Department of Chemistry, University of Modena, Modena, Italy (F.F.,
P.G.DeB.); Consiglio Nazionale delle Richerche Cellular and Molecular
Pharmacology Center, Department of Pharmacology, University of Milan,
Milan, Italy (P.B., D.Z., B.C.)
The aim of this study was to investigate the molecular changes
associated with the transition of the human oxytocin receptor from its
inactive to its active states. Mutation of the conserved arginine of
the glutamate/aspartate-arginine-tyrosine motif located in the
second intracellular domain gave rise to the first known constitutively
active oxytocin receptor (R137A), whereas mutation of the aspartic acid
located in the second transmembrane domain led to an inactive receptor
(D85A). The structural features of the constitutively active and
inactive receptor mutants were compared with those of the wild
type in its free and agonist-bound states. The results suggest
that, although differently triggered, the activation process induced by
the agonist and the activating mutation are characterized by the
opening of a solvent exposed site formed by the 2nd intracellular loop,
the cytosolic extension of helix 5, and the 3rd intracellular
loop; on the contrary, the D85A mutation prevents oxytocin from
triggering the opening of a cytosolic site. On the basis of these
findings, we hypothesize that this cytosolic crevice plays an important
role in G protein recognition. Finally, comparative analysis of the
free- and agonist-bound forms of the wild-type oxytocin receptor and
1B adrenergic receptor suggests that the highly
conserved polar amino acids and the seven helices play similar
mechanistic roles in the different G protein-coupled receptors.
 |
Introduction |
Receptors
of the rhodopsin family use G proteins to transduce signals across the
cell membrane. All of these receptors share the presence of seven
hydrophobic regions, which are believed to form a bundle of -helical
transmembrane domains connected by intracellular and extracellular
loops. Mutational analysis has revealed that the extracellular regions
and transmembrane domains contribute toward the formation of the ligand
binding site, whereas the intracellular loops appear to mediate G
protein coupling (Wess, 1997
). Despite the fact that recent
crystallographic studies of G proteins have begun to show how they
might work (Coleman and Sprang, 1996), a consistent description in
terms of the structure and dynamics of the mechanisms of receptor
activation and receptor-catalyzed nucleotide exchange still remains a
daunting task.
The discovery that mutations in the third intracellular loop of
different adrenergic receptors (ARs) can greatly increase their
constitutive (agonist-independent) activity led to the hypothesis that
G protein-coupled receptors (GPCRs) can exist in equilibrium between
interconvertible inactive (R) and active (R*) allosteric states
(Cotecchia et al., 1990; Samama et al., 1993). Recent studies on
rhodopsin showed that the light-activated conformational changes appear
to involve rigid body motions of helix F relative to helix C (helices C
and F in rhodopsin correspond to helices 3 and 6 in the other
homologous GPCRs) (Farrens et al., 1996) and experiments on
2-AR showed that agonist-induced receptor
activation involves movements of helices 3 and 6 (Gether et al., 1997).
These results emphasize the need to develop a dynamic description of
GPCR proteins to elucidate the structural changes associated with
functionally different states.
The pattern of the microscopic configurations that predominate in each
functional form of the 1B-AR have very
recently been investigated (Scheer et al., 1996, 1997; Fanelli et al.,
1998). These studies suggested that certain highly conserved polar
amino acids located in the seven-helix bundle may drive the motion of the helices that culminates into the rearrangement of the cytosolic domains involved in receptor/G protein recognition. In particular, the
model showed that the aspartate and arginine residues of the glutamate/aspartate-arginine-tyrosine (E/DRY) sequence, as well as of
the N63, D91, N344, and Y348 residues that form a conserved polar
pocket near the cytosol, play a fundamental role in regulating receptor
isomerization into functionally different states. Although it is known
that the E/DRY residues are conserved in almost all GPCRs and are
essential to the coupling process in many different receptors, it is
still not clear whether and how this sequence plays a general role in
controlling the transition from inactive to active states.
The main aim of this study was to investigate whether the control by
the E/DRY region of the transition from R to R*, which has been
proposed to occur in the 1B-AR, is maintained
in the oxytocin receptor (OTR), a peptidergic receptor belonging to the oxytocin (OT)/arginine-vasopressin (AVP) receptor family, which also
includes the V1a, V1b, and
V2 subtypes. A number of functional and
structural aspects of these receptors have recently been elucidated at
the molecular level by means of chimeric receptors (Postina et al.,
1996) and a combination of molecular modeling and site-directed mutagenesis (Chini et al., 1995; Mouillac et al., 1995; Chini et al.,
1996); in particular, several receptor residues and regions have been
shown to be involved in determining the selective affinities and
efficacies of different peptides to the different receptor subtypes;
the receptor regions that specifically interact with G proteins (Liu
and Wess, 1996) have also been identified. Even though a constitutively
active V2 vasopressin receptor has been recently
described (Morin et al., 1998), the structural and dynamic changes
associated with the agonist-independent and agonist-induced transition
from inactive to active states have never been studied in this receptor family.
To investigate the R/R* transition in the human OTR in this study, the
highly conserved amino acids N57 (helix 1), D85 (helix 2), D136 (helix
3), and R137 (helix 3) (the last two of which belong to the highly
conserved E/DRY sequence) were subjected to experimental and
computer-simulated mutagenesis. A comparative analysis of the molecular
dynamics (MD) trajectories of the wild-type (WT) OTR as well as of the
R137A and D85A mutants was performed. This approach led to the
discovery of the activating mutation R137A and the inactivating
mutation D85A. To compare constitutive and agonist-induced activation,
MD of the OT-bound forms of the WT OTR, as well as of the D85A mutant
were also simulated. Finally, a comparative analysis of the active and
the inactive states of OTR with those of the
1B-AR were made to investigate whether the
members of the rhodopsin family of GPCRs might have similar activation mechanisms.
| |
Materials and Methods |
Peptides and Chemicals.
