A Full Biological Response to Autoantibodies in Graves' Disease Requires a Disulfide-bonded Loop in the Thyrotropin Receptor N Terminus Homologous to a Laminin Epidermal Growth Factor-like Domain*

Chun-Rong Chen, Kunihiko Tanaka, Gregorio D. Chazenbalk, Sandra M. McLachlan, and Basil RapoportDagger

From the Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, California 90048

Received for publication, August 31, 2000, and in revised form, February 7, 2001

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INTRODUCTION
MATERIALS AND METHODS
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We observed amino acid homology between the cysteine-rich N terminus of the thyrotropin receptor (TSHR) ectodomain and epidermal growth factor-like repeats in the laminin gamma 1 chain. Thyroid-stimulating autoantibodies (TSAb), the cause of Graves' disease, interact with this region of the TSHR in a manner critically dependent on antigen conformation. We studied the role of the cluster of four cysteine (Cys) residues in this region of the TSHR on the functional response to TSAb in Graves' patients' sera. As a benchmark we also studied TSH binding and action. Removal in various permutations of the four cysteines at TSHR positions 24, 29, 31, and 41 (signal peptide residues are 1-21) revealed Cys41 to be the key residue for receptor expression. Forced pairing of Cys41 with any one of the three upstream Cys residues was necessary for trafficking to the cell surface of a TSHR with high affinity TSH binding similar to the wild-type receptor. However, for a full biological response to TSAb, forced pairing of Cys41 with Cys29 or with Cys31, but not with Cys24, retained functional activity comparable with the wild-type TSHR. These data suggest that an N-terminal disulfide-bonded loop between Cys41 and Cys29 or its close neighbor Cys31 comprises, in part, the highly conformational epitope for TSAb at the critical N terminus of the TSHR. Amino acid homology, as well as cysteine pairing similar to the laminin gamma 1 chain epidermal growth factor-like repeat 11, suggests conformational similarity between the two molecules and raises the possibility of molecular mimicry in the pathogenesis of Graves' disease.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

The thyrotropin receptor (TSHR)1 is the target of the immune system in Graves' disease, and autoantibodies directed against this antigen are the direct cause of clinical hyperthyroidism (reviewed in Ref. 1). Understanding the molecular interaction between TSHR autoantibodies and the TSHR is, therefore, important for advances in the diagnosis and therapy of Graves' disease, one of the most common organ-specific autoimmune diseases affecting humans. Studies of TSHR autoantibody-autoantigen interaction have been difficult because of the very low serum concentrations of the autoantibodies (2, 3) and because of the highly conformational nature of their epitopes. Although the binding sites for TSHR autoantibodies and TSH involve discontinuous segments throughout the TSHR ectodomain (4, 5), the N-terminal region of the ectodomain appears to be particularly important for the functional activity of TSHR autoantibodies (5-9).

Comparison of the primary amino acid sequences of the large ectodomains of the glycoprotein hormone receptors reveals a moderately conserved, central leucine-rich repeat region flanked by segments of much lower homology. The poorly conserved TSHR N-terminal region extends from amino acid residues 22 to 56 (the signal peptide being residues 1-21). This short (35-residue) region contains four of the eleven Cys residues (Cys24, Cys29, Cys31, and Cys41) in the large (397-residue) TSHR ectodomain, supporting the importance of secondary and tertiary structure in TSHR recognition by autoantibodies. Another indicator of the importance of conformation in the TSHR N terminus region is that TSHR autoantibodies and a mouse monoclonal antibody to this segment (3BD10; epitope within amino acid residues 25 to 51) interact reciprocally and exclusively with two different folded forms of the native molecule present in the same TSHR preparation (10).

