THE JOURNAL OF BIOLOGICAL CHEMISTRY
4545–4552, 1996
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Shedding of Human Thyrotropin Receptor Ectodomain
INVOLVEMENT OF A MATRIX METALLOPROTEASE*
(Received for publication, July 12, 1995, and in revised form, October 26, 1995)
Jacques Couet‡, Sokhavut Sar, André Jolivet, Mai-Thu Vu Hai, Edwin Milgrom,
and Micheline Misrahi§
From the Unité de Recherches Hormones et Reproduction, Institut National de la Santé et de la Recherche Médicale,
Unité 135 and the Laboratoire d’Hormonologie et Biologie Moléculaire, Hôpital de Bicêtre, 94270
Le Kremlin-Bicêtre, France
The thyrotropin (TSH) receptor in human thyroid
glands has been shown to be cleaved into an extracellu-
lar a subunit and a transmembrane b subunit held to-
gether by disulfide bridges. An excess of the latter com-
ponent relative to the former suggested the shedding of
the ectodomain.
Indeed we observed such a shedding in cultures of
human thyrocytes and permanently transfected L or
Chinese hamster ovary cells. The shedding was in-
creased by inhibitors of endocytosis, recycling, and ly-
sosomal degradation, suggesting that it was dependent
on receptor residency at the cell surface. It was slightly
increased by TSH and phorbol esters, whereas forskolin
and 8-bromo-cyclic AMP were without effect. Decreas-
ing the serum concentration in cell culture medium en-
hanced the shedding by an unknown mechanism.
The shedding of the TSH receptor a domain is the
consequence of two events: cleavage of the receptor
into a and b subunits and reduction of the disulfide
bridge(s). The complete inhibition of soluble TSH recep-
tor shedding by the specific inhibitor BB-2116 indicated
that the cleavage reaction is catalyzed probably at the
cell surface by a matrix metalloprotease.
This shedding mechanism may be responsible for the
presence of soluble TSH receptor a subunit in human
circulation.
Thyrotropin (TSH)1 is the primary hormone that regulates
thyroid cell growth and function via the G protein-coupled
thyrotropin receptor (1, 2). Members of this receptor family are
characterized by their common structural feature of seven
transmembrane domains (3). However, luteinizing hormone-
choriogonadotropin (LH/CG) (4, 5), follicle-stimulating hor-
* This work was supported by the Institut National de la Santé et de
la Recherche Médicale, the Délégation à la Recherche Clinique (Assist-
ance Publique, Hôpitaux de Paris), the Faculté de Médecine Paris Sud,
the Association pour la Recherche sur le Cancer, the Ligue Nationale
contre le Cancer, and the Fondation pour la Recherche Médicale Fran-
çaise. The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
‡ Recipient of a fellowship from the Medical Research Council of
Canada.
§ To whom correspondence should be addressed: INSERM U. 135,
Hôpital de Bicêtre, 3ème niveau, 94275 Le Kremlin-Bicêtre, France.
1
The abbreviations used are: TSH, thyroid-stimulating hormone;
sTSHR, soluble TSH receptor; cTSHR, cellular TSH receptor; h, human;
b, bovine; LH, luteinizing hormone; CG, choriogonadotropin; LH/CGR,
luteinizing hormone choriogonadotropin receptor; FSH, follicle-stimu-
lating hormone; FSHR, follicle-stimulating hormone receptor; CHO,
Chinese hamster ovary; PBS, phosphate-buffered saline; PMA, phorbol
12-myristate 13-acetate; 8-bromo-cAMP, 8-bromo-cyclic AMP.
4545
Vol. 271, No. 8, Issue of February 23, pp.
Printed in U.S.A.
mone (FSH) (6), and TSH receptors (7–10) form a subgroup in
this family having a large and glycosylated extracellular do-
main specialized in high affinity hormone binding (11, 12).
Interest in the TSH receptor (TSHR) is enhanced by its impli-
cation in autoimmune diseases. Autoantibodies directed
against this receptor have stimulatory (Graves’ disease) or
blocking (idiopathic myxoedema) effects on its function (13).
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From cloning and sequencing of the human TSH receptor
cDNA (7–10), the structure of a preprotein with a calculated
molecular mass of 84.5 kDa was deduced. But the character-
ization of the mature structure of the TSH receptor and of other
receptors of that family awaited the generation of high affinity
specific antibodies (14, 15). Interestingly, while the LH/CG
receptor is expressed in target organs as a monomer (14), the
TSH receptor is expressed in thyroid membranes as a het-
erodimer: an extracellular a subunit (;53 kDa) and a mem-
brane-spanning b subunit (;38 kDa) are held together by
disulfide bridge(s) (15). This observation is in agreement with
one of the models proposed for the structure of the TSH recep-
tor before the cloning (16). The post-translational cleavage in
two subunits is almost complete in human thyroid tissue,
whereas in L cells stably transfected with the TSH receptor, a
small amount of uncleaved mature receptor may still be pres-
ent. In transfected cells, but not in human thyroid tissue, there
is accumulation inside the cells of an unprocessed mannose-
rich monomeric precursor (17).
Pulse-chase experiments performed in L cells and in human
thyrocytes confirmed that the TSH receptor is primarily syn-
thesized as a ;95-kDa mannose-rich monomer, which under-
goes supplementary glycosylation to yield a ;120-kDa mono-
meric precursor (17). The latter is then cleaved into mature a
and b subunits. This processing into two subunits is unique
among G protein-coupled receptors.
