From the Institut de Recherche Interdisciplinaire en
Biologie Humaine et Nucléaire, Université Libre de
Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles,
Belgium, the ** Laboratori de Medicina Computacional, Unitat de
Bioestadística, Facultat de Medicina, Universitat
Autònoma de Barcelona, 08193 Bellaterra, Spain, the
§ Service de Conformation des Macromolécules
Biologiques, Université Libre de Bruxelles, CP 160/16, Avenue F. Roosevelt, 1050 Bruxelles, Belgium, and the
Novasite
Pharmaceuticals, Inc., San Diego, California 92121
Received for publication, March 13, 2001, and in revised form, April 12, 2001
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ABSTRACT |
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The thyrotropin (TSH) receptor is an interesting
model to study G protein-coupled receptor activation as many point
mutations can significantly increase its basal activity. Here, we
identified a molecular interaction between Asp633 in
transmembrane helix 6 (TM6) and Asn674 in TM7 of the TSHr
that is crucial to maintain the inactive state through conformational
constraint of the Asn. We show that these residues are perfectly
conserved in the glycohormone receptor family, except in one case,
where they are exchanged, suggesting a direct interaction. Molecular
modeling of the TSHr, based on the high resolution structure of
rhodopsin, strongly favors this hypothesis. Our approach combining
site-directed mutagenesis with molecular modeling shows that mutations
disrupting this interaction, like the D633A mutation in TM6,
lead to high constitutive activation. The strongly activating N674D
(TM7) mutation, which in our modeling breaks the TM6-TM7 link, is
reverted to wild type-like behavior by an additional D633N mutation
(TM6), which would restore this link. Moreover, we show that the Asn of
TM7 (conserved in most G protein-coupled receptors) is mandatory
for ligand-induced cAMP accumulation, suggesting an active role of this
residue in activation. In the TSHr, the conformation of this Asn
residue of TM7 would be constrained, in the inactive state, by its Asp
partner in TM6.
The TSH,1 LH/CG and FSH
receptors constitute a subfamily of G protein-coupled receptors (GPCR)
characterized by large amino-terminal ectodomains responsible for high
affinity binding of their natural agonists, the glycoprotein hormones
(1, 2). These ectodomains are made essentially of leucine-rich
repeats, a protein fold frequently found to be involved in
protein-protein interactions (3-5). The serpentine portion of these
receptors, responsible for signal transduction, comprises seven
transmembrane helices, showing significant similarity with the
transmembrane portions of rhodopsin-like GPCRs; they are therefore
grouped with them into family 1 of GPCRs.
The glycohormone receptors thus present a clear dichotomy between the
agonist-binding and signal-transduction domains, with the mechanism of
interaction between the two domains, responsible for receptor function,
remaining largely unknown. Sequences displaying strong similarities
with the glycohormone receptors have been identified in
Anthopleura elegantissima, Drosophila, and
Caenorhabditis elegans (6-9). Related receptors have also
been discovered in mammals (10, 11). But for all of these cases, the
nature of the agonists remains to be identified.
Several characteristics make the TSH receptor an interesting system to
study the mechanisms of receptor activation: (i) the wild type receptor
has been shown to display significant basal activity (12-14); (ii)
mutations involving more than 20 different residues have been shown to
increase its constitutive activity, causing autonomous thyroid adenomas
or non-autoimmune hereditary hyperthyroidism (15); (iii) loss of
function mutations have also been described, abolishing basal activity
(16) or affecting agonist-induced response (17, 18).
Interestingly, naturally occurring activating mutations are more rarely
found in the LH/CGr (19), and almost never found the FSHr (20). Indeed,
no spontaneous activating mutation has been identified in the FSH
receptor except for a single case with an unusual phenotype (21). These
data reflect a difference in the structural constraints keeping the
various glycoprotein hormone receptors in the inactive state. The wild
type TSH receptor displaying constitutive activity and being easily
activated by mutations would be the less constrained, followed by the
LH receptor, devoid of constitutive activity but susceptible to
activation by mutations. The FSH receptor would be the most
constrained, as it is silent in the absence of stimulation by agonist
and difficult to activate by mutations.
The high resolution crystal structure of bovine rhodopsin determined
recently (22) provides for the first time a detailed atomic description
of a GPCR molecule in an inactive conformation, and represents a solid
basis for modeling the structures of other rhodopsin-like GPCRs. Such
models can then be used to help rationalize the many observations made
on the relations between conserved sequence feature and functional
properties. Conservation of functionally important sequence motifs
within this receptor family has been interpreted as meaning that the
basic characteristics of the rhodopsin fold, as well as the molecular
mechanisms leading to receptor activation are similar in the different
receptor subtypes. Among such conserved sequence motifs are prolines in
transmembrane helices (TM) 6 and 7 and charged residues in TM2 and TM3
(the Asp and Arg of the canonical "DRY" motif). However,
considering the extreme diversity of the natural agonists of the
different receptors it has also been accepted that different receptor
subtypes may have evolved quite specific structural and functional
features, probably reflected in the specific sequence signatures of
each subtype.
