inhibiting cAMP stimulation by bTSH was: 1) dose dependent; 2) present at a trypsin concentration as low as 3.3 mg/L; 3) fully reversible within 24 h after removal of the enzyme. In accordance with the altered biological activity in human thyroid cells exposed to trypsin the binding of labeled bTSH was reduced (about 40%). On the contrary, in the same cells, the

binding of labeled human TSH was enhanced (about 3-fold). The cAMP response to cholera toxin and forskolin was unaffected in trypsin treated cells, indicating that the tryptic treatment did not alter any other component of the adenylate cyclase complex. The medium obtained from trypsin-treated human thyroid cells was able to neutralize the biological activity of bTSH but not that of hTSH or TSab. Our study demonstrates that in human thyroid cells: 1) trypsin impaires bovine, but not human TSH or TSab biological activity; 2) bovine and human TSH may bind to different components of the TSH receptor. (J Clin Endocrinol Metab 73: 710-716, 1991)

A

VARIETY of autoantibodies reacting with the TSH receptor (TRab) have been demonstrated to play an important pathogenetic role in autoimmune thyroid disorders, by stimulating or blocking thyroid function (1-3). TSH receptor autoantibodies are usually measured by their ability to stimulate some biological effects in thyroid cells (thyroid stimulating antibodies, TSab) or by their ability to inhibit the binding of labeled TSH to its receptor in thyroid tissue (TSH binding inhibiting antibodies, TBIab) (4, 5). It is unclear whether or not TSH, TSab, and TBIab all share identical binding determinants on the TSH receptor. The majority of the studies on the effects of thyroid active antibodies on TSH binding have used bovine TSH (bTSH) as the ligand to probe the human TSH receptor, assuming that the binding and the functional behavior of bovine and human TSH are identical (5-7). In the present study we

have examined in human thyroid cells the interaction of TSH and TSH-mimicking immunoglobulin G(IgG) with the TSH receptor after its partial proteolytic digestion with trypsin. In these cells we have evaluated the functional and binding activities of bTSH, hTSH, and TSab. Our results suggest that trypsin affects differently the biological activity of bovine and hTSH and that TSab behaves functionally like hTSH, rather than bTSH. Materials and Methods Cell culture Human thyroid cells in monolayer culture were obtained by a collagenase digestion procedure from normal thyroid tissue as previously described (8, 9). Cells were frozen and kept in liquid nitrogen until use. One or 2 days before the experiments thyroid cells were thawed and subcultured in culture plates containing 24 2 cm2 wells (Falcon, Becton Dickinson, Milan, Italy) at a density between 5 x 104-106 cells per well for cAMP stimulation studies, and about 5 X 105 cells per well for binding experiments, in M199 medium containing 10% fetal calf serum, 2 mM glutamine, and nonessential amino acids at 37 C in 95% air, 5% CO2.

Received August 2, 1990. Address requests for reprints to: Sebastiano Filetti, M.D., Cattedra di Endocrinologia, Policlinico Mater Domini, Via T. Campanella, 88100 Catanzaro, Italy. * This work was supported by grants (40% and 60%) from MURST (Ministero Universita Ricerca Scientifica e Tecnologica, Italy).

710

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 September 2016. at 00:49 For personal use only. No other uses without permission. . All rights reserved.

