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INTRODUCTION |
The thyrotropin receptor
(TSHR)1 plays a key role in
thyroid growth and function (reviewed in Refs. 1 and 2). This
receptor is the target of stimulating or blocking autoantibodies
produced in patients with autoimmune diseases (reviewed in Refs. 3 and 4).
The TSHR was initially cloned by cross-hybridization with the
luteinizing hormone receptor (5), or by polymerase chain reaction using
degenerate primers (6-8). Expression of the cloned receptor in
Escherichia coli allowed its use as an immunogen to prepare
monoclonal antibodies. These were used for immunoblotting and
immunoprecipitation experiments which showed that the TSH receptor in
human thyroid membranes underwent a post-translational cleavage event
yielding two subunits: a ~53-kDa
extracellular subunit and a
~38-kDa broad
membrane spanning subunit. The subunits are held
together by disulfide bridges (9). This maturation is unique among G
protein-coupled receptors.
In human thyroids, cleavage of the TSHR is almost complete. By
contrast, in heterologous transfected cells monomeric uncleaved precursors may also be observed. They consist either of the mature ~120-kDa uncleaved receptor present on the cell surface or of a
~95-kDa mannose-rich precursor which can be identified by its sensitivity to specific endoglycosidases (10). The latter form accumulates in the endoplasmic reticulum.
The cleavage of the TSHR has been disputed by different authors
(11-16). Indeed, due to the low concentration of the TSHR in thyroid
tissue, nearly all studies have been performed in transfected cells
where the monomeric precursors and specially the mannose-rich form have
in many cases been mistaken for the mature receptor. Recently, however,
a consensus has emerged and most authors now agree on the existence of
a physiological cleavage of the TSH receptor (17, 18).
Very recently the group of Rapoport reported the existence of two
cleavage sites in the TSHR extracellular region, suggesting the
existence of a third polypeptide fragment ("C peptide" by homology
with insulin) released during intramolecular cleavage of the receptor
into two subunits (19-21).
In this work we show that no third fragment of the TSHR is produced
during its maturation, but rather that cleavage occurs initially at a
first site, followed by the processive digestion and excision of a
whole region of the receptor ectodomain. The region which is
deleted is located in an additional segment specific to the TSHR which
shows no homology with the gonadotropin receptors (5). In addition,
besides the similarities that we had previously observed between
pro-TNF
(pro-tumor necrosis factor
) and TSHR convertases (22),
we provide here new information concerning the protease involved in the
maturation of the TSHR and show that it may correspond to a
novel enzyme.
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EXPERIMENTAL PROCEDURES |
Materials--
Phorbol 12-myristate 13-acetate was purchased
from Sigma. Anti-hemagglutinin (HA) monoclonal (12CA5) antibody and the
"Complete" protease inhibitor mixture tablets were obtained from
Boehringer (Mannheim, Germany). Human recombinant tissue inhibitor of
metalloproteases 1 (TIMP-1) was purchased from Valbiotech (Paris,
France). Human recombinant tissue inhibitor of metalloproteases 2 (TIMP-2) was a gift from Dr Agnès Noël (University of
Liège, Belgium).
The wild-type and mutant Chinese hamster ovary (CHO) cell lines
defective in the shedding of several membrane proteins (23, 24) were
kindly provided by Dr. Joaquin Arribas (Val d'Hebron General Hospital,
Barcelona, Spain). The recombinant catalytic domain of tumor necrosis
factor
enzyme (TACE) and the murine TACE expression vectors (25)
were gifts from Dr. Roy Black (Immunex Corp., Seattle, WA). BB-2116
(26) was a gift from British Biotech Co. (Oxford, United Kingdom). The
synthetic peptides used for the localization of Del-Ab (antibodies
recognizing the putative deleted fragment of TSHR ectodomain)
epitope(s) and the study of the action of TACE were purchased from the
microchemistry laboratory of Institut Gustave Roussy (Villejuif, France).
Anti-TSHR Monoclonal Antibodies--
The preparation of Ecto-Ab
(T5-317, an antibody recognizing the TSHR ectodomain) and Endo-Ab
(T3-365, an antibody recognizing the TSHR endodomain) has been
previously described (9).