Precise quantification of each subunit in human thyroid
membranes allowed us to observe a 2.5–3-fold excess of b over
a subunits (15). This observation led us to postulate that the a
subunit might be shed from cell membranes and released into
the extracellular space or bloodstream. Such a phenomenon
could be important in the context of autoimmune diseases. We
report now that spontaneous shedding of the extracellular do-
main of the TSH receptor occurs in stably transfected L and
CHO cell lines as well as in human thyrocytes. This shedding is
also a regulated process involving the cleavage of the TSH
receptor by a matrix metalloprotease.
EXPERIMENTAL PROCEDURES
Materials—Most reagents were purchased from Sigma. Bovine TSH,
monensin, A23187, and ionomycin were from Calbiochem; iodinated
bovine TSH (70 mCi/mg) was from Eria Diagnostics Pasteur (Marnes-
la-Coquette, France); iodinated streptavidin was from Amersham Corp.
BB-2116, a matrix metalloprotease inhibitor, was kindly provided by
British Biotech (Oxford, United Kingdom).
4546 A Soluble Form of the Human TSH Receptor
Culture of an L Cell Line Permanently Expressing the Human TSH
Receptor—This cell line was cultured as described previously (17). Cell
viability tests were performed by the trypan blue exclusion technique.
Primary Cultures of Human Thyrocytes—Human thyroid tissue was
obtained by surgery from patients with benign thyroid diseases. Thy-
roid follicles were prepared as described (18) and cultured for 48 –72 h.
Immunoradiometric Assay of the TSH Receptor—A double-determi-
nant (“sandwich”-type) radioimmunoassay was developed for the spe-
cific quantification of the TSH receptor a subunit. This assay uses two
additive monoclonal antibodies specific for the extracellular domain of
the TSHR (T5-317 and T5-51 (15)). The specificity of these antibodies
has been established by a variety of methods: these antibodies inter-
acted with TSH receptor expressed in Escherichia coli or in mammalian
cells (COS 7, CHO, and L cells) as tested by enzyme-linked immunosor-
bent assay, Western blot, and immunocytochemistry. No signal could be
be detected using all these methods in mock-transfected cells. Finally
antibody 51 has been used to immunopurify 125I-TSH receptor com-
plexes from the Triton X-100 extract of cell membranes. In the assay
T5-317 was used as the “capture” antibody and biotinylated T5–51 in
conjunction with 125I-streptavidin as the “reporter” antibody. Biotiny-
lation of this antibody was performed using the Amersham biotinyla-
tion kit (Amersham). The T5-317 antibody (0.5 mg/well) was coated onto
96-well plates (Maxisorb, Nunc, Rockilde, Denmark) in 0.05 M potas-
sium phosphate buffer (pH 7.4) for 2 h at room temperature. Plates
were washed four times with 300 ml of phosphate-buffered saline (PBS)
(pH 7.4), bovine serum albumin 0.1% (w/v), Tween 0.05% (PBT) and
saturated for 2 h at room temperature with PBS (pH 7.4), bovine serum
albumin 1% (w/v), Tween 0.05%. After washing, samples were then
incubated, directly or diluted in PBT, for 16 h at 4 °C or 2 h at room
temperature. The plates were washed with PBT and the biotinylated
antibody (T5-51) added at a 0.5 mg/ml concentration in PBT for 2 h.
After washing, 125I-streptavidin (75,000 cpm/well) was added for 1 h
and then extensively washed with PBT. Finally, 200 ml of 0.1 N NaOH
was added in each well and an aliquot of 100 ml counted. Samples were
quantified relatively to a standard curve of TSHR immunopurified from
the stably transfected L cell line (17). This cellular TSHR had previ-
ously been quantified comparatively to a hTSHR peptide-b-galactosid-
ase protein fusion standard (15). This assay allowed us to detect rou-
tinely as little as 2 fmol/ml of soluble TSHR (sTSHR). We also developed
a second assay for the determination of full length heterodimeric
(cleaved) and monomeric (uncleaved) forms of the TSHR by replacing
T5-317 by an antibody specific for the intracellular part of the TSHR
(T3-365) (15).
TSHR Determinations in Culture Medium and Cellular Extracts—
For determination of sTSHR concentration, or for the assay of the
full-length receptor in the culture medium (a/b receptor), the medium
was removed, centrifuged at 1500 3 g for 5 min and stored at 220 °C.
Cells were washed with ice-cold PBS, scraped, centrifuged at 1500 3 g
for 5 min, and stored at 270 °C. An aliquot of the Triton X-100 cellular
extract (16) was used for cellular TSHR (cTSHR) determinations or
protein content quantification using the Pierce BCA protein assay re-
agent kit (Pierce).
125
I-TSH Binding to Soluble and Cellular TSHR—TSH receptor
binding assays were performed using the method described by Petersen
et al. (19). Triton X-100 cellular extracts (17) or culture media from
stably transfected L cells (concentrated 30-fold using successive Cen-
triprep-10 and Centricon-10 concentrators (Amicon, Beverly, MA)) were
used for these experiments. Briefly, 2 pmol of soluble or cellular TSHR
were added to iodinated bovine TSH in the absence or in the presence
of increasing concentrations of unlabeled bovine TSH (bTSH) human
FSH (hFSH) or human CG (hCG). The bound complexes were precipi-
tated with polyethylene glycol (molecular weight: 6000).
