THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 8, Issue of February 23, pp.

4545–4552, 1996
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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 mone (FSH) (6), and TSH receptors (7–10) form a subgroup in
glands has been shown to be cleaved into an extracellu- this family having a large and glycosylated extracellular do-
lar a subunit and a transmembrane b subunit held to- main specialized in high affinity hormone binding (11, 12).
gether by disulfide bridges. An excess of the latter com- Interest in the TSH receptor (TSHR) is enhanced by its impli-
ponent relative to the former suggested the shedding of cation in autoimmune diseases. Autoantibodies directed
the ectodomain. against this receptor have stimulatory (Graves’ disease) or
Indeed we observed such a shedding in cultures of blocking (idiopathic myxoedema) effects on its function (13).
human thyrocytes and permanently transfected L or

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

From cloning and sequencing of the human TSH receptor
Chinese hamster ovary cells. The shedding was in- cDNA (7–10), the structure of a preprotein with a calculated
creased by inhibitors of endocytosis, recycling, and ly- molecular mass of 84.5 kDa was deduced. But the character-
sosomal degradation, suggesting that it was dependent
ization of the mature structure of the TSH receptor and of other
on receptor residency at the cell surface. It was slightly
receptors of that family awaited the generation of high affinity
increased by TSH and phorbol esters, whereas forskolin
specific antibodies (14, 15). Interestingly, while the LH/CG
and 8-bromo-cyclic AMP were without effect. Decreas-
ing the serum concentration in cell culture medium en- receptor is expressed in target organs as a monomer (14), the
hanced the shedding by an unknown mechanism. TSH receptor is expressed in thyroid membranes as a het-
The shedding of the TSH receptor a domain is the erodimer: an extracellular a subunit (;53 kDa) and a mem-
consequence of two events: cleavage of the receptor brane-spanning b subunit (;38 kDa) are held together by
into a and b subunits and reduction of the disulfide disulfide bridge(s) (15). This observation is in agreement with
bridge(s). The complete inhibition of soluble TSH recep- one of the models proposed for the structure of the TSH recep-
tor shedding by the specific inhibitor BB-2116 indicated tor before the cloning (16). The post-translational cleavage in
that the cleavage reaction is catalyzed probably at the two subunits is almost complete in human thyroid tissue,
cell surface by a matrix metalloprotease. whereas in L cells stably transfected with the TSH receptor, a
This shedding mechanism may be responsible for the small amount of uncleaved mature receptor may still be pres-
presence of soluble TSH receptor a subunit in human ent. In transfected cells, but not in human thyroid tissue, there
circulation. is accumulation inside the cells of an unprocessed mannose-
rich monomeric precursor (17).
Pulse-chase experiments performed in L cells and in human
Thyrotropin (TSH)1 is the primary hormone that regulates thyrocytes confirmed that the TSH receptor is primarily syn-
thyroid cell growth and function via the G protein-coupled thesized as a ;95-kDa mannose-rich monomer, which under-
thyrotropin receptor (1, 2). Members of this receptor family are goes supplementary glycosylation to yield a ;120-kDa mono-
characterized by their common structural feature of seven meric precursor (17). The latter is then cleaved into mature a
transmembrane domains (3). However, luteinizing hormone- and b subunits. This processing into two subunits is unique
choriogonadotropin (LH/CG) (4, 5), follicle-stimulating hor- 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
* This work was supported by the Institut National de la Santé et de
la Recherche Médicale, the Délégation à la Recherche Clinique (Assist- a subunits (15). This observation led us to postulate that the a
ance Publique, Hôpitaux de Paris), the Faculté de Médecine Paris Sud, subunit might be shed from cell membranes and released into
the Association pour la Recherche sur le Cancer, the Ligue Nationale the extracellular space or bloodstream. Such a phenomenon
contre le Cancer, and the Fondation pour la Recherche Médicale Fran- could be important in the context of autoimmune diseases. We
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
report now that spontaneous shedding of the extracellular do-
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to main of the TSH receptor occurs in stably transfected L and
indicate this fact. CHO cell lines as well as in human thyrocytes. This shedding is
‡ Recipient of a fellowship from the Medical Research Council of also a regulated process involving the cleavage of the TSH
Canada.
receptor by a matrix metalloprotease.
§ To whom correspondence should be addressed: INSERM U. 135,
Hôpital de Bicêtre, 3ème niveau, 94275 Le Kremlin-Bicêtre, France. EXPERIMENTAL PROCEDURES
1
The abbreviations used are: TSH, thyroid-stimulating hormone;
sTSHR, soluble TSH receptor; cTSHR, cellular TSH receptor; h, human; Materials—Most reagents were purchased from Sigma. Bovine TSH,
b, bovine; LH, luteinizing hormone; CG, choriogonadotropin; LH/CGR, monensin, A23187, and ionomycin were from Calbiochem; iodinated
luteinizing hormone choriogonadotropin receptor; FSH, follicle-stimu- bovine TSH (70 mCi/mg) was from Eria Diagnostics Pasteur (Marnes-
lating hormone; FSHR, follicle-stimulating hormone receptor; CHO, la-Coquette, France); iodinated streptavidin was from Amersham Corp.
Chinese hamster ovary; PBS, phosphate-buffered saline; PMA, phorbol BB-2116, a matrix metalloprotease inhibitor, was kindly provided by
12-myristate 13-acetate; 8-bromo-cAMP, 8-bromo-cyclic AMP. British Biotech (Oxford, United Kingdom).