For the preparation of Del-Ab, a cDNA fragment encoding amino acids
19-389 of the human TSHR was introduced into the polylinker of the
vector pNMHUB. A fusion protein of TSHR with ubiquitin and
polyhistidine was produced in E. coli. The protein was
purified from inclusion bodies using chelate chromatography on
nickel-agarose in denaturing conditions. Immunization of BALB/c mice,
preparation and screening of hybridomas, production of ascites, and
purification of antibodies were performed as described previously
(9).
L Cell Line--
The L cell line stably expressing the human TSH
receptor has been previously described (10).
Immunopurification and Immunoblotting--
The TSH receptor was
immunopurified and Western blotting performed as described (10), except
that the secondary antibody used during the Western blots was a mouse
anti-Ig coupled to horseradish peroxidase. Revelation was performed
with the ECL detection reagent (Amersham Corp., Buckinghamshire, United
Kingdom). Ecto-Ab, Endo-Ab, or Del-Ab were used for immunomatrix
preparation and Western blot detection. For the immunopurification of
the shed
subunit from the cell culture medium, the medium was first
concentrated approximately 15-fold using a Minitan apparatus
(Millipore, Bedford, MA) equipped with a filter having a 1,000 Da
molecular mass cut-off.
Immunological Assays--
Enzyme-linked immunosorbent assays
(ELISA) were performed as described previously (9). The concentration
of the TSH receptor molecules was measured by reference to known
concentrations of TSHR fragments expressed in E. coli (hTSHR
19-389 when Ecto-Ab or Del-Ab were used as primary antibodies, hTSHR
640-764 when Endo-Ab was used as primary antibody). The primary
antibodies were used at saturating concentrations (5 µg/ml for
Ecto-Ab or Endo-Ab, 1 µg/ml for Del-Ab) in order to bypass
differences in antibody affinities. It was also verified that the
secondary polyclonal antibody had the same affinity for the three
primary antibodies. The immunoradiometric assay of the TSH receptor and
of its
subunit have been previously described (22). For the assay
of the
subunit, the cell culture medium of wild-type and mutant CHO
cells transfected with TSHR cDNA was concentrated 13-fold using
Centriprep-10 concentrators (Amicon, Beverly, MA).
Preparation of TSHR Enriched in Monomeric Receptor Species and
Incubation with TACE--
L cells expressing the human TSH receptor
were incubated for 2 h at 4 °C with 5 ml/g of
phosphate-buffered saline, 100 mM dithiothreitol, and
protease inhibitors. This procedure provokes the reduction of disulfide
bonds and the release of
subunits (22). The cells were washed three
times with phosphate-buffered saline. TSHR was then extracted as
described (10) and immunopurified on an immunomatrix containing
Ecto-Ab. This procedure leads to the purification of uncleaved
monomeric TSHR since
subunits have previously been released. The
recombinant catalytic domain of TACE (10,000 units) was preincubated
for 30 min at 37 °C in the presence or absence of the BB-2116
inhibitor. The TSH receptor enriched in monomeric forms was then added
and incubation was continued for 2 h at 37 °C. The samples were
then subjected to electrophoresis and Western blots were performed
using various monoclonal antibodies.
Co-transfection of Expression Vectors Encoding TACE and
TSHR--
COS-7 cells (~106 cells per Petri dish) were
seeded 24 h before transfection. TACE expression vector (5 µg)
was then transfected with 5 µg of either human TSHR expression vector
or herring sperm DNA (Promega, Madison, WI). The Superfect reagent
(Qiagen Inc., Hilden, Germany) was used according to the
manufacturer's protocol.
N-terminal Microsequencing of the TSH Receptor
Subunits--
The human TSH receptor was immunopurified with either
Endo-Ab or Del-Ab. Endo-Ab immunopurification and microsequencing were performed twice using two different euthyroid goiters (yielding ~1
pmol of TSHR/g of tissue) or four times with different pools of stably
transfected L cells (yielding ~50 pmol of TSHR/g of cells). Del-Ab
was used to enrich receptor preparation in incompletely processed
subunits present in lower amounts in transfected L cells. A
microsequencing experiment was performed on this material.
