Clin Biochem, Vol. 23, pp. 43--48, 1990 Printed in Canada. All rights reserved.
0009.9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical Chemists.
The Thyroid Stimulating Hormone Receptor in Human Disease L. C. HARRISON and P. J. LEEDMAN Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, 3050, Victoria, Australia The initial step in the action of thyrotropin (TSH) is its binding to the TSH receptor. TSH receptor antibodies are detected in up to 90% of patients with Graves' disease. Serial measurements of TSH receptor antibodies in patients with Graves' hyperthyroidism are helpful in predicting relapse. The TSH receptor was purified using affinity chromatography on wheat germ lectin agarose and TSH-agarose. Using an immunoblotting technique to characterize the TSH receptor, it was found to be an oligomeric glycoprotein consisting of three noncovalently bound subunits of Mr - 70,000, - 50,000 and - 35,000 which on reduction yield a single subunit of Mr - 25,000.
KEY WORDS: thyrotropin; TSH receptor.
Introduction he initial step in the action of thyroid stimulating hormone (thyrotropin, TSH) on thyroid folT licular cells is its binding to the TSH receptor. Binding triggers the activation of membrane-bound adenylate cyclase, the production of cyclic AMP and the activation of protein kinases and other postreceptor processes, leading to the known effects of TSH, viz., iodine uptake, synthesis and release of iodothyronines, DNA synthesis and cell growth (1,2). Although the action of TSH can be described in these general terms, the structure of the receptor and the postreceptor mechanisms are poorly defined in molecular terms. The TSH receptor is, nonetheless, an ideal model for the study of receptors in human physiology and disease, chiefly because it is the target for receptor autoantibodies in the relatively common group of thyroid autoimmune diseases. TSH receptor autoantibodies mimic most of the actions of TSH and are natural reagents for probing the mechanisms of action of TSH and defining the structure-function relationships of the TSH receptor. It is assumed that binding to specific epitopes on the receptor or related molecules medi-
Correspondence: Professor L. C. Harrison, Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, 3050, Victoria, Australia. Manuscript received February 3, 1989; revised July 26, 1989; accepted August 1, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, FEBRUARY 1990
ates the different effects of these polyclonal aut o a n t i b o d i e s - hyperthyroidism, goitre, hypothyroidism, inhibition of thyroid growth, opthalmopathy and other extrathyroid manifestations (reviewed in Refs. 3-6).
Assays for TSH receptor antibodies TSH receptor autoantibodies are designated functionally by in vitro assays, e.g., thyroid stimulating antibodies (TSAb), thyroid growth-stimulating antibodies (TGAb) or TSH binding inhibitory immunoglobulins (TBII) (Table 1). Of these only TBII can be measured by a commercially available assay.
Clinical indications for m e a s u r e m e n t of TSH receptor antibodies Although TSH receptor antibodies are specific markers of thyroid immunopathology, the indications for their measurement in routine clinical practice are limited (Table 2) and are reviewed in Refs. 6 and 27. TSH receptor antibodies are reported in the serum of 70-100% of patients with Graves' hyperthyroidism at the time of diagnosis (27). Receptor antibodies, particularly those that mimic the bioeffects of TSH, are virtually pathognomic for Graves' disease. However, the main clinical application of TSH receptor antibodies is not to confirm the diagnosis of Graves' disease but to attempt to predict a response to antithyroid drug treatment. The outcome of treating hyperthyroidism with antithyroid drugs alone is difficult to predict and there is a high frequency of relapse after treatment is withdrawn. Predicting the outcome of treatment with antithyroid drugs would allow an earlier decision to be reached regarding ablative treatment. In addition to blocking the organification of iodine in the thyroid gland, antithyroid drugs also directly inhibit the production of TSH receptor antibodies by thyroid lymphocytes ex vivo (28). That carbimazole may have an immunosuppressive effect in vivo is evidenced by higher remission rates on carbimazole than on propranolol that correlate with the dose and 43
HARRISON AND LEEDMAN TABLE 1 Assays for TSH Receptor Antibodies
Bioassays Description and Technique
Acronym LATS
Long-acting thyroid stimulator
TSI
Thyroid stimulating immunoglobulins cAMP production
Ref.