Immunopurification of the Soluble TSHR—Immunopurification of
sTSHR from the permanently transfected L cells was performed as
described previously (17) except that a T5-51 Affi-Gel-10 immunoma-
trix was used. For purification of sTSHR culture media containing 1%
fetal calf serum were concentrated using a Minitan apparatus (Milli-
pore, Bedford, MA) equipped with a filter having a 30-kDa molecular
mass cutoff. Deglycosylation of receptors using N-glycanase F and en-
doglycosidase H and immunoblots were performed as described (17).
LH/CG Receptor Measurements in Culture Medium of a Stably
Transfected L Cell Line—A double determinant radioimmunoassay was
developed using two additive monoclonal antibodies against the extra-
cellular part of the porcine LH/CG receptor (LHR 38 and biotinylated
LHR 436) (14). The sensitivity of the assay was ;20 fmol/ml. The
methodology of this new assay was similar to the sTSHR assay. LH/CG
receptor quantification was performed using an L cell line permanently
expressing the porcine LH/CG receptor (12) and an hCG binding assay
as described previously.
Quantification of a/b Heterodimers by Treatment of Cells with Di-
thiothreitol to Release TSHR a Subunit—At the end of incubation, cells
were scraped gently as described above and incubated for 2 h at 4 °C in
500 ml of a PBS (pH 7.4), 100 mM dithiothreitol solution under agitation.
The cells were then centrifuged at 1500 3 g for 5 min. An aliquot of the
supernatant was diluted 100-fold for the assay of released a subunits.
Then the cells were treated with Triton X-100 in order to solubilize the
remaining uncleaved TSHR. An aliquot of this cellular extract was used
for the assay of the cellular receptor.
Statistical Analysis—Statistical differences between groups were an-
alyzed by one way analysis of variance followed by Fisher’s least sig-
nificant difference test for multiple comparison. p , 0.05 was consid-
ered significant. Statview computer program (Abacus Concepts Inc.,
Berkeley, CA) was used for calculations.
RESULTS
Identification of a Soluble Form of the TSHR (sTSHR) Re-
leased from Transfected Cells—To detect possible shedding of
the extracellular domain of the TSH receptor from cells, we
first studied a L cell line stably transfected and expressing high
levels of the human TSH receptor. This model system is more
amenable to experimental analysis and more reproducible than
primary cultures of human thyrocytes. In the latter the con-
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centration of the receptor is known to be very low (15).
A double-determinant (sandwich-type) radioimmunoassay
was developed for the specific quantification of the TSH recep-
tor a subunit. This assay uses two additive monoclonal anti-
bodies specific for the extracellular domain (a subunit) of the
TSHR (T5-317 and T5-51) (15). T5-317 is the capture antibody.
As illustrated in Fig. 1A, the accumulation of a soluble form
of the TSHR was detected in the medium of a mouse L cell line
stably transfected with the full-length human TSH receptor
cDNA (17). This accumulation was time-dependent and
reached 50 pM after 48 h of culture (;25% of total receptor
present in the cells at this time). The same result was obtained
when the medium was filtered through a 0.2-mm syringe filter
or ultracentrifuged at 15,000 3 g to remove cellular debris.
The assay was specific. Competition with an excess of unla-
beled T5-51 antibodies suppressed the binding of the reporter
antibody, while a nonspecific competitor antibody had no effect.
The replacement of one of the TSHR antibodies in the assay by
a nonspecific irrelevant monoclonal antibody or the omission of
TSHR also suppressed the binding of the 125I-streptavidin
(data not shown).
We also examined the possibility that we were detecting the
receptor present in membrane fragments released into the
medium. To verify this point we used an alternative assay in
which the capture antibody recognized the intracellular do-
main (b subunit) of the receptor (T3-365). This assay detects
cleaved or uncleaved whole receptor molecules. No such recep-
tor form was detected in the culture medium (Fig. 1A).
These experiments strongly suggested that spontaneous
shedding of a soluble form of the TSH receptor, corresponding
to its extracellular domain, occurred in the permanently trans-
fected L cell line. The same determinations were performed on
the medium of another TSHR expressing Chinese hamster
ovary (CHO)-derived cell line (a gift from C. Maenhaut and G.
Vassart) (20). Those cells also produced and released a soluble
form of the TSHR into the culture medium in proportions
comparable with the stably transfected L cell line (data not
shown).
As a control, we also studied L cells stably transfected with
the related LH/CG receptor which express high levels of ma-
ture receptors (12). The presence of a functional receptor on the
surface of these cells has been shown by hormone binding and
adenylate cyclase stimulation. A similar assay was developed
using two additive monoclonal antibodies specific for the extra-
cellular domain of the LH/CG receptor. No receptor ectodomain
 A Soluble Form of the Human TSH Receptor
FIG. 1. Shedding of a soluble form of the TSH receptor from a
L cell line permanently expressing the human TSHR. A, an L cell
line expressing the TSH receptor was cultured for various periods of
time. Aliquots of culture medium were used for the assay of the a
subunit (the sandwich assay used T5-317 and biotinylated T5-51, both
antibodies recognizing different epitopes in the ectodomain). As a con-
trol the full-length receptor (a/b receptor) was also assayed using anti-
body T3-365 (epitope in the intracellular domain) and biotinylated
T5–51. B, an identical experiment was performed with a L cell line
expressing LH/CG receptor. Antibodies LHR38 and biotinylated
LHR436, both recognizing the extracellular domain of LH/CG receptor,
were used for the assay. Results are expressed as the mean 6 S.E.
(standard error of the mean) of three independent determinations.
could be detected in the cell culture medium even after a 7-fold
concentration of the medium (Fig. 1B). This is consistent with
the fact that this receptor is expressed as an uncleaved
monomer (14).