BOVINE AND HUMAN TSH BIOLOGICAL ACTIVITY Immunoglobulin preparation IgG, protease-free, were obtained from sera of patients with active Graves' disease by diethylaminoethyl Affy-gel blue column chromatography (Bio-Rad Laboratories, Milan, Italy). IgG was concentrated by ultrafiltration in membrane cones (Centriflo, Amicon Corp., Danvers, MA) to 30-40 g/L as calculated by ultraviolet spectrophotometry at 280 nm, assuming the molar extinction coefficient E 1% 10 mm = 13.5 and stored at —30 C until use. Cell stimulation and cAMP measurement Cells were exposed to bTSH (100 mU/L), hTSH (333m U/ L), or TSab (5 g/L) in 0.4 mL medium Ml99 supplemented with 20 mM HEPES, pH 7.4, and 1 mM 3-isobutyl-l-methylxantine (IBMX). Considering the different relative potency of the stimulators, the indicated concentrations were chosen in order to achieve a similar magnitude in the cellular cAMP stimulation. The incubations were ended by rapidly aspirating the medium and immediately adding 0.6 mL ice-cold 95% ethanol. The ethanol extracts were then transferred to 13 X 100-mm glass tubes and evaporated to dryness. cAMP was then resuspended in 50 mM sodium acetate, pH 6.2, and measured by RIA (10).

711

incubation, cells were washed twice with ice-cold KRH, solubilized in 0.1 N NaOH, and aliquots were counted for radioactivity. Specific binding counts for bTSH studies were 35005000 cpm/well, and nonspecific counts per min were about 10% of total bound. In hTSH experiments we obtained a total binding of 400-700 cpm/well; nonspecific binding counts were 100-200 cpm/well. Neutralization of bTSH, hTSH, TSab biological activity by the medium obtained from cells exposed to trypsin Human thyroid cells (about 50 X 106) cultured in 150 cm2 flasks were exposed to either trypsin alone (10 mg/L (trypsin medium) or to trypsin together with soybean trypsin inhibitor (100 mg/L) (control medium). After 30 min at 22 C, the incubation was terminated by adding an excess of soybean trypsin inhibitor and the medium was collected and centrifuged (100,000 x g for 2 h at 4 C). Subsequently, the medium was concentrated (5-fold) in a speed vac concentrator (Savant Instruments, Inc., Farmingdale, NY), and then was dialyzed overnight against Hanks' balanced salt solution (2 X 4 L). bTSH or hTSH or TSab were first preincubated (2 h at 37 C) with 50 ixL control medium or the trypsin medium, previously concentrated and dialyzed. The preincubation mixtures were then added to different dishes of human thyroid cell and the cAMP production was measured.

Treatment of thyroid cells with trypsin Human thyroid cells in monolayer culture were washed twice with Krebs Ringer-20 mM HEPES solution (KRH), pH 7.4, containing 0.5% BSA. Cells were then incubated in the same solution without BSA but with the indicated concentration of trypsin for 30 min at 24 C. The trypsin proteolytic activity was terminated by rinsing the cells three times with M199 medium containing both 10% fetal calf serum and an excess of soybean trypsin inhibitor. The effect of trypsin on cellular integrity depends on the dose used and on the time length of the cell exposure to the enzyme. Preliminary experiments demonstrated that the exposure of human thyroid cells to trypsin concentration up to 0.5 g/L (30 min, 22 C) did not produce cell damage as judged by [14C]sucrose space distribution (data not shown). In subsequent experiments, therefore, human thyroid cells were incubated in KRH containing trypsin 10 mg/L for 30 min at 22 C. TSH binding study Both highly purified hTSH and bTSH were iodinated using the stoichiometric chloramine-T method of Goldfine et al. (11). The labeled compounds had a specific activity of 70-100 n curies//ig. The binding studies were performed in a NaCl-free isosmotic KRB buffer, as described by Tramontano and Ingbar (12). To minimize the binding of the tracer to the plastic dishes, human thyroid cells were used at confluence (~5 x 105 cells per well). Cells preexposed to trypsin alone or to trypsin together with soybean trypsin inhibitor as described above, were rinsed twice with Krebs Ringer buffer containing sucrose (280 mM) (KRS), pH 7.0, and incubated in the same buffer with [125I]bTSH (40,000-60,000 cpm/well) or with [125I]hTSH (80,000-100,000 cpm/well) for 2 h at 37 C. At the end of the