Comment
Release of 13~I from prelabelled guinea pig or mouse thyroid in vivo
7,8
First assay for TRAb. Laborious, insensitive
Human thyroid membranes
9
Insensitive
Human thyroid slices
10
Sensitive. Requires fresh tissue
Human thyroid cells
11
Sensitive. More convenient
Rat FRTL-5 cell line
12
Sensitive. Most convenient Also measures I uptake and growth
Organification of I
Pig thyroid cells
13
Sensitive. No separate RIA. Not routine
T3 release
Pig thyroid slices
14
Sensitive. Separate RIA. Not routine
Colloid droplet formation
Human thyroid cells
15
Sensitive. Subjective. Not routine
Cytochemical (Lysosomal permeability)
Guinea pig thyroid
16
Very sensitive. Technically difficult
Collagen synthesis
Human fibroblasts
17
Antibodies associated With ophthalmopathy. Not routine
TGI
Thyroid growth-stimulating immunoglobulins
Stimulation of DNA synthesis in guinea pig thyroid slices or rat FRTL-5 cells
18,19
Measures mitogenic antibodies. Not routine
TBI
Thyroid blocking immunoglobulins
Inhibition of TSH-stimulated cAMP production in thyroid membranes or slices
20,21
Antibodies associated with nongoitrous hypothyroidism
Receptor Assays LATS-P
Long-acting thyroid stimulator protector
LATS-negative serum incubated with human thyroid prevents 'inactivation' (absorption) of LATS
22
First receptor assay for TRAb. Laborious
TBII
TSH binding inhibitory immunoglobulins
Inhibition of 125I-TSH binding to intact or solubilized thyroid membranes
23,24
Sensitive, convenient, commercial. Doesn't measure antibody bioeffects
TRPI
TSH receptor precipitating immunoglobulins
Precipitation of 125I-TSHreceptor complex
25,26
Sensitive. Detects antibodies against most epitopes. Not routine
duration of drug t r e a t m e n t (29,30). For this reason, and because they are specific markers of the immunopathogenesis of Graves' disease, TSH receptor antibodies can be used to monitor and predict the outcome of antithyroid drug treatment. Other investigations, including the T3 suppression test, HLA typing,
44
the thyrotropin releasing hormone test and thyroid technetium scanning, are considered unreliable as guides to the outcome of antithyroid drug treatment. We followed 25 patients with Graves' hyperthyroidism (23 women and 2 men) treated with carbimazole, 30-45 mg/day initially, and reduced to 5-10
CLINICALBIOCHEMISTRY,VOLUME23, FEBRUARY 1990
TSH RECEPTOR TABLE 2
TABLE 4
Clinical Indications for the Measurement of TSH Receptor Antibodies (Based on References 6 and 27) Confirm Graves' pathogenesis of hyperthyroidism Predict response of Graves' hyperthyroidism to therapy Confirm diagnosis of euthyroid ophthalmopathy Predict likelihood of neonatal hyperthyroidism
Relapse of Hyperthyroidism in Graves' Disease Patients in Relation to TSH Receptor Antibodies at the Time of Stopping Carbimazole
mg/day subsequently to m a i n t a i n euthyroidism. Treatment was continued for a mean of 13.3 months (range 5-24 months) until patients had been clinically and biochemically euthyroid for at least 3 months. Relapse was defined as the development of persistently elevated thyroid function tests. All patients were followed until relapse or for at least 12 months after t r e a t m e n t had ceased (range: 12-40 months, mean _+ SD 25.3 _+ 8.7 months). TSH receptor antibodies were measured as TBII with the TRAK assay (Henning, Berlin). The results of antithyroid drug t r e a t m e n t in relation to TSH receptor antibodies are presented in Tables 3 and 4. Patients who remained in remission for at least 12 months had, on the average, lower initial levels of TSH receptor antibodies. In contrast, patients who relapsed had higher initial antibody levels t h a t did not suppress significantly (Table 3). Antibody levels remained elevated at the end of t r e a t m e n t in 8 out of the 25 patients; 7 of these patients became thyrotoxic again within 3 months of stopping t r e a t m e n t (Table 4). Overall, the level of TSH receptor antibodies at the end of the course of t r e a t m e n t predicted the clinical status 12 months later in 19 out of 25 patients (76%). The predictability of relapse in patients with high antibody levels after t r e a t m e n t is consistent with the findings of a number of other studies (reviewed in Ref. 6). The predictive value of an antibody level t h a t is within the normal range at the end of t r e a t m e n t is less certain. In agreement with other reports (31,32), 70% of patients with antibody levels in the normal range remained in remission for at least a year. TABLE 3
Clinical Features and TSH Receptor Antibodies (TBII) in Patients with Graves' Hyperthyroidism 12 Months After Stopping Carbimazole
Number
Age (years) Time to relapse (months) Prominent goitre Ophthalmopathy TBII at diagnosis* TBII on stopping carbimazole Months on carbimazole
Relapse
Remission
12
13
30.0 -+ 8.8 41.7 -+ 13.9 2.6 - 2.5 6 6 5 4 24.3 - 27.2 14.8 -+ 18.0 21.5 - 17.6 3.8 - 11.3 12.9 -+ 4.5 13.3 +- 4.6
*TBII reference range: - 15 to + 15 units.
CLINICAL BIOCHEMISTRY, VOLUME 23, FEBRUARY 1990
Duration of Carbimazole
Relapse Rate
TSH Receptor Antibodies: TBII
Treatment
Normal
Elevated
Difference
3 months 12 months
3/17 (18%) 5/17 (29%)
7/8 (88%) 7/8 (88%)
p = 0.003* p = 0.02
*Fisher's exact test.
It is our practice to recommend antithyroid drug t r e a t m e n t for at least 12 months in patients with Graves' hyperthyroidism, and to measure TBH serially. If receptor antibody levels remain elevated, then ablative t r e a t m e n t is recommended after 12 months. In patients in whom receptor antibody levels become normal, t r e a t m e n t is gradually reduced so long as the patient remains euthyroid. Such patients are still monitored regularly as the risk of relapse is still significant. Two approaches could improve the sensitivity, specificity and predictive value of TSH receptor antibody measurements. First, assays t h a t measure the ability of receptor antibodies to mimic the bioeffects of TSH, rather t h a n their ability to inhibit TSH binding to solubilized receptors, would be more likely to reflect disease activity at diagnosis and during treatment. Second, the '%lock-replace" method of t r e a t m e n t with a combination of high-dose carbimazole and replacement t r e a t m e n t with thyroxine should, theoretically, be more effective from the point of view of immunosuppression; indeed, in one study this strategy was shown to result in a higher frequency of posttreatment remissions (33).