After immunopurification, the TSH receptor (~200-500 pmol) was
electrophoresed and transferred onto a polyvinylidene difluoride membrane (ProBlott, Applied Biosystems, Foster City, CA). Proteins were
colored by Coomassie Blue. Bands of interest were localized by
reference to immunoblots, separately cut and sequenced following Edman's method using a multi-sample chemical microsequencer (Procise 494-610A, Applied Biosystems). The sequences observed were at least 12 amino acids long and the yield of the different fragments varied from
0.5 to 8 pmol. In repeated experiments identical results were obtained.
Culture and Transfection of the Wild-type and Mutant CHO Cell
Lines--
These cell lines were grown in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum and 600 µg/ml
geneticin (all reagents were from Life Technologies, Inc., Grand
Island, NY). The DEAE-dextran method was used to transfect the
PSG5-TSHR expression vector.
Processing of Pro-TGF
in Wild-type and Mutant CHO
Cells--
Exponentially growing wild-type and mutant CHO cells
(~2 × 106 cells) expressing HA-tagged pro-TGF
(pro-transforming growth factor
) were incubated for 30 min at
37 °C with labeling medium (cysteine-free modified Eagle's medium,
20 mM Hepes pH 7.5, 1 mg/ml bovine serum albumin (BSA)).
[35S]Cysteine (500 µCi/ml) (NEN Life Science Products,
Boston, MA) was then added to the labeling medium and cells were
further incubated for 1 h at 37 °C. The cells were then washed
twice in Dulbecco's modified Eagle's medium without
NaHCO3, 20 mM Hepes pH 7.5, 2 mg/ml BSA and
incubated for 45 min in chase buffer (Dulbecco's modified Eagle's
medium without NaHCO3, 20 mM Hepes pH 7.5, 10 mg/ml BSA) with or without 1 µM phorbol 12-myristate
13-acetate. Protease inhibitors were added to the collected cell
culture medium. The cells were washed twice with cold chase buffer and
once with cold phosphate-buffered saline. The cells were then gently
scraped and harvested in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 0.2%
BSA, and protease inhibitors). The cells were lysed for 30 min at
4 °C on a rotating wheel. The lysed cells were centrifugated for 30 min at 100,000 × g at 4 °C and the supernatant
collected. Anti-HA monoclonal antibody was added to the cell culture
media and the cell lysates for 1 h. The immune complexes were
incubated with protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden)
for 1 h at 4 °C, washed twice with lysis buffer, twice with 20 mM Tris pH 7.5, 150 mM NaCl, 0.5% Triton
X-100, 5 mM EDTA, 0.1% sodium dodecyl sulfate, 0.2% BSA,
twice with 20 mM Tris pH 7.5, 500 mM NaCl,
0.5% Triton X-100, 0.2% BSA and twice with 50 mM Tris pH
7.5. The samples were then boiled for 10 min in Laemmli buffer and
analyzed on a 14% polyacrylamide gel for the lysate samples and on a
16% polyacrylamide gel for the medium samples.
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RESULTS |
A Fragment Is Excised from the TSHR Ectodomain
Monoclonal Antibodies Recognizing the Excised TSHR Fragment:
Detection of Extended Heterogeneous TSHR
Subunit Precursors--
A
TSH receptor fragment (amino acids 19-389) was expressed in E. coli as a fusion protein with ubiquitin. Immunization of mice with
this antigen allowed the preparation of several monoclonal antibodies.
The study of one group of these monoclonal antibodies (called here
Del-Ab) gave puzzling results. When used for Western blot analysis of
TSHR from human thyroids they reacted neither with the
nor with the
subunits but recognized a group of proteins (~39-44 kDa) larger
than the most abundant
subunit (~38 kDa) (Fig.
1A). The same proteins were
recognized by anti-endodomain antibodies (Endo-Ab), as seen after
overexposure of the corresponding immunoblot (Fig. 1A).
These proteins were thus extended
subunits. The Del-Ab gave no
reaction with the
subunit nor with any protein larger than this
subunit. Traces of the mannose-rich precursor of TSHR present in human
thyroid glands and observed in overexposed immunoblots (10) were also
detected by these antibodies. The same results were obtained when five
different thyroid samples were studied.