Multiple alignments of the glycohormone receptor sequences reveal one
such signature: a conserved Asp residue in TM6 at position 6.44633 (corresponding to residue 633 in the
TSHr sequence; see "Experimental Procedures"). Most other GPCRs
harbor a Phe or a Tyr at this position. Natural mutations of residue
6.44633 in the TSHr and LH/CGr were shown to
lead to constitutive activation, causing autonomous toxic thyroid
adenomas (12) and male limited pseudoprecocious puberty (23),
respectively. This suggested that D6.44633 might
play a role in the mechanism of receptor activation. Recent ex
vivo transfection studies suggested that this residue is important for maintaining the inactive conformation of the LH receptor (24), in
agreement with earlier molecular modeling studies, according to which
the inactive form of the LH receptor would require the formation of
H-bonds between TM6 and TM7, involving residues Thr6.43, Asp6.44, Asn7.45, and Asn7.49
(25).
In the present study, we analyzed the multiple alignments of 44 glycohormone and closely related receptors. This lead to the observation that residues D6.44 and N7.49 (the
Asn of the conserved NPXXY motif in TM7) are both conserved
in glycohormone receptors, except in one of two Drosophila
receptors (6), where they are exchanged (this receptor bearing
N6.44 and D7.49). Since such coordinated
mutations may reflect spatial interactions between the residues (26,
27), our observation was interpreted as indicating the existence of an
interaction between D6.44 and N7.49. Considering
the activating effect of amino acid substitutions at position
6.44, we formulate the hypothesis that this interaction is
required in order to maintain the TSH receptor in an inactive conformation. We then investigated this hypothesis by combining computational and experimental approaches.
Molecular modeling techniques were used to derive the atomic
coordinates of the transmembrane region of the TSHr, using as template
the corresponding region of the high resolution structure of rhodopsin.
To analyze the conformational properties of residues 6.44 and 7.49 and their surroundings in the resulting model,
molecular dynamics simulations were carried out in the presence of
explicit methane molecules, mimicking the non-polar environment of the membrane. This was done for both the wt receptor and for several substitution mutants of residues that are likely to affect the proposed
interaction. In parallel, the same mutants were engineered into human
TSHr using site-directed mutagenesis and their effects on receptor
expression, hormone binding, and activation were measured.
The results provide compelling evidence that D6.44 and
N7.49 do interact in the inactive TSHr and that disruption
of this interaction, by mutating either the 6.44 or the
7.49 residues, results in constitutive activation.
Furthermore, combining our observations with data from other GPCRs is
shown to yield new insights into the general mechanism of GPCR
activation
Numbering Scheme of GPCRs--
The standardized numbering system
of Ballesteros and Weinstein (28) was used throughout to identify
residues in the transmembrane segments of different receptors. Each
residue is identified by two numbers: the first (1 through 7)
corresponds to the helix in which it is located; the second indicates
its position relative to the most conserved residue in that helix,
arbitrarily assigned to 50. For instance, N7.49 is the
asparagine in transmembrane helix 7 (TM7), located 1 residue before the
highly conserved proline P7.50. Residue D2.50
corresponds to Asp460 in the TSHr numbering;
S3.36 to Ser505; S3.39 to
Ser508; T6.43 to Thr632;
D6.44 to Asp633; N7.45 to
Asn670, and N7.49 to Asn674.
Molecular Modeling and Molecular Dynamics Simulation of the
Transmembrane Bundle--
The atomic model of the transmembrane domain
of the TSHr was built by comparative modeling techniques, using as
template the atomic coordinates of the transmembrane domain of bovine
rhodopsin (22). The sequences of the 2 proteins in this region were
aligned so as to equivalence the positions of the following conserved residues: Asn55-Asn1.50432 (the
superscripts represent the residue numbering in rhodopsin structure PDB
code 1F88, and human TSHr sequence, respectively, and 1.50 is the numbering in the standardized nomenclature),
Asp83-Asp2.50460,
Arg135-Arg3.50519,
Trp161-Trp4.50546,
Pro215-N5.50590,
Pro267-Pro6.50639, and
Pro303-Pro7.50675. The conformations
of these side chains were kept as in the rhodopsin crystal structure.
Those of the non-conserved amino acids were built using a rotamer
library specific for
To relieve residual strain resulting from suboptimal positioning of the
side chains, the resulting model was first subjected to energy
minimization (1000 steps), and then to a simulated annealing procedure,
using molecular dynamics. This involved heating to 600 K for 30 ps (1 ps = 10
Molecular models for the mutant receptors containing the single
substitutions D6.44N, D6.44A, N7.49D,
and N7.49A and double substitution
D6.44N-N7.45D and
D6.44N-N7.49D, were built using, as the starting
point the final model derived for the wt TSHr and optimizing the
conformations of the substituted side chains using an analogous
procedure to that described above. This procedure was deemed
reasonable, since the considered substitutions were either isosteric,
replacement of Asn by Asp, or vice versa, or side chain deletion
(replacement by Ala).
To investigate the structural properties of the modeled conformations,
room temperature molecular dynamics simulations were used. In order to
mimic the hydrophobic environment of the membrane, these simulations
were carried out in the presence of explicit methane molecules, and
using periodic boundary conditions. The periodic box was ~73 Å × 63 Å × 52 Å in size, and contained between 4219 and 4241 methane
molecules in addition to the transmembrane domain. Similar conditions
have been recently used to mimic the membrane environment in molecular
dynamics simulations of the potassium channel (30).
To start the simulation protein portion was kept fixed while the
methane molecules were energy minimized (500 steps), then heated to 300 K for 15 ps and equilibrated for another 35 ps. Following this, the
same procedure was repeated on the entire protein-solvent system but
with an equilibration run of 250 ps; a 250 ps production trajectory was
then generated at constant volume using the Particle Mesh Ewald method
for computing electrostatic interactions (31). For analysis purposes,
structures were collected every 2 ps. The simulations were performed
with the Sander module of AMBER 5 (32), the all-atom force field (33),
SHAKE bond constraints on all bonds, a 2 fs integration time step, and
constant temperature of 300 K coupled to a heat bath.