TSH receptor purification and s u b u n l t structure Purification of the TSH receptor, characterization of its subunit structure and definition of its antibody and T lymphocyte receptor epitopes are objectives fundamental to understanding the pathogenesis of autoimmune thyroid disease. Despite attempts over the past few years to characterize the structure of the TSH receptor by various techniques including chemical crosslinking (34-37) and photo-affinity labelling (38) of 125I-TSH, immunoprecipitation using Graves' immunoglobulins (25,39) or monoclonal antibodies (40), target size analysis (41) and affinity purification (25,42-47), there is no firm, consensus structure for the receptor. This is in contrast to a number of other cell surface receptors t h a t are well characterized and cloned. Nevertheless, from the data available it is possible to conclude t h a t the TSH receptor is an oligomeric glycoprotein structure containing several subunits. In addition, there is evi-
45
HARRISON AND LEEDMAN
dence that the receptor contains a ganglioside component involved in signal transduction (48), identified by its reactivity with antireceptor monoclonal antibodies that mimic the bioeffects of TSH (40). A TSH binding subunit of M~ - 50,000 has been identified in thyroid tissue from a number of species (37,38,49), as well as in guinea pig fat cell membranes (47). In addition, nonreduced subunits of Mr - 70,000 and 35,000 have been reported by a number of investigators (25,35,42,44,47,49). Difficulties encountered with the purification and characterization of the TSH receptor can be attributed to a number of factors. These include the lack of a rich source of receptor, the marked lability of the receptor, its apparently complex nature and a Mr 50,000 subunit reported to be hydrophilic and easily dissociable. While Graves' immunoglobulins have been used to precipitate the receptor (25), their application to the identification of receptor subunits by gel electrophoresis and immunoblotting has been limited (50,51). This contrasts with the use of immunoblotting to detect nuclear autoantigens in various cell extracts and cDNA expression libraries (52). Very few membrane receptor molecules are, in fact, easy to identify by immunoblotting, perhaps because of their complex tertiary structure and conformational requirements for antibody binding. Nevertheless, as outlined below, modifications to conventional immunoblotting techniques permit the identification of TSH affinity-purified thyroid autoantigens, presumptively the subunits of the TSH receptor, using Graves' immunoglobulins. We have purified the human TSH receptor by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose (53,54). When TSH affinity-purified receptors were eluted in 3 M NaC1, a single protein of Mr 50,000 was recovered which bound 125I-TSH and could be precipitated by Graves' immunoglobulins. Alternatively, when affinity-purified receptors were eluted in sodium dodecyl sulphate (SDS) sample buffer and directly analyzed by SDS-polyacrylamide gel electrophoresis, three proteins of Mr 70,000, 50,000 and 35,000 were detected. After reduction with 100 mM dithiothreitol, only one protein of Mr - 25,000 was detected. The conventional immunoblotting technique (55) has been systematically modified in order to identify TSH receptor subunits. Essentially, SDS gels containing thyroid proteins are washed in Tris buffer containing 20% glycerol prior to transfer. SDS is omitted from the transfer buffer and a nylon membrane is used in place of nitrocellulose. These modifications enable putative TSH receptor subunits of Mr 70,000 and 50,000 to be identified after electrophoresis of either crude thyroid membranes or affinity-purified receptors. The results of affinity purification and immunoblotting enable us to draw the following conclusions: (i) The nonreduced human TSH receptor is an
46
oligomeric complex comprised of three different subunits ofM r 70,000, 50,000 and 35,000; the reduced receptor would appear to exist as a single subunit of Mr 25,000 which is disulphide-linked to form the high Mr forms. (ii) The Mr 50,000 subunit binds TSH and is linked by noncovalent, ionic bonds to at least one other binding subunit of the receptor complex. (iii) The 70,000 and 50,000 subunits contain epitopes that are recognized by Graves' immunoglobulins, thus confirming directly that the human TSH receptor is a target for Graves' immunoglobulins.
Future perspectives Further studies will define the stoichiometry and function of the disulphide-linked subunits that comprise the TSH receptor oligomer. The ability to purify TSH receptor subunits to homogeneity, and to microsequence picomolar concentrations of protein eluted from SDS gels, should ensure that primary amino acid sequence for the receptor will soon be available. This information can be used to design synthetic oligonucleotides with which to identify clones in cDNA libraries. It is unlikely that immunoscreening of cDNA libraries using autoantibodies will be as sensitive or practical, even though modified immunoblotting allows TSH receptor proteins to be identified by some Graves' immunoglobulins. The uncertainty about the structure of the TSH receptor will be resolved by the cloning of the receptor, opening the way for detailed structurefunction studies to clarify the immune mechanisms of thyroid diseases.
Acknowledgements The clinical and scientific contributions of Dr. T. W. H. Kay are gratefully acknowledged. Financial support was provided by the National Health and Medical Research Council of Australia. We thank Mrs. Margaret Thompson for secretarial assistance.
References 1. Dumont JE, Takeuchi A, Lamy F. Thyroid control: an example of a complex cell regulation network. Adv Cyclic Nucleotide Res 1981; 14: 479-89. 2. Chambard M, Verrier B, Gabrion J. Polarization of thyroid cells in culture: evidence for the basolateral localization of the iodide "pump" and of the thyroidstimulating hormone receptor-adenyl cyclase complex. J Cell Biol 1983; 96: 1172-7. 3. Weetman AP, McGregor AM. Autoimmune thyroid disease: developments in our understanding. Endocr Rev 1984; 5: 309-55.
