Third Medical Department (S.N., S.C., R.P.), University of Leipzig, D-04103 Leipzig, Germany; and Institute for Molecular Pharmacology (G.K.), D-10315 Berlin, Germany
Address all correspondence and request for reprints to: Ralf Paschke, M.D., Third Medical Department, University of Leipzig, Ph.-Rosenthal-Strasse 27, 04103 Leipzig, Germany. E-mail: pasr{at}medizin.uni-leipzig.de
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ABSTRACT |
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INTRODUCTION |
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As for most GPCRs, the precise molecular mechanism of the TSHR
activation is not known. The understanding of the intramolecular
interactions and conformational changes underlying receptor activation
is hindered by a lack of information on the three-dimensional
high-resolution structure of the TSHR. Low-resolution structure data (6
Å) from cryomicroscopy data of frog rhodopsin suggested a tilt of the
transmembrane segments. However, they could not provide structural
information about individual side chains or even atoms (7, 8). Very recently the x-ray crystal structure of the bovine
rhodopsin with a resolution of 2.8 Å provided new, important insights
into side chain orientations of rhodopsin (9). In this
model some helical portions of TM2, TM5, TM6, and TM7 are not organized
in an ideal -helical backbone structure, which may be due to
particular amino acid residues. Noteworthy, in comparison to rhodopsin,
glycoprotein hormone receptors have different residues in almost all
respective critical positions; thus an identical side chain orientation
is very unlikely.
Site-directed mutagenesis and the evaluation of its effects on receptor binding and signal transduction provide a valid means of obtaining insights into intramolecular changes of glycoprotein hormone receptors, as well as GPCRs in general, during activation (5). Moreover, molecular modeling is necessary to integrate experimental observations and biophysical and structural data into a mechanistic three-dimensional receptor model. Models also contribute to reduce the gap of resolution between the 6A and atom level. Homologous receptor models are used to indicate likely conformational differences in the new rhodopsin x-ray structure. They are also important in explaining relations between receptor structure and function. Many models for different GPCRs have been generated from a combination of structural data derived from rhodopsin and mutagenesis data on the receptors themselves (10).
Constitutively activating mutations have been identified in many GPCRs (11). In the TSHR gene, gain of function mutations cause autosomal dominant nonautoimmmune hyperthyroidism and toxic thyroid nodules (12). The identification and functional characterization of naturally occurring mutations in the TSHR provided the first information about mechanisms of receptor signaling. Therefore, naturally occurring mutations that activate the TSHR provide unique hints for further mutagenesis experiments, as has recently been shown (13, 14, 15). However, this approach, until now, has not been employed for TM6.
Figure 1 shows a schematic representation
of the TSHR with the different amino acid exchanges of somatic TSHR
mutations in toxic thyroid nodules collected in a TSHR mutation
database (16). Most of these activating mutations have
been identified in exon 10 of the TSHR, in which TM6 represents a hot
spot for activating mutations. However, none of the previous
site-directed mutagenesis studies of the TSHR has focused on this
domain, despite these indications for its significance in receptor
structure and activation. The central part of TM6 and the intracellular
half of TM7 are highly conserved among GPCRs. Molecular modeling of the
LHR and other GPCRs has revealed consensus residues in TM6 that are
most likely engaged in important interhelical interactions (11, 17). Conformational changes can induce relative movements of the
helicesespecially of helix 6as reported for rhodopsin (18, 19). Recent data from the rat LHR have demonstrated a direct
activation of GS
by TM6 (20).
Therefore, mutations in TM6 of the LHR are likely to directly affect
the coupling of the receptor to GS
. A model of
the LHR revealed several pairs of strong interhelical side chain-side
chain H bonds formed in the midregions of TM6 and TM7
(17). According to this LHR model, the homologous T632 and
D633 residues of the TSHR could be involved in such interhelical H
bonds in the TSHR. Moreover, the highly conserved residue C636 in TM6
could either be hidden in the inactive state or participate in
stabilizing the inactive receptor state by inter- or intrahelical
interactions. This cysteine was observed in several GPCRs to be
accessible for sulfhydryl-reactive agents only in activated receptors
and not in the inactive state (21, 22).