Experimental design Each experiment was repeated at least three times, except the one described in Fig. 2. The data presented (mean ± SD) regard three (or two, for experiments shown in Fig. 2 only) separate experiments using the same IgGs from one Graves' patient and the same cell line preparation. To be sure that no artifacts occurred in our experimental procedures, we performed each experiment (except the one in Fig. 2) with other three cell preparations obtained from different thyroid glands (data not shown). Two more individual Graves' IgG preparations, and a pool of purified IgGs were also tested (data not shown). In both cases, no differences were found. Materials Materials were obtained from the following source: cultured medium, fetal calf serum, 0.25% trypsin EDTA solution, glutamine, and nonessential amino acids were purchased from Grand Island Biological Company, Grand Island, NY; Thytropar (used to study cAMP stimulation and to determine nonspecific binding in binding experiments) from Armour Pharmaceutical Co (Phoenix, AZ); IBMX, HEPES, Trypsin, trypsin soybean inhibitor from Sigma Chemical (London, UK); collagenase from Worthington Biochemical Corp. Na125I carrier free from Radiochemical Center, Amersham (Milan, Italy). All other substances were of analytical grade. Highly purified bTSH (30 U/mg protein) (13) was kindly obtained by Dr. Rapoport (University of California, San Francisco). hTSH coded 68/38 (First IRP hTSH, 150 mlU/ampoule, used to study cAMP stimulation) was kindly provided by Dr. R. Bangham (M.R.C., Mill Hill, UK). For labeling purposes, NIDDK-hTSH-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 September 2016. at 00:49 For personal use only. No other uses without permission. . All rights reserved.

FOTI ET AL.

712

JCE & M • 1991 Vol 73 • No 4

120-j

1-5 (AFP-4370B), kindly provided by NIH, was used (Biopotency: 1.5 IU/mg; RIA potency: 6.2 IU/mg).

100-

Results

T

T

T



Effect of trypsin on cAMP response to bTSH in human thyroid cells As shown in Fig. 1, when human thyroid cells in monolayer culture were exposed for 30 min to trypsin (up to a concentration of 33 mg/L), no significant change in their basal cAMP levels was observed. However, the increase of cell cAMP content in response to bTSH (100 mU/L) was reduced. The effect of trypsin was dose dependent: increasing concentration of trypsin induced a progressive decline in the cAMP response to bTSH. Maximal inhibition (about 60%) was obtained at a trypsin concentration of 10 mg/L; no further inhibition was observed with higher trypsin concentrations (Fig. 1). The inhibitory effect of trypsin was fully reversible; recovery from inhibition was already seen within 4 h after removal of trypsin and a complete recovery was seen after 24 h (Fig. 2). The stimulation of cAMP induced by cholera toxin and forskolin was not affected by trypsin treatment (Table 1). Since these agents do not act through the TSH receptor (14, 15), these data indicate that trypsin specifically alter the TSH receptor but it does not damage the plasma membrane integrity or other components

o a.

8060

"co

40 20

1I

i1 24

4 8 TIME (hrs)

FlG. 2. Reversibility of trypsin effect on the cAMP response to bTSH in culture human thyroid cells. After preincubation (30 min) in either control KRH (open bars) medium or in KRH medium supplemented with trypsin (hatched bars), cultured human thyroid cells were rinsed three times and incubated in fresh Mi99 medium for the indicated times. At each time point a cAMP stimulation after 30-min incubation with bTSH (100 mU/L) was determined. Cellular cAMP levels were determined as described in Materials and Methods. Each bar represents the mean ± SD of individual experiments carried out in triplicate wells. The data are expressed as percentage of cAMP values obtained in paired groups of cells not exposed to trypsin. TABLE 1. Effect of trypsin on cAMP stimulation induced by cholera toxin, forskolin and bTSH in human thyroid cells Additives None Cholera toxin (50 mg/L) Forskolin (10 nM) bTSH (100 mU/L)