CLINICAL BIOCHEMISTRY, VOLUME 23, FEBRUARY 1990
TSH RECEPTOR 4. Harrison LC. Antireceptor antibodies. In: Rose NR, Mackay IR, eds. Autoimmune diseases. Pp. 617-68. London: Academic Press, 1985. 5. Burman KD, Baker JR. Immune mechanisms in Graves' disease. Endocr Rev 1985; 6: 183-232. 6. Rees Smith B, McLachlan SM, Furmaniak J. Autoantibodies to the thyrotropin receptor. Endocr Rev 1988; 9: 106-21. 7. Adams DD, Purves HD. Abnormal responses in the assay of thyrotropin. Proc Univ Otago Med Sch 1956; 34: 11-2. 8. McKenzie JM. The bioassay of thyrotropin in serum. Endocrinology 1958; 63: 372-82. 9. Orgiazzi J, Williams DE, Chopra IJ, Solomon DH. Human thyroid adenyl cyclase stimulating activity in immunoglobulin G of patients with Graves' disease. J Clin Endocrinol Metab 1976; 42: 341-54. 10. McKenzie JM, Zakarija M. A reconsideration of a thyroid stimulating immunoglobulin as the cause of hyperthyroidism in Graves' disease. J Clin Endocrinol Metab 1976; 42: 778-81. 11. Etienne-Decerf J, Winand RJ. A sensitive technique for determination of thyroid-stimulating immunoglobulins (TSI) in unfractionated serum. Clin Endocrinol 1981; 14: 83-91. 12. Vitti P, Valente WA, Ambesi-Impiombato FS, Fenzi G, Pinchera A, Kohn LD. Graves' IgG stimulation of continuously cultured rat thyroid cells: a sensitive and potentially useful clinical assay. J Endocrinol Invest 1982; 5: 179-82. 13. Planells R, Fayet G, Lissitsky S. Bioassay of thyrotropin using isolated porcine thyroid cells. FEBS Lett 1975; 53: 87-91. 14. Atkinson S, Kendall-Taylor P. The stimulation of thyroid hormone secretion in vitro by thyroid-stimulating antibodies. J Clin Endocrinol Metab 1981; 53: 1263-6. 15. Onaya T, Kotani M, Yamada T, Ochi Y. New in vitro tests to detect the thyroid stimulator in sera from hyperthyroid patients by measuring colloid droplet formation and cyclic AMP in human thyroid slices. J Clin Endocrinol Metab 1973; 36: 859-66. 16. Bitensky L, Alaghband-Zadeh J, Chayen J. Studies on thyroid stimulating hormone and the long-acting thyroid stimulating hormone. Clin Endocrinol 1974; 3: 363-74. 17. Rotella CM, Zonefrati R, Tossafondi R, Valente WA, Kohn LD. Ability of monoclonal antibodies to the thyrotropin receptor to increase collagen synthesis in human fibroblasts: an assay which appears to measure exophthalmogenic immunoglobulins in Graves' sera. J Clin Endocrinol Metab 1986; 62: 357-67. 18. Van Der Gaag RD, Drexhage HA, Wieringa WM. Further studies on thyroid growth-stimulating immunoglobulins in euthyroid nonedemic goiter. J Clin Endocrinol Metab 1985; 60: 972-9. 19. Valente WA, Vitti P, Carlo M, et al. Antibodies that promote thyroid growth: a distinct population of thyroid-stimulating autoantibodies. N Engl J Med 1983; 309: 1028-34. 20. Karlsson FA, Dahlberg PA, Ritzen EM. Thyroid blocking antibodies in thyroiditis. Acta Med Scand 1984; 215: 461-6. 21. Drexhage HA, Bottazzo GF, Bitensky L, Chayen J, Doniach D. Thyroid growth-blocking antibodies in primary myxoedema. Nature 1981; 289: 594-6.
22. Adams DD, Kennedy TH. Occurrence in thyrotoxicosis of a gamma globulin which protects LATS from neutralisation by an extract of thyroid gland. J Clin Endocrinol Metab 1967; 27: 173-7. 23. Smith BR, Hall R. Thyroid stimulating immunoglobulins in Graves' disease. Lancet 1974; ii: 427-31. 24. Shewing G, Rees Smith B. An improved radioreceptor assay for TSH-receptor antibodies. Clin Endocrinol 1982; 17: 409-17. 25. Heyma P, Harrison LC. Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins. J Clin Invest 1984; 74: 1090-7. 26. DeBruin TWA, Braverman LE, Brown RS. Further evaluation of an immunoprecipitation assay for TSHreceptor autoantibodies in Graves' disease. Metabolism 1986; 35: 1101-5. 27. Ginsberg J, Von Westarp C. Clinical applications of assays for thyrotropin-receptor antibodies in Graves' disease. Can Med Assoc J 1986; 134: 1141-7. 28. Ratanachaiyavong S, McGregor AM. Immunosuppressive effects of antithyroid drugs. Clin Endocrinol Metab 1985; 14: 449-66. 29. Kendall-Taylor P. Are antithyroid drugs immunosuppressive? Br Med J 1984; 288: 509-10. 30. Weetman AP, McGregor AM, Hall R. Evidence for an effect of antithyroid drugs on the natural history of Graves' disease. Clin Endocrinol 1984; 21: 163-72. 31. Wilson R, McKillip JH, Henderson N, Pearson DW, Thomson JA. The ability of the serum thyrotropin receptor antibody (TRAb) index and HLA status to predict long-term remission of thyrotoxicosis following medical therapy for Graves' disease. Clin Endocrinol 1986; 25: 151-6. 32. Kasagi K, Konishi J, Arai K. A sensitive and practical assay for thyroid-stimulating antibodies using crude immunoglobulin fractions precipitated with polyethylene glycol. J Clin Endocrinol Metab 1986; 62: 85562. 33. Romaldini JH, Bromberg N, Werner RS. Comparison of effects of high and low dosage regimens of antithyroid drugs in the management of Graves' hyperthyroidism. J Clin Endocrinol Metab 1983; 57: 563-70. 34. Buckland PR, Rickards CR, Howells RD, Rees Smith B. Thyrotropin cross-links to the thyrotropin receptor through both ~ and ~ subunits. Biochem J 1986; 235: 879-82. 35. Gennick SE, Thomas CG Jr, Nayfeh SN. Characterisation of the subunit structure of the thyrotropin receptor in the FRTL-5 rat thyroid cell line. Endocrinology 1987; 121: 2119-30. 36. McQuade R, Thomas CG Jr, Nayfeh SN. Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the ~ and ~ subunits of thyrotropin are crosslinked to the receptor. Arch Biochem Biophys 1987; 252: 409-17. 37. Remy JJ, Salamero J, Charreire J. Covalent crosslinking of thyrotropin to its receptor on cloned hybrid human thyroid cells (GEJ). Endocrinology 1987; 121: 1733-41. 38. Kajita Y, Rickards CR, Buckland PR, Howells RD, Rees Smith B. A structure for the porcine TSH receptor. F E B S Lett 1984; 181: 218-22. 39. Parkes AB, Kajita Y, Buckland PR. Immunoprecipitation of TSH-receptor complexes. Clin Endocrinol 1985; 22: 511-20.