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Fig. 1.
Some anti-TSHR monoclonal antibodies
recognize an epitope excised from the majority of receptor molecules:
studies with receptor purified from human thyroid glands. A,
Western blot. TSH receptor was immunopurified from human thyroid glands
(TSHR-T) using an antibody which recognizes the receptor
endodomain. Western blots were performed using antibodies which
recognize the receptor ectodomain (Ecto-Ab), endodomain (Endo-Ab), or
the putative deleted fragment (Del-Ab). Overexposure of the Endo-Ab
immunoblot showed that the receptor species recognized by Del-Ab
represented a minor fraction of those recognized by Endo-Ab. Molecular
size standards (in kilodaltons) are indicated on the left.
B, ELISA. The purified receptor was absorbed onto ELISA plates and
incubated with antibodies recognizing either the receptor endodomain
(Endo-Ab) or the putative deleted epitope (Del-Ab). Results are
expressed as the mean ± S.E. (n = 3).
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These results are compatible with the possibility that the epitope
recognized by Del-Ab antibodies was cleaved-off during maturation of
the TSHR in human thyroids. However, this epitope remained covalently
attached to a minority of
subunits ("extended"
subunits).
There were no extended
subunits carrying the epitope.
We undertook the localization of the epitope recognized by this group
of antibodies. Using bacterially expressed proteins encoding various
segments of the TSHR ectodomain (amino acids 19-389, 19-246, and
246-389) and chemically synthesized peptides corresponding to residues
332-369, 332-356, and 357-369 we localized the epitope to amino
acids 357-369. We also observed that the antibodies we had prepared,
and which gave the pattern described in Fig. 1, were all non-additive
and thus probably recognized the same epitope or very closely
positioned epitopes (data not shown).
Western blot experiments thus suggested that only a minority of human
thyroid TSHR molecules had conserved the epitope recognized by Del-Ab.
We used a quantitative method to measure this population of receptors
more precisely. ELISA tests were performed with purified human thyroid
TSHR, using an antibody which recognizes either the receptor endodomain
(Endo-Ab) or the segment which was cleaved-off (Del-Ab). As shown in
Fig. 1B, about 75% of receptor molecules were devoid of
this segment.
Since human thyroid tissue is difficult to obtain, and since many
studies of the TSH receptor are performed using transfected cells, we
repeated the immunoblot studies using TSHR purified from L cells stably
expressing the receptor. Previous studies (10) have shown that in
transfected cells there is accumulation of a mannose-rich intracellular
precursor (~95 kDa) and incomplete receptor cleavage with persistence
of various amounts of uncleaved full-length mature receptor (~120
kDa). Furthermore, in transfected cells the
subunit has been shown
to be heterogeneous (10). In the receptor prepared from transfected L
cells, Del-Ab antibody reacted as expected with the 2 uncleaved forms
of the receptor (Fig. 2). It also reacted
with the largest forms of the
subunit (extended
subunits).
Again no reaction was seen with the
subunit and there was no
indication of larger forms of the
subunit carrying the Del-Ab
epitope.

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Fig. 2.
Some anti-TSHR monoclonal antibodies
recognize an epitope excised from the majority of receptor molecules:
studies with TSHR purified from transfected L cells. The TSH
receptor was purified from stably transfected L cells
(TSHR-L) using an antibody which recognizes the
receptor endodomain. Western blots were performed using antibodies
directed against the receptor ectodomain (Ecto-Ab), endodomain
(Endo-Ab), or the putative deleted fragment (Del-Ab). Molecular size
standards (in kilodaltons) are indicated on the left.
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Cleaved-off TSHR Fragment Cannot be Recovered from the Cell Culture
Medium or from the Cell Membrane and Does Not Remain Associated with
the Receptor--
The preceding experiments raised the question of the
fate of the excised receptor fragment. Theoretically, it could have
remained bound to the receptor by non-covalent interactions
(constituting a third subunit of the receptor), or it could have
remained attached to the cell membrane, or it could have been released
from the cells.
We examined the latter possibility by searching for the fragment in the
cell culture medium of L cells expressing the TSH receptor. As shown in
Fig. 3 no antigen able to bind Del-Ab
could be detected in the cell culture medium, whereas the shed
subunit (27) was detected by anti-ectodomain antibodies.