4545
4546 A Soluble Form of the Human TSH Receptor
Culture of an L Cell Line Permanently Expressing the Human TSH as described previously.
Receptor—This cell line was cultured as described previously (17). Cell Quantification of a/b Heterodimers by Treatment of Cells with Di-
viability tests were performed by the trypan blue exclusion technique. thiothreitol to Release TSHR a Subunit—At the end of incubation, cells
Primary Cultures of Human Thyrocytes—Human thyroid tissue was were scraped gently as described above and incubated for 2 h at 4 °C in
obtained by surgery from patients with benign thyroid diseases. Thy- 500 ml of a PBS (pH 7.4), 100 mM dithiothreitol solution under agitation.
roid follicles were prepared as described (18) and cultured for 48 –72 h. The cells were then centrifuged at 1500 3 g for 5 min. An aliquot of the
Immunoradiometric Assay of the TSH Receptor—A double-determi- supernatant was diluted 100-fold for the assay of released a subunits.
nant (“sandwich”-type) radioimmunoassay was developed for the spe- Then the cells were treated with Triton X-100 in order to solubilize the
cific quantification of the TSH receptor a subunit. This assay uses two remaining uncleaved TSHR. An aliquot of this cellular extract was used
additive monoclonal antibodies specific for the extracellular domain of for the assay of the cellular receptor.
the TSHR (T5-317 and T5-51 (15)). The specificity of these antibodies Statistical Analysis—Statistical differences between groups were an-
has been established by a variety of methods: these antibodies inter- alyzed by one way analysis of variance followed by Fisher’s least sig-
acted with TSH receptor expressed in Escherichia coli or in mammalian nificant difference test for multiple comparison. p , 0.05 was consid-
cells (COS 7, CHO, and L cells) as tested by enzyme-linked immunosor- ered significant. Statview computer program (Abacus Concepts Inc.,
bent assay, Western blot, and immunocytochemistry. No signal could be Berkeley, CA) was used for calculations.
be detected using all these methods in mock-transfected cells. Finally
antibody 51 has been used to immunopurify 125I-TSH receptor com- RESULTS
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
Identification of a Soluble Form of the TSHR (sTSHR) Re-
conjunction with 125I-streptavidin as the “reporter” antibody. Biotiny- leased from Transfected Cells—To detect possible shedding of
lation of this antibody was performed using the Amersham biotinyla- the extracellular domain of the TSH receptor from cells, we
tion kit (Amersham). The T5-317 antibody (0.5 mg/well) was coated onto first studied a L cell line stably transfected and expressing high
96-well plates (Maxisorb, Nunc, Rockilde, Denmark) in 0.05 M potas- levels of the human TSH receptor. This model system is more
sium phosphate buffer (pH 7.4) for 2 h at room temperature. Plates amenable to experimental analysis and more reproducible than
were washed four times with 300 ml of phosphate-buffered saline (PBS)
primary cultures of human thyrocytes. In the latter the con-