Site-directed Mutagenesis of the TSH Receptor--
Plasmids
encoding the various TSHr mutants were constructed by site-directed
mutagenesis using two subsequent PCR amplifications rounds. This
procedure requires two partially overlapping complementary primers
containing the mutation and two external primers. Two distinct PCRs are
performed on TSHr template by using in one tube the direct mutagenic
primer and the external reverse primer and in the other tube the
reverse mutagenic primer and the external direct primer. One µl of
each PCR product was mixed and used as template in a subsequent PCR
amplification with the two external primers. The resulting amplified
fragment contains the mutation and can be cloned after digestion with
Bsu36I and BamHI into the pSVL expression vector
(Amersham Pharmarcia Biotech, Freiburg, Germany) containing the wild
type TSHr (34) using standard procedures. All PCR-generated receptor
fragments were verified by sequencing before transfection.
Transfection and Assays--
COS-7 cells were grown in
Dulbecco's modified Eagle's medium supplemented with fetal bovine
serum (10%), sodium pyruvate (1 mM), penicillin (100 IU/ml), streptomycin (100 µg/ml), and fungizone (2.5 µg/ml). Cells
were seeded at a density of 150,000 or 300,000 cells/3-cm dish. One day
later, they were transfected (250 or 500 ng of DNA/dish, respectively)
by the DEAE-dextran method followed by a dimethyl sulfoxide shock (35).
Two days after transfection, cells were used for flow
immunocytofluorometry, cAMP and 125I-TSH binding studies.
Duplicate dishes were used for each assay. Each experiment was repeated
at least three times. Cells transfected with pSVL alone were always run
as controls.
Flow Immunocytofluorometry--
Cells were detached from the
plates with phosphate-buffered saline containing EDTA and EGTA (5 mM each) and transferred into Falcon tubes (2052). Cells
were centrifuged at 500 × g, at 4 °C for 3 min and
the supernatant was removed by inversion. They were incubated for 30 min at room temperature with 100 µl of phosphate-buffered saline/bovine serum albumin (0.1%) containing the BA8
monoclonal antibody, obtained from genetic immunization with the wild
type TSH receptor cDNA (36). The cells were washed with 4 ml of
phosphate-buffered saline/bovine serum albumin (0.1%) and centrifuged
as above. They were incubated for 30 min on ice in the dark with
fluoresceine-conjugated cAMP Determination--
Cells were washed with
Krebs-Ringer-Hepes buffer (KRH isotonic, pH 7.4). After a preincubation
in KRH at 37 °C for 30 min, cells were incubated in the same buffer
supplemented with Rolipram 25 µM (a cAMP
phosphodiesterase inhibitor, gift from the laboratory of J. Logeais, Paris, France), in the absence or presence of various bTSH concentrations (Sigma). One hour later, the medium was removed and
0.1 M HCl was added to the cells. The cellular extracts
were dried overnight in a vacuum concentrator (Savant) and
intracellular cAMP was determined exactly as described previously (37).
Basal cAMP was normalized to cell surface expression for each of the constructs. To this end, specific cAMP accumulation (= cAMP of receptor-transfected cells Binding Assays--
Two days after transfection, cells were
washed twice with NaCl-free Hank's buffer supplemented with 280 mM sucrose. Cells were incubated at room temperature in
this medium (supplemented with low fat milk, 2.5%) containing about
100,000 counts/ml 125I-TSH (TRAK Assays, BRAHMS
Diagnostica, Berlin; 58 µCi/µg, 50-60 units/mg) and various
concentrations of unlabeled TSH (Sigma). Four hours later, the cells
were rinsed twice with chilled Hank's buffer and solubilized by 1 N NaOH. Bound radioactivity was determined in a
Sequence Alignment of the Glycohormone Receptor Subfamily
Multiple sequence alignment of the sixth and seventh transmembrane
helices of 44 glycohormone and closely related receptors was performed.
Fig. 1 shows the alignment of
representative sequences, together with that of bovine rhodopsin.
Inspection of this alignment reveals two key sequence motifs
characteristic of the rhodopsin-like family: the P6.50 in
TM6 and the NPXXY motif in TM7. A noteworthy difference is
the presence of Asp at position 6.44 instead of the more
common Phe or Tyr, which represent more than 81% of the rhodopsin-like
receptors found in the GPCRDB (38). D6.44 is completely
conserved throughout the glycohormone family of GPCR, from C. elegans to human, with only a single exception, the
Drosophila DLGR1 sequence (accession number U47005 (6)),
which contains an Asn residue. Interestingly, this change is correlated
with the replacement of the highly conserved Asn at position
7.49 by Asp. Swapped mutations of this kind suggest
interaction between the corresponding residues in the three-dimensional
structures (26, 27). We could indeed verify that in the rhodopsin
structure, the C
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices. (29). Ionizable groups in the helices
were modeled as uncharged except for Asp2.50,
Glu3.37, Glu3.49, Arg3.50,
Asp6.30, and Asp6.44.
12 s), equilibration for 120 ps at 600 K,
cooling to 300 K for 60 ps, and equilibration for 90 ps at 300 K. During this processes the C
atoms and the side chains of
conserved residues were kept fixed at their positions in the rhodopsin
crystal structure.
