Role of cleavage and shedding in human thyrotropin receptor function
and trafficking
Myle
`
ne Quellari
1
, Agne
`
s Desroches
1
, Isabelle Beau
1
, Emmanuelle Beaudeux
1
and Micheline Misrahi
1,2
1
INSERM E120, Re
´
cepteurs, Signalisations et Physiopathologie Thyroı
¨
dienne et de la Reproduction, and
2
Laboratoire
d’Hormonologie et Biologie Mole
´
culaire, Ho
ˆ
pital Bice
ˆ
tre, IFR Bice

activity, showing that the extracellular domain still exerts a
negative effect in the M453T holoreceptor. An increased
internalization of the M453T receptor was observed when
compared with the wild-type receptor, which was increased
further in the corresponding truncated b1-M453T receptor.
Thus cleavage and shedding yield TSHR activation but also
increase internalization of the free b-subunits of the receptor,
the latter mechanism limiting simultaneously excessive
receptor signaling. The combined effects may be responsible
for the limited basal constitutive activation of the cAMP
pathway that is detected for the TSHR.
Keywords: thyrotropin receptor; cleavage; shedding; con-
stitutive activity; traffic.
The thyrotropin receptor (TSHR) plays a key role in
thyroid growth and function [1–3]. This receptor is also the
target of stimulating or blocking autoantibodies in patients
with autoimmune diseases [4,5]. The TSHR belongs to a
particular subgroup of G protein-coupled receptors, inclu-
ding the FSH and LH receptors [6]. They are characterized
by the presence of a seven transmembrane domain and a
large extracellular domain involved in high affinity hormone
binding. These three receptors are mainly coupled to G
s
,
leading to the activation of the adenylate cyclase pathway.
However, unlike the gonadotropin receptors, the TSHR
transduces a signal via adenylate cyclase even in the absence
of ligand, thus having a weak constitutive activity [7].
The TSHR undergoes a unique post-translational mat-
uration among G protein-coupled receptors. In human

G protein-coupled receptor; LH, luteinizing hormone; TSH,
thyrotropin hormone; TSHR, thyroid stimulating hormone receptor.
(Received 25 March 2003, revised 7 June 2003,
accepted 12 June 2003)
Eur. J. Biochem. 270, 3486–3497 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03718.x
most N-terminal site mapped is upstream Ser314 and the
most C-terminal site detected is upstream Leu378 [15]. The
cleavage reaction is sequential and leads to the processive
digestion of the specific extracellular region of the TSHR.
The enzyme involved in the maturation of the TSHR shares
similarities with the ADAM (A Disintegrin and Metallo-
protease) family of metalloproteases [12,18].
The cleavage and shedding of the human TSHR may be
of physiological importance because the precise quantifica-
tion of each subunit in thyroid membranes demonstrated an
approximately 2.5 to 3-fold excess of b-overa-subunits [9].
This observation led us to postulate that the a-subunit
might be shed from cell membranes and released into the
extracellular space or bloodstream. Soluble forms of the
TSHR have been described in human thyroid homogenates
[19], or proposed in human blood [20,21]. Accumulation of
the a-subunit has also been detected in the extracellular
matrix [22].
There have been several difficulties in understanding the
functional role of receptor cleavage and shedding. Some
mutations or small deletions of individual or groups of
amino acids did not prevent receptor cleavage, due to the
multiplicity of the cleavage sites [23–26]. In addition, the
TSHR is expressed in transfected cells in multiple processed
and unprocessed forms [10,17]. Monomeric precursors also

DMEM,
L
-glutamine and gentamycin were from Gibco
BRL (Invitrogen Corporation, Paisley, UK); fetal bovine
serum was from Biochrom. Bovine thyrotropin hormone
(bTSH; 2 IUÆmg
)1
), 3-iso-butyl-1-methylxanthine (IBMX),
gelatin, BSA (bovine serum albumin; fraction V) and
monensin were obtained from Sigma. 2,2¢-Azine-di(ethyl-
benzthiazoline sulfonate) (ABTS) was obtained from Per-
bio. Superfect transfection reagent was from Qiagen.
[
125
I]Streptavidin, cAMP-RIA assay kit, peroxidase-conju-
gated sheep anti-(mouse IgG) Ig and [
125
I]Streptavidin
(specific activity 20–50 lCiÆlg
)1
) were from Amersham
Pharmacia. Alexa488-labeled anti-mouse IgG was from
Molecular Probes (Netherlands).
Anti-TSHR monoclonal Igs
Monoclonal Igs T3-365 and R5T-34 have been described
previously [9,15]. Those Igs were raised against fragments of
the TSHR expressed in Escherichia coli. The T3-365 Ig is
raised against an epitope localized in the intracellular
domain of the TSHR [9], and the R5T-34 Ig recognizes an
epitope localized between amino acids 357 and 369 [15].