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

(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 centration of the receptor is known to be very low (15).
albumin 1% (w/v), Tween 0.05%. After washing, samples were then A double-determinant (sandwich-type) radioimmunoassay
incubated, directly or diluted in PBT, for 16 h at 4 °C or 2 h at room was developed for the specific quantification of the TSH recep-
temperature. The plates were washed with PBT and the biotinylated tor a subunit. This assay uses two additive monoclonal anti-
antibody (T5-51) added at a 0.5 mg/ml concentration in PBT for 2 h. bodies specific for the extracellular domain (a subunit) of the
After washing, 125I-streptavidin (75,000 cpm/well) was added for 1 h
TSHR (T5-317 and T5-51) (15). T5-317 is the capture antibody.
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 As illustrated in Fig. 1A, the accumulation of a soluble form
quantified relatively to a standard curve of TSHR immunopurified from of the TSHR was detected in the medium of a mouse L cell line
the stably transfected L cell line (17). This cellular TSHR had previ- stably transfected with the full-length human TSH receptor
ously been quantified comparatively to a hTSHR peptide-b-galactosid- cDNA (17). This accumulation was time-dependent and
ase protein fusion standard (15). This assay allowed us to detect rou- reached 50 pM after 48 h of culture (;25% of total receptor
tinely as little as 2 fmol/ml of soluble TSHR (sTSHR). We also developed
present in the cells at this time). The same result was obtained
a second assay for the determination of full length heterodimeric
(cleaved) and monomeric (uncleaved) forms of the TSHR by replacing when the medium was filtered through a 0.2-mm syringe filter
T5-317 by an antibody specific for the intracellular part of the TSHR or ultracentrifuged at 15,000 3 g to remove cellular debris.
(T3-365) (15). The assay was specific. Competition with an excess of unla-
TSHR Determinations in Culture Medium and Cellular Extracts— beled T5-51 antibodies suppressed the binding of the reporter
For determination of sTSHR concentration, or for the assay of the antibody, while a nonspecific competitor antibody had no effect.
full-length receptor in the culture medium (a/b receptor), the medium
The replacement of one of the TSHR antibodies in the assay by
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 a nonspecific irrelevant monoclonal antibody or the omission of
for 5 min, and stored at 270 °C. An aliquot of the Triton X-100 cellular TSHR also suppressed the binding of the 125I-streptavidin
extract (16) was used for cellular TSHR (cTSHR) determinations or (data not shown).
protein content quantification using the Pierce BCA protein assay re- We also examined the possibility that we were detecting the
agent kit (Pierce). receptor present in membrane fragments released into the
125
I-TSH Binding to Soluble and Cellular TSHR—TSH receptor
medium. To verify this point we used an alternative assay in
binding assays were performed using the method described by Petersen
et al. (19). Triton X-100 cellular extracts (17) or culture media from which the capture antibody recognized the intracellular do-
stably transfected L cells (concentrated 30-fold using successive Cen- main (b subunit) of the receptor (T3-365). This assay detects
triprep-10 and Centricon-10 concentrators (Amicon, Beverly, MA)) were cleaved or uncleaved whole receptor molecules. No such recep-
used for these experiments. Briefly, 2 pmol of soluble or cellular TSHR tor form was detected in the culture medium (Fig. 1A).
were added to iodinated bovine TSH in the absence or in the presence These experiments strongly suggested that spontaneous
of increasing concentrations of unlabeled bovine TSH (bTSH) human
shedding of a soluble form of the TSH receptor, corresponding
FSH (hFSH) or human CG (hCG). The bound complexes were precipi-
tated with polyethylene glycol (molecular weight: 6000). to its extracellular domain, occurred in the permanently trans-
Immunopurification of the Soluble TSHR—Immunopurification of fected L cell line. The same determinations were performed on
sTSHR from the permanently transfected L cells was performed as the medium of another TSHR expressing Chinese hamster
described previously (17) except that a T5-51 Affi-Gel-10 immunoma- ovary (CHO)-derived cell line (a gift from C. Maenhaut and G.
trix was used. For purification of sTSHR culture media containing 1% Vassart) (20). Those cells also produced and released a soluble
fetal calf serum were concentrated using a Minitan apparatus (Milli-
form of the TSHR into the culture medium in proportions
pore, Bedford, MA) equipped with a filter having a 30-kDa molecular
mass cutoff. Deglycosylation of receptors using N-glycanase F and en- comparable with the stably transfected L cell line (data not
doglycosidase H and immunoblots were performed as described (17). shown).
LH/CG Receptor Measurements in Culture Medium of a Stably As a control, we also studied L cells stably transfected with
Transfected L Cell Line—A double determinant radioimmunoassay was the related LH/CG receptor which express high levels of ma-
developed using two additive monoclonal antibodies against the extra- ture receptors (12). The presence of a functional receptor on the
cellular part of the porcine LH/CG receptor (LHR 38 and biotinylated
surface of these cells has been shown by hormone binding and
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 adenylate cyclase stimulation. A similar assay was developed
receptor quantification was performed using an L cell line permanently using two additive monoclonal antibodies specific for the extra-
expressing the porcine LH/CG receptor (12) and an hCG binding assay cellular domain of the LH/CG receptor. No receptor ectodomain
 A Soluble Form of the Human TSH Receptor 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,