Thus, the existence of a soluble form of the receptor released
into the medium is specific for cells expressing the TSH
receptor.
Identification of a Soluble Form of the TSHR in Primary
Cultures of Human Thyrocytes—In order to confirm the results
obtained with our transfected L cell model, we investigated
TSHR ectodomain shedding in a more physiological system,
namely in human thyrocytes. We observed an accumulation of
an immunoreactive soluble form of the TSHR in primary cul-
tures of human thyrocytes (Fig. 2). The proportion of this
soluble TSHR to the total cellular TSHR concentration reached
about 17% after 48 h of incubation and more than 22% after 72
h. The presence of sTSHR in thyrocyte culture medium was
confirmed in two independent experiments.
Characterization and Purification of the sTSHR from the
Stably Transfected L Cell Line—Preliminary experiments had
shown that when the medium was filtered on Centricon mem-
branes with a molecular cutoff of 30 kDa, most of the immuno-
reactivity (.90%) was retained by the filters (not shown).
To test the ability of sTSHR to bind specifically TSH, con-
centrated (30 3) culture medium from transfected L cells was
incubated with iodinated bovine TSH, in the presence or in the
4547
FIG. 2. Shedding of a soluble form of the TSH receptor from
human thyrocytes. The experiment was performed as described in
the legend to Fig. 1, except that the cell culture medium was concen-
trated 15 times on Centricon-10 filters before receptor assay. Ectodo-
main (a subunit) and full-length receptor (a/b receptor) were assayed as
described in the legend to Fig. 1. The cellular receptor (cTSHR) was
assayed in Triton X-100 membrane extracts. Results are expressed as
the mean ratio (n 5 2) of soluble TSHR (sTSHR) on the cellular amount
of receptor (cTSHR).
absence of increasing concentrations of unlabeled bTSH, hFSH,
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or hCG. The bound complexes were precipitated by polyethyl-
ene glycol. The same experiment was performed in parallel
with cellular TSH receptor present in Triton X-100 extracts. As
shown in Fig. 3, sTSHR specifically bound TSH, in a manner
similar to the cellular receptor. Competition with unlabeled
TSH yielded almost complete displacement, while hFSH and
hCG had no significant effect at these concentrations.
Finally, sTSHR was purified from culture medium using
immunoaffinity chromatography with the T5–51 immunoma-
trix (Fig. 4). More than 90% of the receptor present in the
medium bound to the matrix and was eluted at acidic pH.
Western blots were performed to compare the immunopuri-
fied sTSHR with the cellular TSHR (solubilized from the mem-
branes of the stably transfected L cell line). Antibody T5-317
recognizing the extracellular domain of the receptor was used.
As shown in Fig. 5, sTSHR had an apparent molecular mass of
;53 kDa (sTSHR, lane C) which is slightly smaller than the a
subunit of the TSHR purified from cells (;60 kDa) (cTSHR,
lane C). However after digestion with N-glycanase F, which
removes all oligosaccharides, both proteins migrated as the
same ;35 kDa species (cTSHR (lane F) and sTSHR (lane F)).
Endoglycosidase H (which removes only mannose-rich moieties
of precursor glycoproteins) had no effect on sTSHR or on the a
subunit of the cellular TSHR (cTSHR (lane H) and sTSHR (lane
H)). These results indicate that sTSHR and the a subunit of the
receptor contain the same polypeptide core and differ only
slightly in their mature carbohydrate content. This difference
may be due to the action of glycosidases during accumulation in
the culture medium.
In L cells, a mannose-rich ;95-kDa precursor (cTSHR, lane
C) was present in high concentration. It was converted into an
;80-kDa species after treatment with N-glycanase F or en-
doglycosidases H (cTSHR (lanes F and H)). A small amount of
a ;120-kDa monomeric precursor (cTSHR, lane C) which con-
tains mature oligosaccharides was also observed. The latter
could be converted into the ;80-kDa species by N-glycanase F
(cTSHR, lane F) but was resistant to endoglycosidase H
(cTSHR, lane H). The amount of the mature ;120-kDa species
was variable and seemed to be related to cell growth conditions
and cell confluence (data not shown). In all conditions, it was
markedly less abundant than the cleaved receptor. Pulse-chase
experiments had previously confirmed that both forms corre-
spond to precursors of the TSH receptor (17). The high man-
nose precursor may accumulate inside the transfected cells
because the cellular machinery is unable to process to comple-
4548 A Soluble Form of the Human TSH Receptor
FIG. 3. Comparison of 125I-TSH binding by soluble and cellular
TSH receptor. Triton X-100 membrane extracts from TSHR express-
ing L cells and the corresponding cell culture medium (see “Experimen-
tal Procedures”) were incubated with iodinated bovine TSH (15,000
cpm) in the presence or absence of increasing amounts of unlabeled
bovine TSH (bTSH), human FSH (hFSH), or human CG (hCG) for 16 h
at 4 °C. The incubations were terminated by precipitation of the com-
plexes with polyethylene glycol as indicated under “Experimental Pro-
cedures.” Results are expressed as the ratio of B (iodinated TSH bound
in the presence of unlabeled hormone) on Bo (iodinated TSH bound in
the absence of unlabeled hormone) 3 100.
FIG. 4. Immunopurification of the soluble form of TSHR. The L
cell line stably transfected with the TSH receptor was grown for 48 h as
described under “Experimental Procedures.” After concentration (30-
fold) the cell culture medium was passed through an immunomatrix
containing antibody T5-51 (this antibody recognizes receptor ectodo-
main). After extensive washings elution was performed using a citrate
pH 2 buffer.
tion the overexpressed receptor.