Synthetic OT was obtained from
Bachem Switzerland. The specific OTR antagonist OTA was synthesized in
the laboratory of Dr. M. Manning and iodinated in the laboratory of C. Barberis as described previously (Elands et al., 1987). Guanosine
5'-3-O-(thio)triphosphate (GTP
S) and BSA were purchased
from Sigma Chemical Co. (St. Louis, MO). [3H]OT
(35-45 Ci/mmol), [3H]AVP (35-45 Ci/mmol), and
myo-[2-3H]inositol (10-20 Ci/mmol) were from
Dupont-NEN (Boston, MA).
Construct Preparation.
The human OTR cDNA, a generous gift
of Dr. T. Kimura was subcloned into an M13 vector (M13 mp18) and the
mutants were constructed by means of oligonucleotide-directed
mutagenesis (Sculptor Kit; Amersham Corp., Arlington Heights, IL). WT
and mutant cDNAs were then inserted into an eukaryotic expression
vector under the control of the cytomegalovirus promoter (pRK5). For
checking transfection efficiency by means of fluorescence-activated
cell sorting analysis (FACS) analysis, WT and mutant cDNAs were
subcloned into the internal ribosome entry site enhanced green
fluorescent protein (pIRES-EGFP) vector (Clontech, Palo Alto, CA).
Cell Transfection.
The WT and mutant receptors were
transiently transfected into COS7 cells (American Type Culture
Collection, Rockville, MD) by electroporation. The COS7 cells were
grown in Dulbecco's modified Eagl's medium (Gibco BRL, Gaithersburg,
MD) supplemented with 10% fetal calf serum (Sigma), 100 IU/ml
penicillin, and streptomycin (Gibco BRL), in 5%
CO2 in air, at 37°C. Electroporation (280V, 960 µF, Bio-Rad gene pulser electroporator; Bio-Rad Labs., Hercules, CA)
was performed in a total volume of 300 µl with
107 cells in electroporation buffer (50 mM
KHPO4, 20 mM CH3COOK, and
20 mM KOH) and various amounts of plasmid DNA (vector with a subcloned
insert) and carrier DNA (vector without insert) to reach a total amount
of 20 µg of DNA/transfection. After electroporation, the cells were
split into 6-well clusters [for the determination of inositol
phosphate (InsP) accumulation], 24-well clusters (for intact cell
binding assays), 150-mm petri dishes (for membrane preparation) or
100-mm petri dishes for FACS analysis.
Receptor Binding Assay.
To determine cell surface binding,
the cells were subcultured into 24-well plates at a density of 4 × 105 cells/well; 72 h after transfection,
the cells were washed twice with binding buffer (146 mM NaCl, 4.2 mM
KCl, 0.5 mM MgCl2, 1.0 mM
CaCl2, 10 mM HEPES base, 1% glucose, 0.018%
L-tyrosine, and 1% BSA, pH 7.4) and placed on ice.
Increasing concentrations of [3H]OT were added
to the wells with or without 10
6 unlabeled OT
in a final volume of 200 µl. After incubation for 2 h at 4°C,
the cells were washed three times with cold binding buffer to remove
the unbound radioactivity and then solubilized with 0.5 N NaOH. The
samples were transferred into scintillation vials and counted using a
beta counter after the addition of 3.5 ml of scintillation cocktail
(Ultima Gold; Packard, Meridan, CT). To prepare the membranes,
transfected COS7 cells were homogenized in a glass potter, washed
twice, and resuspended in the binding buffer (50 mM Tris HCl, 5 mM
MgCl, pH 7.4). Homogenates were used immediately or frozen under liquid
nitrogen and stored at 
70°C. For saturation experiments performed
in presence or absence of GTPS only freshly prepared homogenates
were used. When [125I]OTA was used, 1 to
5 µg of membrane proteins were incubated for 60 min at 30°C; when
[3H]OT and [3H]AVP were
used, incubation lasted 30 min in the presence of 5 to 10 µg of
membrane proteins. Nonspecific binding was determined in the presence
of a 250- to 1000-fold excess of unlabeled analogs. Bound and free
radioactivity were separated by filtration over Whatman GF/C filters
presoaked in 10 mg/ml BSA. Binding isotherms were analyzed with the
iterative curve-fitting program LIGAND (Munson and Rodbard; 1980).
InsP Determination.
InsP accumulation was determined as
previously described (Mouillac et al., 1995). Briefly, transfected
cells were grown on six-well dishes for 48 h. The cells were then
labeled for 24 h with myo-[2-3H]inositol
at a final concentration of 2 µCi/ml in a serum-free, inositol-free
medium (Gibco BRL). The cells were washed twice in Krebs buffer (146 mM
NaCl, 4.2 mM KCl, 0.5 mM MgCl2 1.0 mM CaCl2 10 mM HEPES base, and 1% glucose, pH 7.4)
and preincubated at 37°C in the same buffer supplemented with 10 mM
LiCl. After incubation for 20 min in the absence (basal) or presence of
increasing peptide concentrations (from 1015 to
105), the reaction was stopped with percloric
acid, and the InsPs were extracted and separated using a strong anionic
exchange column (Dowex AG1 × 8, formate form, 200-400 mesh;
Bio-Rad). At each concentration, a fraction containing inositol
monophosphates, bisphosphates, and trisphosphates was collected and its
radioactivity determined by means of scintillation counting. This
fraction is referred to as total InsP and expressed as disintegrations
per minute per well. The data were analyzed by means of nonlinear regression using a sigmoidal dose-response equation (Prism, GraphPad, San Diego, CA).
FACS Analysis.
For flow cytometry, cells were scraped and
resuspended in PBS/2.0 mM EDTA at a
concentration of 1 to 2 × 106 cells/ml.
Flow cytometry analysis was performed with a FACScan (Cell Quest
software; Becton Dickinson, Lincoln Park, NJ). Cells were gated for
size and side scatter to exclude dead cells and debris.
Three-Dimensional Model Building of OTR and OT/OTR Complex.
