From the Vascular Biology Research Center and Division of
Hematology, Department of Internal Medicine, The University of
Texas Health Science Center, Houston, Texas 77030
Received for publication, September 11, 2002, and in revised form, January 24, 2003
 |
INTRODUCTION |
Thromboxane A2
(TXA2)1 is a
potent platelet aggregatory and vasoconstrictive mediator (2). The
function of TXA2 is mediated by specific cell surface
receptor, thromboxane A2 receptor (TP) (3). The
understanding of the structure and function of TP receptor can greatly
explain how the ligand binds to its receptor and initiates the
following cell signaling.
TP receptor was first purified from platelet in 1989, and the cDNA
of TP receptor was cloned from placenta in (4, 5). Other human
prostanoid receptor cDNAs have also been cloned by homology
screening. All of the prostanoid receptors belong to the
G-protein-coupled receptor family that share a basic seven transmembrane segments and couple to different signal transduction systems to play diverse physiological and pathological roles (6-14). TXA2 binds to TP receptor and triggers an increase of
intracellular calcium. There were two TP receptor isoforms with
different C-terminal tails, resulting from alternative splicing that
the last 15 amino acids of the C terminus were replaced by 79 amino
acids (15, 16). The two TP receptor isoforms coupled to the same signal transduction, but endothelium expressed only the spliced form and
placenta expressed both types of the TP receptors (15-17).
Based on the sequence alignment, the second extracellular loop (eLP2)
and the third and seventh transmembrane domains of the prostanoid
receptors are highly conserved and are proposed to be involved in
ligand binding (18). Residues 198-205 in the eLP2 of the EP3 receptor
have been reported as an essential determinant of ligand selectivity
(19). These results suggest that the extracellular domains of other
prostanoid receptors are involved in the initial specific ligand
interaction. The residues responsible for specific ligand recognition
within eLP2 of the TP receptor have not been thoroughly examined. The
mutations based on alignment only are controversial and will need
structural information to support. The structures of the transmembrane
domains of prostanoid receptors may be similar, but the specific
recognition sites on extracellular domains will be different because
the ligand structures are different. Thus, structural characterization
of the extracellular functional domains of prostanoid receptors could
help in understanding the specificities of ligand binding. In our
current study, the structure of the highly conserved eLP2 has been
characterized by high resolution NMR using a synthetic eLP2 peptide
with constrained loop ends (1). To identify which residues make up the
ligand recognition site of the receptor, SQ29,548 was added to the
peptide to determine the interaction using high resolution
two-dimensional 1H NMR technique. The residues identified
from the NMR approach for the interaction between the ligand and the
eLP2 were further confirmed by site-directed mutagenesis. Results from
the studies provided an approach of using NMR experiments guided
site-directed mutagenesis for identification of the important residues
of other prostanoid receptors and other G-protein-coupled receptors,
which is more reasonable and close to the fact than those mutations performed only based on alignment.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Ethanol-d6 and
D2O were purchased from Cambridge Isotope Laboratories,
Inc. (Andover, MA). SQ29,548 was from Cayman Chemical (Ann Arbor, MI).
The QIAprep spin miniprep kit was from Qiagen (Valencia, CA). COS-7
cell was purchased from ATCC (Manassas, VA). Medium for culturing
COS-7 cells was from Invitrogen. [3H]SQ29,548 was
purchased from PerkinElmer Life Sciences.
Peptide Synthesis--
A constrained loop peptide mimicking the
sequence of the second extracellular loop of TP receptor (residues
173-193) with homocysteine added at both ends (Fig. 1) was synthesized
for NMR study using fluorenylmethoxycarbonyl-polyamide solid phase
method and cyclized by the formation of disulfide bound as described previously (1, 20-22). Briefly, the peptide was purified to homogeneity by HPLC. For the cyclization, the peptide was dissolved in
1 ml of dimethyl sulfoxide (Me2SO) and added to
H2O at a final concentration of 0.02 mg/ml with pH 8.5 adjusted by triethylamine, and stirred overnight at room temperature.
The cyclic peptide was then lyophilized and purified by HPLC on the C4 column.
NMR Sample Preparation--
The HPLC-purified constrained loop
peptide was dissolved in 20 mM sodium phosphate buffer, pH
6.0, at a final concentration of 5 mM. 1 mg of SQ29,548 was
dissolved in 50 µl of ethanol-d6 and then
added to 0.45 ml of sodium phosphate buffer (20 mM)
containing 10% D2O (1). Any insoluble ligand was removed
by centrifugalization. The concentration for the mixture of peptide and
SQ29,548 was the same as above.