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Fig. 3.
The TSHR fragment cleaved off the receptor
ectodomain is not present in cell culture medium. L cells stably
expressing the TSHR were cultured for 24 h in Dulbecco's modified
Eagle's medium supplemented with 1% fetal calf serum. The cell
culture medium was concentrated approximately 15-fold and
immunopurified using Ecto-Ab or Del-Ab as indicated. Quantification of
purified TSHR molecules containing the Ecto-Ab or Del-Ab epitopes was
performed by ELISA using the corresponding monoclonal antibody. Results
are expressed as the mean ± S.E. (n = 3).
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We then prepared a human thyroid membrane Triton X-100 extract and
subjected it to chromatography on a Del-Ab immunomatrix. Immunoblots
(Fig. 4A) with Del-Ab only
showed the extended forms of
subunit previously described (see Fig.
1A). There was no evidence of a fragment of ~6.5 kDa which
could correspond to the excised fragment (not shown). This experiment
thus suggested that the receptor fragment which was cleaved-off did not
remain attached to the membrane either in a free form or bound to the
receptor.

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Fig. 4.
The TSHR fragment cleaved off the receptor
ectodomain does not remain attached to the receptor. Membrane
fractions were prepared from human thyroid glands (TSHR-T, panel
A) or from L cells stably expressing TSHR (TSHR-L, panel
B). After treatment with Triton X-100, the extracts were subjected
to chromatography on an immunomatrix containing Del-Ab. Western blots
of the immunopurified receptor were performed using antibodies directed
against the receptor ectodomain (Ecto-Ab), endodomain (Endo-Ab), or
excised fragment (Del-Ab). On the right of each panel is
shown an immunoblot (with Endo-Ab) of receptor purified on an
immunomatrix containing Endo-Ab. Molecular size standards (in
kilodaltons) are indicated on the left of each panel.
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Another line of evidence also showed that the excised fragment did not
constitute a third subunit of the receptor. Indeed, if this had been
the case immunopurification using Del-Ab would have yielded all 3 fragments of the receptor. This was not the case: no normal
(non-extended)
subunit of ~38 kDa was purified. Only the larger
subunits of ~39-44 kDa, to which the epitope for Del-Ab remains
covalently attached due to incomplete cleavage, were purified. The
existence of these extended
subunits to which
subunits remain
bound would explain why some of the latter are also retained during
chromatography on the Del-Ab immunomatrix.
Furthermore, when the human thyroid membrane extract was
chromatographed on two successive immunomatrices, first Del-Ab then Endo-Ab, the latter retained the majority of
subunits (of ~38 kDa) and the majority of
subunits (not shown). This experiment shows that there are two populations of receptors: a majority of
-
dimers devoid of the Del-Ab epitope and a minority of
-"extended
" dimers. The latter carry the Del-Ab epitope.
We also used the Del-Ab immunomatrix to purify a Triton X-100 extract
from membranes of L cells expressing the TSHR (Fig. 4B). The
results were similar to those obtained with human thyroid TSHR: absence
of a third fragment and purification of only the extended, largest,
forms of the
subunit.
Determination of Cleavage Sites in the Receptor Ectodomain by
Microsequencing--
The inability to recover the excised fragment
from the receptor, membrane extracts, and cell culture medium suggested
that it was either immediately destroyed or initially excised in small pieces. Microsequencing experiments suggested that the latter explanation was the most probable.
The TSHR was immunopurified from human thyroid glands using an
anti-endodomain antibody. After electrophoresis and transfer to a
polyvinylidene difluoride membrane the region corresponding to the
subunits was excised in successive slices and submitted to
microsequencing (Fig. 5). Three
N-terminal TSHR amino acid sequences were observed in approximately
equimolar amounts, originating at amino acids phenylalanine 366, leucine 370, and leucine 378.

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Fig. 5.