Taken together, these experiments allowed us to conclude
that there is spontaneous shedding of a functional extracellular
domain of the TSH receptor in L cell culture medium, which
corresponds to the a subunit of the receptor. The same immu-
noreactivity was detected in a CHO-derived cell line expressing
the TSHR, as well as in the medium of primary cultures of
human thyrocytes.
Regulation of sTSHR Shedding—At the beginning of these
studies we wondered if the release of soluble receptor could not
be an artifact due to proteolytic enzymes present in the serum
used for cell culture. To investigate this possibility L cells
expressing TSHR were cultured in decreased serum concentra-
tions. However, we observed the opposite effect to that ex-
pected. As illustrated in Fig. 6, the accumulation of sTSHR in
the medium was markedly increased when the fetal calf serum
concentration was reduced in the medium. The ratio of soluble
to cellular receptor was increased 1.6- and 3-fold when serum
concentration was reduced from 10% to 5% and 1%, respec-
tively (Fig. 6, inset). In 1% serum, sTSHR concentration
FIG. 5. Comparison of the structure of the cellular and the
soluble forms of the TSHR. Cellular (cTSHR) and soluble (sTSHR)
receptors were immunopurified from the stably transfected L cell line
and its culture medium as described in the legend to Fig. 4. They were
resolved by SDS-polyacrylamide gel electrophoresis under reducing
conditions and detected by immunoblot with T5-317 (antibody raised
against the ectodomain of the TSHR). C, control receptor preparation;
F, receptor preparation treated with N-glycosidase; H, receptor treated
with endoglycosidase H. The migration of molecular mass markers
(kDa) is indicated on the left.
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FIG. 6. Effect of fetal calf serum concentration on the shedding
of sTSHR. L cells expressing TSHR were cultured for 24 h in a medium
complemented with various concentrations of fetal calf serum (FCS):
respectively, 10% (closed triangles), 5% (open squares), or 1% (closed
circles). Results are expressed as the mean 6 S.E. of three independent
determinations. The inset shows the ratio of soluble (sTSHR) on the
cellular (cTSHR) receptor.
reached ;25% of the cellular receptor content after 24 h of
culture. Addition of serum albumin to cell culture medium
containing 1% serum to keep protein concentration constant
did not prevent the increased shedding (data not shown).
Since serum concentration determines the rate of cell divi-
sion, we wondered if sTSHR shedding might be linked to cell
proliferation. Cells were cultured in presence of reduced serum
concentration (1%), but insulin, fibroblast growth factor, or
epidermal growth factor were added to the culture medium. All
these treatments increased cell proliferation (Fig. 7A) but did
not decrease sTSHR shedding (Fig. 7B). Thus the effect of
serum is not secondary to changes in cell proliferation rates but
is due to the presence of an unidentified component. The latter
is not thermolabile, since heating of fetal calf serum for 30 min
at 56 or 95 °C prior use did not reverse the inhibitory effect.
Effect of TSH and Second Messengers on sTSHR Shedding—
The shedding of the extracellular domain of several other mem-
brane receptors is regulated by their ligand or by the activation
of protein kinase C (reviewed in Refs. 21 and 22). We thus
investigated if sTSHR shedding could be regulated by similar
mechanisms. Table I shows the effect of TSH on sTSHR shed-
ding. A limited (;30%), but statistically significant, stimula-
tory effect was observed. This effect might have been underes-
 A Soluble Form of the Human TSH Receptor
FIG. 7. Effects of growth factors on sTSHR shedding. L cells
expressing TSH receptor were grown for 24 h in a medium containing
10 or 1% fetal calf serum (FCS). Either insulin (INS, 10 mg/ml), fibro-
blast growth factor (FGF, 50 ng/ml), or epidermal growth factor (EGF,
10 ng/ml) were added to the medium of cells grown in presence of 1%
fetal calf serum. Control cells (Cont) were grown in the absence of
growth factors. A, the protein content was determined and used to
evaluate the cell number. It was expressed as the mean 6 S.E. (n 5 3).
B, the ratio of soluble (sTSHR) to cellular (cTSHR) receptor corresponds
to three independent determinations (mean 6 S.E.). *, p , 0.05; **, p ,
0.1 versus 1% FCS control.
TABLE I
Effect of TSH on sTSHR shedding
L cells expressing TSHR were grown in medium containing 1% fetal
calf serum for 24 h at 37 °C in the absence (control) or in the presence
of various amounts of bTSH. Cellular and soluble TSHR and total
protein content in the homogenate were determined as described under
“Experimental Procedures.” sTSHR content was standardized per mg of
protein in the cell homogenate (this corrects for variations in cell num-
bers). It was then expressed as percentage of control in the absence of
TSH. Mean 6 S.E. (n 5 3) are indicated.
Treatment % of control 6 S.E.
Control 100 6 3.6
bTSH, 1 microunit/ml 113 6 4.7
bTSH, 10 microunits/ml 109 6 7.4
bTSH, 100 microunits/ml 126 6 8.3a
bTSH, 1 milliunit/ml 124 6 7.3a
bTSH, 10 milliunits/ml 131 6 3.9a
a
p , 0.01 versus control untreated cells.
timated, since TSH is known to promote receptor
internalization (17, 23). Thus, fewer cell surface receptors were
probably available to release their a subunits.