409–412 was cloned into the pSG5-b1 expression vector,
digested with PstIandBbsI (position +1232).
The same strategy was used to construct the deletion
mutants containing the Met453Thr mutation, starting from
the pSG5-hTSHR-M453T vector, previously described [31].
All the constructs were verified by double-strand DNA
sequencing.
Cell culture and transfection
COS-7 cells were maintained in DMEM with
L
-glutamine
supplemented with 10% fetal bovine serum and 8 lgÆmL
)1
Ó FEBS 2003 Role of cleavage and shedding in TSHR function (Eur. J. Biochem. 270) 3487
gentamycin at 37 °C in a humidified 5% CO
2
atmosphere.
L
cells stably expressing the wild-type and mutant receptors
were maintained in DMEM supplemented with 10% fetal
bovine serum,
L
-glutamine and 200 lgÆmL
)1
G418, as
described [10].
For transient transfection, COS-7 cells were seeded in
six-well plates and grown overnight in DMEM supple-
mented with 10% fetal bovine serum. They were trans-
fected with the Superfect reagent according to the

Cl in NaCl/
P
i
for 20 min. After 1 h saturation and permeabilization
with NaCl/P
i
, 1% BSA, 0,1% saponin, the cells were
incubated for 2 h with the T3-365 monoclonal Ig
(5 lgÆmL
)1
in NaCl/P
i
, 1% BSA, 0,01% saponin). The
cells were then washed with NaCl/P
i
,1%BSA,0,1%
Tween20 and incubated for 1 h with peroxidase-conju-
gated sheep anti-(mouse IgG) Ig (dilution 1 : 1000). After
washing with NaCl/P
i
,400lL ABTS was added into each
well and incubated for 15 min under dark. Absorbances
were read at 450 nm [28].
cAMP assay
COS-7 cells transfected with the expression vectors
encoding the wild-type or the truncated receptors were
washed twice with DMEM medium containing 20 m
M
Hepes, pH 7.4, and gelatin 1 mgÆmL
)1

P
i
. After washing, the aldehyde groups were quenched
with 50 m
M
NH
4
Cl in NaCl/P
i
for20min.After1hof
saturation and permeabilization with NaCl/P
i
,1%BSA,
0.1% saponin, cells were incubated for 2 h with the
monoclonal Ig T3-365 [9] (5 lgÆmL
)1
in NaCl/P
i
,1%
BSA, 0.01% saponin). The cells were then washed with
NaCl/P
i
, 1% BSA, 0.1% Tween20 and incubated for 1 h
with a 1 : 400 dilution of Alexa488-labeled antimouse
IgG. After washing, the cells were mounted with Fluor-
escent Mounting Medium. They were examined with a
Zeiss LSM-510 confocal scanning laser microscope
equipped with a 25 mW Argon laser, using a Plan
Apochromat 63 · objective (NA 1.40, oil immersion).
Green fluorescence was observed with long pass 505 nm

, cells were trypsinized, collected, and the cell-
associated radioactivity, corresponding to receptor-Ig com-
plexes present at the cell surface, was measured using a
c-counter [30]. Experiments were performed at least three
times in duplicate.
Normalization of cAMP accumulation to cell-surface
expression
Basal cAMP accumulation was normalized to cell surface
expression for wild-type, b1, M453T and b1-M453T
receptors. For that purpose, the receptor-dependent
cAMP accumulation (in nmolÆL
)1
) was divided by the
radioactivity measured (in c.p.m.), corresponding to cell
surface receptor-biotinylated Ig complexes, revealed by
[
125
I]Streptavidin: (cAMP in receptor-transfected cells –
cAMP in control pSG5-transfected cells)/(binding of
receptor-transfected cells – binding of the control pSG5-
transfected cells). The values (relative constitutive activities)
3488 M. Quellari et al. (Eur. J. Biochem. 270) Ó FEBS 2003
were then normalized to the value of the specific consti-
tutive activity of the wild-type TSHR, arbitrarily set to 1
[29,33].
Internalization and recycling of receptor-Ig complexes
L cell lines stably expressing the wild-type and the b1, the
M453T and the b1-M453T receptors were obtained using
the calcium phosphate precipitation method and main-
tained as described [10]. Internalization of receptor-Ig

