Exp. Clin. Endocrino!. Vo!. 97, No. 2/3, 1991, pp. 153-159

J. A. Barth, Leipzig

Section on Cell Regulation, Laboratory of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Hea!th, Bethesda/USA

T. Aic&izu, M. SMI, S. IKuYAMA, S. Kosuu, K. TMiA1

and L. D. KOHN

With 1 Figure

Summary. The c!oning approaches of the past two years have opened new doors to the pursuit of our understanding Basedow's disease. The cloning of the TSH receptor is the most dramatic step; nevertheless, all the proteins mentioned in the following appear to be important molecules in the bioactivity of the thyroid cell and are implicated as autoantigeils.

Introduction The signs and symptoms of Basedow's disease are believed to be caused by circulating

autoantibodies to the thyrotropin (TSH) receptor; yet how or why these antibodies develop is still unknown. It is not clear whether this is a thyroid disease or one of abnormal lymphocyte control, what relationship exists between TSH receptor autoantibodies, exophthalmos, and pretibial myxedema, or why there is no simple correlation between goiter and hyperfunction. To resolve some of these questions, our laboratory developed monoclonal antibodies to the TSH receptor (Kohn et al., 1986). We now report on complementary studies on cloning of the TSH receptor and its interaction with these antibodies, the definition of other proteins interacting with the TSH receptor, and the identification, by a cloning approach using patient IgGs, of other autoantigens potentially important to disease expression and autoimmunity.

I. Cloning of the TSH Receptor and Relationship to Receptor Autoantibodies

The cloning approach to define the structure of the TSH receptor has been based on the predicted relationship between receptors for TSH, luteotropin (LII) or chorionic gonadotropin (CG) (Kohn, 1978) and/or the observation that receptors with G-protein interactions have highly homologous transmembrane domains (Lefkowitz and Caron, 1988). The present studies (Akamizu et al., 1990) use the rat FRTL-5 cell TSH receptor cloned in our laboratory (Fig. 1); it is 90o homologous with the human receptor (Libert et al., 1989; Nagayama et al., 1989; Misrahi et al., 1990) and known to react with most human TSH receptor autoantibodies.

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The TSH Receptor in Autoimmune Basedow's Disease

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The TSH receptor cDNA contains a long open reading frame encoding a protein comprised of 764 amino acids, Mr approximately 86,500. The first in-frame ATG is followed by a hydrophobic sequence (Fig. 1, first 21-23 residues) which is a signal peptide important for processing but not TSE! binding. There is a long hydrophilic, extracellular domain followed by a region with 7 hydrophobic, membrane spanning domains (Fig. 1, boxed and numbered). The hydrophilic region contains 5 potential N-linked glycosylation sites (Fig. 1, underlined); the transmembrane region contains one potential protein kinase C phosphorylation site (Fig. 1, broken underlined). The TSH receptor is 64 amino acid residues longer than the LH/CG receptor (Fig. 1) because the extracellular region has one peptide (Fig. 1, bold) not present in the LH/CG glycosylation site is more critical to TSH binding than the unique peptide. Conversely, the

unique peptide has a more critical immunogenic domain. TSH can bind to either the glycosylated or nonglycosylated protein (Akamizu et al., 1990). The gene for the mouse TSH receptor, Tshr, has been assigned to chromosome 12. Linkage relationships among genes on mouse chromosome 12 are conserved on human chromosome 2 and 14. The human TSHR gene was mapped to chromosome 14. Interestingly, the thyroid peroxidase gene also maps to mouse chromosome 12 but is on human chromosome 2 (Akamizu et al., 1990). Northern analyses identifies two mRNA species in rat FRTL-5 thyroid cells, 5.6 and 3.3 kb in size; the 5.6 kb transcript is larger primarily because it contains a longer 3' noncoding region. The same two species of mRNA were barely detected in rat ovary and were not detected in rat testis, brain, liver, lung, or spleen. TSH down-regulates expression of

this gene in FRTL-5 cells. Down regulation is rapid, 3 to 4 fold 8 hours after TSE! challenge, TSH concentration dependent, duplicated by cholera toxin, forskolin, or 8-bromo cAMP, and accompanied by a 60% decrease in TSH binding whether TSH, cholera toxin, forskolin, or 8-bromo AMP is the agent (Akamizu et aI., 1990). As anticipated from the above, thyroid stimulating antibody (TSAb) preparations from patients with Basedow's disease down-regulate TSHR mRNA expression levels. 1gO preparations from patients with primary hypothyroidism, which have potent thyrotropin binding inhibiting antibodies (TBIAbs), but no TSAb activity nor the ability to decrease basal cAMP levels, increase TSHR mRNA levels (Akamizu et al., 1990). The different action of the TBIAb and TSAb on TSH receptor gene expression indicates that the antibodies recognize different epitopes of the TSH receptor, consistent with conclusions from mixing studies using monoclonal antibodies to the TSE! receptor (Akamizu et al., 1990; Kohn et al., 1986).