N-terminal microsequencing of TSHR subunits. The TSHR was immunopurified from human thyroids
(TSHR-T) or from transfected L cells (TSHR-L)
using either an anti-endodomain antibody (Endo-Ab) or an antibody which
recognizes the excised fragment (Del-Ab). Purified receptors were
subjected to electrophoresis in denaturing conditions and transferred
onto a polyvinylidene difluoride membrane. The region of the membrane
containing the subunits was determined by comparison with Western
blots and cut into sections which were used for microsequencing. The
N-terminal amino acids thus determined are shown in the figure.
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We have previously observed that the cleavage of TSHR is incomplete in
transfected cells (10). Furthermore, we have shown here that
immunochromatography with Del-Ab allows receptor preparations to be
enriched with large incompletely cleaved
subunits (see Fig. 4). We
thus immunopurified the receptor from TSHR-expressing L cells, using
either Endo-Ab or Del-Ab. Microsequencing of
subunits from such
preparations showed a variety of species extending upstream from those
observed in the human thyroid, up to Ser-314 (Fig. 5). The most
abundant species started at leucine 370 and threonine 388.
These observations suggested that the excised fragment of the receptor
could not be isolated because it was not produced as a single
polypeptide, but that multiple cleavages occurred which yielded
fragments too small to be detected. Furthermore, the initial cleavage
probably occurred in the N terminus of this region around Ser-314.
Successive cleavages would then progress toward the C terminus up to
phenylalanine 366 and leucines 370 and 378. This conclusion is based on
the fact that the epitope recognized by Del-Ab remains in some
molecules associated with the
subunit, but never in
subunits. Furthermore, whereas the
subunit is known to be of
heterogeneous size, especially in transfected cells, the
subunit
has a discrete size (more conveniently observed after deglycosylation
(10)).
Characterization of the TSHR Cleavage Enzyme
Absence of Inhibition by TIMPs--
We have previously shown that
cleavage of the TSHR, and the ensuing shedding of its
subunit, were
inhibited by BB-2116, a potent specific inhibitor of matrix
metalloproteases (22). Recent studies have shown that the majority of
these enzymes are inhibited by the natural tissue inhibitors of
metalloproteases called TIMPs, whereas some, including TACE (tumor
necrosis factor
-converting enzyme), are insensitive to these
compounds (28).
We thus studied TSHR
subunit shedding in the absence or presence of
either TIMP-1 or TIMP-2. As shown in Fig.
6 there was no significant inhibition of
shedding in the presence of either of these compounds. This experiment
thus suggested that the TSHR cleavage enzyme could be a matrix
metalloprotease-like enzyme, identical or similar to TACE.

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Fig. 6.
TIMP-1 and TIMP-2 do not inhibit shedding of
the TSHR subunit. L cells stably expressing the TSH receptor
were incubated for 24 h in the presence of various concentrations
of human recombinant TIMP-1 (closed circles) or TIMP-2
(open circles). The amount of shed subunit in the
culture medium was assayed, and is expressed as a percentage compared
with that in the absence of either TIMP. Results shown are the
mean ± S.E. (n = 3).
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The TSHR Cleavage Enzyme Is Distinct from TACE--
To study the
effect of TACE on the TSHR, we initially used the purified enzyme. The
TSHR was enriched in monomeric uncleaved forms as described above and
was incubated with the purified recombinant catalytic domain of TACE
(see Fig. 7). Some cleavage was observed, but this yielded a fragment markedly smaller than the physiological
subunit. BB-2116 inhibited this cleavage.

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Fig. 7.
Cleavage of TSHR by purified TACE. A
TSHR preparation enriched in monomeric precursors (see "Experimental
Procedures") was incubated for 2 h at 37 °C with (+) or
without ( ) 10,000 units of the recombinant human catalytic domain of
TACE in the presence (+) or absence ( ) of BB-2116. A control receptor
preparation (not enriched in monomers) is shown on the left.
Western blots were performed using an antibody directed against the
receptor ectodomain. Molecular size standards (in kilodaltons) are
indicated on the left.
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It is thus probable that this cleavage is non-physiological. It is
likely that the very high concentrations of purified TACE used in this
experiment provoked cleavage of the TSHR at a different site to that
recognized in vivo.