Figure 1 shows the transmembrane (t),
extracellular (e), and intracellular (i) domains included in the OTR
model, together with the secondary structures assigned to the input
receptor model. The lengths of the seven helices were chosen on the
basis of multiple sequence alignments and predictions of the topology
of the transmembrane segments (Rost et al., 1996). Helices 3, 5, 6, and
7 were extended into the cytosol on the basis of several lines of
evidence, including NMR and circular dichroism experiments on synthetic
peptides derived from the third and fourth intracellular loops of
rhodopsin, 2-AR, and angiotensin II receptors
(Jung et al., 1995, 1996; Yeagle et al., 1997; Franzoni et al., 1997).
The extracellular ends of the helices were also slightly prolonged.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Amino acids included in the theoretical model of the
human OTR. Model includes the seven helices, the three extracellular
loops (e1, e2, and e3), and the three intracellular loops (i1, i2, and
i3). For the intracellular (i) and the extracellular (e) receptor
domains, the secondary structure (H, helix; T, turns; L, loop) assigned
in the input arrangement is indicated. For the helices, the predicted
topology (e, extracellular; t, transmembrane; i, intracellular) is
indicated.
|
|
In the input arrangement, the seven helices were packed using the
recently published backbone coordinates of the upgraded version of the
1B-AR model as an initial template (Fanelli et al., 1998). The helices were translated and rotated to relieve steric
conflicts and improve the helix-helix packing according to the tilt of
the seven helices derived from the three-dimensional map of frog
rhodopsin (Unger et al., 1997).
The intracellular and extracellular loops (excluding i3) were built
following the same procedure as that previously described for the
1B-AR. The 242 to 270 stretch of amino acids
belonging to i3 was built by restraint-based comparative modeling (Sali and Blundell, 1993), using the NMR structure of the third intracellular loop of the parathyroid hormone receptor as the initial template (Pellegrini et al., 1996). A disulphide bridge was allowed to form
between C112 and C187, in accordance with experimental evidence.
Different input arrangements were built by performing translations and
rotations of the helices and loops, as well as modifications of the
side chain torsion angles. An input structure of the WT OTR was finally
selected among the large number of tested arrangements; upon MD
simulations, this structure produced an average arrangement showing the
best agreement with the experimental data available on GPCRs together
with high quality check scores.
The input structures of the mutant receptor forms were obtained by
substituting the target residue in the selected WT input structure
using the molecular graphics package QUANTA (release 96; Molecular
Simulations Inc., Waltham, MA). The input structure of the OT-WT OTR
complex was obtained by docking OT into the input structure of the WT
OTR. The OT extracted from the crystal structure of the OT-neurophysin
complex (Protein Data Bank code: 1npo) (Rose et al., 1996) was tested,
as was that obtained from deamino OT (Protein Data Bank code: 1xy2)
(Woods et al., 1986). The main orientation of the hormone was chosen
following the suggestions of experimental results obtained using
vasopressin receptors (Kojro et al., 1993; Chini et al., 1995) and
experiments on vasopressin/OT chimeric receptors (Postina et al.,
1996). These experiments indicated that the cyclic part of the hormone
should interact with e2 and e3, whereas the linear C-terminal peptide
should be oriented toward the N-terminal tail and e1. Different initial
orientations of the hormone were tested, as were different initial
conformations of its backbone C-terminal tripeptide and of the amino
acid side chains involved in OT-OTR interactions. The OT-OTR complexes
were submitted to energy minimization and MD simulation. Among the large number of OT-OTR complexes obtained, the model showing the best
agreement with the experimental data mentioned above was selected for
the comparative analysis. The input structure corresponding to this
complex carries the OT extracted from the OT-neurophisin complex (Rose
et al., 1996).
The input structure of the OT-D85A complex was obtained by mutating D85
in the selected input structure of the OT-bound WT OTR.
Computations.
The minimizations and MD simulations were made
using the CHARMM program (Brooks et al., 1983). Minimizations were
carried out using 1500 steps of steepest descent followed by a
conjugate gradient minimization, until the root mean square gradient
was less than 0.001 kcal/mol. A distance-dependent dielectric term (
= 4r) and a 12 Å nonbonded cutoff distance were chosen. The "united atom approximation" was used for computational efficiency (Brooks et al., 1983). The minimized coordinates of the
receptors and hormone-receptor complexes were then used as the starting point for a 150-ps MD run. The systems were heated to 300 K with a
5°C rise per 6000 steps by randomly assigning velocities from the
Gaussian distribution. After heating, the system was allowed to
equilibrate for 34 ps. Velocities were scaled by a single factor. The
system was then subjected to 110 ps MD simulation at a constant temperature (300 K). The reported results were collected every 0.5 ps
from the last 100 ps trajectory. The lengths of the bonds involving
hydrogen atoms were constrained according to the SHAKE algorithm,
allowing an integration time step of 0.001 ps. Newton's equations of
motion were integrated using the Verlet algorithm. The -helix
conformation was preserved by using the nuclear Overhauser effect
constraint with a scaling factor of 10. These constraints were applied
between the backbone oxygen atom of residue i and the backbone nitrogen
atom of residue i + 4, excluding prolines. Different combinations of
distance constraints were tested.
Different prototropic forms of the WT OTR, involving the histidines in
the seven-helix bundle as well as the highly conserved aspartate of the
E/DRY sequence (D136), were simulated. Because H173 (helix 4) and H335
(helix 7) should lie at a pH lower than 7.4 due to the closeness of
phospholipids, and should therefore be suitable for protonation, the
average minimized structure carrying all of the histidines but H173 and
H335 in their neutral form were finally considered in the comparative
analysis. This combination of charged states involving the histidines
was associated with each of the two possible prototropic forms of D136:
the deprotonated (anionic) and protonated (neutral) form. The receptor
structure carrying D136 in its deprotonated form was finally considered for the comparative analysis. The comparative analysis was performed on
the structures averaged over the last 100 ps of the equilibrated time
period of each MD simulation and minimized.
The selected input structure and the computational conditions finally
chosen produced average arrangements of the WT OTR, of the mutants, and
of the OT-bound receptor forms consistent, especially for the active
forms, with the structural features recently inferred from a wide
analysis of the GPCR sequences (Baldwin et al., 1997).
The Protein Health utility implemented in QUANTA (release 96; Molecular
Simulations Inc.) was used to check the quality of the models obtained.
| |
Results |
Expression of WT and mutant OTRs in COS7 cells.