A number of lines of evidence suggest that the four N-terminal cysteines form disulfide bonds with one another rather than with other Cys residues in the TSHR. First, from the current three-dimensional model of the TSHR leucine-rich repeat area (11) based on the known structure of ribonuclease A inhibitor (12), the cluster of four N-terminal cysteine residues is well separated from the seven other cysteines in the TSHR ectodomain. The large size and relatively rigidity of the TSHR leucine-rich repeat segment makes it very unlikely that the N-terminal cysteine cluster can form disulfide bonds with their far downstream counterparts (reviewed in Ref. 1). Second, the N-terminal cysteine cluster cannot form disulfide bridges with either of two cysteines in extracellular loops 1 and 2, because the latter are highly conserved among G protein-coupled receptors and themselves form a disulfide bond (13, 14). Finally, and most important, reduction of disulfide bonds in the TSHR abolishes the conformational epitope of monoclonal antibody 3BD10 (see above), this epitope containing the four N-terminal cysteines. Of the four cysteines, Cys41 is of particular interest in that its substitution or deletion abolishes TSH and TSHR autoantibody binding to the surface of transfected cells (8, 15, 16), reportedly without affecting trafficking and cell surface expression of the TSHR (17). An apparent paradox, however, is that deletion or mutation of any of the potential partners of Cys41 (Cys24, Cys29, or Cys31) is reported to have no effect on ligand or autoantibody binding (8).

In the present study we investigated the role of disulfide bonding among the Cys residues at positions 24, 29, 31, and 41 on the expression of TSHR functional activity in terms of responsiveness to autoantibodies in Graves' patients' sera. In these studies, we were guided by novel observations on the structural relationship between the N terminus of the TSHR and cysteine-rich EGF-like repeats in laminin.

    MATERIALS AND METHODS
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Construction and Expression of TSHR Mutants-- Mutations (C24S, C29S, C31S, C41S, C24S,C29S, C24S,C31S, C29S,C31S, and C24S,C29S,C31S) and a deletion (amino acids 22-30) were created in the TSHR cDNA in the mammalian expression vector pECE-NEO (18) using the QuickChange site-directed mutagenesis kit; Stratagene, San Diego, CA or, for the deletion, by the polymerase chain reaction using overlapping oligonucleotide primers and Pfu DNA polymerase (Stratagene). Note that although we have previously generated a C41S TSHR mutant (16), this mutation was in the 4-kilobase TSHR cDNA in which the 5'- and 3'-untranslated ends greatly reduce the level of TSHR expression (19). All mutants in the present study were, therefore, created in the TSHR with 5'- and 3'-untranslated regions deleted (19). The nucleotide sequences of the mutations and surrounding regions were confirmed by dideoxynucleotide sequencing. Plasmids were stably transfected with LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) into Chinese hamster ovary (CHO) cells cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum and antibiotics (penicillin, 100 units/ml; gentamicin, 50 µg/ml; and amphotericin, 2.5 µg/ml). Selection was performed with 400 µg/ml G418 (Life Technologies). Surviving clones (>100 per 100-mm diameter culture dish) were pooled and propagated for further study.

TSH Binding-- Stably transfected CHO cells were grown to confluence in 24-well culture plates. Cells were then incubated for 2.5 h at 37 °C in 250 µl of binding buffer (Hanks' balanced salt solution with 280 mM sucrose substituting for NaCl to maintain isotonicity and 0.25% bovine serum albumin) containing ~10,000 cpm 125I-TSH (kindly provided by B.R.A.H.M.S., Berlin, Germany) in the presence or absence of increasing concentrations of unlabeled bovine TSH (Sigma). The cells were rinsed carefully 3 times with prechilled binding buffer (4 °C), solubilized with 0.5 ml of 1 N NaOH, and radioactivity was measured in a gamma counter. The Kd values were calculated by Scatchard analysis (20).

TSH Stimulation of Intracellular cAMP-- Transfected CHO cells, grown to near confluence in 24-well plates, were incubated for 2 h at 37 °C in Ham's F-12 medium containing 1% bovine serum albumin and 1 mM isobutylmethylxanthine with or without added bovine TSH (Sigma). The medium was then aspirated, and intracellular cAMP was extracted with 95% ethanol, evaporated to dryness, and resuspended in 0.5 ml of 50 mM sodium acetate buffer, pH 6.2. After acetylation (20 µl of triethlamine and 10 µl of acetic anhydride) cAMP was measured by radioimmunoassay using cAMP, 2'-O-succinyl-[125I]iodotyrosine methyl ester (PerkinElmer Life Sciences) and a rabbit anti-cAMP antibody (Fitzgerald, Concord, MA).

