Báo cáo khoa học: Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor pptx

Proper targeting and activity of a nonfunctioning thyroid-stimulating
hormone receptor (TSHr) combining an inactivating and activating
TSHr mutation in one receptor
Patrizia Agretti
1
, Giuseppina De Marco
1
, Paola Collecchi
2
, Luca Chiovato
3
, Paolo Vitti
1
, Aldo Pinchera
1
and Massimo Tonacchera
1
1
Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro, Universita
`
di Pisa, Pisa, Italy;
2
Dipartimento di Oncologia, Divisione di Anatomia Patologica, Universita
`
di Pisa, Pisa, Italy;
3
Cattedra di Endocrinologia,
Fondazione S Maugeri IRCCS, Pavia, Italy
Activating mutations of the thyroid-stimulating hormone
receptor (TSHr) have been identified as a cause of toxic
adenomas. Germline-inactivating TSHr mutations have

been described as a cause of congenital hypothyroidism. The
effects of combining activating and inactivating mutations
within a single receptor was studied. The double mutant
T477I/P639S contained an activating TSHr mutation
(P639S) together with an inactivating one (T477I). The other
one (I486M/P639S) contained two activating mutations.
Constructs were expressed in COS-7 cells and basal and
TSH-stimulated cyclic AMP (cAMP) accumulation and
inositol phosphate (IP) production were determined. The
expression at the cell surface was studied both with binding
and fluorescence-activated cell scanning analysis. Our results
show that the effect of combining the two activating muta-
tions is an increase in the constitutive activity only for the
cAMP pathway and not for the IP pathway suggesting that
different mutations result in receptor conformations with
different relative abilities to couple to G
s
-alpha or G
q
-alpha.
Surprisingly the double mutant containing the T477I
behaves as an activating receptor with constitutive activity
both for the cAMP and IP pathways. These data show that
an inactive form of the TSHr which is trapped inside a cell
after transfection is able to gain the membrane surface when
combined with an activated form of the receptor.
Keywords: TSH receptor; G-protein-coupled receptors;
constitutive activity; site-directed mutagenesis; somatic
mutations; germline mutations.
G-protein-coupled, seven transmembrane segment recep-

tors comprise the largest superfamily of proteins in the body
[1]. Many G-protein coupled receptors have a certain basal
activity (constitutive activity) and thus can activate
G-proteins in the absence of the agonist [1,2]. Interestingly,
it has been encountered that discrete mutations of these
receptors are able to dramatically increase this constitutive
agonist-independent receptor activity [3]. The thyroid-
stimulating hormone receptor (TSHr), together with the
follicle-stimulating hormone (FSH) and the luteinizing
hormone (LH) receptors, is a member of a subfamily of
seven transmembrane G-protein-coupled receptors, charac-
terized by a large N-terminal extracellular domain involved
in hormone binding [4,5]; the receptor is mainly coupled to
adenylyl cyclase via G
s
-alpha and, in some species including
man, it activates also the inositol phosphate cascade (IP) via
aG
q
-alpha protein [6–8]. Current models of G-protein-
coupled receptor activation consider that binding of the
ligand, within the slit formed by the transmembrane helices
(for biogenic amines), and/or to the extracellular loops (for
peptide ligands), relieves a built-in negative constraint by
stabilizing an active conformation of the receptor [9]. In this
conformation, the new position of the transmembrane
helices translates into an increased affinity of the intracel-
lular loops for G-proteins. Somatic and germline activating
mutations of the TSHr gene have been identified as a major
cause of toxic thyroid adenoma [3,10,11] and hereditary or

sporadic nonautoimmune toxic thyroid hyperplasia [12,13],
respectively. On the contrary, inactivating mutations aboli-
shing basal activity or affecting agonist induced response
have been described in cases of congenital hypothyroidism
with thyroid hypoplasia [14–16].
All activating TSHr mutations have been shown to
activate adenylyl cyclase when expressed in eukaryotic cells
[3,10]. Some of these mutations possess also constitutive
activity for the IP pathway [3,11]. Inactivating mutations of
the TSHr gene may be due to truncated forms of the receptor
or to point mutations [14–16]. It has been demonstrated
in vitro that point mutations can alter the routing of the
receptor to the cell surface, resulting in loss of basal activity
and loss of agonist induced cAMP production [14–16].
In order to study the mechanism of activation of the
TSHr we explored the effects of combining previously
Correspondence to M. Tonacchera, Dipartimento di Endocrinologia,
Via Paradisa 2, 56124 Pisa, Italy. Fax: 050 578772, Tel.: 050 995048,
E-mail:
Abbreviations: bTSH, bovine TSH; FACS, fluorescence-activated
cell scanning; FSH, follicle stimulating hormone; G
q
-alpha,
G-protein q alpha; G
s
-alpha, G-protein s alpha; IP, inositol
phosphate; KRH, Krebs/Ringer/Hepes; LH, luteinizing hormone;
TSH, thyroid-stimulating hormone.
(Received 11 July 2003, accepted 1 August 2003)
Eur. J. Biochem. 270, 3839–3847 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03778.x