duplicate.
Statistics
Statistical significance was assessed by the Mann–Whitney
non-parametric test. Results are expressed as means ± SD.
Results
Generation of truncated mutants corresponding either
to b-subunits of the TSHR, or to a receptor deleted
of almost the entire extracellular domain
To understand the role of receptor cleavage and shedding,
we constructed two deletion mutants, corresponding either
to the longest b1 (starting at Ser314) or to the shortest b2
(starting at Leu378) subunit of the TSHR (Fig. 1A).
Fig. 1. Schematic representation of the trun-
cated mutant receptors. (A) Top panel, human
TSHR. The seven transmembrane segments
(TM) are shown in gray and the signal peptide
is colored in black. The percentage of identity
of the different extracellular regions of the
TSHR to the corresponding regions of the
human LH receptor are indicated above.
The E3 region (residues 289–385) is the most
divergent region and E5 the most conserved
one (residues 403–416). The black arrow
indicates the localization of the constitutive
mutation Met453Thr (M453T) in the second
transmembrane segment of the TSHR [31].
The truncated mutant receptors b1, b2,
TM409 starting, respectively, at Ser314,
Leu378 and Glu409 are schematized. Note
that the N-terminus of the b1andb2mutant

the intracellular domain of the receptor. Figure 1B shows
that the truncated receptors are expressed as single mole-
cular weights species, with respective apparent molecular
weights of approximately 52, 40 and 36 kDa.
Effect of deletions corresponding to cleavage and
shedding comparative to a deletion of almost
the whole extracellular domain of the receptor
on its basal activity
COS-7 cells transfected with expression vectors encoding
either the wild-type or the truncated receptors were
incubated with various concentrations of bTSH (0–
10 IUÆL
)1
), and the basal or hormone-induced cAMP levels
were measured (Fig. 2A). Transfection efficiencies were
verified by immunocytochemistry, using the T3-365 mono-
clonal Ig. In addition, total receptor expression was verified
using a cellular ELISA ([28] and see Methods) on per-
meabilized cells. Figure 2B shows a similar total expression
of the wild-type and the truncated b1, b2 and TM409
receptors.
As shown in Fig. 2A, the b1andb2 mutant receptors
displayed a similar approximately 2.5-fold higher total basal
(not normalized) accumulation of cAMP (P <0.01)when
compared with the wild-type receptor. This experiment was
repeated at least three times with similar results.
In contrast, the basal cAMP levels detected in transfected
cells expressing the TM409 mutant receptor were signifi-
cantly lower when compared with cells expressing the wild-
type receptor (P < 0.05) and not significantly different

(h), b2(r), and TM409 (s) receptors. Forty-eight hours later, they
were incubated for 1 h with 0–100 IUÆL
)1
of bTSH, and cAMP
accumulation was measured. The data presented are expressed as raw
values (intracellular cAMP accumulation in nmolÆL
)1
), and represent
the mean ± SD of triplicate wells from a representative experiment.
3490 M. Quellari et al. (Eur. J. Biochem. 270) Ó FEBS 2003
basal constitutive activity detected for the wild-type
receptor.
In all cases, incubation of cells transfected with expression
vectors encoding the truncated b1, b2orTM409mutants
with bTSH (0.1, 1, 10 or 100 IUÆL
)1
), did not enhance the
accumulation of cAMP (Fig. 2C), contrary to the wild-type
receptor.
Cell-surface expression of the TM409 mutant receptor
As the TM409 mutant exhibited a complete loss in receptor
function, we also verified the cell surface expression of this
truncated receptor, comparatively to the wild-type receptor.
For that purpose, we performed indirect immunofluores-
cence using a T3-365 monoclonal Ig directed against the
intracellular domain of the receptor. Cell surface expression
was observed using confocal microscopy and the Nomarski
differential interference contrast. As shown in Fig. 3A (top
panel), in COS-7 cells expressing the wild-type receptor,
staining is observed at the cell surface at the leading edge of

