(Received for publication, July 12, 1995; and in revised form, October 26, 1995)
From the
The thyrotropin (TSH) receptor in human thyroid glands has been
shown to be cleaved into an extracellular subunit and a
transmembrane
subunit held together by disulfide bridges. An
excess of the latter component 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 increased by inhibitors of endocytosis, recycling, and lysosomal 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. Decreasing the serum concentration in cell culture medium enhanced the shedding by an unknown mechanism.
The shedding of the TSH receptor domain is
the consequence of two events: cleavage of the receptor into
and
subunits and reduction of the disulfide bridge(s). The complete
inhibition of soluble TSH receptor 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
subunit in human circulation.
Thyrotropin (TSH) ()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 hormone (FSH)(6) , and TSH receptors (7, 8, 9, 10) form a subgroup in
this family having a large and glycosylated extracellular domain
specialized in high affinity hormone binding(11, 12) .
Interest in the TSH receptor (TSHR) is enhanced by its implication in
autoimmune diseases. Autoantibodies directed against this receptor have
stimulatory (Graves' disease) or blocking (idiopathic myxoedema)
effects on its function(13) . From cloning and sequencing of
the human TSH receptor
cDNA(7, 8, 9, 10) , the structure of
a preprotein with a calculated molecular mass of 84.5 kDa was deduced.
But the characterization 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 heterodimer: an extracellular
subunit (
53
kDa) and a membrane-spanning
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
receptor 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 present. 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
synthesized as a 95-kDa mannose-rich monomer, which undergoes
supplementary glycosylation to yield a
120-kDa monomeric
precursor(17) . The latter is then cleaved into mature
and
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 over
subunits (15) . This observation led us to
postulate that the
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 domain 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.
A double-determinant
(sandwich-type) radioimmunoassay was developed for the specific
quantification of the TSH receptor subunit. This assay uses two
additive monoclonal antibodies specific for the extracellular domain
(
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-µm syringe
filter or ultracentrifuged at 15,000
g to remove
cellular debris.
Figure 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 subunit (the sandwich assay used T5-317 and
biotinylated T5-51, both antibodies recognizing different epitopes in
the ectodomain). As a control the full-length receptor (
/
receptor) was also assayed using antibody 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 ± S.E. (standard error of the
mean) of three independent determinations.
The assay was specific. Competition with an excess
of unlabeled 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 I-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 domain ( subunit) of the receptor
(T3-365). This assay detects cleaved or uncleaved whole receptor
molecules. No such receptor 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 transfected 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 mature 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 extracellular domain of the LH/CG receptor. No receptor ectodomain 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.
Figure 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 concentrated 15 times on Centricon-10 filters before
receptor assay. Ectodomain ( subunit) and full-length receptor
(
/
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 = 2) of soluble TSHR (sTSHR) on the
cellular amount of receptor (cTSHR).
To test the ability of sTSHR to
bind specifically TSH, concentrated (30 ) culture medium from
transfected L cells was incubated with iodinated bovine TSH, in the
presence or in the absence of increasing concentrations of unlabeled
bTSH, hFSH, or hCG. The bound complexes were precipitated by
polyethylene 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.
Figure 3:
Comparison of I-TSH binding
by soluble and cellular TSH receptor. Triton X-100 membrane extracts
from TSHR expressing L cells and the corresponding cell culture medium
(see ``Experimental 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 complexes with
polyethylene glycol as indicated under ``Experimental
Procedures.'' Results are expressed as the ratio of B (iodinated
TSH bound in the presence of unlabeled hormone) on B
(iodinated TSH bound in the absence of unlabeled hormone)
100.
Finally, sTSHR was purified from culture medium using immunoaffinity chromatography with the T5-51 immunomatrix (Fig. 4). More than 90% of the receptor present in the medium bound to the matrix and was eluted at acidic pH.
Figure 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 ectodomain). After extensive washings elution was performed using a citrate pH 2 buffer.