described mutations within a single receptor. We decided to
combine two particularly potent activating TSHr mutations
and to combine an activating mutation together with an
inactivating one.
Double mutant receptors (containing two activating TSHr
mutations) harboring the amino acid substitution P639S (6th
transmembrane segment) [17] and I486M (1st extracellular
loop) [3], named I486M/P639S, and the double mutant
receptor (containing an activating together with an inacti-
vating TSHr mutation) P639S and T477I (1st extracellular
loop) [16], named T477I/P639S, were constructed. Con-
structs were subcloned in the expression vector pSVL and,
after transient expression in COS-7 cells, basal and TSH-
induced cAMP and IP production were determined. Cell-
surface expression was evaluated with [
125
I]bTSH (bovine
TSH) binding, an enzyme immunosorbent assay (EIA) and a
fluorescence-activated cell scanning (FACS) analysis using
different monoclonal antibodies against the TSHr.
Our results show that the effect of combining the two
activating mutations is an increase in the constitutive
activity only for the cAMP pathway and not for the IP
pathway suggesting that different mutations result in
receptor conformations with different relative abilities to
couple to G
s
-alpha or G
q
-alpha. Surprisingly the double

mutant containing the T477I behaves as an activating
receptor with constitutive activity both for the cAMP and
IP pathways. These data show that an inactive form of the
TSHr which is trapped inside a cell after transfection is able
to gain the membrane surface when combined with an
activated form of the receptor.
Materials and methods
Construction of mutant TSH receptors
The constructs harboring the single mutated TSH receptors
T477I, I486M and P639S have been described [11,16,17]. In
brief, a SpeI-CvnI segment (1660–1932) or a CvnI-BstEII
segment (1932–2450) in the cDNA of the wild type (WT)
TSHr in the expression vector pSVL, was replaced by a
homologous segment harboring the mutation in position
477 and 486 or 639, respectively. These mutated sequences
were cloned directly from DNA extracted from nodular
tissues obtained from patients with toxic multinodular
goiter (I486M, P639S) or from the blood of a patient with
a germline inactivating TSHr mutation (T477I). The SpeI
restriction site in the WT-TSHr was created by site-directed
mutagenesis (the change in the coding region does not
modify the encoded amino acid sequence).
For the construction of the double mutants, a CvnI-
BstEII fragment in the T477I and I486M TSHr was
substituted by homologous segments harboring the P639S
mutation, yielding the T477I/P639S or I486M/P639S dou-
ble mutant, respectively (Fig. 1). The resulting constructs
were sequenced directly by an ABI PRISM 310 Genetic
Analyzer (PE Applied Biosystems, Foster City, CA, USA)
to verify the presence of the mutations.

Expression in eukaryotic cells of mutated genes
For transient expression, COS-7 cells were seeded at the
concentration of about 150 000 cells per 3-cm dish for
binding, EIA and FACS analysis, cAMP and IP
determination. COS-7 cells were grown in DMEM sup-
plemented with 10% fetal bovine serum, penicillin
100 IUÆmL
)1
, streptomycin 100 lgÆmL
)1
, fungizone 2.5
lgÆmL
)1
and 1 m
M
sodium pyruvate. One day after
seeding, cells were transfected with the DEAE–dextran
method followed by a 2-min 10% dimethylsulfoxide
shock [18].
For functional assays, 48 h after transfection cells were
used for cAMP or IP determinations and for EIA, FACS
analysis and [
125
I]bTSH binding studies. Triplicate dishes
were used for each condition and each experiment was
repeated at least three times. Results were expressed as
mean ± SEM from one representative experiment. When
not shown, SEM values were so small that they fall within
the symbols.
cAMP assay