NMR Experiments--
Proton NMR experiments were carried out on
a VARIAN Unity Plus 500 spectrometer, which was equipped with Z-pulsed
field gradient. Two-dimensional NMR experiments (DQF-COSY, TOCSY, and
NOESY) were performed for eLP2 only (1), SQ29,548 only, and their
mixture at 298 K. The WATERGATE method was used to suppress the signal of water. NOESY spectra were recorded with mixing time of 200 ms. TOCSY
spectra was carried out with decoupling in the presence of scalar
interactions spin-lock sequence with a total mixing time of 50 ms. 512 t1 increments were used in F1 with 32 scans per t1 increment
and composed of 2048 complex points in F2 in all experiments.
Quadrature detection was achieved in F1 by the states-time proportional
phase increment method. The NMR data were processed using Felix
program. All free induction decays were zero-filled to 2048 × 2048 before Fourier transformation, and 0° (for DQF-COSY), 70°
(for TOCSY), or 90° (for NOESY) shifted sinbell2 window
function was used in both dimensions.
PCR Cloning of the TP Receptor--
PCR cloning was used to
isolate the full-length cDNA of the TP receptor from human lung
cDNA obtained from Invitrogen. The PCR primers were designed based
on human TP cDNA with some modifications (5, 23). The primer
sequences were: 5'-CGGAATTCATGTGGCCCAACGGCAGTTC-3' (forward) and
5'-GAAGATCTCGCTCTGTCCACTTCCTACTG-3' (reverse), with EcoRI and BglII sites on the ends. The
full-length cDNA of the TP receptor was obtained from standard PCR
amplification that was performed in 50 µl of reaction mixture
containing 1 unit of Vent polymerase and buffer (New England Biolabs,
Beverly, MA), a 0.4 µM concentration of each
primer, 2 µl of human lung cDNA for 30 cycles of 98 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min. The amplified products
were isolated from agarose gel and subcloned into the
EcoRI/BglII sites of pAcSG. Correct cDNA
sequence of the receptor was confirmed by restriction enzyme digestions and DNA sequencing analysis using Sanger dideoxy chain termination method (24).
Site-directed Mutagenesis--
pAcSG-TP wild-type cDNA was
first subcloned into EcoRI/XbaI sites of
pcDNA3.1(+) expression vector. The TP receptor mutants were then
constructed using standard PCR. The procedure utilized pcDNA3.1(+)
vector with wild-type TP receptor as template and two synthetic
oligonucleotide primers containing the desired mutation for the
reaction. The primers, which were complementary to opposite strands of
the template, extended during the temperature cycling of 95 °C for
30 s, 53 °C for 1 min 30 s, and 68 °C for 13 min for a
total of 25 cycles with an additional extension cycle of 68 °C for
10 min using Pfu DNA polymerase from Stratagene (La Jolla,
CA). The mutant products were treated with DpnI endonuclease (Stratagene) to digest the parental DNA template and confirmed by DNA
sequencing. The plasmids were then prepared using Midiprep kit (Qiagen)
for the transfection into COS-7 cells for expression.
Expression of TP Receptor Wild-type and Mutants in COS-7
Cells--
COS-7 cells were cultured at 37 °C in a humidified 5%
CO2 atmosphere in high glucose Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, antibiotics, and
antimycotics. The cells placed on 100-mm dishes at a density of
1.0 × 106 were cultured overnight and then
transfected with 10 µg of purified cDNA of pcDNA3.1(+)/TP
wild-type or mutants by DEAE-dextran method. Approximately 48 h
after transfection, the cells were harvested in ice-cold
phosphate-buffered saline buffer and collected by centrifuge for
further protein determination.
Ligand Binding Assay--
Ligand binding assay for TP receptor
was performed using the method as described by Tai's group (25). The
cell pellets of 800 µg in 25 mM Tris-HCl buffer, pH 7.4, containing 5 mM CaCl2 were incubated with 3 nM [3H]SQ29,548 (30,000 cpm, 30 Ci/mol,
PerkinElmer Life Sciences) in the presence or absence of 5 µM of unlabeled (cold) SQ29,548 in the 0.1-ml reaction
volume with vigorous shaking at room temperature for 60 min. The
reaction was then terminated by adding 1 ml of ice-cold washing buffer
(25 mM Tris-HCl, pH 7.4). The unbound ligand was filtered
through an ice-cold washing buffer presoaked Whatman GF/C glass
filter (Whatman, Clifton, NJ) under vacuum. The radioactivity of the TP
receptor-bound [3H]SQ29,548 remained on the glass filter
was counted in 4 ml of scintillation mixture using a Beckman
counter (Fullerton, CA).
 |
RESULTS |
NMR Study of TP eLP2 Interacted with SQ29,548--
The
constrained synthetic eLP2 peptide (Fig.