-chain-specific goat anti-mouse IgG (Sigma)
in the same buffer. Propidium iodide (10 µg/ml) was used for
detection of damaged cells which were excluded from the analysis. Cells
were washed once again and resuspended in 250 µl of
phosphate-buffered saline/bovine serum albumin (0.1%). The
fluorescence of 10,000 cells per tube was assayed by a FACScan Flow
Cytofluorometer (Beckton Dickinson, Erembodegem, Belgium).
cAMP of the pSVL-transfected cells) is divided by the specific FACS value (= fluorescence of
receptor-transfected cells
fluorescence of pSVL-transfected
cells), which can be summarized as: specific basal activity = (cAMP(receptor)
cAMP(pSVL))/(FACS(receptor)
FACS
(pSVL)). The values are then expressed as percentage of specific
basal activity of the wt TSHr.
-scintillation counter. Under our conditions, the radioactivity nonspecifically bound to COS cells expressing the TSH receptor (defined
as the radioactivity bound to the dish in the presence of 100 milliunits/ml unlabeled TSH) was identical to that bound to
mock-transfected cells. In the absence of a consensus about the
bioactivity of pure bovine TSH, we have expressed all TSH or TSH
receptor concentrations in milliunits/ml, assuming a 1:1 stoichiometry
for TSH binding to its receptor. The competition binding curves have
been fitted by nonlinear regression, assuming a single receptor site,
using the Prism3 program (GraphPad Softwares).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
of Phe6.44 and
Asn7.49 (the conserved N of the NPXXY motif) were
facing each other at a distance of 11 Å, sufficient to allow direct
interaction between the side chains. Given that a series of amino acid
substitutions at D6.44 found in thyroid adenomas result in a
significant increase in the constitutive activity of the TSHr, we then
made the hypothesis that interactions between the D6.44 and
N7.49 side chains are important in maintaining the receptor
in its inactive conformation.
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Fig. 1.
Multiple sequence alignment of TM6-ECL3-TM7
of glycohormone receptors and bovine rhodopsin. For the sake of
clarity, representative sets of sequences from mammals and bird
glycohormone receptors are shown together with related sequences from
fish, insects, sea anemone, and C. elegans. The generalized
numbering scheme (see "Experimental Procedures") is used to label
the alignment. Residues 6.44 and 7.49 are in
bold characters, the residue exchange is
highlighted. The corresponding sequence of bovine rhodopsin
is also aligned. The limits of the helices have been defined according
to those observed in the rhodopsin structure (22). Accession numbers
for the non-mammalian sequences are: Q90674 (LHr chicken), P79763 (FSHr
chicken), AB005587 (fshr newt), AB030005 (gnII salmon), AF239761 (tshr
bass), T20123 (C50H2 C. elegans), P35409 (GLHR sea anemona),
P46023 (GRL101 snail), AF142343 (LGR2 Drosophila), and
U47005 (LGR1 Drosophila).
Molecular Dynamics Simulation of the Transmembrane Region of the TSHr
In order to investigate the proposed interaction between D6.44 and N7.49 in the context of the entire helix bundle, the molecular model of the transmembrane domain of the TSHr was built, based on the high-resolution structure of bovine rhodopsin. This model was then subjected to unrestrained molecular dynamics simulations in the presence of explicit methane molecules mimicking the apolar membrane environment (see "Experimental Procedures"). Having ascertained that the helical segments conserved their secondary structure and that the bundle remained well packed and maintained the rhodopsin fold throughout the trajectory, we analyzed the polar interactions made between TM6 and TM7. In particular, we computed the average hydrogen-acceptor distances in conformations along the trajectory, and the fraction of the conformations in which the bond was formed.
All throughout the simulation D6.44 and N7.49
were seen to form a hydrogen bond between the D6.44
O1 and N7.49 N
2H atoms, with
an average O-H distance of 1.9 Å (see Fig.
2 for a representative structure). The
TM6-TM7 interaction involved in addition a more complex hydrogen bond
network. N7.45, located one helix turn prior to
N7.49, formed 2 H-bonds. One, with D6.44 (N
27.45H-O
26.44;
average distance 2.0 Å) and another with N7.49
(N
27.45H-O
17.49;
average distance of 1.9 Å). The hydrogen bond between D6.44 and N7.45, thus provides a second polar interaction between
TM6 and TM7. In addition, the D6.44 O
2 was
seen to form and H-bond with O
H group of the conserved
S3.39, in TM3 (average distance of 1.8 Å), establishing a
polar interaction between TM3 and TM6 as well.
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Experimental Probing of the 6.44-7.49 Interaction
The existence and functional role of this predicted TM6-TM7 interaction was probed by mutating the residues involved and testing the functional consequences after transfection of the mutant constructs in COS-7 cell. The following mutants were engineered: (i) the D6.44N/N7.49D double mutant, made in order to mimic the situation of the Drosophila receptor in the context of the human TSHr; (ii) D6.44N and N7.49D single mutants, introduced in order to explore the effects of the individual mutations; (iii) D6.44A and N7.49A side chain deletion mutants engineered to test the effects of eliminating altogether the interactions between these side chains and any other residue. Following observations made from our molecular dynamics simulation of the TSHr and other studies on the LH/CG receptor (24) we designed two additional mutants, the single N7.45D substitution and the double substitution D6.44N/N7.45D. This was done in order to explore the possibility of a direct interaction between D6.44 and N7.45.