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

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.
FIG. 1. Shedding of a soluble form of the TSH receptor from a Finally, sTSHR was purified from culture medium using
L cell line permanently expressing the human TSHR. A, an L cell immunoaffinity chromatography with the T5–51 immunoma-
line expressing the TSH receptor was cultured for various periods of trix (Fig. 4). More than 90% of the receptor present in the
time. Aliquots of culture medium were used for the assay of the a medium bound to the matrix and was eluted at acidic pH.
subunit (the sandwich assay used T5-317 and biotinylated T5-51, both
antibodies recognizing different epitopes in the ectodomain). As a con- Western blots were performed to compare the immunopuri-
trol the full-length receptor (a/b receptor) was also assayed using anti- fied sTSHR with the cellular TSHR (solubilized from the mem-
body T3-365 (epitope in the intracellular domain) and biotinylated branes of the stably transfected L cell line). Antibody T5-317
T5–51. B, an identical experiment was performed with a L cell line recognizing the extracellular domain of the receptor was used.
expressing LH/CG receptor. Antibodies LHR38 and biotinylated
LHR436, both recognizing the extracellular domain of LH/CG receptor, As shown in Fig. 5, sTSHR had an apparent molecular mass of
were used for the assay. Results are expressed as the mean 6 S.E. ;53 kDa (sTSHR, lane C) which is slightly smaller than the a
(standard error of the mean) of three independent determinations. subunit of the TSHR purified from cells (;60 kDa) (cTSHR,
lane C). However after digestion with N-glycanase F, which
could be detected in the cell culture medium even after a 7-fold removes all oligosaccharides, both proteins migrated as the
concentration of the medium (Fig. 1B). This is consistent with same ;35 kDa species (cTSHR (lane F) and sTSHR (lane F)).
the fact that this receptor is expressed as an uncleaved Endoglycosidase H (which removes only mannose-rich moieties
monomer (14). of precursor glycoproteins) had no effect on sTSHR or on the a
Thus, the existence of a soluble form of the receptor released subunit of the cellular TSHR (cTSHR (lane H) and sTSHR (lane
into the medium is specific for cells expressing the TSH H)). These results indicate that sTSHR and the a subunit of the
receptor. receptor contain the same polypeptide core and differ only
Identification of a Soluble Form of the TSHR in Primary slightly in their mature carbohydrate content. This difference
Cultures of Human Thyrocytes—In order to confirm the results may be due to the action of glycosidases during accumulation in
obtained with our transfected L cell model, we investigated the culture medium.
TSHR ectodomain shedding in a more physiological system, In L cells, a mannose-rich ;95-kDa precursor (cTSHR, lane
namely in human thyrocytes. We observed an accumulation of C) was present in high concentration. It was converted into an
an immunoreactive soluble form of the TSHR in primary cul- ;80-kDa species after treatment with N-glycanase F or en-
tures of human thyrocytes (Fig. 2). The proportion of this doglycosidases H (cTSHR (lanes F and H)). A small amount of
soluble TSHR to the total cellular TSHR concentration reached a ;120-kDa monomeric precursor (cTSHR, lane C) which con-
about 17% after 48 h of incubation and more than 22% after 72 tains mature oligosaccharides was also observed. The latter
h. The presence of sTSHR in thyrocyte culture medium was could be converted into the ;80-kDa species by N-glycanase F
confirmed in two independent experiments. (cTSHR, lane F) but was resistant to endoglycosidase H
Characterization and Purification of the sTSHR from the (cTSHR, lane H). The amount of the mature ;120-kDa species
Stably Transfected L Cell Line—Preliminary experiments had was variable and seemed to be related to cell growth conditions
shown that when the medium was filtered on Centricon mem- and cell confluence (data not shown). In all conditions, it was
branes with a molecular cutoff of 30 kDa, most of the immuno- markedly less abundant than the cleaved receptor. Pulse-chase
reactivity (.90%) was retained by the filters (not shown). experiments had previously confirmed that both forms corre-
To test the ability of sTSHR to bind specifically TSH, con- spond to precursors of the TSH receptor (17). The high man-
centrated (30 3) culture medium from transfected L cells was nose precursor may accumulate inside the transfected cells
incubated with iodinated bovine TSH, in the presence or in the 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 FIG. 5. Comparison of the structure of the cellular and the
TSH receptor. Triton X-100 membrane extracts from TSHR express- soluble forms of the TSHR. Cellular (cTSHR) and soluble (sTSHR)
ing L cells and the corresponding cell culture medium (see “Experimen- receptors were immunopurified from the stably transfected L cell line
tal Procedures”) were incubated with iodinated bovine TSH (15,000 and its culture medium as described in the legend to Fig. 4. They were
cpm) in the presence or absence of increasing amounts of unlabeled resolved by SDS-polyacrylamide gel electrophoresis under reducing
bovine TSH (bTSH), human FSH (hFSH), or human CG (hCG) for 16 h conditions and detected by immunoblot with T5-317 (antibody raised
at 4 °C. The incubations were terminated by precipitation of the com- against the ectodomain of the TSHR). C, control receptor preparation;
plexes with polyethylene glycol as indicated under “Experimental Pro- F, receptor preparation treated with N-glycosidase; H, receptor treated
cedures.” Results are expressed as the ratio of B (iodinated TSH bound with endoglycosidase H. The migration of molecular mass markers
in the presence of unlabeled hormone) on Bo (iodinated TSH bound in (kDa) is indicated on the left.
the absence of unlabeled hormone) 3 100.

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

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
FIG. 4. Immunopurification of the soluble form of TSHR. The L
circles). Results are expressed as the mean 6 S.E. of three independent
cell line stably transfected with the TSH receptor was grown for 48 h as
determinations. The inset shows the ratio of soluble (sTSHR) on the
described under “Experimental Procedures.” After concentration (30-
cellular (cTSHR) receptor.
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 reached ;25% of the cellular receptor content after 24 h of
pH 2 buffer. culture. Addition of serum albumin to cell culture medium
containing 1% serum to keep protein concentration constant
tion the overexpressed receptor. did not prevent the increased shedding (data not shown).
Taken together, these experiments allowed us to conclude Since serum concentration determines the rate of cell divi-
that there is spontaneous shedding of a functional extracellular sion, we wondered if sTSHR shedding might be linked to cell
domain of the TSH receptor in L cell culture medium, which proliferation. Cells were cultured in presence of reduced serum
corresponds to the a subunit of the receptor. The same immu- concentration (1%), but insulin, fibroblast growth factor, or
noreactivity was detected in a CHO-derived cell line expressing epidermal growth factor were added to the culture medium. All
the TSHR, as well as in the medium of primary cultures of these treatments increased cell proliferation (Fig. 7A) but did
human thyrocytes. not decrease sTSHR shedding (Fig. 7B). Thus the effect of
Regulation of sTSHR Shedding—At the beginning of these serum is not secondary to changes in cell proliferation rates but
studies we wondered if the release of soluble receptor could not is due to the presence of an unidentified component. The latter
be an artifact due to proteolytic enzymes present in the serum is not thermolabile, since heating of fetal calf serum for 30 min
used for cell culture. To investigate this possibility L cells at 56 or 95 °C prior use did not reverse the inhibitory effect.
expressing TSHR were cultured in decreased serum concentra- Effect of TSH and Second Messengers on sTSHR Shedding—
tions. However, we observed the opposite effect to that ex- The shedding of the extracellular domain of several other mem-
pected. As illustrated in Fig. 6, the accumulation of sTSHR in brane receptors is regulated by their ligand or by the activation
the medium was markedly increased when the fetal calf serum of protein kinase C (reviewed in Refs. 21 and 22). We thus
concentration was reduced in the medium. The ratio of soluble investigated if sTSHR shedding could be regulated by similar
to cellular receptor was increased 1.6- and 3-fold when serum mechanisms. Table I shows the effect of TSH on sTSHR shed-
concentration was reduced from 10% to 5% and 1%, respec- ding. A limited (;30%), but statistically significant, stimula-
tively (Fig. 6, inset). In 1% serum, sTSHR concentration tory effect was observed. This effect might have been underes-
 A Soluble Form of the Human TSH Receptor 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