However, it is possible that TACE can only act specifically on its
substrate in the context of a membrane. To examine this hypothesis, we
co-transfected cells with expression vectors encoding TSHR and TACE. In
these conditions we found a fragment larger than the physiological
subunit released from the cells and present in the cell culture medium
(Fig. 8). Immunopurification of the receptor from cells co-transfected with TACE showed that there was a
decrease in receptor concentration (probably reflecting nonspecific
proteolysis) but no increased concentration of
subunits.

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Fig. 8.
Cleavage of the TSHR in cells co-transfected
with an expression vector encoding TACE. Cos-7 cells were
co-transfected with expression vectors encoding the human TSHR and
either herring sperm DNA ( ) or murine TACE (+). The shed subunit
was then immunopurified using an antibody directed against the
extracellular domain of the TSH receptor. Molecular size standards (in
kilodaltons) are indicated on the left.
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Finally, to further approach this problem we used a mutant CHO cell
line obtained by Dr. Joaquin Arribas (23), which has been shown to be
deficient in the cleavage and shedding of a variety of membrane protein
ectodomains. The most extensively studied reaction to date has been the
inability of these cells to convert pro-TGF
into TGF
. Recently,
this cell line has also been shown not to express active TACE (29). We
thus transfected wild-type and mutant CHO cells with TSHR. As shown in
Fig. 9, A and B,
cleavage of the TSHR occurred in the mutant cells, and shedding of the
subunit was identical to that observed in wild-type CHO cells. We
verified that the cells we have used were indeed unable to process
pro-TGF
into TGF
(Fig. 9C).

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Fig. 9.
The TSH receptor is cleaved in mutant CHO
cells. Wild-type (WT) and mutant (Mut) CHO
cell lines were transiently transfected with the TSH receptor
expression vector. A, the receptor was immunopurified using
an antibody directed against its intracellular domain and analyzed on a
Western blot using an antibody recognizing the ectodomain. Molecular
size standards (in kilodaltons) are indicated on the left.
B, the amount of TSHR ectodomain in cell membrane extracts and
cell culture medium was quantified by immunoradiometric assays. Results
are expressed as percent of TSHR ectodomain shed into the cell culture
medium (mean ± S.E., n = 3). C, as a
control, wild-type or mutant CHO cells transfected with HA-tagged
pro-TGF , were metabolically labeled with 35S-cysteine
and then chased for 45 min in the presence or absence of phorbol
12-myristate 13-acetate (phorbol 12-myristate 13-acetate
(PMA) activates pro-TGF cleavage). Cell lysates and
medium samples were immunoprecipitated with anti-HA monoclonal antibody
and analyzed by polyacrylamide gel electrophoresis and autoradiography.
Molecular size standards (in kilodaltons) are indicated on the
left.
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DISCUSSION |
A group of anti-TSHR monoclonal antibodies were obtained which
recognized an epitope cleaved-off from the majority of molecules of the
mature receptor. This epitope was localized in a specific segment of
the TSHR ectodomain characterized by its non-homology with gonadotropin
receptors. The excised TSHR fragment was not shed into the cell culture
medium, and did not remain associated with the cell membrane or the
mature receptor. These observations suggest that the excision produced
multiple small undetectable fragments.
This hypothesis was confirmed by the determination of TSHR cleavage
sites. Mature
subunits were isolated from human thyroid glands and
from transfected L cells, and extended
subunits (incompletely processed) were purified from transfected L cells. In transfected cells
cleavage of the receptor has previously been shown to be incomplete
(10). Multiple cleavage sites located within the excised region were
detected. The most N-terminal site observed was located at Ser-314
while a group of C-terminal sites recognized in thyroid tissue were
located at Phe-366, Leu-370 and -378. Furthermore, the existence of a
single discrete
subunit in the thyroid, or in transfected L cells,
in contrast to the heterogeneity of the
subunits strongly suggested
sequential cleavages. A first N-terminal cleavage reaction probably
occurs around Ser-314, followed by a series of downstream cleavages up
to amino acids 366-378.