Thyroid-stimulating Antibody (TSAb) Assay-- Crude IgG fractions of sera from patients with Graves' disease were precipitated with polyethylene glycol 4,000 and resuspended in modified Hanks' buffer lacking NaCl (21) and supplemented with 20 mM HEPES, pH 7.4, 1 mM isobutylmethylxanthine, and 0.3% bovine serum albumin. Transfected CHO cells, grown to confluence in 96-well plates, were incubated for 3 h at 37 °C in this buffer containing patients' IgG. The plates were then frozen for 1 h at -80 °C (without aspirating the medium), thawed, and total (intracellular and released) cAMP was measured by radioimmunoassay as described above.

Flow Cytometry-- Intact CHO cells transfected with the wild-type and mutated TSHR were harvested from 10-cm diameter dishes using 1 mM EDTA, 1 mM EGTA in Dulbecco's phosphate-buffered saline, pH 7.5. After washing twice with phosphate-buffered saline containing 10 mM HEPES, pH 7.4, 2% fetal bovine serum, and 0.05% NaN3, the cells were incubated for 1 h at 4 °C in 100 µl of the same buffer containing 1 µg of mouse monoclonal antibody 2C11 to the TSHR ectodomain (Serotec, Oxford, United Kingdom) (22). As a negative control, cells were incubated in 100 µl of normal mouse sera (1:100). After rinsing, the cells were incubated for 45 min with 100 µl of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:100) (Caltag, Burlingame, CA), washed, and analyzed using a Beckman FACScan flow cytofluorimeter. Cells stained with propidium iodide (1 µg/ml final concentration) were excluded from analysis.

For selected TSHR mutants, intracellular receptors were also detected in permeabilized cells. For this purpose, before adding the 2C11 monoclonal antibody, the cells were first fixed for 10 min in chilled 1% paraformaldehyde, resuspended in saponin buffer (0.01 mM phosphate-buffered saline, pH 7.4, containing 0.5% bovine serum albumin, NaN3, and 0.1% saponin; Sigma). The cells were then processed as described above except that they were rinsed in buffer containing saponin and incubated for 45 min with the goat anti-mouse IgG in saponin buffer.

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INTRODUCTION
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Insight into the TSHR N-terminal Structure from Homology Searches with Other Proteins-- The N-terminal region of the TSHR upstream of the leucine-rich repeats bears little homology to the other members of the glycoprotein hormone receptor family. However, we searched the protein data bases for homology with other, unrelated proteins. Not recognized previously, homology was found between TSHR amino acid residues 23-43 and amino acid residues 979-997 of the human laminin gamma 1 chain (23) (accession number 126369). Of 21 TSHR amino acid residues, 10 are identical, and 2 are conserved (Fig. 1A). The corresponding region of the laminin gamma 1 chain contains the C terminus of EGF-like repeat 10 and the N terminus of EGF-like repeat 11. 


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Fig. 1.   Amino acid homology between the N terminus of the TSHR and EGF-like repeats in the human laminin gamma 1 chain. Panel A, of 21 amino acids in the region of homology (TSHR residues 23-43 and laminin gamma 1 chain residues 979-997), 10 residues are identical (vertical line), and 2 are conserved (colon). The dashes indicate gaps in the laminin sequence. Disulfide bonding in the laminin gamma 1 chain EGF-like repeats are indicated by the brackets. EGF-like repeats 10 and 11 include laminin residues 935-982 and 983-1030, respectively (parentheses). Panel B, based on the known disulfide bonding of the laminin gamma 1 chain EGF-like repeats, we hypothesized that TSHR Cys41, known to be critically important in TSH and TSHR autoantibody binding, was likely to be linked with either Cys29 (left) or with Cys31 (right). Because, from the EGF-like structure, a Cys41-Cys29 bond was more likely, we hypothesized that the reciprocally exclusive interaction between TSHR autoantibodies and mouse mAb 3BD10 to this region could involve a Cys41-Cys29 bond and a Cys41-Cys31 bond, respectively.