to the wild-type receptor, COS-7 cells were transfected with
each expression vector and incubated at 4 °Cwiththe
Fig. 3. Confocal microscopy of COS-7 cells expressing either the wild-type or the TM409 receptors. Transfected cells were fixed, permeabilized,
incubated with the T3-365 monoclonal Ig that recognizes an intracellular epitope of the TSHR, and indirect immunofluorescence was performed
(see Experimental procedures). Top panel, wild-type TSHR (WT); lower panel, TM409 receptor. (A) Confocal microscopy study; (B) Nomarski
optics was used to study cell morphology; (C) fluorescence image was overlaid on Nomarski image to generate merged image.
Ó FEBS 2003 Role of cleavage and shedding in TSHR function (Eur. J. Biochem. 270) 3491
biotinylated R5T-34 anti-TSHR Ig during 30 min. After
washing the cells, the concentration of receptor-Ig com-
plexes at the cell surface was quantified by measuring the
binding of [
125
I]Streptavidin [30]. No Ig binding was
observed in cells transfected with the vector alone (Fig. 4B).
As shown in Fig. 4B, quantification of cell surface
expression of COS-7 cells expressing the b1 mutant receptor
revealed an approximate threefold decrease in cell surface
expression when compared with the wild-type receptor.
Therefore, the increase in the specific constitutive activity of
the b1 receptor, after normalization to cell surface expres-
sion, is at least approximately eightfold (mean of three
independent experiments, including the data presented in
Fig. 2A, see also Fig. 4C).
Increased basal internalization of the b1
mutant receptor
A decreased cell surface expression of the b1mutant
receptor was observed when compared with the wild-type
receptor (Fig. 4B). We wondered whether shedding of the
ectodomain would modify the trafficking of the receptor.
Therefore, we studied the comparative internalization of the

Effect of cleavage and shedding on a constitutively
activated receptor
Natural point mutations of the TSHR have been described
in familial hyperthyroidism or toxic adenomas [7,34,35].
These mutations are mainly located in the transmembrane
domain of the receptor and lead to a constitutive activation
of the receptor.
We evaluated the functional consequences of receptor
shedding in a mutant harboring a constitutive natural
Fig. 4. Specific constitutive activity of the wild-type and the truncated b1
receptors. (A) Total basal cAMP accumulation was measured in
COS-7 cells transfected with the control pSG5 expression vector or
vectors encoding the wild-type (WT) and the b1 mutant receptors. The
data are expressed as raw values (intracellular cAMP accumulation in
nmolÆL
)1
)andrepresentthemean±SDoftriplicatewellsfroma
representative experiment of three independent experiments. (B)
Quantification of receptor-bound Ig complexes was performed using
R5T-34 monoclonal Ig as described [30]. Briefly, transfected cells were
incubated at 4 °C with the biotinylated R5T-34 Ig. The receptor-Ig
complexes present at the cell surface were quantified using
[
125
I]Streptavidin. The data, expressed in percentage of cell surface
expression, represent the mean ± SD of triplicate wells from a rep-
resentative experiment of three independent experiments. (C) Relative
specific constitutive activity of the wild-type TSHR (WT) (arbitrarily
setto1)andoftheb1 mutant receptor: normalization of cAMP
accumulation to cell-surface expression was performed (see Experi-

)1
) did not enhance the accumulation of
cAMP (Fig. 6).
When the cell surface expression of the wild-type, the
M453T and the b1-M453T truncated receptors was studied
using the R5T-34 anti-TSHR monoclonal Ig, a diminished
expression of the M453T receptor at the cell membrane of
approximately 1.7-fold when compared with the wild-type
receptor was observed (Fig. 7B). Its normalized constitutive
activity is thus increased at least approximately eightfold
(mean of three independent experiments), when compared
with the wild-type receptor. Likewise, the expression of the
truncated b1-M453T mutant receptor was strongly reduced
by 7.7-fold (Fig. 7B), and thus the specific constitutive
activity is increased by at least approximately fourfold,
when compared with the M453T receptor (Fig. 7C).
Increased basal internalization of the M453T receptor,
further enhanced by deletion of its ectodomain
We also studied the trafficking of the constitutive M453T
receptor, which also exhibited a diminished cell surface
expression. Therefore, we established
L
-cell lines expressing
either the M453T holoreceptor or the truncated b1-M453T
mutant receptor.
As shown in Fig. 8, in the absence of hormone, the
constitutive M453T receptor exhibited an increased basal
internalization when compared with the wild-type receptor,
with approximately 40% of receptor molecules being
internalized after 20 min at 37 °C, and approximately

M
)
(m) were also added to the incubation medium. After removal of
unbound Ig, cells were incubated for the indicated times at 37 °C.
Surface-bound Ig was quantified as described [30] by measuring the
binding of [
125
I]Streptavidin. Specific binding at each point was nor-
malized with reference to specific binding before the incubation at
37 °C (0-min point) to derive the fraction of initial total receptor-Ig
complexes remaining at the cell surface. Bars, SD of duplicate points.
The assay shown is representative of an experiment repeated at least
three times.
Ó FEBS 2003 Role of cleavage and shedding in TSHR function (Eur. J. Biochem. 270) 3493
periods up to 120 min. In keeping with this observation,
incubation with monensin did not modify the trafficking of
the receptor (Fig. 8).
Deletion of the ectodomain of the receptor (b1-M453T
mutant receptor) led to a supplementary increase in the
basal internalization, approximately 50% of receptor mole-
cules being internalized after 15 min, and approximately
70% after 60 min (Fig. 8). No receptor recycling was
observed. bTSH and monensin had no effect on the
intracellular traffic of the mutant receptor (data not shown).
Discussion
In this study, we focused on the role of TSHR cleavage and
shedding, by constructing mutant receptors corresponding
to the longest (b1) and shortest (b2) b-subunits that we had
previously mapped in thyroid and L cells [15]. Altogether,
our results show that mutants corresponding to cleaved and