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

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
FIG. 7. Effects of growth factors on sTSHR shedding. L cells viability, incubations with these agents were limited to 8 h.
expressing TSH receptor were grown for 24 h in a medium containing Under these conditions, only small effects on total cellular
10 or 1% fetal calf serum (FCS). Either insulin (INS, 10 mg/ml), fibro-
receptor content were observed (,10% decrease).
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% The weak amine chloroquine, which increases the pH of
fetal calf serum. Control cells (Cont) were grown in the absence of acidic vacuoles, impedes the traffic through acidified compart-
growth factors. A, the protein content was determined and used to ments and disturbs lysosomal function (24), enhanced sTSHR
evaluate the cell number. It was expressed as the mean 6 S.E. (n 5 3). shedding (50% over untreated cells). Bafilomycin, an inhibitor
B, the ratio of soluble (sTSHR) to cellular (cTSHR) receptor corresponds
to three independent determinations (mean 6 S.E.). *, p , 0.05; **, p , of the H1-ATPase of acidic vacuoles (25), also enhanced TSHR
0.1 versus 1% FCS control. shedding. Monensin, a monovalent carboxylic ionophore which
dissipates transmembrane proton gradient and blocks the re-
TABLE I cycling pathway (26), had a stimulatory effect on sTSHR shed-
Effect of TSH on sTSHR shedding
ding. These results suggested that endocytosis and lysosomal
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 proteolysis are not implicated in sTSHR shedding and that all
of various amounts of bTSH. Cellular and soluble TSHR and total processes which enhance receptor residency on the plasma
protein content in the homogenate were determined as described under membrane increase shedding.
“Experimental Procedures.” sTSHR content was standardized per mg of Finally brefeldin A, an agent which inhibits Golgi function
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
(27), did not modify TSHR shedding, suggesting that this proc-
TSH. Mean 6 S.E. (n 5 3) are indicated. ess is not linked to any event occurring during the progression
of the receptor from the Golgi complex to the cell membrane.
Treatment % of control 6 S.E.
(The 8-h incubation was insufficient to decrease receptor con-
Control 100 6 3.6 centration on the cell surface).
bTSH, 1 microunit/ml 113 6 4.7 Together, these experiments strongly supported the concept
bTSH, 10 microunits/ml 109 6 7.4
bTSH, 100 microunits/ml 126 6 8.3a that sTSHR shedding was dependent upon events occurring at
bTSH, 1 milliunit/ml 124 6 7.3a or near the cell membrane.
bTSH, 10 milliunits/ml 131 6 3.9a Effect of Protease Inhibitors on sTSHR Shedding—To iden-
a
p , 0.01 versus control untreated cells. tify the protease involved in TSHR maturation, a variety of
inhibitors was examined for their effect on the accumulation of
timated, since TSH is known to promote receptor sTSHR in L cell culture medium (Table III). The majority of the
internalization (17, 23). Thus, fewer cell surface receptors were protease inhibitors tested did not modify the accumulation of
probably available to release their a subunits. 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.