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HARRISON AND LEEDMAN 40. Chan J, Santisteban P, deLuca M, Isozaki O, Grollman E, Kohn L. TSH receptor structure. Acta Endocrinol 1987; Suppl. 281: 166-72. 41. Nielsen TB, Totsuka Y, Kempmer ES, Field JB. Structure of the thyrotropin receptor and thyroid adenylate cyclase system as determined by target analysis. Biochemistry 1984; 23: 6009-16. 42. Tate RL, Holmes JM, Kohn LD, Winand RJ. Characteristics of a solubilized thyrotropin receptor from bovine thyroid plasma membranes. JBiol Chem 1975; 250: 6527-33. 43. Drummond RW, McQuade R, Grunwald R, Thomas CG Jr, Nayfeh SN. Separation of two thyrotropin binding components from porcine thyroid tissue by affinity chromatography. Characterization of high and low affinity sites. Proc Natl Acad Sci USA 1982; 79: 2202-6. 44. Koizumi Y, Zakarija M, McKenzie JM. Solubilization, purification and partial characterization of thyrotropin receptor from bovine and human thyroid glands. Endocrinology 1982; 11{}: 1381-91. 45. Iida Y, Konishi J, Kasagi K. Partial purification and properties of the TSH receptor from human thyroid plasma membranes. Acta Endocrinol 1983; 103: 198204. 46. Krees BC, Spiro RG. Studies on the glycoprotein nature of the thyrotropin receptor: interaction with lectins and purification of the bovine protein with the use of Bandeiraea (Griffonia) simplicifolia I affinity chromatography. Endocrinology 1986; 118: 974-9. 47. Iida Y, Amir SM, Ingbar SH. Stabilization, partial
48
48. 49.
53. 51.
52. 53.
54. 55.
purification and characterization ofthyrotropin receptors in solubilized guinea pig fat cell membranes. Endocrinology 1987; 121: 1627-36. Gardas A, Nauman J. Presence of gangliosides in the structure of membrane receptor for thyrotropin. Acta Endocrinol 1981; 98: 549-55. Kohn LD, Alvarez E, Marcocci C, et al. Monoclonal antibody studies defining the origin and properties of autoantibodies in Graves' disease. Ann N Y Acad Sci 1986; 475: 157-73. Furmaniak J, Bradbury J, Smith BR. Antibodies to membrane antigens in autoimmune thyroid disease. Acta Endocrinol 1987; 116: 13-20. Weetman AP, Nutman TB, Burman KD, Baker JR, Volkman DJ. Heterogeneity of thyroid autoantigens identified by immunoblotting. Clin Immunol Immunopathol 1987; 43: 333--42. Whittingham S, McNeilage I.J. Antinuclear antibodies as molecular and diagnostic probes. Mol Cell Probes 1988; 2: 169-79. Leedman PJ, Newman JD, Harrison LC. Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and Western blotting. J Clin Endocrinol Metab 1989; 69:137 {1. Leedman PJ, Harrison LC. In: G. Litwack, ed. Thyrotropin receptor purification. Receptor purification. Clifton, NJ: Humana Press (in press). Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4.
CLINICAL BIOCHEMISTRY,VOLUME 23, FEBRUARY 1990
0009.9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical Chemists.
The Thyroid Stimulating Hormone Receptor in Human Disease L. C. HARRISON and P. J. LEEDMAN Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, 3050, Victoria, Australia The initial step in the action of thyrotropin (TSH) is its binding to the TSH receptor. TSH receptor antibodies are detected in up to 90% of patients with Graves' disease. Serial measurements of TSH receptor antibodies in patients with Graves' hyperthyroidism are helpful in predicting relapse. The TSH receptor was purified using affinity chromatography on wheat germ lectin agarose and TSH-agarose. Using an immunoblotting technique to characterize the TSH receptor, it was found to be an oligomeric glycoprotein consisting of three noncovalently bound subunits of Mr - 70,000, - 50,000 and - 35,000 which on reduction yield a single subunit of Mr - 25,000.
KEY WORDS: thyrotropin; TSH receptor.
Introduction he initial step in the action of thyroid stimulating hormone (thyrotropin, TSH) on thyroid folT licular cells is its binding to the TSH receptor. Binding triggers the activation of membrane-bound adenylate cyclase, the production of cyclic AMP and the activation of protein kinases and other postreceptor processes, leading to the known effects of TSH, viz., iodine uptake, synthesis and release of iodothyronines, DNA synthesis and cell growth (1,2). Although the action of TSH can be described in these general terms, the structure of the receptor and the postreceptor mechanisms are poorly defined in molecular terms. The TSH receptor is, nonetheless, an ideal model for the study of receptors in human physiology and disease, chiefly because it is the target for receptor autoantibodies in the relatively common group of thyroid autoimmune diseases. TSH receptor autoantibodies mimic most of the actions of TSH and are natural reagents for probing the mechanisms of action of TSH and defining the structure-function relationships of the TSH receptor. It is assumed that binding to specific epitopes on the receptor or related molecules medi-
Correspondence: Professor L. C. Harrison, Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, 3050, Victoria, Australia. Manuscript received February 3, 1989; revised July 26, 1989; accepted August 1, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, FEBRUARY 1990
ates the different effects of these polyclonal aut o a n t i b o d i e s - hyperthyroidism, goitre, hypothyroidism, inhibition of thyroid growth, opthalmopathy and other extrathyroid manifestations (reviewed in Refs. 3-6).
Assays for TSH receptor antibodies TSH receptor autoantibodies are designated functionally by in vitro assays, e.g., thyroid stimulating antibodies (TSAb), thyroid growth-stimulating antibodies (TGAb) or TSH binding inhibitory immunoglobulins (TBII) (Table 1). Of these only TBII can be measured by a commercially available assay.