Cells were washed with Krebs/Ringer/Hepes buffer (KRH)
and preincubated for 30 min at 37 °C. This was followed
by a 1-h incubation at 37 °C in the presence of 0.5 m
M
isobutylmethyl xanthine as a cAMP phosphodiesterase
inhibitor, in the absence of bTSH (basal values), or in the
presence of various concentrations of bTSH (Sigma
Chemical Co). At the end of the incubation the medium
was removed and replaced by 0.1
M
HCl. The cell extracts
were dried in a vacuum concentrator and cAMP was
determined as described [17] and expressed as picomoles
per dish.
Fig. 1. Schematic representation of the double mutants TSHr (I486M/
P639S; T477I/P639S).
3840 P. Agretti et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Inositol phosphate assay
For IP determinations, 24 h after transfection cells were
incubated with 20 lCiÆmL
)1
[
3
H]inositol (Amersham Phar-
macia Biotech Europe, Germany). The next day, dishes
were washed three times with KRH, preincubated in KRH
plus LiCl 10 m
M
for 30 min at 37 °C and incubated for
18 min at 37 °C in the presence of KRH plus LiCl 10 m

M
(basal values) or in the same medium plus different
concentrations of bTSH. The incubation was stopped by
addition of ice cold 3% HClO
4
,and
3
H-labeled IP were
isolated and assayed by stepwise chromatography on
AG1 · 8 resin [19]. The cell debris in the bottom of the
dishes was dissolved in 1
M
NaOH and counted as phos-
phatidyl inositols. Results were expressed as percentage
radioactivity incorporated in inositol phosphates (IP
1
+
IP
2
+IP
3
) over the sum of radioactivity in inositol
phosphates and phosphatidyl inositols [19].
Binding assay
Forty-eight hours after transfection, cells were washed once
with Hanks’ solution in which NaCl was replaced by
sucrose 280 m
M
containing 0.2% bovine serum albumin
(BSA) and 2.5% low fat milk. Binding studies were

performed by incubating cells in that same medium at
room temperature for 4 h in the presence of about
90 000 c.p.m. [
125
I]bTSH (a gift of BRAHMS, Berlin,
Germany) and the appropriate concentrations of cold
bTSH. At the end of the incubation cells were rinsed twice
with ice cold Hanks’ medium, solubilized with 1
M
NaOH
and bound radioactivity was determined in a gamma-
counter. In the absence of a consensus about the bioactivity
of pure bovine TSH [20], we have expressed all TSH or
TSHr concentrations in mUÆmL
)1
, assuming a 1/1 stechio-
metry for TSH binding to its receptor. The competition
binding curves have been fitted by nonlinear regression
assuming a single receptor site [21].
Enzyme immunosorbent assay (EIA)
EIA measurements were carried out with transfected cells,
nonpermeabilized and in suspension. Forty-eight hours
after transfection cells were detached from the plates with
NaCl/P
i
containing 5 m
M
EDTA and EGTA. Cells were
then pelleted and incubated with a mouse anti-(human
TSHr Ig) (Novocastra Laboratories Ltd, UK) diluted at

1 lgÆmL
)1
in NaCl/P
i
containing 0.5% BSA. After two
washes with NaCl/P
i
, cells were incubated for 1 h at 4 °C
with peroxidase-conjugated anti-(mouse IgG) as secondary
antibody (Sigma Chemical Co.) diluted 1 : 70 000 in NaCl/
P
i
containing 0.5% BSA. The cells were washed three times
with NaCl/P
i
and, finally, incubated with the o-phenylene-
diamine dihydrochloride substrate (Sigma Chemical Co.)
for 30 min at 37 °C. The reaction was stopped by adding
1
M
H
2
SO
4
and color development was measured at
492 nm.
FACS analysis
Cells were detached from culture dishes with 5 mmolÆL
)1
each of ethylenediamine tetraacetate and ethyleneglycol-