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RESULTS |
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The central cavities of glycoprotein hormone receptors including the D633 residue are quite polar (17). Moreover, D has a negative charge, is strongly acidic, and is characterized by the lowest hydrophobicity of all amino acids. To induce significant physicochemical changes, amino acids with qualities strongly differing from D were selected. D was therefore replaced by the amino acids R, C, W, and F. R is also polar, but it is positively charged, strongly basic, and weakly hydrophobic. In contrast to D and R, C has no charge. However, this amino acid residue is weakly polar but also hydrophobic. In contrast to these residues W and F are apolar, strongly hydrophobic, and not charged. Moreover, both W and F introduce bulky aromatic side chains at position 633. In addition, D633 was deleted.
Functional Assessment of the TSHR Mutants at Position D633 in
TM6
The effects of all mutations at position D633 on basal and
TSH-stimulated cAMP and inositol phosphate (IP) production are
summarized in Table 1. As a control for
our assay system, D633H, a naturally occurring activating mutation at
position 633 (25, 26), was also investigated. Apart from
the D633R substitution, all other initial amino acid substitutions
resulted in constitutive activation of the cAMP signaling cascade (Fig. 2
). Substitution of D633 with F or W
induced the highest constitutive activity despite lower cell surface
expression. In comparison to the wild-type (wt) TSHR, most of the
substitutions resulted in a decreased cell surface expression.
Therefore, we reduced the cell surface expression of the wt TSHR and
the TSHR mutants to the level of the D633W mutant, which showed the
lowest cell surface expression (16 ± 1.2% of wt TSHR, set at
100%, Table 1
) apart from the deletion of D633. The wt TSHR and the
mutated TSHR constructs were cotransfected with various amounts of
plasmids containing the human V2 receptor (V2R). The cell surface
expressions of these cotransfected TSHR constructs in comparison to the
wt TSHR transfected alone were as follows: wt TSHR set at 100%; wt
TSHR/V2R, 14.7 ± 1.0%; D633C/V2R, 11.7 ± 1.4%; D633F/V2R,
12.7 ± 1.5%; and D633H/V2R, 15.2 ± 3.1%. Measurement of
cAMP accumulation under these conditions in three independent
experiments confirmed the constitutive activity of the TSHR mutants
D633C (3.2 ± 0.3-fold over wt TSHR basal), D633F (7.6 ±
0.4-fold), D633W (10.3 ± 0.5-fold), and D633H (7.0 ±
1.0-fold) for identical cell surface expressions of these TSHR
constructs. The TSH-stimulated cAMP accumulation (wt TSHR, 14.5 ±
1.8-fold over wt TSHR basal; D633C, 22.9 ± 1.2-fold; D633F,
14.4 ± 3.0-fold; D633W, 14.8 ± 1.9-fold; and D633H,
14.6 ± 1.4-fold) was comparable or only slightly increased
compared with data determined for different expression levels (Table 1
).
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Cells transfected with the TSHR deleted at position 633 (D633) were
characterized by low cell surface expression compared with the wt TSHR
(Table 1
and Fig. 4A
). However, D633
was clearly detectable within
the cells measured by fluorescence-activated cell sorting (FACS)
analysis on permeabilized cells (Fig. 4B
) suggesting increased
accumulation of this receptor mutant within the cells. This mutant
showed hardly detectable binding for labeled TSH (Fig. 3
). Neither the adenylate cyclase nor the
PLC signaling cascade are activated by this TSHR mutant (Table 1
).
