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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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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RESULTS |
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
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
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
1 chain. Panel A, of 21 amino acids in
the region of homology (TSHR residues 23-43 and laminin 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 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
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.
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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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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.
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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.
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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.
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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).
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DISCUSSION |
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
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.