bis-(beta-aminoethyl ether)-N,N,N¢,N¢-tetraacetic acid in
NaCl/P
i
and transferred to Falcon tubes (2052, Falcon
Labware, Cockeysville, MD, USA). Cells were washed with
NaCl/P
i
plus 0.1% BSA, centrifuged at 500 g at 4 °Cfor
3 min, and treated appropriately for the nonpermeabilized
or permeabilized cell assay as described previously [16].
Nonpermeabilized cells were incubated at room tempera-
ture for 30 min with 200 lL of a monoclonal antibody
directed against the TSHr (BA8 gently gifted from Dr
Sabine Costagliola) diluted in NaCl/P
i
plus 0.1% BSA. A
blank sample was prepared by incubating cells with 200 lL
NaCl/P
i
plus 0.1% BSA. For the permeabilized cell assay,
cells were fixed with 2% NaCl/P
i
/paraformaldehyde (UCB,
Brussels, Belgium) and then treated for 30 min with NaCl/P
i
plus 0.1% BSA and 0.2% saponin (Sigma Chemical Co.,
St. Louis, MO, USA); all subsequent steps with antibodies
were performed in 0.2% saponin. Cell-bound monoclonal
antibodies were detected washing the cells with NaCl/P
i

plus
0.1% BSA and then incubating them for 30 min at 4 °Cin
the dark with a goat anti-(mouse IgG) fluorescin-conjugated
(Becton Dickinson and Co., San Jose, CA, USA) diluted
1:20inNaCl/P
i
with 0.1% BSA containing 10 lgÆmL
)1
propidium iodide (Sigma). Propidium iodide (PI) was used
to detect and exclude from the analysis damaged cells. Flow
cytometric analysis was performed using a FACSort flow
cytometer (Becton Dickinson and Co.) equipped with a
laser for an excitation at 488 nm to detect monoclonal
antibodies conjugated with fluorescin 5-isothiocyanate and
PI. Fluorescence emission of fluorescin 5-isothiocyanate
and PI from single cells were separated and measured using
the standard optics of the FACSort. The
CELLQUEST
software program (Becton Dickinson and Co.) was used
to acquire and analyze data. A minimum of at least 10 000
cells was analyzed.
Computation of specific constitutive activity (SCA)
and relative SCA (RSCA)
Given that the transfection efficiency for each construct is
constant for a given batch of cells, the SCA was calculated
by:
SCA ¼ðAr À AvÞ=ðFr À FvÞ
where Ar and Av are the cAMP (or IP) of cells transfected
with the mutant constructs and vector, respectively, and Fr
and Fv are the corresponding mean fluorescence unit

obtained with FACS analysis. RSCA, which is normalized
to the WT-TSHr, was obtained by:
RSCA ¼ SCAr=SCA-WT:
Results
cAMP cascade
The effects of each TSH receptor construct harboring one
or two mutations have been investigated after transient
expression in COS-7 cells.
For basal cAMP determinations, 250 ng per dish of the
DNA of the various constructs, giving maximal cAMP
stimulation [3], were used to transfect COS-7 cells. As
expected, cells transfected with the WT-TSHr exhibited a
Ó FEBS 2003 Double mutant TSHr mutations (Eur. J. Biochem. 270) 3841
threefold increase in basal cyclic AMP accumulation with
respect to cells transfected with vector alone, showing
constitutive activity (Table 1, Fig. 2A). Cells transfected
with two of the constructs harboring a single mutation
(I486M; P639S) displayed higher level of basal cAMP as
compared to cells transfected with the WT-TSHr (Table 1,
Fig. 2A), while cells transfected with the construct harbor-
ing the T477I mutation showed levels of cAMP that were
very similar to those exhibited from the cells transfected
with the empty vector alone. The I486M/P639S double
mutant showed a further increase of cAMP accumulation
with respect to the single parental mutated receptors.
Surprisingly the combination of the activating P639S with
the inactivating T477I yielded a receptor T477I/P639S
with a strong constitutive activity similar to that obtained
with P639S.
The biological response to bTSH of the cells transfected