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Molecular Modeling of the Human TSHR
To investigate the alterations of TSHR activation caused by the
substitution of aspartate with arginine, a computer model, based on the
TSHR model published by Biebermann et al. (13),
was developed. This model suggests the following potential interaction
partners for R633 to maintain the inactive receptor conformation (Fig. 6A):
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Theoretically, and in contrast to all other amino acid substitutions at
this position, there are several simultaneous possibilities for R633 to
form H bonds with TM7 or TM3 on the basis of available hydrogens. For
the prediction of a possible H bond pattern, it was necessary to
determine whether the 633 residue must be charged in the TSHR or
whether it can be uncharged as in the D578N mutant of the LHR
(29). To further delineate the possible interaction
partners of R633, which contribute to the formation of H bonds and
preserve a completely inactive receptor conformation, the following
single and double substitutions were generated. For the identification
of functionally relevant interactions, the D633 residue was substituted
with lysine (D633K). In addition, the introduction of this amino acid
residue led to a shortening of the side chain at position 633 and
therefore tested potential long-range interaction partners such as D460
in TM2 of the TSHR. Double mutantsR633 in combination with
substitutions of N674 (D633R/N674D, D633R/N674A, D633R/N674S) and of
N670 (D633R/N670A, D633R/N670S)were generated with the aim of
rescuing the receptor signaling by destroying possible H bonds
suggested by computer modeling and thereby releasing the completely
blocked inactive conformations. To evaluate the contribution of the
second partner within the double mutants, the single mutations (N674D,
N674A, N674S, N670A, N670S) were also tested. Moreover, the computer
simulations also suggested Ser508 in TM3 as an alternative interaction
site for R633 (Fig. 6A). Therefore, the double mutant D633R/S508G was
created to abrogate a potential interaction of R633 with TM3.
Among the polar amino acid residues present in the cytoplasmatic half of TM3 are S505 and E506, which point toward the intracellular receptor part but are too far away from D633 for interhelical interactions in the native inactive TSHR state. Y510 (TM3) very likely interacts with a hydrophobic cluster between TM4 and TM5. T632 and C636 of TM6 and C672 of TM7 are clearly oriented toward TM7 and TM1, respectively. While C636 and C672 are involved in aromatic/hydrophobic interactions and are contributing to interhelical stabilization, T632 forms a hydrogen bond toward the TM6 backbone and thus appears very likely to stabilize TM6 in an intrahelical manner.
Experimental Testing of R633 Interactions Maintaining the Inactive
State
Similarly to the D633R mutant cells transfected with the TSHR
mutant, D633K showed a lower basal cAMP activity in comparison to the
wt TSHR at comparable receptor density on the cell surface (Table 1).
In contrast to D633R, cells transfected with the D633K construct showed
a slight increase in cAMP production after stimulation with high TSH
concentrations of 10 to 100 mU/ml TSH, which do not occur in
physiological in vivo conditions. The substitution at
position 633 with an uncharged asparagine did not result in a
constitutive activation of the cAMP pathway (Table 1
). Receptor density
on the cell surface, dissociation constant (KD)
values, and basal and stimulated cAMP levels were similar for D633N and
the wt TSHR receptor (Table 1
). The substitution of S508 in TM3 with G
(S508G) had a significant effect on the cell surface expression (23%
of the wt). The basal cAMP level was slightly below the wt TSHR level.
However, the agonist-dependent cAMP activation corresponds to the wt
TSHR. In contrast, the TSHR double mutant S508G/D633R, like D633R, was
completely inactive for cAMP and IP production at a similar cell
surface expression as the wt TSHR (Table 1
).
Partial Rescue of TSHR Signaling by the Double Mutation
D633R/N674D
COS-7-cells transfected with the substitutions N674D or N674S
exhibited an increase in basal cAMP accumulation in comparison to the
wt TSHR at a significantly reduced cell surface expression. Both
constitutively active mutants N674D and N674S showed a reduced cAMP
accumulation after bovine TSH (bTSH) stimulation compared with the wt
TSHR. Cells transfected with the N674A TSHR mutant showed a strongly
impaired TSHR function. The cAMP accumulation after bTSH stimulation
was extremely reduced. Both double mutants D633R/N674A and D633R/N674S
were inactive with respect to the adenylate cyclase pathway, despite a
slightly increased cell surface expression compared with the wt TSHR
(Table 1). The double mutants D633R/N670A and D633R/N670S were also
completely inactive as regards the cAMP pathway. The TSHR substitution
N670S resulted in constitutive activation as previously described
(30) (Table 1
).