Clinical indications for m e a s u r e m e n t of TSH receptor antibodies Although TSH receptor antibodies are specific markers of thyroid immunopathology, the indications for their measurement in routine clinical practice are limited (Table 2) and are reviewed in Refs. 6 and 27. TSH receptor antibodies are reported in the serum of 70-100% of patients with Graves' hyperthyroidism at the time of diagnosis (27). Receptor antibodies, particularly those that mimic the bioeffects of TSH, are virtually pathognomic for Graves' disease. However, the main clinical application of TSH receptor antibodies is not to confirm the diagnosis of Graves' disease but to attempt to predict a response to antithyroid drug treatment. The outcome of treating hyperthyroidism with antithyroid drugs alone is difficult to predict and there is a high frequency of relapse after treatment is withdrawn. Predicting the outcome of treatment with antithyroid drugs would allow an earlier decision to be reached regarding ablative treatment. In addition to blocking the organification of iodine in the thyroid gland, antithyroid drugs also directly inhibit the production of TSH receptor antibodies by thyroid lymphocytes ex vivo (28). That carbimazole may have an immunosuppressive effect in vivo is evidenced by higher remission rates on carbimazole than on propranolol that correlate with the dose and 43
HARRISON AND LEEDMAN TABLE 1 Assays for TSH Receptor Antibodies
Bioassays Description and Technique
Acronym LATS
Long-acting thyroid stimulator
TSI
Thyroid stimulating immunoglobulins cAMP production
Ref.
Comment
Release of 13~I from prelabelled guinea pig or mouse thyroid in vivo
7,8
First assay for TRAb. Laborious, insensitive
Human thyroid membranes
9
Insensitive
Human thyroid slices
10
Sensitive. Requires fresh tissue
Human thyroid cells
11
Sensitive. More convenient
Rat FRTL-5 cell line
12
Sensitive. Most convenient Also measures I uptake and growth
Organification of I
Pig thyroid cells
13
Sensitive. No separate RIA. Not routine
T3 release
Pig thyroid slices
14
Sensitive. Separate RIA. Not routine
Colloid droplet formation
Human thyroid cells
15
Sensitive. Subjective. Not routine
Cytochemical (Lysosomal permeability)
Guinea pig thyroid
16
Very sensitive. Technically difficult
Collagen synthesis
Human fibroblasts
17
Antibodies associated With ophthalmopathy. Not routine
TGI
Thyroid growth-stimulating immunoglobulins
Stimulation of DNA synthesis in guinea pig thyroid slices or rat FRTL-5 cells
18,19
Measures mitogenic antibodies. Not routine
TBI
Thyroid blocking immunoglobulins
Inhibition of TSH-stimulated cAMP production in thyroid membranes or slices
20,21
Antibodies associated with nongoitrous hypothyroidism
Receptor Assays LATS-P
Long-acting thyroid stimulator protector
LATS-negative serum incubated with human thyroid prevents 'inactivation' (absorption) of LATS
22
First receptor assay for TRAb. Laborious
TBII
TSH binding inhibitory immunoglobulins
Inhibition of 125I-TSH binding to intact or solubilized thyroid membranes
23,24
Sensitive, convenient, commercial. Doesn't measure antibody bioeffects
TRPI
TSH receptor precipitating immunoglobulins
Precipitation of 125I-TSHreceptor complex
25,26
Sensitive. Detects antibodies against most epitopes. Not routine
duration of drug t r e a t m e n t (29,30). For this reason, and because they are specific markers of the immunopathogenesis of Graves' disease, TSH receptor antibodies can be used to monitor and predict the outcome of antithyroid drug treatment. Other investigations, including the T3 suppression test, HLA typing,
44
the thyrotropin releasing hormone test and thyroid technetium scanning, are considered unreliable as guides to the outcome of antithyroid drug treatment. We followed 25 patients with Graves' hyperthyroidism (23 women and 2 men) treated with carbimazole, 30-45 mg/day initially, and reduced to 5-10
CLINICALBIOCHEMISTRY,VOLUME23, FEBRUARY 1990
TSH RECEPTOR TABLE 2
TABLE 4
Clinical Indications for the Measurement of TSH Receptor Antibodies (Based on References 6 and 27) Confirm Graves' pathogenesis of hyperthyroidism Predict response of Graves' hyperthyroidism to therapy Confirm diagnosis of euthyroid ophthalmopathy Predict likelihood of neonatal hyperthyroidism
Relapse of Hyperthyroidism in Graves' Disease Patients in Relation to TSH Receptor Antibodies at the Time of Stopping Carbimazole
mg/day subsequently to m a i n t a i n euthyroidism. Treatment was continued for a mean of 13.3 months (range 5-24 months) until patients had been clinically and biochemically euthyroid for at least 3 months. Relapse was defined as the development of persistently elevated thyroid function tests. All patients were followed until relapse or for at least 12 months after t r e a t m e n t had ceased (range: 12-40 months, mean _+ SD 25.3 _+ 8.7 months). TSH receptor antibodies were measured as TBII with the TRAK assay (Henning, Berlin). The results of antithyroid drug t r e a t m e n t in relation to TSH receptor antibodies are presented in Tables 3 and 4. Patients who remained in remission for at least 12 months had, on the average, lower initial levels of TSH receptor antibodies. In contrast, patients who relapsed had higher initial antibody levels t h a t did not suppress significantly (Table 3). Antibody levels remained elevated at the end of t r e a t m e n t in 8 out of the 25 patients; 7 of these patients became thyrotoxic again within 3 months of stopping t r e a t m e n t (Table 4). Overall, the level of TSH receptor antibodies at the end of the course of t r e a t m e n t predicted the clinical status 12 months later in 19 out of 25 patients (76%). The predictability of relapse in patients with high antibody levels after t r e a t m e n t is consistent with the findings of a number of other studies (reviewed in Ref. 6). The predictive value of an antibody level t h a t is within the normal range at the end of t r e a t m e n t is less certain. In agreement with other reports (31,32), 70% of patients with antibody levels in the normal range remained in remission for at least a year. TABLE 3
Clinical Features and TSH Receptor Antibodies (TBII) in Patients with Graves' Hyperthyroidism 12 Months After Stopping Carbimazole
Number
Age (years) Time to relapse (months) Prominent goitre Ophthalmopathy TBII at diagnosis* TBII on stopping carbimazole Months on carbimazole
Relapse
Remission
12
13
30.0 -+ 8.8 41.7 -+ 13.9 2.6 - 2.5 6 6 5 4 24.3 - 27.2 14.8 -+ 18.0 21.5 - 17.6 3.8 - 11.3 12.9 -+ 4.5 13.3 +- 4.6
*TBII reference range: - 15 to + 15 units.