4549
TABLE II
Effect of TSH, cAMP, forskolin, PMA, and calcium ionophores on
sTSHR shedding
L cells expressing TSHR were grown in a medium containing 1% fetal
calf serum for 24 h at 37 °C in the absence (control) or in the presence
of bTSH, 8-bromo-cAMP, forskolin, PMA, A23187, and ionomycin. Cel-
lular TSHR and total protein content in the homogenate were deter-
mined and expressed as described in Table I and under “Experimental
Procedures.”
Treatment % of control 6 S.E.
Control 100 6 5.0
bTSH, 10 milliunits/ml 128 6 7.8a
8-Bromo-cAMP, 500 mM 117 6 3.2
Forskolin, 10 mM 115 6 8.1
PMA, 200 nM 143 6 12.0b
A23187, 1 mM 146 6 14.3b
Ionomycin, 500 nM 146 6 10.6b
a
p , 0.05 versus control untreated cells.
b
p , 0.01 versus control untreated cells.
In order to better discriminate the pathways (adenylate cy-
clase or phospholipase C) implicated in this hormonal effect, we
studied forskolin, 8-bromo-cAMP, phorbol 12-myristate 13-ac-
etate (PMA), as well as calcium ionophores (Table II). Only
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PMA (43% over untreated control) and the calcium ionophores
A23187 (46%) and ionomycin (46%) (all p , 0.01) mimicked the
effect of bTSH on sTSHR shedding. Forskolin and 8-bromo-
cAMP had no significant effect. These results suggest that the
protein kinase C pathway and intracellular calcium concentra-
tion are important parameters in the regulation of sTSHR
shedding.
Effect of Inhibitors of Intracellular Traffic on sTSHR Shed-
ding—We used various inhibitors to analyze the role of recep-
tor cellular trafficking on the shedding. Cleavage of the recep-
tor might have occurred either during its internalization and
its recycling or during its biosynthetic progression through the
Golgi apparatus. Initially we analyzed the effect of inhibitors
interfering with internalization, recycling, and degradation in
lysosomes (Fig. 8). In order to prevent adverse effects on cell
viability, incubations with these agents were limited to 8 h.
Under these conditions, only small effects on total cellular
receptor content were observed (,10% decrease).
The weak amine chloroquine, which increases the pH of
acidic vacuoles, impedes the traffic through acidified compart-
ments and disturbs lysosomal function (24), enhanced sTSHR
shedding (50% over untreated cells). Bafilomycin, an inhibitor
of the H1-ATPase of acidic vacuoles (25), also enhanced TSHR
shedding. Monensin, a monovalent carboxylic ionophore which
dissipates transmembrane proton gradient and blocks the re-
cycling pathway (26), had a stimulatory effect on sTSHR shed-
ding. These results suggested that endocytosis and lysosomal
proteolysis are not implicated in sTSHR shedding and that all
processes which enhance receptor residency on the plasma
membrane increase shedding.
Finally brefeldin A, an agent which inhibits Golgi function
(27), did not modify TSHR shedding, suggesting that this proc-
ess is not linked to any event occurring during the progression
of the receptor from the Golgi complex to the cell membrane.
(The 8-h incubation was insufficient to decrease receptor con-
centration on the cell surface).
Together, these experiments strongly supported the concept
that sTSHR shedding was dependent upon events occurring at
or near the cell membrane.
Effect of Protease Inhibitors on sTSHR Shedding—To iden-
tify the protease involved in TSHR maturation, a variety of
inhibitors was examined for their effect on the accumulation of
sTSHR in L cell culture medium (Table III). The majority of the
protease inhibitors tested did not modify the accumulation of
sTSHR, including inhibitors of serine proteases (aprotinin),
4550 A Soluble Form of the Human TSH Receptor

FIG. 8. Effects of inhibitors of cellular traffic on sTSHR shed-

ding. L cells expressing TSH receptor were grown for 8 h in a culture
medium complemented with 1% serum in the absence (Cont) or in the
presence of chloroquine (CQ), 10 mM; bafilomycin (Baf), 1 mM; monensin
(Mon), 10 mM; or brefeldin A (BFA), 20 mg/ml. The ratio of soluble
(sTSHR) on cellular (cTSHR) receptor was determined and expressed
as percent of control. The results of three independent determinations
are shown (mean 6 S.E.) **, p , 0,01 versus untreated control.

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TABLE III
Effect of protease inhibitors on sTSHR accumulation
L cells expressing TSHR were cultured for 24 h in 1% serum in the
presence of the inhibitors. The soluble TSHR was assayed in the cell
culture medium. It was compared with the concentration of soluble
receptor in culture medium of cells grown in the absence of inhibitors
(mean of three determinations). TLCK, tosyl-L-lysine chloromethyl ke-
tone; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; TAME,
tosylarginyl methyl ester; pCMB, p-chloromercuribenzoate.
Protease inhibitors sTSHR shedding

% of control
Serine and cysteine (thiol) proteases
Aprotinin, 1.6 mg/ml 100
Leupeptin, 3 mM 76
Phenylmethylsulfonyl fluoride, 2 mM 100
TLCK, 70 mM 100 FIG. 9. Effect of a matrix metalloproteinase inhibitor
TPCK, 1 mM 100 (BB-2116) on sTSHR shedding. L cells expressing TSHR were cul-
E64, 700 mM 100 tured for 18 h in the presence of various concentrations of BB-2116. A,
TAME, 3 mM 100 the soluble TSHR (sTSHR) was assayed in the culture medium. B, the
pCMB, 100 mM 100 a subunits remaining attached by disulfide bridges to the b subunits in
the cell membranes were assayed. The cells were scraped and treated
Metalloproteases
with 100 mM dithrotreitol (DTT) (see “Experimental Procedures”), and
Phosphoramidon, 100 mM 100
the concentration of released a subunits was determined. Results are
EDTA, 3 mM 70
expressed in pM (A) or pmol of a subunits released (B) as the mean 6
EGTA, 3 mM 81
S.E. (n 5 4).