It has previously been proposed that two cleavage sites in the TSHR
extracellular domain release a C peptide by a mechanism similar to the
processing of pro-insulin (19). We show here that no C peptide is
actually produced, but that a series of sequential cleavage reactions
lead to the excision of this segment of the TSHR ectodomain. The exact
location of the TSHR cleavage sites could not be determined by
mutagenesis (20, 21). This concords with the hypothesis that several
sites, in close proximity to each other, can be cleaved. These could
thus be used alternatively if one of them was mutated. The cleavage of
amyloid protein precursor has also been shown to occur at several
sites in a region of the precursor close to the cell surface (30). A
sequential proteolysis of the tyrosine kinase ErbB4 receptor has been
described (31). However, sequential cleavages of the ErbB4 receptor are due both to an exofacial metalloprotease, inhibited by hydroxamic acids, and to the cytoplasmic proteasome (31).
The cleavage sites within the THSR do not exhibit primary sequence
similarity. It is possible that their location is dictated by their
position relative to the cell membrane. Indeed, for several membrane
proteins cleavage occurs at a site located a fixed distance from the
membrane (32-36). Furthermore, mutational analysis has shown a lack of
strict sequence specificity for the cleavage of
amyloid precursor
protein (32), the TNF
receptor (33), the interleukin-6 receptor
(37), L selectin (35), and pro-TNF
(36). In addition, a mechanism
involving a recognition of secondary structure in the juxtamembrane
region has been suggested by the domain swap experiments of J. Arribas
(38).
The precise determination of the most N-terminal cleavage site of the
TSHR would necessitate the determination of the C-terminal sequence of
the
subunit. We have submitted the purified
subunit to cyanogen
bromide digestion, chromatographic separation of peptides, and
microsequencing. However, the amounts of protein which could be
purified have proved insufficient to date to give meaningful results.
The TSHR
subunits produced by receptor cleavage are very
heterogeneous in size. This is especially true in transfected cells where cleavage is incomplete. But even in human thyroid glands, 25% of
receptor molecules undergo incomplete cleavage and are of larger
molecular weight. In the majority of receptor molecules the N termini
of
subunits cluster from amino acids 366 to 378.
We have shown that TSHR cleavage is inhibited by BB-2116 (22), a
synthetic hydroxamic acid which was initially claimed to inhibit
specifically matrix metalloproteases (MMP) (26). This compound also
inhibits the cleavage of pro-TNF
. This led to the conclusion that
the enzymes involved in the maturation of TNF
(TACE) and TSHR belong
to the MMP family. However, pro-TNF
(28) and also interleukin 6 receptor (39) and TSHR (present study) convertases are not inhibited by
the natural physiological inhibitors of MMPs, i.e. TIMPs,
which are secreted by cells to control MMP action (40). These
observations suggest that pro-TNF
and TSHR convertases are not bona
fide MMPs. Furthermore, TACE has recently been cloned (25, 41) and
shown to be a new member of the adamalysin family of the metzincin
metalloproteases (or ADAMS). These enzymes are characterized by the
presence of a metalloprotease domain and of a disintegrin domain. The
latter may interact with integrins and in some mammalian adamalysins
may mediate cell fusion (42).
The recombinant catalytic domain of TACE failed to cleave
immunopurified TSHR at the physiological site. It could be that the
correct action of this enzyme depends on its location, and that of its
substrate, in the cell membrane. This has proven to be the case for the
angiotensin-converting enzyme secretase (43). To examine this
possibility we performed co-transfection experiments with TACE and TSHR
expression vectors. However, this did not result in cleavage of the
TSHR at the physiological site.
Another argument suggesting that TACE and TSHR convertase are different
enzymes was provided by the use of a mutant CHO cell line. This cell
line is defective in the shedding of several membrane proteins (23, 24)
including TNF
(29). It is not known if the defect is due to the
alteration of a single protease that acts on all these proteins, or if
it is due to an alteration of a common regulatory mechanism necessary
for the activation of several proteases. In either case, these cells do
not express active TACE but they do properly cleave the TSHR.
We conclude that TACE and TSHR convertases are very likely to be
distinct enzymes, although related to each other as shown by their
common location at the cell surface, their inhibition by
hydroxamic-based metalloprotease inhibitors and their lack of
inhibition by TIMPs. Further experiments will be needed to establish
the functional role of the cleavage of the TSHR, its possible
involvement in receptor autoimmunity, and to identify precisely the
corresponding protease.