The most important finding was the partial rescue of the impaired
signaling of D633R by the double mutant D633R/N674D, as demonstrated by
TSH stimulation of the mutants. The cell surface expression of 117%
for D633R/N674D was similar to the cell surface expression of the
inactive D633R substitution (124%). The basal cAMP production for the
D633R/N674D double mutant was below the wt TSHR level. However, after
stimulation with 100 mU/ml bTSH, the D633R/N674D TSHR mutant reached
36% of the stimulated wt TSHR cAMP accumulation (Fig. 5 and Table 1
).
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DISCUSSION |
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Recent in vivo findings for the TSHR and the LHR
support the hypothesis that this aspartate residue in TM6 could play a
pivotal role in the activation process of the glycoprotein hormone
receptors. Four different naturally occurring constitutively activating
mutations of the D633 residue of the TSHR are known (Fig. 1). Analogous
to the TSHR D578 in the LHR is the only position with four different
amino acid substitutions (D578G, D578Y, D578H, D578E) (27, 32, 33, 34, 35). Kosugi et al. (29) suggested
that the D578 side chain could serve as a properly positioned hydrogen
bond acceptor and could thus be important for stabilizing the inactive
state of the LHR. Furthermore, Lin et al. (17)
proposed that the H bond interactions between TM6 and TM7 could
constrain the inactive receptor state and that release or weakening of
the H bonds could cause reorientation of TM6 as an important step in
LHR activation. Rearrangements of TM6 during the activation of the TSHR
were concluded from mutations disturbing the positions of the tightly
packed hydrophobic helices of TM5 and TM6 (13). An
additional rearrangement of TM7 in the LHR was recently suggested by
data obtained with different gain or loss of function mutants of the
two highly conserved asparagines in TM7 (N670 and N674 in the TSHR)
(36). Moreover, recent evidence demonstrates that the
activation of GPCRs is accompanied by a rigid body movement of the
intracellular portion of TM6 and especially of the TM6 region relative
to the other TMs (18, 19, 37). Investigations, based on
the protein structure of bovine and frog rhodopsin (38, 39), and experimental data, obtained for the LHR
(17), suggest that the activation signal is propagated
from the ligand binding site along the axis TM2TM3TM7 and further
to TM6 toward the intracellular part of the receptor.
Based on the observation that D633 is the only amino acid with four
different amino acid exchanges in vivo (Fig. 1), this study
focused on the pivotal role of the D633 residue in the TSHR for
interhelical interactions, receptor activation, and signaling. The
further in vitro substitutions at this position led to
constitutive activation (D633Y, F, C) as well as to inactivation of the
TSHR without changes of membrane expression or binding (D633R).
Substitution of D633 with F or W caused the highest constitutive
activity of the cAMP pathway as well as constitutive activation of the
IP pathway. These findings are in accordance with the previously
reported data for the LHR (29). The high constitutive
activity for the cAMP pathway is very likely due to the introduction of
bulky hydrophobic side chains. Phenylalanine and tryptophan require not
only a larger space at position 633 but also tend to escape from the
strong hydrophilic proximity of TM7 toward hydrophobic patches either
between TM6 (F634), TM3 (e.g. L512), and TM5
(e.g. F594) or between TM6 and TM7 (L677). Both
possibilities result in a rearrangement of TM6 (e.g. by
pushing the helices apart). A further likely consequence is a partial
destabilization of the transmembrane segments and their packing. These
strong conformational rearrangements obviously release conformational
constraints, most likely enabling the receptor to interact with
heterotrimeric Gq/11 proteins and thus initiating
the constitutive activation of the IP pathway (Table 1
). The same
effect seems to be true for the D633H substitution. At physiological pH
conditions, histidine has H acceptor and also H donator properties.