Captopril, 100 mM 100
Calpain inhibitor I, 100 mM 100
Aspartyl proteases medium which became undetectable at 100 mg/ml of BB-2116.
Pepstatin, 100 mg/ml 100 The concentrations of BB-2116 suppressing TSHR a subunit
Aminopeptidase shedding matched those previously described for the inhibition
Bestatin, 250 mM 100 of pro-tumor necrosis factor a cleavage (28). However the shed-
ding of TSHR a subunit is a two-step process. The first step
consists in the cleavage of the receptor and the second step in
cysteine proteases (E64), aspartyl proteases (pepstatin), and the reduction of its disulfide bond(s). It was necessary to verify
aminopeptidase (bestatin). A limited (;24%) inhibition of that BB-2116 was indeed acting on the cleavage. We thus
TSHR shedding was observed only at high concentrations (3 measured the concentration of a subunits linked by disulfide
mM) of leupeptin. EDTA and EGTA (3 mM for each) also signif- bonds to b subunits and which thus remain attached to cell
icantly inhibited sTSHR shedding. Higher concentrations could membranes. This was done by treating cell membranes with
not be used because of their deleterious effects on cells. Al- dithiotreitol and measuring the released a subunits. If inhibi-
though limited, this inhibition led us to postulate that a met- tion occurs at the level of the cleavage the concentration of
alloprotease might be involved in the maturation of TSHR. “releasable” a subunit should decrease, whereas if inhibition
Inhibitors of nonmatrix metalloproteinases (captopril, phos- occurs at the level of reduction of disulfide bridge(s) it should
phoramidon) had no effect. increase.
We thus investigated the effect of a recently described potent A marked decrease in the concentration of membrane-at-
inhibitor of matrix metalloproteases, the synthetic hydroxamic tached cleaved receptors was produced by incubation with BB-
acid BB-2116 (28). As shown in Fig. 9A, we observed a dose- 2116 (Fig. 9B). Thus the inhibition occurs at the level of the
dependent inhibition of sTSHR accumulation in cell culture cleavage of the receptor. However at concentrations of inhibitor
 A Soluble Form of the Human TSH Receptor
which totally suppressed a subunit shedding there was only a
55% decrease of cleaved receptors present on the membranes.
This suggests that a critical threshold of the a/b heterodimer
must be reached on the cell surface to allow shedding to occur.
DISCUSSION
Using a quantitative immunoradiometric assay performed
with two monoclonal antibodies directed against the extracel-
lular domain of the TSH receptor, we have detected an immu-
noreactive protein in the culture medium of L cells and CHO
cells stably transfected with the TSH receptor. The same im-
munoreactivity was released from human thyrocytes. This ac-
cumulation was time-dependent and reached ;25% of the total
cellular receptor by 48 h.
This soluble TSHR is a glycosylated protein which is re-
tained by concanavalin A-Sepharose (not shown) and specifi-
cally binds its ligand TSH. Immunopurification of sTSHR from
the L cell line conditioned media demonstrated that its
polypeptide core corresponds to the a subunit (;35 kDa).
These experiments strongly suggested that there is a spon-
taneous shedding of the extracellular domain of the TSH re-
ceptor in stably transfected L and CHO cells and in human
thyrocytes. The shedding is specific for TSHR-expressing cells
and does not occur in a L cell line stably transfected with the
LH/CG receptor. This observation is concordant with the fact
that nearly all of the TSH receptors in human thyroid tissue
and most of these receptors in L cells (17) have a heterodimeric
structure, while the LH/CG receptors are expressed at the cell
surface as uncleaved monomers (12, 14).
The structure of the TSH receptor has been debated. The
hypothesis that artifactual proteolysis occurs during cell ho-
mogenization has been proposed by some authors (29 –31). This
possibility was not supported by our previous experiments (15,
17) and is strongly opposed by the present observation of spon-
taneous shedding of the extracellular domain of the TSH re-
ceptor by intact cells in culture.
In addition, we have detected the presence of sTSHR in
normal human serum using the same assay.2 Complete char-
acterization of this circulating sTSHR will allow us to deter-
mine whether it corresponds to the sTSHR detected in the
supernatant of cultured cells. There have been some prelimi-
nary reports of TSHR related peptide-like immunoreactivity
(32) and TSH-binding protein (33) in human serum. Our pre-
liminary data suggest that the sTSHR that we detect in human
serum is generated by shedding. Alternatively spliced TSHR
mRNAs also have been cloned from normal and Graves’ disease
thyroids (34, 35). Further experiments will allow us to deter-
mine whether a correlation can be established between the
concentration of sTSHR in the serum and specific pathological
conditions. This shed a subunit could be implicated in the
pathogenesis of certain autoimmune diseases.
Various cell surface receptors are known to be shed into the
extracellular milieu (reviewed in Refs. 21 and 22). In several
cases receptor shedding was shown to be increased by its cog-
nate ligand or by phorbol esters (21, 22, 36). We thus examined
the effect of TSH and of various signal mediatory molecules on
TSHR shedding. TSH induced a significant increase in the
shedding, PMA and calcium ionophores mimicked the effect of
TSH, while agents acting on the cAMP pathway had no effect.