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

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 4551
which totally suppressed a subunit shedding there was only a possibly the reduction of disulfide bond(s) joining the a and b
55% decrease of cleaved receptors present on the membranes. subunits.
This suggests that a critical threshold of the a/b heterodimer Surprisingly, lowering serum concentration from 10 to 1%
must be reached on the cell surface to allow shedding to occur. greatly enhanced TSHR shedding. This inhibitory effect of se-
DISCUSSION
rum was not mediated by an effect on cell growth as it could not
be reproduced by insulin or other growth factors. The mecha-
Using a quantitative immunoradiometric assay performed nism of this inhibition is not yet understood and needs further
with two monoclonal antibodies directed against the extracel- study.
lular domain of the TSH receptor, we have detected an immu- The use of various inhibitors of intracellular trafficking of
noreactive protein in the culture medium of L cells and CHO
the receptor allowed us to conclude that neither receptor inter-
cells stably transfected with the TSH receptor. The same im-
nalization, recycling nor degradation in lysosomes were in-
munoreactivity was released from human thyrocytes. This ac-
volved in sTSHR shedding. Moreover all agents that increased
cumulation was time-dependent and reached ;25% of the total
the residence time of the receptor at the cell surface increased
cellular receptor by 48 h.
sTSHR shedding. By contrast, inhibition of Golgi function did
This soluble TSHR is a glycosylated protein which is re-
not modify receptor shedding. Taken together, these experi-
tained by concanavalin A-Sepharose (not shown) and specifi-
ments strongly suggested that receptor modifications involved
cally binds its ligand TSH. Immunopurification of sTSHR from
in shedding occur at (or very near) the cell membrane.
the L cell line conditioned media demonstrated that its
We also investigated the nature of the protease involved in
polypeptide core corresponds to the a subunit (;35 kDa).
TSHR maturation. The best known convertases involved in the
These experiments strongly suggested that there is a spon-
maturation of precursor proteins belong to the subtilisin family
taneous shedding of the extracellular domain of the TSH re-
of serine proteases (37). Their most common cleavage site com-
ceptor in stably transfected L and CHO cells and in human
prises a pair of basic residues. Such basic sequences are found