with the DNA of the different constructs was explored in
terms of cAMP accumulation (Table 1, Fig. 2A). Cells
transfected with the constructs harboring a single activating
mutation (P639S; I486M) exhibited a maximal stimulation
to bTSH that was similar to that observed with the
WT-TSHr. Cells transfected with the inactivating mutation
T477I showed a very low response to bTSH with values of
cAMP production at 100 mUÆmL
)1
of bTSH about seven-
fold lower than the WT-TSHr. The I486M/P639S double
mutant showed an increase of maximal cAMP accumula-
tion after bTSH challenge with respect to the single mutated
receptors. The T477I/P639S maintained a cAMP response
to bTSH similar to P639S alone.
Inositol phosphate cascade
As expected from previous studies [3,11], no significant
increase of basal levels of IP production was observed in the
cells transfected with the WT-TSHr (Table 1, Fig. 2B); a
slight increase over values obtained with an empty vector or
the wild-type receptor construct, was observed in cells
transfected with the P639S or I486M single mutant,
showing constitutive activity for the IP production (Table 1,
Table 1. Functional characteristics of TSHr mutants transfected in COS-7 cells as described in materials and methods. cAMP values, expressed as
percentage basal cAMP levels of WT-TSHr, were measured in basal conditions and after stimulation with 10 and 100 mUÆmL
)1
bTSH. IP values,
expressed as percentage basal IP levels of WT-TSHr, were measured in basal conditions and after stimulation with 100 mUÆmL
)1
bTSH. Results are

mean ± SEM of three independent experiments.
cAMP IP
Basal 10 mUÆmL
)1
100 mUÆmL
)1
Basal 100 mUÆmL
)1
WT-TSHr 100 1070 ± 46 1087 ± 37 100 338 ± 12
T477I 40 ± 8 95 ± 11 149 ± 16 96 ± 11 107 ± 10
I486M 430 ± 22 1053 ± 29 1214 ± 39 170 ± 10 592 ± 26
P639S 476 ± 17 1137 ± 35 1241 ± 27 238 ± 21 700 ± 31
T477I/P639S 403 ± 12 970 ± 18 1024 ± 69 123 ± 13 423 ± 18
I486M/P639S 586 ± 21 1186 ± 34 1671 ± 35 215 ± 15 600 ± 24
Fig. 2. Basal and bTSH-induced stimulation of cAMP and IP levels in COS-7 cells transfected with the single or double mutated receptors. Values are
mean ± SEM from one representative experiment in which triplicate dishes were used. (A) Basal intracellular cAMP values (picomoles per dish)
and levels of cAMP production after stimulation with 10 and 100 mUÆmL
)1
bTSH in COS-7 cells transfected with saturating concentrations of
DNA (250 ng per dish), of single mutated receptors (T477I, I486M, P639S) or double mutant receptors (T477I/P639S; I486M/P639S), WT-TSHr
and pSVL alone, are shown. (B) Basal IP values, expressed as percentage of (IP + PI) and IP levels after bTSH stimulation (100 mUÆmL
)1
)in
COS-7 cells transfected with saturating concentrations of DNA (250 ng per dish), of single mutated receptors (T477I, I486M, P639S) or double
mutant receptors (T477I/P639S; I486M/P639S), WT-TSHr and pSVL alone, are shown.
3842 P. Agretti et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 2B). Cells transfected with the T477I mutation showed
basal IP values similar to those obtained with pSVL or WT-
TSHr alone. Cells transfected with the I486M/P639S double
mutant showed basal IP values intermediate to those

obtained in cells transfected with the single parental
mutants, showing that the double mutant promoted the
same higher basal IP values. Cells transfected with the
T477I/P639S double mutant showed only a slight increase
in basal IP production with respect to WT-TSHr or T477I
alone, and a clear decrease with respect to P639S.
Stimulation of IP accumulation by 100 mUÆmL
)1
of
bTSH exhibited a threefold stimulation of IP production in
cells transfected with the WT-TSHr (Table 1, Fig. 2B). The
I486M and P639S mutated receptors showed an increased
production of IP with respect to the WT-TSHr after bTSH
stimulation. The T477I completely lost the ability to
respond to bTSH. The I486M/P639S maintained a similar
production of IP after bTSH challenge with respect to the
parental TSH receptors. The T477I/P639S was able to
produce a significant IP stimulation after bTSH challenge,
which was lower to that produced by P639S alone.
Binding of [
125
I]bTSH
To measure the total number of receptors (or TSH binding
capacity, B
max
) expressed at the surface of the cells
transfected with the different constructs, and their relative
dissociation constants (K
d
), binding studies were performed