However, histidine also has a bulky shape similar to phenylalanine. Our
results demonstrate that the substitution of D633 with H results in a
strong constitutive activity for both the cAMP and the IP signaling
cascades. To our knowledge, this is the first time that constitutive
activity for the IP pathway could also be demonstrated for the
naturally occurring D633H TSHR mutation (40). Recently, in
a patient with a Leydig cell tumor, the homologous D578H mutation
in the LHR was identified, which was also constitutively active for
both signaling pathways (33).
In cells transfected with the D633 mutant, neither the adenylate
cyclase nor the PLC cascade could be stimulated by TSH (Fig. 2
and
Table 1
). This could be due to the strongly decreased cell surface
expression of this receptor mutant (Fig. 4A
). Detection of the D633
receptor
within the cells suggested that this receptor mutant was synthesized
(Fig. 4B
). Obviously, this receptor variant does not reach the cell
surface, most likely because of strong alterations of the receptor
folding. In the vasopressin V2 receptor, a deletion of the V278 residue
in TM6 was reported in a patient with diabetes insipidus
(41). This V278
mutant totally abolished
receptor-ligand binding and subsequent adenylyl cyclase stimulation
(42). These identical phenotypes of two deletion mutants
in TM6 in different GPCRs suggest that a deletion of a central
transmembrane residue probably causes a strong perturbation of the
helix packing within the shifted part by interrupting interactions of
complementary side chains between the helices (e.g. TM6 and
TM5).
Our first major experimental finding was that the substitution D633R
holds the TSHR tightly in a completely inactive conformation since this
receptor does not respond to TSH stimulation. This is the first
mutation in TM6 that inactivates the TSHR without impairing cell
surface expression or ligand binding. Others have proposed that the
central aspartate in TM6 in the LHR (D633 in the wt TSHR) functions as
an H acceptor. D633, together with T632, is therefore most likely
involved in the H bonding network between the central portions of TM6
and the two asparagines (N670, N674 in the wt TSHR) of TM7
(17). This H bonding network has been proposed to be
essential to preserve the inactive receptor conformation. Molecular
dynamics simulations (MD) of our wt TSHR model suggested conformations
in which D633 primarily forms an interhelical H bond with N674 (Fig. 7). In the hydrogen bonding network constraining the inactive state,
N674 also forms an intrahelical H bond to N670 in TM7. In addition to
establishing the strong interhelical H bond to TM7 during the MD runs,
D633 was also observed to simultaneously form intrahelical H bonds with
TM6. H bonds have been observed between D633 and the side chains of
C636 and/or T632, but with somewhat weaker occurrence. Both of these
residues are highly conserved within the glycoprotein receptors and
have been reported to be affected by mutation T632 in the TSHR
and mutation C581 in the LHR (TSHR C636) (12, 27).
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Our second major experimental finding is the partial rescue of the TSHR
activity by the double mutant D633R/N674D (Fig. 5). Interestingly, aspartate at position
674 contains two oxygen atoms in the side chain, where according to our
model one oxygen is involved in the H bond with R633. Therefore, the
other oxygen is available in TM7 as an H acceptor for the activation
process (Fig. 6C
). Additional
arginine double mutants containing serine or alanine in position 674,
and thus missing a freely available carboxylate oxygen as a strong H
bond acceptor, were not able to rescue any TSHR activity. Results for
mutants like N674A (see Table 1
) prompted us to assume (an) additional
partner(s) for R633. Based on our experimental data, we suggest the
following scenario. Since S508 and N670 are not involved in R633
interactions, it appears likely that the completely inactive D633R
mutant locks the receptor by H bonds between N674 in TM7 and the
backbone of TM6. N674 in TM7 forms H bonds with TM6 in the unstimulated
wt (D633) (Figs. 6
and 7
) and the mutant
(R633) TSHR as well. However, the rotatable amide group allows N674 in
the wt TSHR (D633) to act as a hydrogen donator, whereas in the mutant
TSHR (R633) it acts as a hydrogen acceptor. Thus in the wt TSHR there