E c

1.0 ± 23.2 ± 31.0 ± 17.6 ±

0.3 1.5 1.9 1.1

0.9 ± 26.0 ± 30.3 ± 8.5 ±

0.1 1.8 2.0 0.7

Incubation was for 1 h. Values are means ± SD (n = 3).

bTSH —a—

of the adenylate cyclase complex such as the N-protein and the catalytic unit.

control

20

Control cells Trypsin-treated cells cAMP (pmol/well)

30

Trypsin (mg/L) during the preincubation FIG. 1. Effect of trypsin on the cAMP response to bTSH in cultured human thyroid cells. Cultured thyroid cells were preincubated (30 min) in KRH medium containing the indicated concentrations of trypsin (as described in Materials and Methods). Then the medium was aspirated, and the cells were washed three times with medium containing 10% fetal calf serum and soybean trypsin inhibitor (100 mg/L). Thereafter, the cells were incubated (1 h) in fresh medium containing 1 mM IBMX without (control) or with bTSH (100 mU/L). Cellular cAMP was extracted and measured by RIA as described in Materials and Methods. Each point represents the mean ± SD from three separate experiments, each performed in triplicate wells.

Effect of trypsin on the time course of the cAMP response to bTSH and TSab stimulation Since trypsin alters the TSH receptor, one would expect that also the immunoglobulins known to interact with the TSH receptor (TSab) have a reduced effect on the cAMP stimulation in trypsin-treated human thyroid cells. Surprisingly, however, this was not the case. bTSH and TSab exerted their typical pattern of cAMP stimulation in control cells (Fig. 3). However, in paired experiment using trypsin-treated cells, cAMP stimulation induced by bTSH was clearly reduced whereas, in contrast, no decrease in the cAMP response to TSab was observed.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 September 2016. at 00:49 For personal use only. No other uses without permission. . All rights reserved.

713

BOVINE AND HUMAN TSH BIOLOGICAL ACTIVITY

30

hTSH

TSAb

bTSH

60

90

120

30

SO

00

30

120

80

00

120

TIME (mln)

FIG. 3. Effect of trypsin on the time course of the cAMP response to bTSH, TSab, and hTSH. Human cultured thyroid cells were preincubated (30 min) in KRH medium without or with trypsin (10 mg/L) as described in Materials and Methods. The medium was, then, aspirated and the cells were washed three times in medium containing 10% fetal calf serum and soybean trypsin inhibitor (100 mg/L). Thereafter, the cells were incubated for the indicated time in fresh medium containing 1 mM IBMX with bTSH (100 mU/L or TSab (5 mg/ml), or hTSH (333 mU/L). Cellular cAMP was extracted and measured by RIA as described in Materials and Methods. Each point represents the mean ± SD of data obtained in three separate experiments, each performed in triplicate wells.

Because of the possibility that TSab biological activity might be correlated with hTSH rather than bTSH, the effect of trypsin on the time course of cAMP stimulation in response to hTSH was also examined. Similarly to TSab, cAMP stimulation by hTSH was unaffected by trypsin treatment. The difference between bTSH and hTSH in cAMP stimulation before and after trypsin treatment of human thyroid cells was present at all hormone concentrations tested, ranging from 0.033-1 mU TSH/mL (Fig. 4). These data indicate, therefore,

Effect of trypsin on TSH binding in human thyroid cells Exposure of cultured thyroid cells to trypsin induces a decrease in the TSH binding (16-18) and the simultaneous release of a component of the TSH receptor into

bTSH

30" FIG. 4. Effect of trypsin on the cAMP response to increasing doses of bovine and human TSH in cultured human thyroid cells. Cells were preincubated (30 min) in KRH medium without (open bars) or with trypsin (10 mg/L) (hatched bars) as described in Materials and Methods. The medium was then aspirated and the cells were washed three times in medium containing 10% fetal calf serum and soybean trypsin inhibitor (100 mg/L). Thereafter, the cells were incubated (1 h) in fresh medium containing 1 mM IBMX and the indicated doses of bTSH or hTSH. Cellular cAMP was extracted and measured by RIA as described in Materials and Methods. Each point represents the mean ± SD of data obtained in three individual experiments, each carried out in triplicates.

that proteolysis allows us to discriminate the thyroid response to either bTSH or hTSH and TSab, and suggest that in human thyroid cells bTSH has different binding sites than hTSH and TSab.