The effect of TSH might be due to an increase in the concen-
tration or activity of the cleaving enzyme. Ligand binding or
cellular activation might also induce changes in the receptor
(phosphorylation, conformational changes) that make it more
susceptible to enzymatic cleavage. Alternatively TSH might
activate another mechanism involved in receptor shedding:
2
J. Couet, E. Milgrom, and M. Misrahi, unpublished results.
4551
possibly the reduction of disulfide bond(s) joining the a and b
subunits.
Surprisingly, lowering serum concentration from 10 to 1%
greatly enhanced TSHR shedding. This inhibitory effect of se-
rum was not mediated by an effect on cell growth as it could not
be reproduced by insulin or other growth factors. The mecha-
nism of this inhibition is not yet understood and needs further
study.
The use of various inhibitors of intracellular trafficking of
the receptor allowed us to conclude that neither receptor inter-
nalization, recycling nor degradation in lysosomes were in-
volved in sTSHR shedding. Moreover all agents that increased
the residence time of the receptor at the cell surface increased
sTSHR shedding. By contrast, inhibition of Golgi function did
not modify receptor shedding. Taken together, these experi-
ments strongly suggested that receptor modifications involved
in shedding occur at (or very near) the cell membrane.
We also investigated the nature of the protease involved in
TSHR maturation. The best known convertases involved in the
maturation of precursor proteins belong to the subtilisin family
of serine proteases (37). Their most common cleavage site com-
prises a pair of basic residues. Such basic sequences are found
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in the extracellular domain of the TSHR, and we, and others,
have previously thought that TSHR convertase might belong to
this family (15, 38). One member of these proteases, furin, has
been shown to be involved in the maturation of the insulin
proreceptor (39). This maturation occurs in the late Golgi com-
partment. In a previous work, pulse-chase experiments indi-
cated that the cleavage mechanism seemed different between
TSH and insulin receptors (17).
We examined a variety of protease inhibitors for their effects
on sTSHR production. Most of them were ineffective. The di-
valent ion chelators EDTA and EGTA inhibited TSHR process-
ing (30 and 20% inhibition, respectively). Higher concentra-
tions could not be used in our whole cell system as these
compounds had a cytotoxic effect. However these results sug-
gested that the TSHR convertase could be a metalloprotease.
Finally we used a potent and specific inhibitor of zinc-depend-
ent matrix metalloproteases. This synthetic hydroxamic acid,
BB-2116, inhibits in vitro the maturation of tumor necrosis
factor a (28). In our system, this agent totally suppressed
sTSHR accumulation in culture medium. This effect was sec-
ondary to a strong inhibition of TSHR cleavage into a/b
heterodimers.
BB-2116 inhibits in vitro the activity of collagenases, strome-
lysins, gelatinases, and PUMP I, which all belong to the matrix
metalloprotease family (28). These enzymes are secreted by the
cells and their primary role is to degrade extracellular matrix
proteins such as collagen, laminin, and proteoglycan. They are
zinc- and calcium-dependent enzymes secreted as zymogens
and are activated in situ by different mechanisms, particularly
proteolysis (reviewed in Refs. 40 and 41). These enzymes are
regulated by numerous growth factors, hormones, and tissue
inhibitors and are implicated in the physiological remodeling
of connective tissue, in destructive inflammatory pathologic
processes and in tumour invasion (reviewed in Ref. 42). Very
recently matrix metalloproteases have also been implicated in
the maturation of tumor necrosis factor a, a potent pro-inflam-
matory and immunomodulatory cytokine produced in inflam-
matory conditions (27, 43). The sites of cleavage are difficult to
predict as matrix metalloproteases exhibit broad substrate and
sequence specificities (44 – 46). The finding that matrix metal-
loprotease-like enzymes are implicated in TSHR maturation
also supports our observation that the cleavage occurs at the
cell membrane.
The shedding of TSH receptor is a two-step process: cleavage
4552 A Soluble Form of the Human TSH Receptor
of the receptor and reduction of disulfide bond(s). It is unknown
if the second step may also be regulated. (We cannot, however,
eliminate the possibility that in a small fraction of receptor
molecules no disulfide bonds are formed and that the cleavage
alone is sufficient for the shedding to occur). Further experi-
ments will be necessary to understand the physiological signif-
icance of the shedding process. We do not know yet if receptor
cleavage is necessary for receptor biological activity. The low
concentration of the 120-kDa precursor did not allow to study
its hormone binding ability. Purification of the b subunit of the
receptor and sequencing of its N-terminal part will allow to
define the site of cleavage. Mutagenesis experiments will then
lead to the understanding of the role of the cleavage in the
function of the TSH receptor. Other questions remain unan-
swered: is shedding of the a subunit a mechanism limiting the
transmission of the signal? Is the shed a subunit able to bind
hormone in the plasma and does it influence its biological
activity?
Acknowledgments—We thank British Biotech for the kind gift of
BB-2116. We thank Dr. G. Vassart and C. Maenhaut for providing the
stably transfected CHO cell line. We thank B. Saunier for his help in
primary culture of human thyrocytes. We also thank V. Coquendeau for
typing the manuscript.
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 Shedding of Human Thyrotropin Receptor Ectodomain: INVOLVEMENT OF A
MATRIX METALLOPROTEASE
Jacques Couet, Sokhavut Sar, André Jolivet, Mai-Thu Vu Hai, Edwin Milgrom and
Micheline Misrahi
J. Biol. Chem. 1996, 271:4545-4552.
doi: 10.1074/jbc.271.8.4545

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