CLINICAL BIOCHEMISTRY, VOLUME 23, FEBRUARY 1990
Duration of Carbimazole
Relapse Rate
TSH Receptor Antibodies: TBII
Treatment
Normal
Elevated
Difference
3 months 12 months
3/17 (18%) 5/17 (29%)
7/8 (88%) 7/8 (88%)
p = 0.003* p = 0.02
*Fisher's exact test.
It is our practice to recommend antithyroid drug t r e a t m e n t for at least 12 months in patients with Graves' hyperthyroidism, and to measure TBH serially. If receptor antibody levels remain elevated, then ablative t r e a t m e n t is recommended after 12 months. In patients in whom receptor antibody levels become normal, t r e a t m e n t is gradually reduced so long as the patient remains euthyroid. Such patients are still monitored regularly as the risk of relapse is still significant. Two approaches could improve the sensitivity, specificity and predictive value of TSH receptor antibody measurements. First, assays t h a t measure the ability of receptor antibodies to mimic the bioeffects of TSH, rather t h a n their ability to inhibit TSH binding to solubilized receptors, would be more likely to reflect disease activity at diagnosis and during treatment. Second, the '%lock-replace" method of t r e a t m e n t with a combination of high-dose carbimazole and replacement t r e a t m e n t with thyroxine should, theoretically, be more effective from the point of view of immunosuppression; indeed, in one study this strategy was shown to result in a higher frequency of posttreatment remissions (33).
TSH receptor purification and s u b u n l t structure Purification of the TSH receptor, characterization of its subunit structure and definition of its antibody and T lymphocyte receptor epitopes are objectives fundamental to understanding the pathogenesis of autoimmune thyroid disease. Despite attempts over the past few years to characterize the structure of the TSH receptor by various techniques including chemical crosslinking (34-37) and photo-affinity labelling (38) of 125I-TSH, immunoprecipitation using Graves' immunoglobulins (25,39) or monoclonal antibodies (40), target size analysis (41) and affinity purification (25,42-47), there is no firm, consensus structure for the receptor. This is in contrast to a number of other cell surface receptors t h a t are well characterized and cloned. Nevertheless, from the data available it is possible to conclude t h a t the TSH receptor is an oligomeric glycoprotein structure containing several subunits. In addition, there is evi-
45
HARRISON AND LEEDMAN
dence that the receptor contains a ganglioside component involved in signal transduction (48), identified by its reactivity with antireceptor monoclonal antibodies that mimic the bioeffects of TSH (40). A TSH binding subunit of M~ - 50,000 has been identified in thyroid tissue from a number of species (37,38,49), as well as in guinea pig fat cell membranes (47). In addition, nonreduced subunits of Mr - 70,000 and 35,000 have been reported by a number of investigators (25,35,42,44,47,49). Difficulties encountered with the purification and characterization of the TSH receptor can be attributed to a number of factors. These include the lack of a rich source of receptor, the marked lability of the receptor, its apparently complex nature and a Mr 50,000 subunit reported to be hydrophilic and easily dissociable. While Graves' immunoglobulins have been used to precipitate the receptor (25), their application to the identification of receptor subunits by gel electrophoresis and immunoblotting has been limited (50,51). This contrasts with the use of immunoblotting to detect nuclear autoantigens in various cell extracts and cDNA expression libraries (52). Very few membrane receptor molecules are, in fact, easy to identify by immunoblotting, perhaps because of their complex tertiary structure and conformational requirements for antibody binding. Nevertheless, as outlined below, modifications to conventional immunoblotting techniques permit the identification of TSH affinity-purified thyroid autoantigens, presumptively the subunits of the TSH receptor, using Graves' immunoglobulins. We have purified the human TSH receptor by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose (53,54). When TSH affinity-purified receptors were eluted in 3 M NaC1, a single protein of Mr 50,000 was recovered which bound 125I-TSH and could be precipitated by Graves' immunoglobulins. Alternatively, when affinity-purified receptors were eluted in sodium dodecyl sulphate (SDS) sample buffer and directly analyzed by SDS-polyacrylamide gel electrophoresis, three proteins of Mr 70,000, 50,000 and 35,000 were detected. After reduction with 100 mM dithiothreitol, only one protein of Mr - 25,000 was detected. The conventional immunoblotting technique (55) has been systematically modified in order to identify TSH receptor subunits. Essentially, SDS gels containing thyroid proteins are washed in Tris buffer containing 20% glycerol prior to transfer. SDS is omitted from the transfer buffer and a nylon membrane is used in place of nitrocellulose. These modifications enable putative TSH receptor subunits of Mr 70,000 and 50,000 to be identified after electrophoresis of either crude thyroid membranes or affinity-purified receptors. The results of affinity purification and immunoblotting enable us to draw the following conclusions: (i) The nonreduced human TSH receptor is an
46
oligomeric complex comprised of three different subunits ofM r 70,000, 50,000 and 35,000; the reduced receptor would appear to exist as a single subunit of Mr 25,000 which is disulphide-linked to form the high Mr forms. (ii) The Mr 50,000 subunit binds TSH and is linked by noncovalent, ionic bonds to at least one other binding subunit of the receptor complex. (iii) The 70,000 and 50,000 subunits contain epitopes that are recognized by Graves' immunoglobulins, thus confirming directly that the human TSH receptor is a target for Graves' immunoglobulins.