To investigate
the role played by highly conserved residues in determining the active
and inactive states of the human OTR, the N57A, D85A and D136A, D136Q,
and R137A mutations were introduced into the WT OTR receptor by
site-directed mutagenesis. The binding and coupling properties of all
of the receptor mutants were assayed upon transient transfection in
COS7 cells as described in Materials and Methods. The cells
transfected with the N57A, D136A, and D136Q mutants did not show any
specific binding when probed in saturation binding assays with labeled
agonists ([3H]OT and
[3H]AVP) or antagonists
([125I]OTA); furthermore, they were completely
unresponsive when assayed for InsP production upon stimulation with
high doses of OT and AVP (105
M; data not shown). This indicates that these
receptor mutants are either completely functionally inactive or that
they are trapped inside the intracellular compartments due to folding,
sorting, and/or recycling defects. On the contrary, the R137A and the
D85A mutants were sorted to the plasma membrane, as indicated by
binding analysis of intact cells; however, we had to increase the
amount of specific DNA transfected (plasmid with a subcloned receptor cDNA) by a factor of 10 to achieve a level of expression similar to
that of the WT. The total amount of DNA transfected (20 µg/electroporation) was maintained constant by decreasing the carrier
DNA (plasmid without insert). Under these conditions, the
Bmax values for the WT and the R137A
mutant, as measured by means of [3H]OT
saturation experiments with intact cells, were 426,300 ± 157,400 (n = 3) and 435,000 ± 50,140 (n = 3) sites/cell, respectively. The KD values
were also similar: 2.99 ± 0.41 nM (n = 3). and
5.42 ± 1.69 nM (n = 3), respectively. In the case
of the D85A mutant, the level of expression was determined by means of
[3H]AVP homologous competition because no
specific binding was detected when [3H]OT was
used in saturation and competition experiments. Our data indicate an
expression of 298,600 ± 172,700 sites/cells (n = 2) and a very large decrease in AVP affinity
(Ki = 1350 ± 826 nM; n = 2) in comparison with the WT receptor
(Ki = 1.65 ± 0.49 nM; n = 2).
Coupling Properties of R137A (Constitutively Active) and D85A
(Inactive) Mutants.
To determine the coupling properties of the
R137A and D85A mutants, the basal production of total InsP was measured
in transiently transfected COS7 cells expressing similar levels of WT
or mutant OTRs (0.4-0.5 pmol/mg of proteins). As shown in Fig.
2A, our data indicate that the R137A
induced a significant increase in the basal production of total InsP;
if the basal level of total InsP in mock-transfected cells is taken as
100%, this increase was estimated to be 145% (n = 5, p > .001). At this level of expression, there was no
difference in the basal InsP production by the WT receptor and that by
the mock-transfected cells. Furthermore, the maximum level of InsP
production induced by treatment with 105
M OT was similar for the two receptors (3-5
times more than basal; Fig. 2B), whereas no detectable increase in InsP
production was observed in the D85A mutant under basal or stimulated
conditions. Because the D85A mutant retained a measurable affinity for
AVP, we also checked InsP production after treatment with
105 M AVP; once again, no
detectable variation in InsP accumulation was detected (not shown).
These data indicate that we obtained one constitutively active mutant
(R137A) that is still responsive to OT, and one that is unable to
stimulate phospholipase C and is functionally inactive (D85A).
View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
Basal and stimulated activation of WT, R137A, and
D85A OTR receptors. WT, R137A, and D85A receptors were transiently
expressed in COS7 cells and their coupling to Gq/G11 was evaluated by
measuring the accumulation of total InsP over a period of 15 min. A,
basal InsP levels. Graph shows the mean values of five different
experiments; in each experiment, performed in triplicate, the basal
InsP production of the WT and R137A mutant was expressed as the
percentage of increase over that of mock-transfected cells (taken as
100%). B, agonist-stimulated InsP levels. In these experiments, the
maximum InsP production was measured by applying a high dose of OT
(105 M). For each receptor, the basal level
of InsP production was fixed to the value of 1 and the ratio between
the maximum InsP production obtained with 105
M OT and basal InsP production was calculated. This ratio
is shown on the ordinate axis as the fold increase over basal.
|
|
To evaluate whether variations of transfection efficiency between the
WT and the R137A mutant may have influenced our findings, we subcloned
the WT OTR and the constitutively active R137A mutant into the
multicloning site of the pIRES-EGFP vector (Clontech). This vector
contains the IRES sequence of the encephalomyocarditis virus between
the multicloning site and the EGFP; the IRES sequence permits the
translation of two open reading frames from a single mRNA. The
transfection of our constructs (WT-OTR-pIRES and R137A-pIRES) led to
the coexpression of the receptor and EGFP in the same cells, thus
allowing transient transfection efficiency to be evaluated by means of
FACS analysis. In preliminary experiments, we investigated whether
transfection efficiency varied when increasing amounts of pIRES-EGFP
DNA were electroporated. As shown in Fig.
3A, for amounts of specific DNA ranging
from 0.75 to 10 µg, transfection efficiency was between 17 and 37%;
at lower DNA concentrations, transfection efficiency dropped (3% for
0.075 µg of transfected DNA). These data indicate that transfection
efficiency remains fairly constant for quantities of specific DNA
ranging from 0.75 to 10 µg and with carrier DNA added to reach 20 µg of DNA/electroporation. We then determined the amount of specific
DNA needed to obtain a Bmax of
approximately 1 pmol of receptor/mg of protein (not shown). These
amounts of DNA, 0.75 µg of WT-OTR-pIRES, and 7.5 µg R137A-pIRES,
are approximately 10 times more than that used with our constructs in
the pRK5 vector (0.09 and 0.9 µg for the WT and R137A, respectively);
although both plasmids use a cytomegalovirus promoter, they have
different enhancer, synthetic-splicing, and polyadenilation
signals, and these differences may explain their different ability to
drive heterologous protein expression. Figure 3, B and C, shows the
results of one of two experiments in which we used flow cytometry to
measured the percentage of cells transfected with WT-OTR-pIRES and
R137A-pIRES. In these experiments, the transfection efficiencies were
highly comparable (30 and 28% in the first experiment, 16 and 18% in
the second) as were their Bmax values (1.05 and 1.31 pmol/mg protein in the first experiment, 1.34 and 1.65 pmol/mg protein in the second). At this level of expression, the basal activity
of the R137A and WT OTR had increased to 177% (n = 2) and 116% (n = 2) that of the mock-transfected cells.