of unbound Ig, cells were incubated for the indicated times at 37 °C.
Surface-bound Ig was quantified as described ([30] and see Fig. 7) by
measuring the binding of [
125
I]Streptavidin. Bars, SD of duplicate
points. The assay shown is representative of an experiment repeated at
least three times.
Fig. 7. Specific constitutive activity of the wild-type, M453T and
b1-M453T receptors. (A) Total basal cAMP accumulation was meas-
ured in COS-7 cells transfected with the control pSG5 expression
vector or vectors encoding the wild-type (WT), M453T and b1-M453T
receptors. The data are expressed as raw values (intracellular cAMP
accumulation in nmolÆL
)1
)andrepresentthemean±SDoftriplicate
wells from a representative experiment of three independent experi-
ments. (B) Quantification of receptor-bound Ig complexes was per-
formed using R5T-34 monoclonal Ig as described [30], and see Fig. 4.
The data, expressed in percentage of cell surface expression, represent
the mean ± SD of triplicate wells from a representative experiment of
three independent experiments. (C) Relative specific constitutive
activity of wild-type (WT) (arbitrarily set to 1), M453T and b1-M453T
receptors: normalization of cAMP production to cell-surface expres-
sion was performed (see Experimental procedures). Presented data are
the mean ± SD of three independent experiments.
3494 M. Quellari et al. (Eur. J. Biochem. 270) Ó FEBS 2003
M453T receptor, when this region is deleted (TM409-
M453T receptor), also strongly supports a role for this
juxtamembrane region in receptor function and cell surface
targeting. We could detect the presence of the TM409

wild-type TSHR exhibited a very limited basal internal-
ization [30], deletion of the ectodomain in the b1 receptor
led to a marked increased basal internalization of the
receptor. Thus, the extracellular domain of the TSHR
negatively modulates receptor internalization probably
through a conformational change transmitted to the
b-subunit of the receptor. Deletion of the ectodomain, or
addition of TSH, relieves constrained conformations and
increases internalization. As the b1 mutant is constitu-
tively activated, this observation suggests a link between
the conformations necessary for receptor activation and
for internalization. A similar situation has been described
for other G protein-coupled receptors [41]. However, no
recycling was observed for the b1 receptor. Its intracel-
lular traffic is very similar to the one of the wild-type
receptor activated by TSH, but in the presence of
monensin, which inhibits the recycling. This observation
strongly argues for a different conformation between the
receptor activated by cleavage and shedding on the one
hand, and the receptor activated by the ligand on the
other hand. This may be due to different post-transla-
tional maturation of the receptors, leading to different
conformations of the sequence(s) implicated in the
recycling of the receptor.
The TSHR can also be activated by natural constitutive
point mutations found mainly in the transmembrane
domain of the receptor [7,34,35]. The current hypothesis
maintains that constitutively activated receptors release the
conformational constraints of the GPCR inactive state that
normally keep the ligand-free receptor silent. Therefore, we

phosphorylation and/or interaction with arrestins, or due
to an easier clustering of the activated receptors in coated
pits [44].
It has to be noted that in the case of the M453T mutant
receptor, contrary to the wild-type receptor, no recycling
was observed. This was confirmed by the use of monensin,
which did not modify the trafficking of this mutated
receptor.
These observations strongly suggest a difference in the
conformation of the M453T receptor when compared with
the hormone-activated receptor.
Deletion of the ectodomain of the M453T receptor,
mimicking receptor shedding, led to a supplementary
increase in receptor internalization, indicating probably a
supplementary change in receptor conformation. It has been
proposed that each function of the receptor (G protein
coupling, internalization, recycling) is not triggered by only
one well-defined conformation, but by a continuum of
independent conformations [41,45].
In conclusion, cleavage and shedding yield TSHR
activation but also increase receptor downregulation
through an increased internalization of the b-subunits
of the receptor, the latter mechanism limiting simulta-
neously excessive receptor signaling. The combined effects
may be responsible for the limited basal constitutive
activation of the cAMP pathway that is detected for the
TSHR.
Further studies are necessary to discover whether the
shedding may be regulated [46], which might be a novel way
to modulate receptor activity.

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