with a bovine [
125
I]TSH tracer as described in Materials
and methods.
Cells transfected with the constructs harboring the single
activating mutations I486M and P639S, exhibited a lower
level of expression as compared to the WT-TSHr, indicating
that the increased cAMP production (measured in the same
experiment) was not due to overexpression of the mutated
receptors (Fig. 3). When compared with the WT-TSHr, the
level of receptor expression in COS-7 cells transfected with
the T477I inactivating mutant was about four times lower
(Fig. 3). The I486M/P639S double mutant showed a similar
level of expression with respect to the single mutated
receptors. Surprisingly, also the T477I/P639S showed a
good ability to be expressed at the surface of the cells. As
already noted earlier [3], constitutively active receptors
showed a higher affinity for bTSH binding than the
WT-TSHr (Fig. 3).
EIA and FACS analysis
The level of receptor expression on the cell surface for the
different constructs was independently measured by EIA,
using a monoclonal antibody directed against the extracel-
lular domain of the TSH receptor (NCL-TSH-R2, Novo-
castra Laboratories Ltd, Newcastle, UK) (data not shown),
and by FACS analysis using BA8, a monoclonal antibody
directed against the TSHr (Fig. 4). COS-7 cells transfected
with the inactivating mutant T477I showed a very low level
of expression of the mutated receptor at the cell surface
(Fig. 4A). The T477I was however, clearly detectable within

the cells after saponin permeabilization (Fig. 4B), suggest-
ing that the T477I receptor was synthesized and recognized
by the antibody, but might have a defect in folding of the
mutant protein resulting in abnormal routing to the plasma
membrane. The level of expression at the cell surface of the
single mutated receptors (I486M, P639S) and the I486M/
P639S double mutant was similar and was the same
according to the binding experiments (Table 2). The FACS
analysis also confirmed that the T477I/P639S double
mutant was able to reach the membrane surface. The
comparison of WT-TSHr and mutated receptors expression
at cell surface by using the three methods are shown in
Table 2. The level of receptor expression on the cell surface
was statistically different for all the single and double
mutant receptors with respect to the WT-TSHr. The
Student’s t-test with 95% confidence was used to establish
significance (P £ 0.05).
Fig. 3. Binding characteristics of the single and double mutants
expressedinCOS-7cells.[
125
]IbTSH binding to COS-7 cells transfected
with 250 ng per dish of the construct harboring the single mutated
receptors (T477I, I486M, P639S) or double mutant receptors (T477I/
P639S; I486M/P639S) and WT-TSHr. The B
max
, total receptor
amount (binding capacity; expressed as milliunits of TSH per milliliter)
and K
d
(alsoexpressedinmUÆmL

)1
of TSH) were computed as des-
cribed in Materials and methods (the SEM values are so small that
they fall within the symbols).
Ó FEBS 2003 Double mutant TSHr mutations (Eur. J. Biochem. 270) 3843
Specific constitutive activity
The data obtained from the FACS analysis allowed
measurement of efficiency of transfection and computation
of the increase in cAMP and IP within the effectively
transfected cells. Assuming that the mutations did not affect
recognition by the monoclonal antibodies, these data also
allowed normalization of the increase in cAMP and IP
accumulation to the amount of receptor expressed at the cell
surface, yielding an estimation of specific constitutive
activity. When transfecting COS-7 cells with increasing
amounts of wild-type and mutant cDNAs we observed that
the constitutive activity of the WT and mutated receptors
was linear over the range of cell surface expression (data not
shown). The I486M showed to be eightfold and the P639S
10-fold more active than the WT-TSHr. The combination
of the two activating TSHr mutations produced a I486M/
P639S double mutant that showed an increase of SCA with
respect to the parental constructs (11-fold with respect to the
WT-TSHr) (Fig. 5A). The combination of the activating/
inactivating T477I/P639S double mutant produced a recep-
tor that was 7.5 more active than the WT-TSHr but less
active with respect to the P639S alone.
These data were also used to compute IP accumulation
with respect to the amount of receptor expressed at the cell
surface. The data clearly showed that both the single