Cell Surface Expression of the Mutants
Cell surface expression of the mutated receptors was measured by
FACS analysis using the BA8 monoclonal antibody (36). As the
epitope of the BA8 antibody is located in the NH2-terminal region of the TSHr (1), we do not expect a modification of its affinity
following mutations in the transmembrane region. Also, similar results
were obtained with another monoclonal antibody (3G4), recognizing a
different epitope in the ectodomain (not shown). Hence, it is assumed
that observed fluorescence changes will be in direct relation with the
number of receptors present at the cell surface. As shown in Fig.
3, mutants D6.44N,
D6.44N/N7.49D, and N7.49A are
expressed at levels comparable (above 50%) to that of the wild type
TSHr. In contrast, mutants N7.49D, D6.44A, and the D6.44N/N7.45D double mutant display a reduced
expression, between 15 and 30% of the wild type receptor expression.
The level of expression of N7.45D is too low (specific
fluorescence of about 3% of wt TSHr) to allow reliable normalization
of functional results, it will therefore not be considered further.
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Basal and TSH-stimulated cAMP Accumulation in COS-7 Cells Transfected with the Mutants
The constitutive activity of the wt and mutant receptors was characterized by measuring intracellular cAMP accumulation in transiently transfected COS-7 cells. As constitutive activity is linearly dependent on the number of receptors at the cell surface (39), we have normalized the basal activity of each construct using cell surface expression data yielded by FACS analyses (see "Experimental Procedures"). This allows to compute constitutive activity on a per receptor basis, and to compare it to that of the wild type receptor.
Mutants D6.44N, D6.44N/N7.49D, and
N7.49A display basal activity similar to that of the wild
type (Fig. 4A). Strikingly,
the single mutants N7.49D and D6.44A show a
dramatic increase in constitutive activity, reaching more than 15 times
that of wild type TSHr, when normalized to the level of expression
(Fig. 4B). Although the N7.49D mutant, bearing
aspartate residues at both positions 6.44 (as in wild type)
and 7.49 (introduced by mutation), is among the strongest
constitutive mutant of the TSHr ever identified (37), it is remarkable
that addition of the D6.44N mutation on the
N7.49D background reverses completely the phenotype back to
a wild type-like behavior. The D6.44N/N7.45D can
also be considered as a constitutive mutant as its basal activity is
more than five times that of wt TSHr (Fig. 4B).
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We then tested the ability of TSH to stimulate the various constructs.
Fig. 5 illustrates typical
concentration-response curves and the corresponding EC50
are summarized in Table I.
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All mutants except N7.49A are stimulated by TSH in a way similar to the wild type. While starting from a higher basal level, the N7.49D mutant is activated efficiently by TSH, reaching comparable Emax (not shown). All EC50 values were in the range 0.4-1 milliunits/ml of TSH (Fig. 5, A and B, and Table I). Although the N7.49A mutant displays wt-like constitutive activity, TSH is unable to elicit signal transduction, as only very moderate stimulation if any can be observed at 100 milliunits/ml, a saturating concentration for wt TSHr (Fig. 5C).
Hormone Binding
The binding properties of the different receptors were tested by
homologous competition using 125I-bovine TSH as tracer. As
shown in Fig. 6, all constructs tested were able to bind TSH efficiently.
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Mutants D6.44N and D6.44N/N7.49D bind TSH with similar affinity as the wild type receptor, as measured by the IC50 test (IC50 of about 3 milliunits/ml, see Table I and Fig. 6A). Noticeably, the various constitutive mutants display a significantly lower IC50 (below 1 milliunits/ml, see Table I and Fig. 6B). Increase in apparent affinity with no significant modification of hormone potency has been observed in most constitutive mutants of the TSH receptor (40). Interestingly, mutant N7.49A binds TSH with high affinity, even slightly higher than the wt receptor (Fig. 6A and Table I), although this mutant is completely unable to be activated by the hormone.
It should be noted that the residues identified here as key actors in the activation mechanism of the TSHr have also been studied by several groups on the LH receptor (24, 41). Overall these studies are in agreement with the results presented here. Note, however, that these studies did not consider double mutants in different TMs, which in our view are crucial to the identification of TM6-TM7 interactions.
Molecular Dynamics Simulations of the Transmembrane Region of Mutant Receptors
In an effort to interpret, in structural terms, the results of the site-directed mutagenesis experiments, the molecular dynamics trajectories of the transmembrane region of TSHr featuring the different mutants were analyzed, with focus on the region between TM6 and TM7, surrounding the mutated residues. In particular, we monitored all possible interactions between the various polar groups in the vicinity of 6.44 and 7.49, which were involved in the local hydrogen bond network in the wt TSHr trajectory. Representative structures of the mutant trajectories are shown in Fig. 2.
The D6.44N Mutant Receptor--
Notwithstanding the absence of the
acid group, the D6.44N mutant was found to maintain polar
interactions between TM6 and TM7 (see Fig. 2). Residue N6.44
interacts with N7.49 through two hydrogen bonds. One forms
persistently between the O1 atom of N6.44 and
the N
2H group of N7.49 (average O-H distance of 2.0 Å). The other forms in 40% of the conformations, between the
N
2H group of N6.44 and the O
1
atom of N7.49 (average O-H distance of 2.2 Å).
The same N
2H group also H bonds to the backbone oxygen
in the preceding turn of the TM6 helix. We find moreover that the
TM6-TM7 link is reinforced by an additional H-bond, between the
O
1H of T6.43 and the O
1 of
N7.49 (average O-H distance, 1.8 Å).