These data confirm that the R137A mutant is characterized by a basal
activity higher than that of the wild-type receptor.
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Evaluation of transfection efficiency. In our
experiments, the total DNA transfected (20 µg/electroporation) was
maintained constant by adjusting the amount of carrier DNA. A, various
amounts of pIRES vector DNA (Clontech) were electroporated into COS7
cells. Percentage of fluorescent transfected cells was determined by
FACS analysis. B, FACS profiles of COS7 cells transfected with 0.75 and
7.5 µg of WT-OTR-pIRES and R137A-pIRES DNA. Dotted line represents
mock-transfected cells, straight line represents the population of
cells electroporated with specific DNA, filled histogram represents
fluorescent-transfected cells, the percentage of which is reported on
the bottom right. C, basal InsP production of cells transfected with
0.75 and 7.5 µg of WT-OTR-pIRES and R137A-pIRES DNA. In this
experiment, performed in triplicate, the basal InsP production of the
WT and R137A mutant was expressed as the percentage of increase over
that of mock-transfected cells (taken as 100%).
|
|
To better analyze the coupling properties of the R137A mutant, we drew
the dose-response curves of total InsP accumulation upon agonist
stimulation. As shown in Fig. 4A, R137A
was able to stimulate phospholipase C in a dose-dependent manner with a calculated EC50 for OT of 57.4 ± 24.4 nM
(n = 3); this value differs by approximately a factor
10 from that of the WT receptor expressed in the same cells (4.8 ± 0.8 nM; n = 3). Finally, the inverse agonist
properties of the specific peptidic antagonist OTA were assayed in
cells transfected with the R137A mutant. As shown in Fig. 4B, this
analog had inverse agonist properties with a measured IC50 of 0.2415 pM (n = 2).
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Agonist and inverse agonist properties of OT and OTA
in the WT OTR and R137A mutant. InsP production was measured after the
stimulation of transiently transfected COS7 cells with increasing
concentrations of peptides. Curves are representative of at least two
independent assays performed in triplicate. Each point represents the
total amount of InsPs expressed as dpm/well. A, effects of increasing
concentrations of OT; in these experiments, we calculated an
EC50 value of 1.32 ± 0.13 nM and 66.67 ± 1.20 nM for the WT OTR and the R137A mutant, respectively. B, effects of
increasing concentrations of the specific antagonist OTA; in this
experiment, the IC50 value for R137A was 0.1 ± 0.71 pM.
|
|
Finally, to correlate the basal InsP production with the level of
receptor expression of the WT and R137A mutant, we performed a set of
experiments in which the number of receptors expressed on the cell
surface was measured by means of [3H]OT
saturation binding on intact cells. In this case, because differences
between the desensitization process of the WT and that of the mutant
receptor cannot be excluded, the binding assay was performed at 4°C
over ice to prevent any ligand-induced endocytosis. Shown in Fig.
5, our data indicate a linear correlation
between the increase in cell surface receptors and basal InsP
production for both the WT and the R137A mutant OTRs. Even though we
were unable to reach very high levels of expression with the R137A mutant, these data confirm that at comparable level of expression, the
R137A produced more InsP than the WT receptor.
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
WT and R137A basal activities as a function of
receptor expression levels. WT and R137A receptors were transiently
expressed at variable levels in COS7 cells by transfecting increasing
amounts of specific plasmid cDNA; basal InsP production and
[3H]OT binding were determined as described in
Materials and Methods. For each transfection, basal InsP
production and [3H]OT binding sites were expressed per
million of cells present in the well at the time of the
determination.
|
|
Binding Properties of Constitutively Active R137A Mutant.
Cell
homogenates were used to obtain a more detailed pharmacological
characterization of the R137A mutant. When assayed with the tritiated
agonist [3H]OT, no significant difference in OT
affinity between the WT (0.46 ± 0.047 nM; n = 5)
and the R137A mutant (1.2 ± 0.81 nM; n = 3) was
observed in saturation experiments. Similarly, no changes were found
when AVP affinity was measured by means of
[3H]OT competitive binding (1.65 ± 0.49 nM; n = 3 and 2.5 ± 0.42 nM; n = 3 for the WT and R137A, respectively). However, the R137A mutant showed
a marked reduction (20-fold) in the affinity of the specific cyclic
peptidic antagonist OTA, as determined in saturation experiments
(0.095 ± 0.033 nM, n = 5; and 1.95 ± 0.81 nM, n = 4 for the WT and R137A, respectively).
We also performed [3H]OT saturation binding
experiments in the presence and absence of GTPS to compare the
coupling state of R137A with that of the WT receptor. As shown in Fig.
6, the WT receptor displayed an expected
4-fold increase in the KD value for OT in
the presence of GTPS, thus indicating that GTPS is able to
uncouple the receptor from the G protein and give rise to a receptor
population with lower affinity for OT. On the contrary, in the R137A
mutant, no significant change in OT affinity was detected in the
presence of GTPS. These findings support the evidence that the R137A
mutant is characterized by (a) particular precoupling state(s) that
is/are quite different from that of the WT receptor and that
determine(s) its increased basal activity.
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of GTPS treatment on [3H]OT
affinity in the WT and R137A. Membrane from COS7 cells expressing the
WT or the R137A mutant receptors were prepared and preincubated for 10 min in the presence of 100 mM GTPS; [3H]OT saturation
experiments were then performed as described in Materials and
Methods. Figure shows a Scatchard analysis that is
representative of three separate experiments, each performed in
triplicate. Mean ± S.E. of the KD
values are shown in the inset.
|
|
Computer Simulation of WT and R137A OTRs.