Western
blots were performed to compare the immunopurified sTSHR with the
cellular TSHR (solubilized from the membranes 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
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
subunit of the cellular TSHR (cTSHR (lane
H) and sTSHR (lane H)). These results indicate that sTSHR
and the
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.
Figure 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.
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 endoglycosidases H (cTSHR (lanes F and H)). A small amount of a
120-kDa monomeric
precursor (cTSHR, lane C) which contains 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 correspond to precursors of the TSH
receptor(17) . The high mannose precursor may accumulate inside
the transfected cells because the cellular machinery is unable to
process to completion 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 subunit of the
receptor. The same immunoreactivity was detected in a CHO-derived cell
line expressing the TSHR, as well as in the medium of primary cultures
of human thyrocytes.
Figure 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 ± S.E. of three independent determinations. The inset shows the ratio of soluble (sTSHR) on the cellular (cTSHR) receptor.
Since serum concentration determines the rate of cell division, 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.
Figure 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 µg/ml), fibroblast 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 ± S.E. (n = 3). B, the ratio of soluble (sTSHR) to cellular (cTSHR) receptor corresponds to three independent determinations (mean ± S.E.). *, p < 0.05;**, p < 0.1 versus 1% FCS control.
In order to better discriminate the pathways (adenylate cyclase or phospholipase C) implicated in this hormonal effect, we studied forskolin, 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA), as well as calcium ionophores (Table 2). Only 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 concentration are important parameters in the regulation of sTSHR shedding.
Figure 8: Effects of inhibitors of cellular traffic on sTSHR shedding. 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 µM; bafilomycin (Baf), 1 µM; monensin (Mon), 10 µM; or brefeldin A (BFA), 20 µg/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 ± S.E.)**, p < 0,01 versus untreated control.
The weak amine chloroquine, which increases the pH of acidic vacuoles, impedes the traffic through acidified compartments and disturbs lysosomal function(24) , enhanced sTSHR shedding (50% over untreated cells). Bafilomycin, an inhibitor of the H+-ATPase of acidic vacuoles (25) , also enhanced TSHR shedding. Monensin, a monovalent carboxylic ionophore which dissipates transmembrane proton gradient and blocks the recycling pathway(26) , had a stimulatory effect on sTSHR shedding. 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 process 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 concentration 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.
We thus investigated the effect of a recently
described potent inhibitor of matrix metalloproteases, the synthetic
hydroxamic acid BB-2116(28) . As shown in Fig. 9A, we observed a dose-dependent inhibition of
sTSHR accumulation in cell culture medium which became undetectable at
100 µg/ml of BB-2116. The concentrations of BB-2116 suppressing
TSHR subunit shedding matched those previously described for the
inhibition of pro-tumor necrosis factor
cleavage(28) .
However the shedding of TSHR
subunit is a two-step process. The
first step consists in the cleavage of the receptor and the second step
in the reduction of its disulfide bond(s). It was necessary to verify
that BB-2116 was indeed acting on the cleavage. We thus measured the
concentration of
subunits linked by disulfide bonds to
subunits and which thus remain attached to cell membranes. This was
done by treating cell membranes with dithiotreitol and measuring the
released
subunits. If inhibition occurs at the level of the
cleavage the concentration of ``releasable''
subunit
should decrease, whereas if inhibition occurs at the level of reduction
of disulfide bridge(s) it should increase.
Figure 9:
Effect of a matrix metalloproteinase
inhibitor (BB-2116) on sTSHR shedding. L cells expressing TSHR were
cultured for 18 h in the presence of various concentrations of BB-2116. A, the soluble TSHR (sTSHR) was assayed in the
culture medium. B, the subunits remaining attached by
disulfide bridges to the
subunits in the cell membranes were
assayed. The cells were scraped and treated with 100 mM dithrotreitol (DTT) (see ``Experimental
Procedures''), and the concentration of released
subunits
was determined. Results are expressed in pM (A) or
pmol of
subunits released (B) as the mean ± S.E. (n = 4).