activated TSHr receptors had constitutive activity with
respect to the WT-TSHr (Fig. 5B) being 15-fold for the
I486M and 33-fold for the P639S. The combination of the
two activating TSHr mutations produced a receptor which
Fig. 4. Expression analysis of the single and double mutants by FACS analysis using the BA8 monoclonal antibody. Fluorescence intensity is expressed
in arbitrary units, as a function of cell number plotted on a logarithmic scale. (A) Nonpermeabilized cells assayed after transfection with pSVL,
TSHr WT, the T477I, I486M and P639S single mutants, and the T477I/P639S and I486M/P639S double mutants. BA8 immunoreactivity
(arbitrary units): vector, 3.20; TSHr WT, 19.67; T477I, 5.04; I486M, 15.00; P639S, 14.20; T477I/P639S, 15.52; I486M/P639S, 16.21. (B) Saponin-
permeabilized cells identically transfected. BA8 immunoreactivity (arbitrary units): vector, 6.00; TSHr WT 11.80; T477I, 15.13; I486M, 17.70;
P639S, 22.37; T477I/P639S, 20.16; I486M/P639S, 17.68.
Table 2. The level of receptor expression on the cell surface for the
different constructs after transfection was measured by [
125
I]bTSH
binding, EIA and FACS analysis. Data are represented as a percentage
of the values corresponding to WT-TSHr minus the nonspecific
absorbance readings for EIA, the non-specific binding and the non-
specific fluorescence for FACS analysis. The nonspecific data were
obtained transfecting COS-7 cells with the empty vector. Values are
mean ± SEM of three independent experiments.
[
125
I]bTSH binding EIA FACS
WT-TSHr 100 100 100
T477I 23 ± 3.8 44 ± 7.5 26 ± 3.9
I486M 81 ± 13.2 80 ± 13.8 77 ± 10.2
P639S 70 ± 8.4 65 ± 6.8 72 ± 9.9
T477I/P639S 77 ± 10.8 67 ± 7.1 78 ± 12.1
I486M/P639S 77 ± 9.5 72 ± 9.5 81 ± 8.8
3844 P. Agretti et al. (Eur. J. Biochem. 270) Ó FEBS 2003

was less potent that the P639S alone (24-fold over the
WT-TSHr) while the combination of the activating/inacti-
vating T477I/P639S produced a constitutive activity of
sixfold with respect to the WT-TSHr.
Discussion
According to the extension of the ternary complex model
[22] for activation of G-proteins by serpentine receptors, the
receptor is assumed to exist in two interconvertible confor-
mations: the ÔinactiveÕ one, enforced by a built-in negative
constraint, corresponds to the major unliganded form of the
receptor; the active conformation capable of promoting
GDP/GTP exchange on the G-protein is achieved by a
fraction of the unliganded receptors only, but is stabilized by
binding of its ligand. The model predicts that unliganded
receptors may display a basal activity (corresponding to the
fraction of active receptors) which can be antagonized by
inverse agonists, the effect of which is to stabilize the
inactive conformation [22]. The TSH receptor offers a series
of characteristics which make it an interesting model to
study in this context: (a) it is capable of activating both
G
s
-alpha and G
q
-alpha [6–8]; (b) it displays an easily
measurable constitutive activity towards adenylyl cyclase
after transfection in COS cells [1,3,10,11]; (c) it can be
activated by a surprisingly diverse spectrum of point
mutations, or (d) by stimulating autoantibodies; (e) loss-
of-function mutations have also been described, abolishing

basal activity or affecting agonist-induced response and
(f) the mechanism by which binding of the hormone to the
N-terminal domain of the receptor translates into activation
of its serpentine domain is unknown.
With the aim of improving our understanding of the
mechanisms underlying some of these characteristics, we
have explored the effects of combining activating and
inactivating mutations of the TSHr within a single receptor
on its constitutive activity towards G
s
-alpha and G
q
-alpha
and its binding characteristics and responsiveness to bTSH.
When two strong activating mutations of the TSHr gene
(both possessing constitutive activity for both cAMP and IP
pathways) have been combined a further increase of basal
and bTSH-stimulated cAMP production was observed with
respect to the single parental receptors. The expression of
the I486M/P639S on the cell surface was similar to the single
mutated receptors. These results suggest that the double
mutant I486M/P639S may be one step further in a scale of
constraint release and the two mutations cooperate to
potentiate the activity of the receptor. The increase of basal
activity by combining activating mutations is well known
for glycoprotein hormone receptors [23,24]. No further
increase of IP was evident in the double mutant I486M/
P639S with respect to the parental receptors. This observa-
tion suggests the possible existence of multiple conformation
states with different abilities to interact with G