The D6.44N/N7.49D Double Mutant Receptor--
The representative
structure for the trajectory of this mutant receptor TM region features
a hydrogen bond network opposite to that seen in the wild type
simulation (see Fig. 2). The negatively charged D7.49
interacts with the neutral N6.44, through a
N26.44
H-O
17.49 hydrogen
bond (average distance, 1.8 Å). In addition, its O
1 and
O
2 atoms take turns in forming an H-bond with the
N
1H group of N7.45 (average minimum distance
of 1.9 Å). Furthermore the O
1 of N6.44 forms
hydrogen bonds to both the N
2H of N7.45 (2.2 Å), and the O
H of S3.39 (1.9 Å), mimicking the hydrogen bond network seen in the wt simulations.
The N7.49D Mutant Receptor--
The molecular dynamics simulation
of the constitutive N7.49D mutant receptor shows a dramatic
change in the hydrogen bond network linking TM6 and TM7 (see Fig. 2).
Due to the repulsion between the 6.44 and 7.49 side chains, both an aspartate in this mutant, the D6.44
side chain has moved away from TM7 and toward TM3, forming two new
persistent H-bonds. One with S3.36
(O3.36H-O
16.44,
average distance 1.7 Å), and one with S3.39
(O
3.39H-O
26.44,
average distance 2.1 Å). On the other hand, D7.49 forms
H-bonds with the N
2H of N7.45 (average
distance 1.8 Å) and with the O
1H of T6.43
(average distance 2.2 Å), via its O
1 and O
2 atoms, respectively.
The D6.44N/N7.45D Double Mutant Receptor--
In this mutant
receptor polar interactions between helices TM6 and TM7 are maintained
throughout the trajectory, but their pattern differs from that observed
in the wt. They are formed between residues N6.44 and
T6.43 on the one hand, and D7.45 and N7.49 on the other. An H-bond forms persistently between the
N6.44 N2H and the D7.45
O
1 (average distance, 1.9 Å). N6.44 also
H-bonds to N7.49 in a sizable fraction of the conformations, but not as persistently as in the wt simulations. In addition, the
D7.45 O
1 hydrogen bonds persistently to the
T6.43 O
1H (average distance, 1.7 Å). To form
this pattern of interactions, TM6 and TM7 have undergone a small local
rearrangement, which now enables H-bond formation between the
N7.49 N
2H and D2.50 O
1 (average distance 2.5 Å). The latter residue is the
highly conserved Asp in TM2, found in most rhodopsin-like GPCRs (see "Discussion").
The D6.44A Mutant Receptor--
In the absence of a polar side
chain at 6.44, the interactions of TM6 with N7.45
and N7.49 in TM7 can no longer be made. As a consequence,
the latter polar side chains rearrange their respective conformations,
so as to find new H-bonding partners. N7.49 rotates toward
TM2, forming an H-bond between its N2H group and the D2.50 O
2 (average distance 2.0 Å) (Fig. 2).
This interaction too is facilitated by a local rearrangement of TM7
which brings 7.49 closer to 2.50 than in the wt
simulation (data not shown).
The N7.49A Mutant Receptor--
Unlike for the D6.44A
mutant, in this mutant, polar interactions between TM6 and TM7 are
maintained through H-bonds of D6.44 to N7.45
(N27.45H-O
26.44,
average distance, 1.8 Å) and to T6.43
(O
16.43H-O
16.44,
average distance, 1.9 Å) (see Fig. 2).
In summary, the nonconstitutive mutant receptors (D6.44N,
D6.44N/N7.49D, and N7.49A)
maintain the TM6-TM7 interaction through a complex hydrogen bond
network involving the side chains at positions 3.39,
6.43, 6.44, 7.45, and 7.49.
Moreover, as in the wt simulation, the side chain of residue
7.49 is always maintained close to TM6 (except in the
N7.49A mutant), essentially through direct partnership with
residue 6.44. The least active of the constitutive mutant receptors (D6.44N/N7.45D) maintains the TM6-TM7
link but there is a clear re-orientation of the N7.49 side
chain toward D2.50. The most constitutive mutant receptors
(N7.49D and D6.44A) do not maintain the TM6-TM7
interaction, and especially the orientation of residue 7.49,
which moves away from TM6.
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DISCUSSION |
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---|
A 6.44-7.49 Link Maintains the Inactive State of the TSHr-- On the basis of multiple alignment of glycohormone receptors, we have identified in the sequence of a putative Drosophila receptor (6) an exchange between two highly conserved residues: Asp6.44 and Asn7.49. These two residues are spatially close to each other in the rhodopsin template, suggesting a possible direct interaction. In addition, the observation that a variety of spontaneous mutations affecting D6.44 in both the TSHr and LHr increase their constitutive activity, pointed to a possible role of D6.44 (and thus of the postulated 6.44-7.49 interaction) in the activation mechanism.
In this context, mutation of N7.49 to Asp is expected to abrogate this putative interaction between residues 6.44 and 7.49, owing to electrostatic repulsion between the 2 negatively charged side chain, which should be rescued by the complementary mutation of D6.44 to Asn. The functional characteristics of these mutants fulfill nicely these expectations when assayed in COS cells: the N7.49D mutant displays a very strong increase in basal activity, reaching more than 15 times that of the wild type receptor, whereas the D6.44N/N6.49D double mutant behaves as the wild type receptor. Together with the reciprocal amino acid exchange observed at these positions in the Drosophila receptor the simplest explanation is that these two residues are close in space and do interact with each other. Similar reasoning and experimental approaches have been used in the study of other receptors (42-44). Interestingly, they supported the idea that the same N7.49 residue would interact with D2.50, one of the most conserved residues in rhodopsin-like GPCRs. The apparent incompatibility between these two mutually exclusive interactions find an explanation in the study of additional mutants (see below).