is a free carboxylate oxygen at position 674 available as an H acceptor
(TM7), which mediates the TSHR activation by H bond release upon TSH
stimulation (Figs. 6
and 7
). However, R633 enforces an inverted H bond
with N674 and causes a reverse effect, since in N674 the only side
chain oxygen is already involved in an H bond with R633 and no longer
available in TM7 for the activation process. Moreover, the mutant
receptor R633 presumably not only interacts with N674 but also with
TM6. As a consequence, the receptor is constrained in an inactive
conformation not responsive to TSH stimulation. Due to the rotatable
midgroups of the two asparagines, it is conceivable that the
activation signal is propagated along TM7 by a mechanism inducing an
inversion of the H bond network between the highly conserved residues
N670 and N674, thereby causing a release of interhelical H bonds
between TM6 and TM7. The availability of a side chain carboxylate
oxygen in position 674 is obviously essential to serve as H acceptor
for this activation mechanism along TM7 for the glycoprotein hormone
receptors. Interestingly, not only the cAMP pathway is affected by this
phenomenon. Of all our single mutants, only those containing aspartate
or asparagine either in position 633 or in position 674 are able to
stimulate the IP pathway. Therefore, the activation of the IP pathway
also appears to be initiated by the release of the H bonds between TM6
and TM7. Alternatively, there could be an intersection of both pathways
at position N674 within the highly conserved (N/D)PxxY motif. This
hypothesis is supported by the description of the IP sensitive mutation
N391A (homologous to N674 in the TSHR) in the cholecystokinin B
receptor, which also abolishes Gq protein
activation without affecting binding or expression of the receptor
(43).
Moreover, the interactions between positions 633 and 674 and the
importance of a free H acceptor in position 674 (Fig. 7) could be of
relevance for all glycoprotein hormone receptors since nearly all
glycoprotein hormone receptors contain aspartate in position 633
and asparagine in position 674. Only the FSH receptor-TSHR of
Drosophila melanogaster (EMBL accession no. No. AAB07030)
differs from all other glycoprotein hormone receptors by amino acid
exchanges in both positions (asparagine in position 633 and aspartate
in position 674, according to TSHR cDNA) (44). Our results
and the suggested scenario for the intramolecular TSHR signaling are
also complementary to recently reported mutations in TM7 of the rat
LHR. Transferred to our TSHR model, the gain of function induced by the
point mutations N593R and N597Q (equivalent to N670, N674 in the TSHR)
in the rat LHR (36) is most likely caused by the
introduction of the larger hydrophilic side chains. They allow a
spontaneous rearrangement of TM6 by inducing larger distances while
still maintaining a hydrogen bond network between TM7 and TM6.
Moreover, at the mutated side chain in position 674 in TM7 (N597Q rat
LHR), there is an H acceptor atom available for the activation process.
Mutants of the rat LHR with loss of function are reported for N597R
(position 674 TSHR) and N593A, Q (position 670 TSHR). These results fit
perfectly with our hypothesis postulating an essential H acceptor at
this position together with an H bond network along the TM7 segment.
Only the reported loss of function for the rat LHR N593S mutant
(position 670 in the TSHR) differs from our findings and the findings
of others (30) showing a constitutive activity for N670S
in the TSHR. This disagreement might illustrate the slightly different
activation process of the rat LHR compared with the human TSHR, which
remains to be elucidated.
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MATERIALS AND METHODS |
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Cell Culture and Transfection
COS-7 cells were grown in DMEM supplemented with 10% FBS, 100
U/ml penicillin, and 100 µg/ml streptomycin (Life technologies, Paisley, UK) at 37 C in a humidified 7%
CO2 incubator. Cells were transiently
transfected in 12-well plates (1 x
105 cells per well) with 1 µg DNA/well using
the FuGENE6 reagent (Roche, Basel, Switzerland).
Radioligand Binding Assay
Competitive binding studies were performed as previously
described (15). Data were analyzed assuming a one-site
binding model using the fitting module of SigmaPlot 2.0 for Windows
(47).