As illustrated in
Fig. 7, different interaction patterns
involving polar and charged amino acids characterize the average minimized structures of the WT and R137A mutant OTRs.
View larger version (47K):
[in this window]
[in a new window]
|
Fig. 7.
Average minimized structures of the free and OT-bound
forms of WT and mutant OTRs. Intracellular and extracellular sides are
at the top and bottom of the helix bundles, respectively. All views are
parallel to the membrane surface. The seven helices 1, 2, 3, 4, 5, 6, and 7 are, respectively, colored in blue, orange, green, pink, yellow,
sky blue, and violet; i1, i2, and i3 are, respectively, colored in
green, white, and rose. The side chains of some amino acids
(colored according to their location) located in the environment of
D85, D136, and R137 of the E/DRY, as well as of some of the ionic amino
acids in the cytosolic loops that contribute to the closing/opening of
a cytosolic site in the inactive/active receptor forms, are shown in
the figure. Backbone -carbon atoms of the cytosolic half of the
receptor models are also displayed.
|
|
In the average minimized structure of WT OTR several salt bridge
interactions contribute toward stabilizing the receptor "ground state". As shown in Fig. 7, R137 is subjected to the attractive effects of the electrostatic fields generated by the two highly conserved aspartates on helices 2 and 3: D85 and D136, respectively. D136 is also involved in a charge-reinforced H-bonding interaction with
R150 (i2). Other charge-reinforced H-bonding interactions constitute
structural features of the OTR "ground state". In particular, 1)
the D136 (helix 3)/R150 (i2)/D153 (helix 4) interactions produce a
structural link between the cytosolic ends of helices 3 and 4; and 2)
the R73 (i1)/D251 (i3), the R146 (i2)/E339 (helix 7), and the R149
(i2)/E242 (i3) interactions provide more strength to the intramolecular
interactions that connect i1 and i2 with i3 and the cytosolic extension
of helix 7. Although these interactions are overemphasized in our
structures, which are the results of simulations performed in vacuo, it
is conceivable that the attraction between most of these charged
residues still operates in the solvated proteins, because these
residues should not be separated by a bulk of water molecules.
Moreover, the highly conserved amino acids forming a polar pocket near
the cytosol, (N57, D85, N325, and Y329) are involved in the following
interaction pattern: 1) D85 (helix 2) performs van der Waals attractive
interactions with N57 (helix 1) and N325 (helix 7); and 2) N325 (helix
7) and Y329 (helix 7) of the highly conserved NPXXY sequence contribute
toward constraining the motion of R137 by means of H-bonding and van der Waals attractive interactions, respectively, with its side chain.
A rearrangement of the interaction patterns involving the "polar
pocket" amino acids occurs in the constitutively active R137A mutant
structure (Fig. 7). In particular, D85 (helix 2) performs H-bonding
interactions with both N57 (helix 1) and N325 (helix 7). Moreover, Y329
is shifted toward helix 2, being directed toward helix 6 in the WT OTR.
The differences in the interaction patterns of the polar pocket amino
acids in the WT and R137A mutant are associated with differences in the
arrangement of helices and loops (Figs. 7 and 8b). In particular, one of the structural
peculiarity of the R137A mutant is the detachment of helices 4 and 5 and the approaching of the cytosolic end of helix 4 to that of helix 2 (Fig. 8a). Moreover, many of the salt bridge interactions, as well as
the bulk of the other weaker nonbonding interactions, connecting i1 and
i2 with i3 and the cytosolic extension of helix 7 in the WT OTR, are
released in the structure of the active mutant (Fig. 7), thus promoting
the opening of a solvent accessible site in between i1 and i2, on one
hand, and i3, on the other one (Figs. 7 and 8c). In fact, as it can be
seen in Fig. 8c, i1 and i2 in the active mutant are characterized by a
larger solvent accessible surface than in the WT OTR.
View larger version (61K):
[in this window]
[in a new window]
|
Fig. 8.
Average minimized structures of the free and OT-bound
forms of WT and mutant OTRs. The intracellular and extracellular sides
are at the top and bottom of the helix bundles, respectively. a,
cylinder representation of the seven helices of WT OTR superimposed on
the structures of the R137A active mutant (top left), the OT-D85A
complex (bottom left), and the OT-WT OTR complex (bottom right). Helix
bundle is seen in a direction parallel to the membrane surface. WT
helices are colored in white, whereas the helices in the other
structures are colored as described in Fig. 7. b, cylinder
representation like that in point a but seen from the intracellular
side in a direction perpendicular to the membrane surface. Side chain
of W288 is also displayed. c, two views of the average minimized
structure, represented by cartoons. i1, i2, and i3 are, respectively,
colored in green, white, and rose, whereas e1, e2, and e3 are,
respectively, colored in light blue, red, and carnation. Receptors are
seen in a direction parallel to the membrane surface.
Solvent-accessible surfaces (represented by dots) computed on i1, i2,
i3 and the cytosolic extensions of helices 5 and 6 are shown on the
receptor structures in one of the two views. Hormone in the OT-WT OTR
and in OT-D85A complexes is represented by liquorices and is
colored by atom types.
|
|
Computer Simulation of WT and D85A OT/OTR Complexes.
To
investigate the structural and dynamic features of the agonist-induced
active states of OTR, OT was docked into the input structures of the WT
OTR and of the agonist-unactivatable D85A mutant.
Among the large number of OT- WT OTR complexes obtained, the average
minimized structure showing the best agreement with the experimental
data (Fig. 9 and Table
1) was selected for the comparative analysis. However, given the low resolution level of the experimental data available and the consequent absence of any structural constraint, at the atomic level, for choosing the appropriate OT-OTR interaction model, the chosen complex might represent one of the possible models.
In this complex, the cyclic part of the peptide (1-6 sequence) docks
into the site formed by e2 and the extracellular ends of helices 3, 4, 5, and 6, whereas the linear C-terminal tripeptide (7-9 sequence)
mainly docks into the site formed by the amino acid residues of e1, e2,
and the extracellular ends of helices 3 and 7 (Figs. 8 and 9). The
OT-OTR interaction model presented in this work differs from the
previously published model (Chini et al., 1996) as a result of a number
of concomitant factors: 1) the arrangement of the seven helices and the
conformation of the loops were obtained by means of different
methodologies and contain different experimental information, i.e., the
present model takes into account the structural information coming from the recently published three-dimensional map of frog rhodopsin (Unger
et al., 1997); 2) the input conformation of the hormone is different in
the two models; and 3) the amino terminus of the hormone in our model
is protonated and not neutral.