From the known three-dimensional structure of EGF-like repeats in other regions of the laminin molecule (24, 25), we hypothesized that the critical conformational structure of the TSHR N-terminal region interaction with autoantibodies was unlikely to involve Cys24 but was more likely to involve disulfide bonding between Cys41 and Cys29 or, alternatively, between Cys41 and Cys31 (Fig. 1B). As mentioned above, this region of the TSHR contains the epitope for a mouse monoclonal antibody (3BD10). However, in a purified, native TSHR ectodomain preparation, this epitope is only present in molecules not recognized by human autoantibodies. Conversely, an alternatively folded form of the molecule in the same preparation only interacts with human autoantibodies and not with mouse monoclonal antibody (mAb) 3BD10 (10). We, therefore, considered that these reciprocal interactions could be explained by conformational differences resulting from altered disulfide bonding (Fig. 1B).

TSH Binding to and Activation of TSHR with N-terminal Cysteine Replacements or Deletions-- To understand better the role in receptor conformation of two putative disulfide bonds among the four Cys residues in the TSHR N terminus, we substituted or deleted these Cys residues in different permutations (Fig. 2). The rationale for these mutations was that removal of either cysteine in a linked pair should produce the same "phenotype," either altered or unchanged ligand and antibody binding. As observed previously (8, 16), replacement of Cys41 totally abolished TSH binding to intact cells stably transfected with the mutant receptor (Table I). However, against the expectation that mutation of a cysteine coupled with Cys41 should also lead to a defective receptor, the individual substitutions of Cys24, Cys29, and Cys31 were all associated with high affinity TSH binding (Kd range of 1.5-3.6 × 10-10 M) (Table I).


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Fig. 2.   Schematic representation of amino acid substitution and deletions involving the cluster of cysteine residues in the TSHR N terminus. Amino acid residue 22 becomes the N terminus after removal of the signal peptide (residues 1-21). Where indicated, Cys residues were replaced with Ser. Deleted 22-30 consists of a TSHR with an N terminus at Cys31.

                              
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Table I
TSH binding affinity and TSHR level of expression for stably transfected cell lines

In a second series of mutations guided by homology with the laminin EGF-like domains (Fig. 1B), we replaced or deleted multiple pairs of Cys residues in the TSHR N terminus not involving Cys41, as well as all three cysteines other than Cys41 (Table I). Paired replacements of Cys24 and Cys31 and Cys29 and Cys31, as well as deletion of residues 22-30 (which includes Cys24 and Cys29), also did not prevent the cell surface expression of TSHR capable of high affinity TSH binding (Table I). On the other hand, the simultaneous replacement of Cys24, Cys29, and Cys31 produced a TSHR that bound very poorly to TSH at a level too low to determine affinity.

The cAMP response of the TSHR mutants to TSH stimulation was consistent with the TSH binding data. All mutants responded vigorously to TSH with the exceptions of those with the C41S and the triple (C24S,C29S,C31S) substitutions that had very small responses (Fig. 3).


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Fig. 3.   TSH stimulation of intracellular cAMP levels in CHO cells stably expressing TSHR N-terminal cysteine mutants. Monolayers of CHO cells expressing the indicated TSHR mutants (see Fig. 2) were incubated without (Basal) or with TSH (100 milliunits/ml). Cellular cAMP was extracted and measured by radioimmunoassay (see "Materials and Methods"). Each bar represents the mean ± S.D. of duplicate wells, and each well was assayed in duplicate. WT, wild-type.

Antibody Recognition of TSHR N-terminal Cysteine Mutants on the Cell Surface-- Previously, it was reported that the TSHR with a C41S substitution expressed normally on the cell surface (17). Because of the important implications of this observation on the role of Cys41 in the TSH binding site, we examined cell surface expression of the TSHR N-terminal cysteine mutants independent of ligand binding. For this purpose we performed flow cytometry on stably transfected CHO cells using a mouse mAb (2C11) to a distal region of the molecule (22, 26). Consistent with the TSH binding and functional data (see Table I and Fig. 3), cell surface expression was comparable for the wild-type TSHR and all mutant receptors except for those with the C41S and the combined C24S, C29S, and C31S substitutions (Fig. 4). Flow cytometry with mAb 2C11 on permeabilized cells revealed levels of TSHR expression in these two mutants similar to that of the wild-type TSHR (Fig. 5, clear histograms). Therefore, absent or minimal cell surface expression of TSHR with the C41S and the combined C24S, C29S, and C31S replacements was the result of faulty trafficking rather than of a diminished level of expression.