Future perspectives Further studies will define the stoichiometry and function of the disulphide-linked subunits that comprise the TSH receptor oligomer. The ability to purify TSH receptor subunits to homogeneity, and to microsequence picomolar concentrations of protein eluted from SDS gels, should ensure that primary amino acid sequence for the receptor will soon be available. This information can be used to design synthetic oligonucleotides with which to identify clones in cDNA libraries. It is unlikely that immunoscreening of cDNA libraries using autoantibodies will be as sensitive or practical, even though modified immunoblotting allows TSH receptor proteins to be identified by some Graves' immunoglobulins. The uncertainty about the structure of the TSH receptor will be resolved by the cloning of the receptor, opening the way for detailed structurefunction studies to clarify the immune mechanisms of thyroid diseases.
Acknowledgements The clinical and scientific contributions of Dr. T. W. H. Kay are gratefully acknowledged. Financial support was provided by the National Health and Medical Research Council of Australia. We thank Mrs. Margaret Thompson for secretarial assistance.
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22. Adams DD, Kennedy TH. Occurrence in thyrotoxicosis of a gamma globulin which protects LATS from neutralisation by an extract of thyroid gland. J Clin Endocrinol Metab 1967; 27: 173-7. 23. Smith BR, Hall R. Thyroid stimulating immunoglobulins in Graves' disease. Lancet 1974; ii: 427-31. 24. Shewing G, Rees Smith B. An improved radioreceptor assay for TSH-receptor antibodies. Clin Endocrinol 1982; 17: 409-17. 25. Heyma P, Harrison LC. Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins. J Clin Invest 1984; 74: 1090-7. 26. DeBruin TWA, Braverman LE, Brown RS. Further evaluation of an immunoprecipitation assay for TSHreceptor autoantibodies in Graves' disease. Metabolism 1986; 35: 1101-5. 27. Ginsberg J, Von Westarp C. Clinical applications of assays for thyrotropin-receptor antibodies in Graves' disease. Can Med Assoc J 1986; 134: 1141-7. 28. Ratanachaiyavong S, McGregor AM. Immunosuppressive effects of antithyroid drugs. Clin Endocrinol Metab 1985; 14: 449-66. 29. Kendall-Taylor P. Are antithyroid drugs immunosuppressive? Br Med J 1984; 288: 509-10. 30. Weetman AP, McGregor AM, Hall R. Evidence for an effect of antithyroid drugs on the natural history of Graves' disease. Clin Endocrinol 1984; 21: 163-72. 31. Wilson R, McKillip JH, Henderson N, Pearson DW, Thomson JA. The ability of the serum thyrotropin receptor antibody (TRAb) index and HLA status to predict long-term remission of thyrotoxicosis following medical therapy for Graves' disease. Clin Endocrinol 1986; 25: 151-6. 32. Kasagi K, Konishi J, Arai K. A sensitive and practical assay for thyroid-stimulating antibodies using crude immunoglobulin fractions precipitated with polyethylene glycol. J Clin Endocrinol Metab 1986; 62: 85562. 33. Romaldini JH, Bromberg N, Werner RS. Comparison of effects of high and low dosage regimens of antithyroid drugs in the management of Graves' hyperthyroidism. J Clin Endocrinol Metab 1983; 57: 563-70. 34. Buckland PR, Rickards CR, Howells RD, Rees Smith B. Thyrotropin cross-links to the thyrotropin receptor through both ~ and ~ subunits. Biochem J 1986; 235: 879-82. 35. Gennick SE, Thomas CG Jr, Nayfeh SN. Characterisation of the subunit structure of the thyrotropin receptor in the FRTL-5 rat thyroid cell line. Endocrinology 1987; 121: 2119-30. 36. McQuade R, Thomas CG Jr, Nayfeh SN. Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the ~ and ~ subunits of thyrotropin are crosslinked to the receptor. Arch Biochem Biophys 1987; 252: 409-17. 37. Remy JJ, Salamero J, Charreire J. Covalent crosslinking of thyrotropin to its receptor on cloned hybrid human thyroid cells (GEJ). Endocrinology 1987; 121: 1733-41. 38. Kajita Y, Rickards CR, Buckland PR, Howells RD, Rees Smith B. A structure for the porcine TSH receptor. F E B S Lett 1984; 181: 218-22. 39. Parkes AB, Kajita Y, Buckland PR. Immunoprecipitation of TSH-receptor complexes. Clin Endocrinol 1985; 22: 511-20.
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HARRISON AND LEEDMAN 40. Chan J, Santisteban P, deLuca M, Isozaki O, Grollman E, Kohn L. TSH receptor structure. Acta Endocrinol 1987; Suppl. 281: 166-72. 41. Nielsen TB, Totsuka Y, Kempmer ES, Field JB. Structure of the thyrotropin receptor and thyroid adenylate cyclase system as determined by target analysis. Biochemistry 1984; 23: 6009-16. 42. Tate RL, Holmes JM, Kohn LD, Winand RJ. Characteristics of a solubilized thyrotropin receptor from bovine thyroid plasma membranes. JBiol Chem 1975; 250: 6527-33. 43. Drummond RW, McQuade R, Grunwald R, Thomas CG Jr, Nayfeh SN. Separation of two thyrotropin binding components from porcine thyroid tissue by affinity chromatography. Characterization of high and low affinity sites. Proc Natl Acad Sci USA 1982; 79: 2202-6. 44. Koizumi Y, Zakarija M, McKenzie JM. Solubilization, purification and partial characterization of thyrotropin receptor from bovine and human thyroid glands. Endocrinology 1982; 11{}: 1381-91. 45. Iida Y, Konishi J, Kasagi K. Partial purification and properties of the TSH receptor from human thyroid plasma membranes. Acta Endocrinol 1983; 103: 198204. 46. Krees BC, Spiro RG. Studies on the glycoprotein nature of the thyrotropin receptor: interaction with lectins and purification of the bovine protein with the use of Bandeiraea (Griffonia) simplicifolia I affinity chromatography. Endocrinology 1986; 118: 974-9. 47. Iida Y, Amir SM, Ingbar SH. Stabilization, partial
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