1

hTSH

0 — 20

o

E a

Q.10-

n 0.01

0.033 0.1 0.333

1.0

0.033

I

0 . 1

0.333

U/L

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 September 2016. at 00:49 For personal use only. No other uses without permission. . All rights reserved.

1 . 0

FOTI ET AL.

714

the supernatant (16,17). To determine whether the effect of trypsin on cAMP stimulation reflected an action on the TSH receptor, we examined directly the binding of radiolabeled hTSH and bTSH to human thyroid cells before and after proteolytic treatment. As expected, preexposure to trypsin decreased by about 40% the specific binding of [125I]bTSH (Fig. 5). In striking contrast, in similar trypsin treated cells, a 3-fold enhancement of [125I]hTSH binding was observed (Fig. 5). This finding, therefore, supports the possibility that trypsin acts on the TSH receptor causing modifications that oppositely affect bTSH and hTSH binding and biological activity. Effect of the medium obtained from trypsin-treated cells on the biological activity of bTSH, hTSH, and TSab Tate et al. (16) have demonstrated in thyroid plasma membranes that trypsin does not destroy the TSH receptor but, instead, releases a low molecular weight component able to bind TSH. In the light of our aforementioned results, this component should bind and neutralize bovine TSH but not human TSH or TSab activities. This was indeed the case. Thus, the preincubation (2 h at 37 C) with the medium obtained from thyroid cell exposed to trypsin, induced a clearly decreased cellular cAMP response to bTSH (10 mU/L). Under the same experimental conditions, cAMP response to either hTSH or TSab was unaffected (Fig. 6). In parallel experiment,

the preincubation with control medium (obtained from thyroid cells exposed simultaneously to trypsin plus trypsin inhibitor) did not alter the cAMP response to either bTSH or hTSH and TSab. This finding suggests the possibility that trypsin may cleave a component of the TSH receptor specifically involved in the binding of bTSH.

Discussion The TSH receptor, freely mobile in the lipid bilayer of the plasma membrane, is responsible for mediating the actions of the TSH hormone on thyroid cells. In the serum of patients with autoimmune thyroid diseases, autoantibodies that interact with the TSH receptor (13) and that may be measured as either thyroid-stimulating (TSab) or TSH binding inhibiting (TBIab) activities have been described (4, 5). However, these two activities are not always well correlated (19, 20). It is unclear whether TSab and TBIab represent two different classes of antibodies with different binding sites on the TSH receptor. In fact, antibodies able to inhibit TSH binding without having any agonist activity and responsible for thyroid hypofunction, have also been described (3, 2123). Virtually all studies on the effects of thyroid active antibodies on TSH binding have used bTSH as the ligand to probe the human TSH receptor, assuming that the binding and the functional behavior of bTSH and hTSH

125 120 -•

125

bTSH BINDING

1

.