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Fig. 4.   Flow cytometric detection of the TSHR on the surface of intact, stably transfected CHO cells. The wild-type (WT) TSHR and TSHR mutants described in Fig. 2 were detected with mouse mAb 2C11 whose epitope is near the C terminus of the TSHR ectodomain and, therefore, uninfluenced by the mutations at the N terminus. Panels A and B show two experiments involving different TSHR mutants. For each cell line, controls (not shown) included omission of the first antibody (2C11), omission of both first and second antibodies, and use of normal mouse serum in place of first antibody.


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Fig. 5.   Flow cytometric detection of TSHR in permeabilized, stably transfected CHO cells. Untransfected cells (black histograms) and cells expressing either the wild-type (WT) TSHR or the indicated TSHR mutants (overlapping clear histograms) were pre-treated with saponin (see "Materials and Methods"), thereby allowing detection of intracellular TSHR. TSHR-C24S,C29S,C31S and TSHR-C41S, which have absent or minimal cell surface expression, are clearly expressed intracellularly to a similar degree as the wild-type TSHR.

Mouse mAb 3BD10, whose epitope includes the N-terminal region with the Cys residue cluster, was generated by immunization with a soluble, truncated TSHR ectodomain (10). As described above, this molecule exists in two folded forms, with 3BD10 recognizing only the form that is not bound by TSHR autoantibodies (and vice versa). 3BD10 does not bind to the full-length TSHR expressed on the cell surface (27). Two possible reasons for this phenomenon are as follows: (i) the 3BD10 epitope is sterically inaccessible with the membrane-associated TSHR, and (ii) the 3BD10 epitope on the membrane-associated TSHR is in the form recognized only by autoantibodies. In the latter case, it was possible that rearrangement of disulfide bonds could restore recognition by 3BD10 of TSHR on the cell surface. For example, removal of Cys29 could force formation of a Cys41-Cys31 bond, or removal of Cys31 could force formation of a Cys41-Cys29 bond (Fig. 1B). However, on flow cytometry, 3BD10 recognition of cell surface TSHR did not emerge with any of the cysteine variants in the receptor N terminus (data not shown). These data favor the alternative that the 3BD10 epitope is obscured in the cell-surface TSHR.

Divergent Effects of N-terminal Cysteine Mutations on TSH and TSAb Action-- Finally, we explored the effect of mutations in the TSHR N-terminal cysteine cluster on autoantibody activation of the TSHR. With the exception of a few uniquely potent sera, serum TSHR autoantibodies cannot be detected by flow cytometry because of their extremely low level in serum, and because the TSHR is "sticky" and produces an elevated background even with sera from normal individuals lacking TSHR autoantibodies (3). On the other hand, TSAb to the TSHR can be detected in the majority of Graves' sera by their ability to activate the TSHR with consequent cAMP generation. The functional activity of panels of Graves' sera was tested on the mutant TSHR and compared with the activity of the same sera on the wild-type TSHR. More than one panel of Graves' sera was required, because insufficient volumes were available to test all TSHR mutants with the same sera.

Three patterns of response were observed with cells expressing the N-terminal cysteine mutants. First, consistent with absent or poor cell-surface expression of TSHR mutants C41S and C24S,C29S,C31S, TSAb activity was not detected when Graves' IgG were incubated with cell lines stably transfected with these receptors (Fig. 6, A and B). Second, and conversely, TSAb activity with cells expressing C24S and C24S,C31S was comparable with values obtained with the wild-type TSHR. In a third pattern, TSHR-C29S, TSHR-C31S, and TSHR-C29S,C31S, as well as the receptor lacking Cys24 and Cys29, elicited cAMP responses lower than those observed with the wild-type TSHR.