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 9.
Agonist binding sites in the OT-bound forms of the WT
OTR (top) and the D85A mutant (bottom). Top and bottom left view,
docking of the hormone in the extracellular halves of OTRs. Top and
bottom right views, details of the interactions of OT with the WT OTR
(top) and the D85A mutant (bottom). Color of the side chains of the
receptor in the right part of the figure matches that used for the
helices and the extracellular regions in the left part. Amino acid
residues of the hormone have been also labeled.
|
|
The comparison of the free and the OT-bound forms of the WT OTR shows
that the agonist induces a structural rearrangement propagating from
the extracellular to the intracellular domains (Figs. 7 and 8). In the
OT-OTR-simulated complex, the interaction of the hormone with E307 (e3;
Fig. 9) induces the breakage of the intramolecular salt bridge found in
the free receptor form between K116 (helix 3) and E307 (e3). The
anchoring of the hormone to E307 (e3) by means of the protonated
N-terminal nitrogen atom, followed by the interaction with helices 3 and 6, promotes outward motions of the extracellular extensions of the
two helices (Fig. 8b). The motion of helix 6 leads to a shift of W288
(helix 6) from the core of the helix bundle (directed toward helix 3)
to a position toward helix 5 and the membrane (Fig. 8b). The shift of
W288 (helix 6) is consistent with the results of ultraviolet resonance
Raman study of the light-induced protein structural changes in
rhodopsin activation (Kochendoerfer et al., 1997).
Similar to the R137A active mutant, the OT-bound OTR structure is
characterized by the approaching of the cytosolic ends of helices 2 and
4, as compared with WT OTR (Fig. 8b). Another structural peculiarity of
the OT-bound OTR structure is the weakening of the attractive effect
exerted by both D85 and D136 on R137 in the average structure of the
free WT OTR that results into the shift of R143 away from the polar
pocket (Fig. 7). Similar to the constitutively active structure R137A,
the motions of helices 3, 4, 5, and 6 promote the release of the
majority of the salt bridge interactions and of the other weaker
interactions connecting i1 and i2 with i3 and the cytosolic extension
of helix 7 in the free form of WT OTR (Fig. 7). One consequence of this
rearrangement is that i1, i2, i3, and the cytosolic extension of helix
5 become more exposed to the solvent than in the unbound form of WT
OTR, as it clearly results from the comparison of their solvent
accessible surfaces in the free and the OT-bound forms of WT OTR (Fig.
8c).
The simple substitution of alanine for D85 in the input structure of
the OT-OTR complex produces an average arrangement that differs from
that of the corresponding complex between OT and the WT receptor. Some
of the many structural differences involve the conformation of the
hormone (Fig. 9); in fact, the root mean square deviations of the main
chain -carbon atoms of the whole peptide and the cyclic part are,
respectively, 1.58 Å and 0.64 Å. Moreover, the interaction patterns
involving OT in the complex with the D85A mutant differ from those in
the corresponding complex with the WT receptor (Table 1 and Fig. 9).
One of these differences consists of the lack of the salt bridge
interaction between E307 and the protonated N-terminal nitrogen atom of
the hormone (Fig. 9). The OT-D85A complex is also characterized by the
attractive effect exerted by D136 on R137 that contributes toward
constraining the motion of this conserved arginine. One important
consequence is that OT is no longer capable of triggering the opening
of the solvent-exposed site in the cytosolic domains of the D85A mutant (Figs. 7 and 8c) because i1 and i2 are buried by i3, even more than in
the free form of WT OTR (Figs. 7 and 8c).
| |
Discussion |
In this study, we analyzed the role of a number of human OTR
residues that may be of fundamental importance in the process of
receptor activation. By targeting the highly conserved amino acids N57
(helix 1), D85 (helix 2), D136 (helix 3), and R137 (helix 3), we found
that mutation of the arginine of the E/DRY sequence into alanine
(R137A) leads to a form of the OTR characterized by an increased basal
activity. It is worth noting that mutations involving the arginine of
the E/DRY sequence in the vasopressin V2 receptor (Rosenthal et al.,
1993), as well as in other GPCRs (Zhu et al., 1994; Scheer et al.,
1996; Acarya and Karnik; 1996; Arora et al., 1997; Lu et al., 1997),
lead to uncoupled receptor forms. However, very recent results of
mutagenesis experiments involving the 1B-AR
targeting the arginine of the E/DRY sequence have shown that mutations
of R143 (corresponding to R137 in OTR) into lysine, histidine, and
aspartate induces constitutive activation, whereas mutations into
alanine, isoleucine, asparagine, and glutamate lead to a loss of
coupling (S. Cotecchia, personal communication). Similarly, mutations
of the arginine of the E/DRY sequence of the 2-AR did not abolish
coupling (Seibold et al., 1998). Together these data support the
hypothesis that this arginine plays a fundamental role in promoting
receptor isomerization into functionally different states.
This hypothesis is consistent with the results of comparative MD
simulations of the WT and mutant OTRs. On the basis of these results,
R137 should play a fundamental role in constraining the OTR in the
ground state, as well as in allowing the transition toward active
states. In the WT ground state, the motion of R137 is constrained by
the attractive electrostatic field generated by D85 (helix 2) and D136
(helix 3), and by persistent H-bonding interactions with N325 (helix
7). This configuration allows the formation of salt bridges and van der
Waals-attractive interactions that keep closed the cytosolic side of
the central pore of the helix bundle (Figs. 7 and 8). Mutation R137A
triggers rigid body motions that involve the whole helix bundle,
leading to the opening of a cytosolic site involving i2, the cytosolic
extension of helix 5 and i3 (Fig. 8c). We hypothesize that this
cytosolic crevice may play a fundamental role in G protein recognition,
consistent with some experimental findings (Wess, 1997).