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

thyrocytes. The shedding is specific for TSHR-expressing cells
and does not occur in a L cell line stably transfected with the in the extracellular domain of the TSHR, and we, and others,
LH/CG receptor. This observation is concordant with the fact have previously thought that TSHR convertase might belong to
that nearly all of the TSH receptors in human thyroid tissue this family (15, 38). One member of these proteases, furin, has
and most of these receptors in L cells (17) have a heterodimeric been shown to be involved in the maturation of the insulin
structure, while the LH/CG receptors are expressed at the cell proreceptor (39). This maturation occurs in the late Golgi com-
surface as uncleaved monomers (12, 14). partment. In a previous work, pulse-chase experiments indi-
The structure of the TSH receptor has been debated. The cated that the cleavage mechanism seemed different between
hypothesis that artifactual proteolysis occurs during cell ho- TSH and insulin receptors (17).
mogenization has been proposed by some authors (29 –31). This We examined a variety of protease inhibitors for their effects
possibility was not supported by our previous experiments (15, on sTSHR production. Most of them were ineffective. The di-
17) and is strongly opposed by the present observation of spon- valent ion chelators EDTA and EGTA inhibited TSHR process-
taneous shedding of the extracellular domain of the TSH re- ing (30 and 20% inhibition, respectively). Higher concentra-
ceptor by intact cells in culture. tions could not be used in our whole cell system as these
In addition, we have detected the presence of sTSHR in compounds had a cytotoxic effect. However these results sug-
normal human serum using the same assay.2 Complete char- gested that the TSHR convertase could be a metalloprotease.
acterization of this circulating sTSHR will allow us to deter- Finally we used a potent and specific inhibitor of zinc-depend-
mine whether it corresponds to the sTSHR detected in the ent matrix metalloproteases. This synthetic hydroxamic acid,
supernatant of cultured cells. There have been some prelimi- BB-2116, inhibits in vitro the maturation of tumor necrosis
nary reports of TSHR related peptide-like immunoreactivity factor a (28). In our system, this agent totally suppressed
(32) and TSH-binding protein (33) in human serum. Our pre- sTSHR accumulation in culture medium. This effect was sec-
liminary data suggest that the sTSHR that we detect in human ondary to a strong inhibition of TSHR cleavage into a/b
serum is generated by shedding. Alternatively spliced TSHR heterodimers.
mRNAs also have been cloned from normal and Graves’ disease BB-2116 inhibits in vitro the activity of collagenases, strome-
thyroids (34, 35). Further experiments will allow us to deter- lysins, gelatinases, and PUMP I, which all belong to the matrix
mine whether a correlation can be established between the metalloprotease family (28). These enzymes are secreted by the
concentration of sTSHR in the serum and specific pathological cells and their primary role is to degrade extracellular matrix
conditions. This shed a subunit could be implicated in the proteins such as collagen, laminin, and proteoglycan. They are
pathogenesis of certain autoimmune diseases. zinc- and calcium-dependent enzymes secreted as zymogens
Various cell surface receptors are known to be shed into the and are activated in situ by different mechanisms, particularly
extracellular milieu (reviewed in Refs. 21 and 22). In several proteolysis (reviewed in Refs. 40 and 41). These enzymes are
cases receptor shedding was shown to be increased by its cog- regulated by numerous growth factors, hormones, and tissue
nate ligand or by phorbol esters (21, 22, 36). We thus examined inhibitors and are implicated in the physiological remodeling
the effect of TSH and of various signal mediatory molecules on of connective tissue, in destructive inflammatory pathologic
TSHR shedding. TSH induced a significant increase in the processes and in tumour invasion (reviewed in Ref. 42). Very
shedding, PMA and calcium ionophores mimicked the effect of recently matrix metalloproteases have also been implicated in
TSH, while agents acting on the cAMP pathway had no effect. the maturation of tumor necrosis factor a, a potent pro-inflam-
The effect of TSH might be due to an increase in the concen- matory and immunomodulatory cytokine produced in inflam-
tration or activity of the cleaving enzyme. Ligand binding or matory conditions (27, 43). The sites of cleavage are difficult to
cellular activation might also induce changes in the receptor predict as matrix metalloproteases exhibit broad substrate and
(phosphorylation, conformational changes) that make it more sequence specificities (44 – 46). The finding that matrix metal-
susceptible to enzymatic cleavage. Alternatively TSH might loprotease-like enzymes are implicated in TSHR maturation
activate another mechanism involved in receptor shedding: also supports our observation that the cleavage occurs at the
cell membrane.
2
J. Couet, E. Milgrom, and M. Misrahi, unpublished results. 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 Vannier, B., and Milgrom, E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,
3765–3769
if the second step may also be regulated. (We cannot, however, 16. Furmaniak, J., Nakajima, Y., Hashim, F. A., Creagh, F. M., Davies Jones, E.,
eliminate the possibility that in a small fraction of receptor Howells, R. D., McLachlan, S. M., and Rees Smith, B. (1987) Acta
Endocrinol. Suppl. 281, 157–165
molecules no disulfide bonds are formed and that the cleavage
17. Misrahi, M., Ghinea, N., Sar, S., Saunier, B., Jolivet, A., Loosfelt, H., Cerutti,
alone is sufficient for the shedding to occur). Further experi- M., Devauchelle, G., and Milgrom, E. (1994) Eur. J. Biochem. 222, 711–719
ments will be necessary to understand the physiological signif- 18. Roger, P. P., and Dumont, J. E. (1987) Biochem. Biophys. Res. Commun. 149,
707–711
icance of the shedding process. We do not know yet if receptor 19. Petersen, V. B., Dawes, P. J. D., Rees Smith, B., and Hall, R. (1977) FEBS Lett.
cleavage is necessary for receptor biological activity. The low 83, 63– 67
concentration of the 120-kDa precursor did not allow to study 20. Perret, J., Ludgate, M., Libert, F., Gerard, C., Dumont, J. E., Vassart, G., and
Parmentier, M. (1990) Biochem. Biophys. Res. Commun. 171, 1044 –1050
its hormone binding ability. Purification of the b subunit of the 21. Ehlers, M. R. W., and Riordan, J. F. (1991) Biochemistry 30, 10065–10074
receptor and sequencing of its N-terminal part will allow to 22. Tedder, F. T. (1991) Am. J. Respir. Cell. Mol. Biol. 5, 305–306
define the site of cleavage. Mutagenesis experiments will then 23. Heldin, N. E., Gustavsson, B., Hermansson, A., and Westermark, B. (1994)
Endocrinology 134, 2032–2036
lead to the understanding of the role of the cleavage in the 24. Zuppan, A. A., and Johnson, E. M., Jr. (1991) J. Biol. Chem. 266, 15384 –15390
function of the TSH receptor. Other questions remain unan- 25. Bowman, E. J., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci.
U. S. A. 85, 7972–7976
swered: is shedding of the a subunit a mechanism limiting the 26. Tartakoff, A. M. (1983) Cell 32, 1026 –1028
transmission of the signal? Is the shed a subunit able to bind 27. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell
hormone in the plasma and does it influence its biological Biol. 116, 1071–1080
28. Gearing, A. J. H., Beckett, P., Christodoulou, M., Churchill, M., Clements, J.,
activity? Davidson, A. H., Drummond, A. H., Galloway, W. A., Gilbert, R., Gordon,
J. L., Leber, T. M., Mangan, M., Miller, K., Nayee, P., Owen, K., Patel, S.,
Acknowledgments—We thank British Biotech for the kind gift of Thomas, W., Weils, G., Wood, L. M., and Wooley, K. (1994) Nature 370,
BB-2116. We thank Dr. G. Vassart and C. Maenhaut for providing the 555–557
stably transfected CHO cell line. We thank B. Saunier for his help in 29. Ban, T., Kosugi, S., and Kohn, L. D. (1992) Endocrinology 131, 815– 829
primary culture of human thyrocytes. We also thank V. Coquendeau for 30. Russo, D., Chazenbalk, G. D., Nagayama, Y., Wadsworth, H. L., Seto, P., and