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Fig. 6.   TSAb activity assayed using CHO cells expressing the indicated TSHR. CHO cells stably transfected with the wild-type (WT) TSHR or TSHR mutants were incubated with IgG prepared from sera of patients with Graves' disease, and total intracellular and released cAMP production was measured by radioimmunoassay (see "Materials and Methods"). Because the large volume of serum required in these studies precluded assaying all cell lines with the same serum, we tested different panels of 11, 10, and 10 Graves' sera. To facilitate comparison between cell lines and as a benchmark, the wild-type TSHR was repeated with each panel of sera, and TSHR-C24S,C31S was also repeated (A and B). TSAb assays included serum from normal individuals lacking TSHR autoantibodies. Basal cAMP values obtained with normal sera were defined as 100% (horizontal lines). Each bar represents the mean ± S.D. of cAMP values determined in duplicate wells, and each well was assayed in duplicate. Note the different scales on the ordinates, reflecting the varying potency of the TSAb in the different sera.

TSHR lacking Cys24 and Cys29 has the N-terminal 9 amino acids deleted (residues 22-30, the signal peptide being residues 1-21). We initially performed this deletion because of a previous report that this region could be deleted without affecting the response to TSHR autoantibodies (8). However, it was possible that the deletion altered the TSHR autoantibody epitope(s) independent of the presence or absence of disulfide bonds involving Cys residues within the region. We, therefore, generated a TSHR mutant C24S,C29S in which only these two residues were involved. The level of expression of this receptor on the cell surface was similar to that of the wild-type TSHR on flow cytometry with monoclonal antibody 2C11 (data not shown), as was its affinity for TSH (4 × 10-10 M). Indeed, with TSHR-C24S,C29S (unlike with the TSHR with residues 22-30 deleted) the cAMP response to TSHR autoantibodies was not reduced relative to that with the wild-type TSHR (Fig. 6C).

    DISCUSSION
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The cysteine-rich N terminus of the TSHR (amino acid residues 22-57, upstream of the leucine rich repeats) is an important component of the epitope for thyroid-stimulating autoantibodies that cause Graves' disease (reviewed in Ref. 1). The structural segregation of the four N-terminal Cys residues from the other seven Cys residues in the ectodomain suggested that any disulfide bonds involving the four upstream cysteines were among themselves (see Fig. 4 in Ref. 1). Proof of at least one disulfide bond involving the four N-terminal cysteines was obtained with mouse mAb 3BD10, which has a conformational epitope encompassing residues 25-51 in the TSHR N terminus (10). Our present evidence that disulfide bonding between TSHR Cys41 and either Cys29 or Cys31 (but not Cys24) is important for TSHR autoantibody function is consistent with the known three-dimensional structure of cysteine-rich EGF-like repeats in the laminin gamma 1 chain (24, 25). Remarkable homology exists between TSHR amino acid residues 23-43 and this region of EGF (10 identical and 2 conserved amino acids).

The ability of two different ligands (TSH and TSHR autoantibodies) to activate the same receptor provides a powerful and necessary control for interpreting mutations introduced into the TSHR. Regarding TSH binding and action, TSHR amino acid Cys41 is the key cysteine in that it is the only one in the cluster of four whose mutation alters TSH binding. In principle, removal of the cysteine paired with Cys41 should produce a similar phenotype. However, only the simultaneous mutation of all three potential Cys41 partners reproduces the C41S phenotype. These surprising data indicate that Cys41 has the option of pairing with any one of Cys24, Cys29, or Cys31 and still producing a functional receptor with high affinity TSH binding. Moreover, our data do not support the previous concept that mutation of Cys41 is compatible with normal TSHR trafficking and expression on the cell surface (8, 17). To the contrary, neither TSHR-C41S nor TSHR-C24S,C29S,C31S (its phenotypic pair) reaches the cell surface, thereby explaining the lack of TSH binding and TSH functional activity. On the other hand, TSHR cell surface expression appears to be dependent on a disulfide-bonded loop in the TSHR N-terminal region. Examination of TSH binding to the mutant TSHR retained within the cell would be of interest. However, it is not possible to detect intracellular TSH ligand binding even to the wild-type TSHR.