A change in the interaction pattern of R137 with respect to WT OTR is
also one of the structural features of the agonist-induced active
state. We found that, to allow the effective transfer of structural
information from the extracellular to the intracellular side of the
receptor, interactions between the cyclic part of the hormone and the
receptor residues on the extracellular ends of helices 3, 5, and 6 needed to be stabilized by the formation of a salt-bridge interaction
between the protonated N-terminal amino group of the hormone and E307
(e3) of the receptor (Fig. 9). The establishment of these interactions
induces rigid helix body motions that primarily involve helices 3 and
6, and that propagate to the whole helix bundle. As a consequence, the
interactions that constrain the motion of R137 and connect i1 and i2
with i3 in the WT "ground state" structure are released, thus
promoting the opening of the solvent-exposed site in the cytosolic
domains that has been hypothesized to be involved in G protein
recognition (Fig. 8c).
The comparative analysis of the average minimized structures also
suggests that the highly conserved aspartate located on helix 2 has to
stay in its deprotonated (anionic form) to allow the rearrangement of
the cytosolic domains and the exposure of the G protein docking site to
occur. In agreement with this hypothesis, the experimental results show
that the D85A mutant is completely uncoupled. Furthermore, computer
simulations revealed that the irreversible neutralization of D85
following its mutation into alanine, increases the attractive effect
exerted by D136 on R137, thus preventing the agonist-induced structural
changes observed in the WT structure (Figs. 7 and 8).
Our results suggest that, although differently triggered, the motion of
the helices in the OT-OTR complexes and in the R137A active mutant
leads to the opening of a cytosolic crevice in between i2 and i3.
However, although the mutation-induced and agonist-induced active forms
have a number of structural similarities, there are also some clear
differences between them. First, the opening of cytosolic sites, which
are formed by the same receptor portions but show slightly different
shapes, suggests that different active forms of the same receptor
should recognize the G protein with the same domains by establishing
different interaction patterns. These differences in receptor/G protein
recognition may be responsible for the particular coupling properties
of the R137A mutant. Second, several differences between the WT and the
R137A active mutant involve the arrangement of the extracellular
domains, including the extracellular ends of the helix bundle. These
differences in the domains involved in the formation of the agonist
binding site should imply a different ability to interact with the
agonists. In this respect, it is worth noting that, unlike the great
majority of constitutively active receptor mutants, the constitutively active R137A does not show any measurable increase in agonist affinity.
Because examples of constitutively active mutants with unchanged
affinities for agonists, including a recently identified constitutively
active V2 receptor (Morin et al., 1998) have been reported in the
literature (Groblewski et al., 1997; Hjorth et al., 1998; Zhao
et al., 1998), this property may characterize a subset of
constitutively active receptors.
Finally, comparative analysis of the free- and agonist-bound forms of
the WT OTR and 1B-AR suggest that the highly
conserved polar amino acids and the seven helices should play similar
mechanistic roles in the different GPCRs. The ground and inactive
states of both receptors are characterized by the constraining effects
exerted on the motions of the E/DRY arginine by the conserved
aspartates on helices 2 and 3, as well as by the other "polar
pocket" amino acids. These effects are weakened in the
mutation-induced or agonist-induced active states. Moreover, the
helices of both receptors have the fundamental roles of transferring
the structural information between the two poles of the helix-bundles
and dictating the arrangements of the flexible loops. In the different
active forms of both receptors helix motions induce the opening of a
solvent-exposed site involving i2, the intracellular extension of helix
5 and i3. Our results showed that, although mutation of the E/DRY
arginine into alanine induces opposite structural effects in the OTR
and the 1B-AR (Scheer et al., 1996), this
fully conserved arginine plays similar mechanistic roles in the
agonist-induced activation of these two receptors.
In conclusion, this article reports the first steps in the molecular
analysis of the amino acids forming the "polar pocket" of the human
OTR and the identification of the first constitutively active mutation
of this peptidergic receptor. We proposed a possible docking of the
peptide into the receptor that allowed us to describe the similarities
and the differences between a mutation-induced and an agonist-induced
active state at the molecular level; furthermore, the use of the R137A
mutant made it possible to demonstrate that the specific antagonist
OTA, one of the most extensively used analogs for characterizing the
pharmacological and functional aspects of human OTR, behaves as an
inverse agonist. Finally, by comparing the activation process of the
OTR with that of the 1B-AR, we found that the
receptor domains involved in G protein recognition should be mainly the
same in these two members of the rhodopsin family; this finding should
allow the planning of future experiments aimed at elucidating the
specificity of receptor/G protein recognition.
We are indebted to C. Barberis and M. Manning for proving us
with the labeled and unlabeled OTA antagonist, to T. Kimura for the
gift of the human OTR cDNA, and to S. Cotecchia and D. Fesce for
critically reading the manuscript. We are grateful to S. Saini for
technical help, to Centro Interdipartimentale di Calcolo Automatico ed
Informatica Applicata (University of Modena) for technical help
and for allowing us to use its computer facilities, to S. Citterio and
M. Rescigno for help with FACS analysis.
This work was supported by grants from Consiglio Nazionale
delle Richerche (CNR) and Associazione Italiana Ricerca sul Cancro to
B.C.
AR, adrenergic receptor;
WT, wild-type;
OTR, oxytocin receptor;
OT, oxytocin;
G protein, guanine nucleotide
binding protein;
GPCR, G protein-coupled receptor;
R, inactive receptor
conformation;
R*, active receptor conformation;
E/DRY, glutamate/aspartate-arginine-tyrosine;
AVP, arginine-vasopressin;
OTA, d(CH2)5[Tyr(Me)2,Thr4,Tyr-9-NH2]-OVT;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
InsP, inositol phosphate;
EGFP, enhanced green fluorescent protein;
MD, molecular dynamics;
t, transmembrane;
e, extracellular;
i, intracellular.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Summary
Introduction
mutations of aspartate 122 and tyrosine 124 decrease receptor expression but do not abolish signaling.
Mol Pharmacol
51:
234-241