Our molecular dynamics study of the helix bundle of the TSHr based on the crystal structure of rhodopsin strongly supports the D6.44-N7.49 interaction. In simulations of the wild type TSHr structure, the D6.44-N7.49 interaction was observed in all conformations of the trajectory, indicating that this putative partnership fits well the rhodopsin template, under our simulation conditions. In agreement with experimental observations, simulation of the D6.44N/N7.49D double mutant shows that the interaction between these amino acids is preserved when the two side chains are exchanged, as found in the Drosophila receptor. It should be noted that our modeling study predicts that a 6.44-7.49 interaction would be preserved in the D6.44N single mutant, and, indeed, when tested experimentally, this mutant showed a wild type-like behavior. This observation is of particular significance, considering that most if not all other mutations at this position induce constitutive activity in the TSH or LH receptors (35, 41). The structural rationale explaining why only Asn is tolerated at position 6.44 in the glycohormone receptors gives support to the local structure proposed here and suggests an explanation as to how the D6.44N/N6.49D double substitution may have occurred stepwise in evolution in the Drosophila receptor.
Our modeling points to additional partners to account for the phenotype of some of the constitutive mutants identified here. The strongest constitutive activity is associated with a complete lack of the 6.44-7.49 interaction (like the N7.49D and D6.44A mutants). Weakening of this bond as observed in the D6.44N/N7.45D double mutant causes increase in constitutive activity but to a smaller extent, which indicates that this mutant is not completely released from its ground state constraints. In our N7.49D simulation, D6.44 interacts with serine residues S3.36505 and S3.39508, in TM3. Interestingly mutations of S3.36 into Arg or Asn were found to constitutively activate the receptor (40, 45). It is likely that the 3.36-6.44 interaction observed in the N7.49D mutant would be strongly favored in a S3.36505R mutant. An ionic R3.36-D6.44 interaction would be more stable than the D6.44-N7.49 (wt situation), therefore redirecting the side chain of D6.44 toward TM3 and releasing N7.49.
Sequestration of N7.49 Is Implicated in the Inactive Conformation of the TSHr-- Overall the modeling and experimental data converge toward the idea that, in the TSHr, a direct D6.44-N7.49 interaction is required to maintain the inactive state of the receptor, and that breaking this link results in high constitutive activity. How does this view agree with current notions of activation of other rhodopsin-like GPCRs?
Despite the fact that residue D6.44 is not conserved throughout the family (in most cases it is a Phe), mutation of 6.44 into Ala leads also to constitutive activation in other rhodopsin-like receptors (46, 47). Moreover, in the opsins, 6.44 has been shown to be linked to the activation process, being involved in color sensitivity (48, 49). The mechanism by which Asp (in the glycohormone receptors) or Phe (in most of the others) might exert similar functions cannot be determined from the present study. A provocative explanation could be that sequestration of the side chain of N7.49 by H-bonding to D6.44 in the inactive TSHr would be replaced by the existence of a steric shield in the other GPCRs.
An Active Role of N7.49 in Signal Transduction-- Mutation of N7.49 into Ala casts another light on the role of the TM6-TM7 motif in the activation of the TSHr. Although the N7.49A mutant binds TSH with wild type-like affinity, it is totally refractory to activation by the hormone. This indicates that disrupting the 6.44-7.49 interaction (which is de facto absent here) is not sufficient to trigger activation, but that N7.49 is fulfilling a specific role during activation, which cannot be achieved by an Ala side chain. Work on various rhodopsin-like GPCRs have suggested that, upon activation, N7.49 would interact with the highly conserved Asp of TM2 (D2.50) and also with the perfectly conserved Asn of TM1 (N1.50) (42, 43). As already mentioned, using a similar experimental strategy as the one described here, it has been shown in these studies that inactivation by a D2.50N mutation could be rescued by an additional N7.49D mutation. Moreover, it has been proposed that D2.50 undergoes a change in H-bonding upon light activation of rhodopsin (50).
A Model of the Early Steps of Activation-- We can summarize the different points discussed above in a simple sequence of intramolecular events that would be necessary to trigger the conformational changes leading to activation.
(i) In the inactive state of the receptor, N7.49 is locked away from TM2. In the glycohormone receptor family, this conformational constraint is achieved by a direct interaction with D6.44.
(ii) A necessary step in activation would be the release of this interaction. This could happen either upon ligand binding, or due to mutation of a key residue. For instance, the D6.44A mutation abrogates completely the interaction. Noticeably, this mutation leads to almost maximal activation (2/3 of activity after full stimulation by the hormone).
(iii) N7.49 would then reorient toward the 1.50-2.50 motif, where it could modify the polar equilibrium and subsequently cause a reorganization of the inter-helical H-bond network. This rearrangement would in turn induce the conformational changes of the TM bundle known to be associated with activation (i.e. motions of TM3 and TM6 (51, 52)). In the N7.49A mutant, the side chain of A7.49 would not be able to establish the interaction(s) normally fostered by the Asn, and no transition to the active state is observed. Whether this scenario applies to the physiological activation of the receptor by its natural agonist remains to be demonstrated.