1

BINDING

20O 60 -

g

40 -

h

20

i

0 ^

hTSH BINDING 300-

T

J

oo

JCE & M • 1991 Vol 73 • No 4

n CONTROL

I

WITH TRYPSIN PREINCUBATION

100

I CONTROL

WITH TRYPSIN PREINCUBATION

FIG. 5. Effect of trypsin treatment on the binding of [125I]bTSH and [125I]hTSH to cultured thyroid cells. Confluent thyroid cells were preincubated (30 min) in control KRH medium containing both trypsin and an excess of the soybean trypsin inhibitor (100 mg/L) (control cells, open bars) or in medium containing only trypsin (10 mg/L) [hatched bars) as described in Materials and Methods. Then the medium was aspirated, and the cells were washed three times with modified KRB containing soybean trypsin inhibitor (100 mg/L). Thereafter, binding studies were carried out in modified KRB using [125I]bTSH or [ m I]hTSH. Each bar represents the mean ± SD of data obtained from three separate experiments carried out in triplicate wells of cells. For bTSH binding, specific bound counts were 3500-5000 cpm; nonspecific counts were 10% of total binding. For hTSH binding studies, specific bound counts were 300-500; nonspecific counts were 100-200 cpm. Data are expressed as the percent of maximal binding in control cells.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 September 2016. at 00:49 For personal use only. No other uses without permission. . All rights reserved.

BOVINE AND HUMAN TSH BIOLOGICAL ACTIVITY prelncubation

prelncubatlon In medium obtained from cells exposed to trypsin

30 ., a> 5 ^ o

In control medium

T

20 -|

E Q.

10 . < control

bTSH

hTSH

TSAb

FIG. 6. Effect of the preincubations of bTSH, hTSH, or TSab with the medium obtained (as described in Materials and Methods) from trypsin-treated thyroid cells on their ability to elicit a cAMP response in human thyroid cells. bTSH (10 mU/L), human TSH (33 mU/L), or TSab (3.3 mg/mL) were incubated (2 h at 37 C) either with medium from thyroid cells exposed to trypsin plus trypsin inhibitor (control medium, open bars) or with medium from thyroid cells exposed to trypsin alone (hatched bars). At the end of this period the ability of these mixtures (containing either bTSH, or hTSH or TSab) to stimulate cAMP (1 h) was assessed in human thyroid cells. Each bar represents the mean ± SD of data obtained in three experiments, each performed in triplicates.

are identical (5, 7). The reason for this choice is primarily the higher specific biological activity of bTSH and, most important, the better binding of bTSH in respect to hTSH in the radioreceptor assay (7, 13). The present study provides new information regarding the interaction of bTSH, hTSH, and TSab with human thyroid cells and suggests the assumption that bTSH and hTSH do not necessarily behave as identical ligands for the hTSH receptor. The major finding of the present report is that trypsin treatment of human thyroid cells decreases their ability to stimulate cAMP in response to bTSH. In similar trypsin treated cells, the cAMP response to TSab stimulation is fully maintained. Also, the cAMP response to human TSH, like that observed after TSab, is trypsin insensitive. The inhibition of thyroid cell response to bTSH induced by trypsin is dose dependent, present even at a very low trypsin concentration (3.3-10 mg/L), and fully reversible within 24 h. In the same cells, trypsin treatment does not alter the cAMP response to either cholera toxin or forskolin, indicating that two postreceptorial components of the adenylate cyclase complex are not damaged by the enzyme. The most likely explanation for these results is that trypsin cleaves a component of the thyroid cell plasma membrane necessary for the binding and for the subsequent biological activity of bTSH, without affecting the activity of hTSH or TSab. Two different lines of evidence support this explanation. First, trypsin treatment of human thyroid cells decreases bTSH binding, as previously reported (16-18), whereas