FACS Analysis
Forty eight hours after transfection nonpermeabilized cells were
detached from the dishes using 1 mM EDTA and 1
mM EGTA in PBS and transferred in Falcon 2052 tubes. Before
incubation with the primary antibody, cells were washed once with PBS
containing 0.1% BSA and 0.1% NaN3.
For permeabilized cell assays, cells were first fixed with 1% paraformaldehyde for 10 min on ice following an incubation with PBS containing 0.1% BSA, 0.1% NaN3, and 0.2% Saponin for 30 min. Saponin was supplemented in all subsequent buffers.
Afterward, cells were incubated with a mouse antihuman TSHR antibody (2C11, Serotec, Oxford, U.K.; 10 µg/ml) in the same buffer. Tubes were washed and incubated for 1 h on ice in the dark with fluorescein-conjugated F(ab')2 rabbit antimouse IgG (Serotec, dilution 1:1000). Before FACS analysis (FACscan Becton Dickinson and Co., Franklin Lakes, NJ), cells were washed twice and fixed with 1% paraformaldehyde. Receptor expression was determined by the fluorescence intensity, whereas the percentage of signal positive cells corresponds to the transfection efficiency.
cAMP Accumulation Assay
Forty eight hours after transfection, measurement of
cAMP accumulation was performed as previously described
(15). Moreover, TSHR single mutants at position 633 were
expressed at the same level, to additionally determine the cAMP
accumulation at comparable cell surface expressions. To decrease the
cell surface expression of the TSHR constructs expressed at a higher
level (wt TSHR, D633C, D633H, D633F) to the level of the D633W mutant,
we cotransfected these constructs with various amounts of plasmids
containing the human V2 vasopressin receptor (48).
Stimulation of IP Formation
Transfected COS-7 cells were incubated with 2 µCi/ml of
[myo-3H] inositol (18.6 Ci/mmol),
Amersham Pharmacia Biotech, Braunschweig, Germany) for
6 h. Thereafter, cells were preincubated with serum free DMEM
without antibiotics containing 10 mM LiCl for 30 min.
Stimulation with TSH was performed with the same medium supplemented
with 100 mU/ml TSH for 1 h. Basal and TSH-induced increases in
intracellular IP levels were determined by anion exchange
chromatography as described (49). IP values are expressed
as the percentage of radioactivity incorporated from
(3)[H]-inositol phosphates (IP13) over the sum of
radioactivity incorporated in IPs and phosphatidylinositols.
Molecular Modeling
The approach used to construct the TSHR model was the same as
previously described (13). Packing of the transmembrane
helices was based on electron density maps of frog rhodopsin
(8). The TSHR structure model was computed with special
emphasis on the transmembrane and intracellular portions, without the
large amino-terminal domain but including the extra- and intracellular
loops. The starting conformation of the intracellular loops i1, i2, and
the first portion of the C-terminal tail comprising the putative i4
loop of the TSHR were adopted from the nuclear magnetic resonance
structure of the rhodopsin cytosolic loop peptide complex
(50) as described elsewhere for the V2 receptor
(51). For the remaining parts of the intracellular loops
as well as for the extracellular loops, fragments of four to seven
residues were selected and tested against the three-dimensional protein
database. Only fragments occurring more than once with a similar
backbone conformation in the database were used for assembling the
loops. All model components were assembled with the biopolymer module
of the SYBYL program package (TRIPOS Inc., St. Louis, MO) using the
AMBER 5.0 force field (52). MD simulations were performed
at 300 K for 200 psec, where only the helix stability was
maintained by restraints for hydrogen bonds of the TM backbones.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: bTSH, bovine TSH; FACS, fluorescence-activated cell sorting; GPCR, G protein-coupled receptor; IP, inositol phosphate; LHR, LH receptor; MD, molecular dynamics simulations; TSHR, TSH receptor; TM, transmembrane domain; V2R, human V2 receptor; wt, wild type
Received for publication October 23, 2000. Accepted for publication April 4, 2001.
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REFERENCES |
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