The 2.50-7.49 interaction is well supported by numerous studies on various receptors. However, a survey of the GPCR data base yields about 100 sequences of rhodopsin-like receptors with an Asp residue at both the 2.50 and 7.49 positions. In this case a direct 2.50-7.49 interaction is impossible. It has been shown that an Asn to Asp substitution at position 7.49 leads to modification of the signaling characteristics in GPCRs: changing the N7.49PXXY motif for D7.49PXXY leads to changes in the coupling specificity of the GnRH receptor (53). This indicates that the active conformation achieved by receptors bearing an Asp in 7.49 may be somewhat different from that of those bearing an Asn, in agreement with the current concept that GPCRs may achieve multiple active states with the potential to activate different G proteins (52).
When integrated with the current literature, the present results point
to the 1.50-2.50-7.49 motif as an
important actor in the activation of rhodopsin-like GPCRs. Additional
players are likely to be involved as well, which remain to be
identified. Among these are positively charged residue(s),
participation of structural water molecules and specific protonations.
In this context, it was suggested that D2.50 would interact
with the conserved R3.50 of the DRY motif at the base of TM3
(54, 55). However, since the C of these two residues are
separated by more than 24 Å in the crystal structure of rhodopsin, we
do not currently favor such a possibility. Positively charged residues
are often found at the cytoplasmic end of TM6 and could possibly
interact with the 1.50-2.50-7.49 motif
after a rigid body motion of TM6 of the kind which is believed to occur
upon activation (56-58). It is noteworthy that a water molecule is
present in close vicinity of D2.50 in one of the two
monomers of the rhodopsin crystal structure (22). Also, it is known
that protonation events do take place upon activation of GPCRs. One of
the protonation site is most probably the highly conserved
D3.49 of the DRY motif (59). Recent studies on the
2-adrenergic receptor indicate that protonation of an
additional negatively charged residue is most probably implicated in
activation (60). D2.50 is a good candidate for the
"missing" protonated residue in adrenergic receptors and has been
proposed to be protonated in rhodopsin (50, 61). Protonation of
D2.50 could be a necessary step in the activation process,
as it has been shown for several receptors that a D2.50N
mutation severely impairs the signaling properties, while a
D2.50E mutation can be tolerated (44). Protonation of
D2.50 would account for the observation that the
N1.50-D2.50-N7.49 motif tolerates
N7.49D substitutions in many systems, including the TSH
receptor. As we were not able to include the various putative
additional players in the molecular dynamics simulations, we could not
observe a 2.50-7.49 interaction in our model of
the N7.49D mutant. In all fairness, the absence of these
players in our simulations and for that matter, also that of the entire
extracellular domain and of an explicit lipid environment, are
limitations that must be kept in mind also when considering other
conclusions deduced from the simulations.
The Active Conformation of the Wild Type Receptor May Be Different from that of D6.44 Mutants-- Of potential interest is the observation that the N7.49A mutant, while it cannot be activated by the hormone, still displays constitutive activity similar to the wild type TSHr. This implies that the interactions achieved by the side chain of N7.49 would be dispensable for the basal activity of the wild type receptor, whereas they are required for that of the ligand induced activity. The existence of multiple active conformations of GPCRs has been well documented in the case of TSHr mutants (16, 62) as well as in many other GPCRs (see Ref. 52 for review). The present observation suggests that "noisy" receptors like the unliganded wild type TSHr might achieve an active conformation via a route involving residues distinct from those implicated in full agonist-induced activation.
In conclusion, we have identified a conserved motif central to the
activation of the TSH receptor. Our molecular modeling provides a
structural framework for understanding how the
D6.44-N7.49 link, by sequestrating the
side chain of N7.49, would keep it in an
"inactive" conformation. Upon activation, this side chain would be
released, and free to adopt its "active" conformation, which
involves interactions with the N1.50-D2.50
motif. Further rearrangements of the intramolecular network, which
remain to be defined, lead to exposure of surfaces allowing interaction between the receptor and its cognate G protein(s). We suggest that in
other GPCRs, a similar mechanism might operate, but involving a
different type of partner for N7.49 in the inactive
state. Now that rhodopsin provides a reliable template for modeling the
inactive conformation of GPCRs, it is our hope that further molecular
dynamics simulations coupled to site-directed mutagenesis experiments
will help in elucidating their active conformation(s).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Claude Massart for expert technical assistance and Jean Richelle for valuable help with the computer systems. We are grateful to BRAHMS Diagnostica (Berlin, Germany) for providing radiolabeled bovine TSH. We thank the Center de Computació i Comunicacions de Catalunya for use of the computer facilities.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Belgian State, Prime Minister's office, Service for Sciences, Technology, and Culture, grants from the Fonds de la Recherche Scientifique Médicale, Fonds National de la Recherche Scientific, Association Recherche Biomédicale et Diagnostic, and BRAHMS Diagnostics, Comision Interministerial de Ciencia y Tecnologia Grant SAF99-073, Fundació La Marató TV3 Grant 0014/97, Improving Human Potential of the European Community Grant HPRI-CT-1999-00071.
¶ Fellow of the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture.
To whom correspondence should be addressed: IRIBHN,
Université Libre de Bruxelles, Campus Erasme, 808 route de
Lennik, B-1070 Bruxelles, Belgium. Tel.: 32-2-555-41-71; Fax:
32-2-555-46-55; E-mail: gvassart@ulb.ac.be.
Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M102244200
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ABBREVIATIONS |
---|
The abbreviations used are: TSH, thyroid-stimulating hormone; GPCR, G protein-coupled receptor; TM, transmembrane helix; LH, lutenizing hormone; CG, chorionic gondadotropin; FSH, follicle stimulating hormone; wt, wild type; PCR, polymerase chain reaction.
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