715

hTSH binding is enhanced. The increased hTSH binding is an effect similar to that already described for other ligand-receptor interactions (24), and is possibly due to unmasking of hTSH binding sites by the proteolytic treatment. Second, and more direct evidence, is that the medium from trypsin treated cells, supposedly containing the enzymatically cleaved fragment from the TSH receptor, is able to specifically neutralize the activity of bTSH, but not that of either hTSH or TSab. Our results are consistent with the data of Smith et al. (25, 26) showing that a soluble fragment of the TSH receptor can be released by freeze-thawing plasma membrane. This fragment also can neutralize TSab bioactivity (27). Furthermore, it has a trypsin cleavage site that releases a smaller-fragment (28); however, no functional studies on this subunit have been reported. Thus, these lines of evidence suggest that the fragment released by trypsin is a component of the TSH receptor, and it contains a binding site primarily specific for bTSH. bTSH and TSab, therefore, might interact with the cell membrane at different sites, or with different components of the TSH receptor. However, to settle these and other issues, the definition and the knowledge of the TSH receptor structure is fundamental. Thus, the recent molecular cloning of the hTSH receptor now provides the opportunity for understanding the physical characteristics of the TSH receptor and the possibility to recognize the TRab epitopes (29, 30, 31). In conclusion: (1) trypsin impairs bTSH but not hTSH or TSab cAMP response in cultured human thyroid cells; (2) TSab behaves functionally like hTSH, rather than bTSH; (3) our studies suggest that the TSH and hTSH have different binding sites and that TSab is related to the hTSH binding site.

Acknowledgment We wish to thank Dr. R. Vigneri for his encouragement and helpful discussions during this study.

References 1. Burman KD, Baker JR. Immune mechanisms in Graves' disease. Endocr Rev. 1985;6:183-232. 2. Zakarija M, McKenzie JM. The spectrum and significance of autoantibodies reacting with the thyrotropin receptor. Endocrinol Metab Clin North Am. 1987;16:343-63. 3. Rees Smith B, McLachlan SM, Furmaniak J. Autoantibodies to the thyrotropin receptor. Endocr Rev. 1988;9:106-21. 4. McKenzie JM, Zakarija M. Assay of the thyroid-stimulating antibodies (TSAb) of Graves' disease. In: Ingbar SH, Braverman LE eds. Werner's: the thyroid. Philadelphia: Lippincott 1986;559-75. 5. McKenzie JM, Zakarija M. Assays of thyroid-stimulating antibody. Methods Enzymol. 1985;109:677-91. 6. Rees Smith B, Hall R. Thyroid-stimulating immunoglobulins in Graves' disease. Lancet. 1974;2:427-30. 7. Kermode JC, Thompson BD, Edmonds CJ. Comparison of binding of bovine and human thyroid-stimulating hormone to receptor sites on human thyroid membranes. J Endocrinol. 1981;88:205-17. 8. Rapoport B, Filetti S, Takai N, Seto P, Halverson G. Studies on the cyclic AMP response to thyroid stimulating immunoglobulin

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 September 2016. at 00:49 For personal use only. No other uses without permission. . All rights reserved.

716

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21.

FOTI ET AL. (TSI) and thyrotropin (TSH) in human thyroid cell monolayers. Metabolism. 1982;31:1159-67. Filetti S, Vetri M, Damante G, Belfiore A. Thyroid autoregulation: effect of iodine on glucose transport in cultured thyroid cells. Endocrinology 1985;118:1395-400. Rapoport B. Dog thyroid cells in monolayer culture: adenosine 3',5'cyclic monophosphate response to thyrotropic hormone. Endocrinology. 1976;98:1189-97. Goldfine ID, Amir SM, Petersen AW, Ingbar SH. Preparation of biologically active 125I-TSH. Endocrinology. 1974;95:1228-33. Tramontano D, Ingbar SH. Properties and regulation of the thyrotropin receptor in the FRTL5 rat thyroid cell line. Endocrinology. 1986;118:1945-51. Rapoport B, Seto P. Bovine thyrotropin has a specific bioactivity 5-10 fold that of previous estimate for highly purified hormone. Endocrinology. 1985;116:1379-82. Fradkin JE, Cook GM, Kilhoffer MC, Wolff J. Forskolin stimulation of thyroid adenylate cyclase and cyclic 3',5'-adenosine monophosphate accumulation. Endocrinology. 1982; 111:849-56. Rapoport B, Filetti S, Takai N. Studies on the desensitization of the cyclic AMP response to thyrotropin in thyroid tissue. FEBS Lett. 1982;146:23-7. Tate RL, Schwartz HI, Holmes JM, Kohn LD, Winand RJ. Thyrotropin receptors in thyroid plasma membranes. J Biol Chem. 1975;250:6509-15 Winan RJ, Kohn LD. Thyrotropin effect in thyroid cells in culture. J Biol Chem. 1975;250:6534-40 Karsenty G, Michel-Bechet M, Charreire J. Monoclonal human thyroid cell line GEJ expressing human thyrotropin receptors. Proc Natl Acad Sci USA 1985;82:2120-24. Macchia E, Fenzi GF, Monzani F, et al. Comparison between thyroid stimulating and TSH-binding inhibiting immunoglobulins of Graves' disease. Clin Endocrinol (Oxf.) 1981;15:175-82. Sugenoya A, Kidd A, Row VV, Volpe' R. Correlation between thyrotropin-displacing activity by immunoglobulin from patients with Graves' disease and other thyroid disorders. J Clin Endocrinol Metab. 1974;48:398-402. Takasu N, Yamada T, Katakura M, Yamauchi K, Shimizu Y, Ishizuki Y. Evidence for thyrotropin (TSH)-blocking activity in goitrous Hashimoto's thyroditis with assays measuring inhibition