s
-alpha and or
G
q
-alpha. The mutational dissociation of functional con-
formations have also been described for glycoprotein
hormone receptors [25–27]. The T477I has been described
as an inactivating mutation in a patient with congenital
hypothyroidism and thyroid hypoplasia [16]. Similarly, an
inactivating mutation of the FSH receptor gene that is
located in the close vicinity of the amino acid 477 of the
TSHr was found in a patient affected by primary ovarian
failure [28]. When the T477I was transiently expressed in
COS-7 cells we observed the complete loss of basal
constitutive activity for the cAMP pathway, a strong
reduction in bTSH stimulated cAMP production and
absent bTSH stimulated IP production. The level of
receptor expression in COS-7 cells transfected with the
T477I measured both by binding with bTSH and by using a
monoclonal antibody in FACS analysis directed against the
TSHr was very low. The very low expression of the mutated
receptor on the cell membrane is probably due to the poor
routing of the receptor at the cell surface. Surprisingly the
effect of combining this T477I with the activating P639S
produced a double mutant with a similar level of expression
at the cell surface with respect to the parental P639S. The
T477I/P639S behaves like the P639S alone, in terms of basal
and bTSH stimulated cAMP accumulation. Only a slight
decrease in the basal and bTSH stimulated IP production
was noticed transfecting this T477I/P639S with respect to

P639S alone.
These data clearly show that an inactivated G protein-
coupled receptor was trapped intracellularly after transient
expression in eukaryotic cells and when combined with an
activated form of the receptor reconstituted a functional
membrane receptor. Similarly, a D578H mutant LH
receptor was shown to increase the cell surface expression
of poorly expressed vasopressin-LH receptor’s chimera [29].
However, contrary to what has been observed in our model
where the P639S has a lower expression at the cell surface
Fig. 5. RSCA of single and double mutant receptors for cAMP (A) and
IP (B) computed as described in Materials and methods. Results are the
mean ± SEM from one representative experiment in which triplicate
dishes were used.
Ó FEBS 2003 Double mutant TSHr mutations (Eur. J. Biochem. 270) 3845
with respect to the WT-TSHr, the D578H mutant alone was
shown to be expressed at the cell surface four times more
than the WT-LH receptor [29]. Maya-Nunez et al.[30]
showed that a mutant (E90K) gonadotropin-releasing
hormone receptor (GnRHr) was rescued either by deleting
K191 or by adding a C-terminal sequence. Either of these
approaches alone supported high-membrane expression of
the GnRHr. It has been proposed that the E90K mutation
itself while resulting in protein misfolding does not
irreversibly destroy the intrinsic ability of the mutant to
bind ligand or to couple effectors. The rescued receptor,
now stabilized in the plasma membrane, was able to activate
the appropriate effector system. The proper folding and
assembly of polypeptides occur in the endoplasmic reti-
culum. If polypeptides cannot fold correctly, mechanisms

of quality control ensure that the aberrant protein is not
further processed along the secretory pathway [31]. The
quality control mechanisms are mediated by a family of
proteins called molecular chaperones. Recently, a class of
compounds called chemical chaperones were shown to
reverse the intracellular retention of several misfolded
proteins [31]. Besides it has been described that small cell-
permeable molecules can act as either chemical chaperones
or pharmacological (ligand-mediated) chaperones to rescue
mutant protein function [31]. Thus, the development of
strategies aimed at promoting proper folding and matur-
ation of mutant proteins could provide new therapies for a
wide spectrum of diseases [31].
In conclusion, the combination of two activating muta-
tions of the TSHr determined an increase in the activity only
for the cAMP pathway and not for the IP pathway
suggesting that different activating mutations result in
receptor conformations with different relative abilities to
stimulate the cAMP or IP regulatory cascades. Surprisingly
the combination of a strong inactivating mutation with an
activating one produced a receptor that was able to be
expressed at the cell surface with high constitutive activity.
Acknowledgements
This work was supported by the following grants: Ministero dell’Uni-
versita
`
e della Ricerca Scientifica (MURST), Programma di Ricerca: Le
malattie della tiroide: dalle basi molecolari alla clinica. Ministero
dell’Universita
`

e della Ricerca Scientifica (MURST), Programma di
Ricerca: Strategie per la valutazione degli effetti disruptivi dei
contaminanti ambientali sul sistema endocrino degli animali e
dell’uomo. CNR Progetto Biotecnologie CTB 99.00.224.PF 31: Basi
molecolari delle neoplasie benigne e maligne della tiroide. Istituto
Superiore di Sanita
`
: Basi Molecolari dell’ipotiroidismo congenito:
predizione, prevenzione ed intervento I.S.S. Centro Eccellenza Ambi-
SEN, Pisa.
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