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

typing the manuscript. Rapoport, B. (1991) Mol. Endocrinol. 5, 1607–1612
31. Russo, D., Nagayama, Y., Chazenbalk, G. D., Wadsworth, H. L., and Rapoport,
REFERENCES B. (1992) Endocrinology 130, 2135–2138
32. Murakami, M., Miyashita, K., Yamada, M., Iriuchijima, T., and Mori, M.
1. Vassart, G., and Dumont, J. E. (1992) Endocr. Rev. 13, 596 – 611 (1992) Biochem. Biophys. Res. Commun. 186 1074 –1080
2. Misrahi, M., Loosfelt, H., Gross, B., Atger, M., Jolivet, A., Savouret, J. F., and 33. Hunt, N., Willey, K. P., Jahner, D., Ivell, R., Northemann, W., Castel, M. A.,
Milgrom, E. (1994) Curr. Opin. Endocrinol. & Diabetes 1, 175–183 and Leidenberger, F. (1992) Exp. Clin. Endocrinol. 100, 22–27
3. Dohlman, H. G., Caron, M. G., and Lefkowitz, R. J. (1987) Biochemistry 26,
34. Graves, P. N., Tomer, Y., and Davies, T. F. (1992) Biochem. Biophys. Res.
2657–2664
Commun. 187, 1135–1143
4. Loosfelt, H., Misrahi, M., Atger, M., Salesse, R., Vu Hai, M. T., Jolivet, A.,
35. Takeshita, A., Nagayama, Y., Fujiyama, K., Yokoyama, N., Namba, H.,
Guiochon-Mantel, A., Sar, S., Jallal, B., Garnier, J., and Milgrom, E. (1989)
Yamashita, S., Izumi, M., and Nagataki, S. (1992) Biochem. Biophys. Res.
Science 245, 525–528
Commun. 188, 1214 –1219
5. MacFarland, K. C., Sprengel, R., Phillips, H. S., Kohler, M., Rosembilt, N.,
36. Mui, A. L. F., Kay, R. J., Humphries, R. K., and Krystal, G. (1992) Proc. Natl.
Nikolics, K., Segaloff, D. L., and Seeburg, P. H. (1989) Science 245, 494 – 499
6. Sprengel, R., Braun, T., Nikolics, K., Segaloff, D. L., and Seeburg, P. H. (1990) Acad. Sci. U. S. A. 89, 10812–10816
Mol. Endocrinol. 4, 525–530 37. Seidah, N. G., and Chretien, M. (1992) Trends Endocrinol. Metab. 3, 133–140
7. Libert, F., Lefort, A., Gerard, C., Parmentier, M., Perret, J., Ludgate, M., 38. Chazenbalk, G. D., and Rapoport, B. (1994) J. Biol. Chem. 269, 32209 –32213
Dumont, J. E., and Vassart, G. (1989) Biochem. Biophys. Res. Commun. 39. Bravo, D. A., Gleason, J. B., Sanchez, R. L., Roth, R. A., and Fuller, R. S. (1994)
165, 1250 –1255 J. Biol. Chem. 269, 25830 –25837
8. Nagayama, Y., Kaufman, K., Seto, P., and Rapoport, B. (1989) Biochem. 40. Van Wart, H. E., and Birkedal-Hansen, H. (1990) Proc. Natl. Acad. Sci.
Biophys. Res. Commun. 165, 1184 –1190 U. S. A. 87, 5578 –5582
9. Misrahi, M., Loosfelt, H., Atger, M., Sar, S., Guiochon-Mantel, A., and 41. Woessner, J. F. (1991) FASEB J. 5, 2145–2154
Milgrom, E. (1990) Biochem. Biophys. Res. Commun. 166, 394 – 403 42. Stetler-Stevenson, W. G., Liotta, L. A., and Kleiner, D. E. (1993) FASEB J. 7,
10. Frazier, A. L., Robbins, L. S., Stork, P. J., Sprengel, R., Segaloff, D. L., and 1434 –1441
Cone, R. D. (1990) Mol. Endocrinol. 4, 1264 –1276 43. McGeehan, G. M., Becherer, J. D., Bast, R. C., Boyer, C. M., Champion, B.,
11. Wadsworth, H. L., Chazenbalk, G. D., Nagayama, Y., Russo, D., and Rapoport, Connolly, K. M., Conway, J. G., Furdon, P., Karp, S., Kidao, S., McElroy,
B. (1990) Science 249, 1423–1425 A. B., Nichols, J., Pryzwansky, K. M., Schoenen, F., Sekut, L., Truesdale, A.,
12. Vu Hai Luu Thi, M. T., Misrahi, M., Houllier, A., Jolivet, A., and Milgrom, E. Verghese, M., Warner, J., and Ways, J. P. (1994) Nature 370, 558 –561
(1992) Biochemistry 31, 366 –373 44. Netzel-Arnett, S., Fields, G., Kirkedal-Hansen, H., and Van Wart, H. E. (1991)
13. Smith, B. R., McLachlan, S. M., and Furmaniak, J. (1988) Endocr. Rev. 9, J. Biol. Chem. 266, 6747– 6755
106 –121 45. Fosang, A. J., Neame, P. J., Last, K., Hardingham, T. E., Murphy, G., and
14. VuHai-Luuthi, M. T., Jolivet, A., Jallal, B., Salesse, R., Bidart, J. M., Houllier, Hamilton, J. A. (1992) J. Biol. Chem. 267, 19470 –19474
A., Guiochon-Mantel, A., Garnier, J., and Milgrom, E. (1990) Endocrinology 46. Sires, U. I., Griffin, G. L., Broekelmann, T. J., Mecham, R. P., Murphy, G.,
127, 2090 –2098 Chung, A. E., Welgus, H. G., and Senior, R. M. (1993) J. Biol. Chem. 268,
15. Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., 2069 –2074
 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

Access the most updated version of this article at http://www.jbc.org/content/271/8/4545

Alerts:
• When this article is cited
• When a correction for this article is posted

Downloaded from http://www.jbc.org/ by guest on September 4, 2019

Click here to choose from all of JBC's e-mail alerts

This article cites 45 references, 14 of which can be accessed free at

http://www.jbc.org/content/271/8/4545.full.html#ref-list-1