22.

23. 24.

25.

26.

27.

28. 29.

30.

31.

JCE & M • 1991 Vol 73 • No 4

of TSH receptor binding and TSH-stimulated thryoid adenosine 3',5'-monophosphate responses/cell growth by immunoglobulins. J Clin Endocrinol Metab. 1987;65:239-45. Konishi J, Iida Y, Endo K, et al. Inhibition of thyrotropin-induced adenosine 3'5' monophosphate increase by immunoglobulins from patients with primary mixedema. J Clin Endocrinol Metab. 1983;57:544-9. Zakarija M, McKenzie JM, Eidson MS. Transient neonatal hypothyroidism: characterization of maternal antibodies to the thyrotropin receptor. J Clin Endocrinol Metab. 1990;70:1239-46. Liscovitch I, Ben-Aroya N, Meidan R, Koch Y. A differential effect of trypsin on pituitary gonadotropin releasing hormone receptors from intact and ovariectomized rats. Eur J Biochem. 1984;140:1917. Davies Jones E, Rees Smith B. A water-soluble fragment of the thyroid stimulating hormone receptor which binds both thyroidstimulating hormone and thyroid-stimulating hormone receptor antibodies. J Endocrinol. 1984;100:113-8. Kajita Y, Rickards CR, Buckland PR, Howells RD, Rees Smith B. Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane. Biochem J. 1985;227:413-20. Davies Jones E. Hashim FA, Creagh FM, Williams SE, Rees Smith B. The interaction between the TSH receptor and Graves' sera with TSH agonist or antagonist properties. Mol Cell Endocrinol. 1985;42:257-61. Buckland PR, Howells RD, Rickards CR, Rees Smith B. Affinitylabelling of the thyrotropin receptor. Characterization of the photoactive ligand. Biochem J. 1985;225:753-60 Nagayama Y, Kaufman KD, Seto P, Rapoport B. Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun. 1989; 165:1184-90. Libert F, Lefort A, Gerard C. et al. Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem Biophys Res Commun. 1989;165:1250-5. Filetti S, Foti D, Costante G, Rapoport B. Recombinant human thyrotropin (TSH) receptor in a radioreceptor assay for the measurement of TSH receptor antibodies. J Clin Endocrinol Metab. 1991;72:1096-101.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 September 2016. at 00:49 For personal use only. No other uses without permission. . All rights reserved.

The biological activity of bovine and human thyrotropin is differently affected by trypsin treatment of human thyroid cells: thyroid-stimulating antibody is related to human thyrotropin.

Pretreatment of cultured human thyroid cells with trypsin decreased the cAMP response to bovine TSH (bTSH) (by 50-60%). In striking contrast, in tryps...
860KB Sizes 0 Downloads 0 Views