Reactivity of both types of antibodies with FRTL-5 cells is associated with the presence of the TSH receptor in the cell. Thus, TSAbs and TBIAbs, monoclonal or patient preparations, react with FRTL-5 rat thyroid cells, as does TSH, but not to FRT rat thyroid cells. FRT cells, a continuously growing line which like FRTL-5 cells are derived from Fisher rats, have an apparently normal adenylate cyclase complex sensitive to cholera toxin and forskolin but no expressed TSE! mRNA (Akamizu et al., 1990). In sum current data establish that the multiplicity of monoclonal antibodies described in previous studies interact with the receptor and confirm that they interact with different epitopes which encode, in turn, different bioactivities. Studies in progress should be able to map these epitopes more definitively as well as define the role of the carbohydrate moieties on the receptor which appear important for full expression of TSAb

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T. AKAMIZU et al., Autoimmunity and TSH Receptor 155

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activity. They support conclusions concerning the multiplicity of TSH receptor antibodies, the multiplicity of signals, and the multiplicity of individual activities with respect to function, growth, and nonthyroidal actions. IL Microsequencing Suggests the TSH Receptor is a Complex of Several Proteins

These results in no way explain why multiple laboratories, using a wide range of

preted to suggest a TSH receptor with multiple subunits, modified by proteolytic degradation, and/or undergoing processing. They did not, however, exclude the possible existence of more than one TSH receptor or binding protein, coupled to different signals or actions. They did not eliminate the possible existence of receptor associated proteins of potential importance to its structure, function, and autoimmunity. A microsequencing approach has been used to resolve these possibilities (Akamizu et al., 1989). Radiolabeled FRTL-5 thyroid cell proteins were solubilized, "affinitypurified" on TSH-Sepharose, identified by SDS PAGE, and microsequenced. Thus far in these studies, two proteins, 43 and 70 Kd, which exhibit TSH-dependent bonding to TSHSepharose, have been identified. The former is gamma-actin; the latter is a member of the 70 Kd heat shock protein family. The identification of the 43 kd protein as an actin was confirmed by Western blotting, reaction with antiactin and immunostaining. Identification of the 70 kd protein as a Hsp7O family member was supported by showing that it was able to specifically bind ATP, a characteristic of heat shock 70 proteins. Both proteins appear to be associated with the TSH receptor in the membrane and/or in its proximity. Thus, one of the monoclonal antibody reactive with the TSH receptor, 52A8, identified the 43 kd protein after Western blotting and immunostaining. A different anti-TSH receptor monoclonal, 11E8, immunoadsorbed the 7Okd protein. Im-

munoreactivity wasprevented by free TSH, but not albumin or insulin, and was not duplicated by substituting normal mouse IgG for monoclonal anti-receptor IgG. Monoclonal 52A8 and 1 1E8 are competitive agonists and antagonists of TSH, respectively (Kohn et al., 1986). Gamma actin, though a minor component of non-muscle actin, is a component of the

membrane-associated cytoskeleton which is linked to membrane clustering and endocytosis of TSH into FRTL-5 thyroid cells (Avivi et al., 1982). Proteins in the 70 kd fami-

ly interact with other proteins; this interaction is hydrophobic, ATP dependent, and argued to be important in membrane translocation and the proper folding of the protein (Lindquist, 1986); this possibility must be considered in the case of the TSH receptor. Hsp7O family members have also been shown to be important in the immune response. Thus the conserved nature of heat shock proteins has been argued to increase the probability of autoimmune Basedow's (De Groot and Quintans, 1989) and to the development of antireceptor autoantibodies. Proteins in the 70 kd family are known to be influenced by stress and developmental signals (Lindquist, 1986). The microsequencing approach thus suggests that the TSH receptor is a complex structure with associated proteins which may be important in its structure, function and autoimmunity. It is presumed that some TSH receptor monoclonal antibodies recognize

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techniques centered around TSH-binding, had identified a multiplicity of proteins which appeared to interact with the hormone (Chan et al., 1987). These results had been inter-