A marked decrease in the
concentration of membrane-attached cleaved receptors was produced by
incubation with BB-2116 (Fig. 9B). Thus the inhibition
occurs at the level of the cleavage of the receptor. However at
concentrations of inhibitor which totally suppressed subunit
shedding there was only a 55% decrease of cleaved receptors present on
the membranes. This suggests that a critical threshold of the
/
heterodimer must be reached on the cell surface to allow
shedding to occur.
Using a quantitative immunoradiometric assay performed with
two monoclonal antibodies directed against the extracellular domain of
the TSH receptor, we have detected an immunoreactive protein in the
culture medium of L cells and CHO cells stably transfected with the TSH
receptor. The same immunoreactivity was released from human thyrocytes.
This accumulation was time-dependent and reached 25% of the total
cellular receptor by 48 h.
This soluble TSHR is a glycosylated
protein which is retained by concanavalin A-Sepharose (not shown) and
specifically binds its ligand TSH. Immunopurification of sTSHR from the
L cell line conditioned media demonstrated that its polypeptide core
corresponds to the subunit (
35 kDa).
These experiments strongly suggested that there is a spontaneous shedding of the extracellular domain of the TSH receptor 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 homogenization has been proposed by some authors(29, 30, 31) . This possibility was not supported by our previous experiments (15, 17) and is strongly opposed by the present observation of spontaneous shedding of the extracellular domain of the TSH receptor by intact cells in culture.
In addition, we have
detected the presence of sTSHR in normal human serum using the same
assay. ()Complete characterization of this circulating sTSHR
will allow us to determine whether it corresponds to the sTSHR detected
in the supernatant of cultured cells. There have been some preliminary
reports of TSHR related peptide-like immunoreactivity (32) and
TSH-binding protein (33) in human serum. Our preliminary 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 determine whether a correlation
can be established between the concentration of sTSHR in the serum and
specific pathological conditions. This shed
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 (21) and (22) ). In
several cases receptor shedding was shown to be increased by its
cognate 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 concentration 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: possibly the reduction
of disulfide bond(s) joining the and
subunits.
Surprisingly, lowering serum concentration from 10 to 1% greatly enhanced TSHR shedding. This inhibitory effect of serum was not mediated by an effect on cell growth as it could not be reproduced by insulin or other growth factors. The mechanism 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 internalization, recycling nor degradation in lysosomes were involved 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 experiments 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 comprises a pair of basic residues. Such basic sequences are found 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 compartment. In a previous work, pulse-chase experiments indicated 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 divalent ion chelators EDTA and EGTA
inhibited TSHR processing (30 and 20% inhibition, respectively). Higher
concentrations could not be used in our whole cell system as these
compounds had a cytotoxic effect. However these results suggested that
the TSHR convertase could be a metalloprotease. Finally we used a
potent and specific inhibitor of zinc-dependent matrix
metalloproteases. This synthetic hydroxamic acid, BB-2116, inhibits in vitro the maturation of tumor necrosis factor
(28) . In our system, this agent totally suppressed sTSHR
accumulation in culture medium. This effect was secondary to a strong
inhibition of TSHR cleavage into
/
heterodimers.
BB-2116
inhibits in vitro the activity of collagenases, stromelysins,
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 (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 (42) ). Very recently matrix metalloproteases have
also been implicated in the maturation of tumor necrosis factor ,
a potent pro-inflammatory and immunomodulatory cytokine produced in
inflammatory conditions(27, 43) . The sites of
cleavage are difficult to predict as matrix metalloproteases exhibit
broad substrate and sequence
specificities(44, 45, 46) . The finding that
matrix metalloprotease-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 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 experiments will be
necessary to understand the physiological significance 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 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
unanswered: is shedding of the
subunit a mechanism limiting the
transmission of the signal? Is the shed
subunit able to bind
hormone in the plasma and does it influence its biological activity?