Regarding TSAb functional activity (the affinity or binding of these autoantibodies cannot be measured), most informative were the three double cysteine mutations not involving Cys41 (TSHR-C24S,C29S, TSHR-C24S,C31S, and TSHR-C29S, C31S). Mutation of each of these cysteine pairs would force Cys41 to couple with Cys31, Cys29, or Cys24, respectively, to form the N-terminal loop necessary for TSHR cell surface expression. All retained high affinity TSH binding and function similar to the wild-type TSHR, yet only TSHR-C29S,C31S displayed a reduced TSAb response relative to the wild-type receptor. These data suggest that Cys41 can pair with either Cys29 or its close neighbor Cys31 to provide the optimal conformation for the TSAb epitope (Fig. 1B). These results, as well as the lack of effect of the single Cys24 mutation, are also consistent with the homology comparison with laminin suggesting that TSHR Cys24 is part of a separate domain. Reduced TSAb responses in TSHR with single substitutions at Cys29 or Cys31 are difficult to explain. In principle, such single mutations would permit Cys41 pairing with either Cys31 or Cys29, respectively, without impairing TSAb function. It is possible that conformational changes and an odd number (three) of remaining Cys residues may lead to abnormal disulfide bonding, such as Cys41-Cys24.

An interesting comparison with the present data is provided by previous evidence obtained with chimeric lutropin-TSHR receptors. A chimeric substitution of TSHR amino acid residues 25-30 (SSPPCE) with the homologous residues of lutropin (PEPCD) also attenuated TSAb activity despite a normal TSH response (28) but with one interesting difference. In the chimeric receptor study, the diminished response was observed only with half of the 10 Graves' sera whereas in the present study, the response was diminished in all sera tested. One possible reason for this difference is that, in the chimeric receptor study, TSHR residue Cys29 is retained, because it is conserved within the lutropin receptor. However, the altered proline alignment (including a gap of one amino acid residue) suggests a significant conformational difference between this region of the two receptors. These data support the notion that the amino acids adjacent to Cys29 contribute to this component of the TSAb epitope, as does the present finding of reduced TSAb activity in the TSHR with amino acid residues 22-30 deleted.

Regarding mouse mAb 3BD10, our hypothesis that the exclusive and reciprocal interaction between it and TSHR autoantibodies was consequent to alternative (e.g. Cys41-Cys29 and Cys41-Cys31) disulfide bonding was not confirmed. 3BD10 recognized none of the TSHR mutants created in the present study. However, although not confirmed, neither can the hypothesis be excluded. 3BD10 was generated against a secreted form of truncated TSHR ectodomain, and this antibody does not recognize the wild-type, membrane-associated TSHR. It is possible that its epitope, available on a soluble TSHR fragment, is inaccessible because of steric hindrance caused by the relationship of the TSHR ectodomain to the plasma membrane.

Finally, besides homology with a module in laminin, striking homology (8/11 amino acids identical, 2 conserved) also exists between residues 23-33 (GCSSPPCECGQ) in the critical TSHR N-terminal region and residues 1103-1113 of host cell factor (GCSNPPCETHE; accession number P51610), a protein that interacts with a Herpes simplex virus protein upon lytic infection of permissive cells. Further studies are necessary to investigate the possibility of cross-reactivity with laminin or with other as yet unrecognized antigens, including infectious organisms (reviewed in Ref. 29), in the pathogenesis of Graves' disease.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Joachim Struck of B.R.A.H.M.S., Berlin, Germany for generously providing radiolabeled TSH.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK19289.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Cedars-Sinai Medical Center, 8700 Beverly Blvd., Suite B-131, Los Angeles, CA 90048. Tel.: 310-423-0555; Fax: 310-423-0221; E-mail: rapoportb@cshs.org.

Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M008001200

    ABBREVIATIONS

The abbreviations used are: TSHR, thyrotropin receptor; TSH, thyrotropin; EGF, epidermal growth factor; CHO, Chinese hamster ovary; TSAb, thyroid-stimulating autoantibodies; mAb, monoclonal antibody.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

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