T. AKAMIZU et al., Autoimmunity and TSH Receptor

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these proteins because they are related by the three-dimensional array while others immunoprecipitate them as a part of a larger complex, since no direct interactions are evident on Western blots. III. Immunoscreening Identjfies Other Potentially Important Autoantigens in Basedow 'S Disease

An Autoantigen Related to Thyroid Cell Growth and Autoimmune Lupus

One autoántigen discovered this way, M 69,812, has two potential N-linked glycosylation sites and one hydrophobic region compatible with being a membrane spanning domain (Chan et al., 1989). The protein reacts with the sera of 60% of Basedow's patients tested, but not with normal sera similarly diluted. More importantly, it binds to Sepharose-TSH; this binding is inhibited by free TSH but not by free insulin, ACTH, prolactin, cholera toxin, or even hCG (Kohn et al., 1989 a, 1989 b). Transient transfection experiments confirmed that the protein could bind TSH and could, produced in excess, appear on the cell surface (Kohn et al., 1989a, 1989b; Allaway et al., 1990). In no case, however, was there an associated ability for TSH to increase cAMP levels. The TSH binding site has been located to residues 212 through 228, since this peptide inhibited the ability of TSH to both increase cAMP levels in FRTL-5 thyroid cells as well as increase tritiated thymidine incorporation into FRTL-5 cell DNA (Kohn et al., 1989 a, 1989 b). The TSH binding did, however, appear to be specific in that it was not inhibited by prolactin, albumin, thyroglobulin, insulin, or glucagon. The 70 K protein is however primarily present in the nuclei of transfected cells and is a DNA binding protein (Allaway et al., 1990). Further, separate evidence has been accumulated that this clone is the Ku autoantigen in some patients with Lupus (Reeves and Sthoeger, 1989) or the Ki antigen known to be related to cell growth and transformation.

Consistent with this, nuclear expression of Ki increases in parallel with increases in mitotic index of FRTL-5 cells stimulated with TSH. The presumptive functional chromosome, 22q1 1-13, is associated with the Sis proto-oncogene. In mice the gene is mapped to a single locus on chromosome 15 and also is associated with the Sis protooncogene. Of interest in this respect, recent studies have suggested there is a significant risk of exophthalmos in patients with P blood group by comparison to patients with Basedow's who do not have exophthalmos (Kendall-Taylor et al.,

1988). P blood group antigen is associated the alpha-4-Gal transferase converting paragloboside to the P1 antigen. Current studies (W. O. McBride, personal communication) indicate that it maps to the same locus 22q1 1-13 and is probably near and closely linked, but not identical to, the gene coding for the cDNA described in these studies.

Paragloboside and the P1 antigen are glycolipids which can be viewed as structural analogs of higher order gangliosides implicated in TSH receptor structure and function (Kohn, 1978).

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Immunoscreening of thyroid expression libraries with IgG preparations from patients with active Basedow's disease has identified several autoantigens other than the TSH receptor that may have relevance to disease expression.

Exp. Clin. Endocrinol. 97 (1991) 2/3

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The sum of these results thus raise the possibility that the 70 Kd protein is an important TSH-binding and DNA-binding autoantigen in Basedow's disease. Transcript and protein expression in thyroid cells increases with mitogen stimulation or transformation, suggesting the autoantigen had an important role in growth. It may also be a relevant autoantigen in exophthalmos patients. Since it is an autoantigen associated with Lupus. Further understanding of its role could provide important new understanding to the pathogenesis of autoimmunity in general.

A second clone characterized in this manner (Zarrilli et al., 1989) contained a highly conserved 1.05 Kb open reading frame. Its mRNA was present in human and rat thyroid cells and at lower levels in liver and differentiated myoblasts but not in lymphocytes such as 1M9 cells. The cDNA hybridized with a single copy genomic sequence on human

chromosome 10. The predicted amino acid sequence of this autoantigen exhibited a

strong homology with the mitochondrial ADP/ATP translocator and two other mitochondrial carrier proteins, the phosphate carrier and the hydrogen ion carrier. These results thus define, for the first time, a mitochondrial protein as an autoantigen in Basedow's disease. This autoantigen has been recognized to be important in autoim-

mune myocarditis and liver disease (Schultheiss et al., 1986) and is an important modulator of thyroid hormone action in mitochondria (Sterling, 1986). Autoantibodies to this antigen may contribute to the myopathy of Basedow's, perhaps explaining the failure to correlate thyroid hormone levels with myopathy.

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