1) mimicking the second extracellular
loop of the native human TP receptor, which showed the conformational
change upon the interaction with the receptor antagonist SQ29,548 in
the circular dichroism (CD) and fluorescent spectroscopic studies (1),
was used for the two-dimensional 1H NMR
experiments. To observe the interaction between the constrained peptide
and SQ29,548 at the atomic level, NOESY spectra for the eLP2 peptide,
SQ29,548, and the mixture of the peptide with SQ29,548 were recorded
separately under the same conditions as described under "Experimental
Procedures." Resonance assignments were made using a standard
approach (Table I) (26). To determine which residues of the constrained
TP eLP2 interact with the ligand SQ29,548, the NOESY spectra were used
to identify the intermolecular contact between the peptide and SQ29,548
(Fig. 2). The results indicate that
Val176, Leu185, Thr186, and
Leu187 interacted with SQ29,548 and predict that the
residues are involved in the TP receptor initial ligand
recognition.

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Fig. 1.
Topology model of the TP receptor. The
amino acid sequence of the second extracellular loop (eLP2)
synthesized is shown. The constrained eLP2 was synthesized with a
connection between the N and C termini by a disulfide bond using
additional homocysteine (hC) residues. eLP,
extracellular loop; iLP, intracellular loop; NT,
N-terminal region; CT, C-terminal region.
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Fig. 2.
Expanded region of TOCSY spectrum of
SQ29,548 (A), NOESY spectra of TP eLP2 peptide
(B), and the mixture of TP eLP2 peptide and SQ29,548
(C). The two-dimensional 1H NMR
spectra were recorded and assigned using the same conditions as
described under "Experimental Procedures." Three cross-peaks
((1), H2/V176HG2; (2), H7/L185HD1; H7/L187HD1,
and (3) H8/T186HG) showing the contacts between the TP eLP2
peptide and SQ29,548 are indicated in C.
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Confirmation of the NMR Experiment-based Prediction for the
Residues Involved in TP Receptor Ligand Recognition Using Recombinant
TP Receptors--
To test whether the residues Val176,
Leu185, Thr186, and Leu187
identified by the NMR experiments using the constrained eLP2 peptide
are involved in the ligand recognition for the native TP receptor, a
series of recombinant protein of the human TP receptor with point
mutation at the four residues were constructed. These four residues
Val176, Leu185, Thr186, and
Leu187 were first replaced with glycine to eliminate the
side chains of the residues. After transfection of the cDNA of the
recombinant TP receptors into COS-7 cells, the similar expression level
of the TP receptors were confirmed by Western blot (Fig.
3A). The binding of the
recombinant receptors to its ligand was then performed using
[3H]SQ29,548, and unlabeled (cold) SQ29,548 was used as a
competitive ligand (Fig. 3B). All of the mutants with
glycine replacement showed decreased or lost binding activity to the
receptor antagonist, SQ29,548, as compared with the TP wild type (Fig.
3B). These data indicate that the side chains of the
residues Val176, Leu185, Leu187,
and Thr186 of the native TP receptor are important to the
ligand binding via a direct contact in the ligand-binding site or an
indirect induced structural effect. These results also support the
conclusion based on the NMR experiments in which the four residues of
the TP receptor play important roles on the receptor ligand
interaction.

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Fig. 3.
A, Western blot of the recombinant human
TP receptors expressed on COS-7 cells. 50 µg of COS-7 cells
transfected with wild-type (TPwt) or mutant TP receptor
cDNA were subjected to SDS-PAGE and transferred onto a
nitrocellulose membrane. The membrane was probed with rabbit anti-TP
peptide antibody. The line on the left side
showed the position of TP receptor protein. B, the ligand
binding activities of wild-type and mutant TP receptors. The
[3H]SQ29,548 binding was assayed for COS-7 cell
transfected with wild-type or mutant TP receptors. Unlabeled (cold)
SQ29,548 was added as a competitive ligand for the assay. The binding
activity of wild-type receptor was considered as 100%.
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To further identify what determines the ligand binding, the four
residues were then mutated to either the same type residues, residues
with different structures, or residues with different charged (Fig.
4). Val176 was mutated to
residue Asp, Leu, or Arg. Leu185 and Leu187
were mutated to residue Ala, Asp, or Arg. Thr186 was
mutated to residue Ala, Arg, or Ser. The cDNAs of the mutated receptors were obtained using standard PCR approach and then
transfected into COS-7 cells. The expression of the recombinant
TP receptors was confirmed by Western blot (Fig.
5A). The binding of the
mutated TP receptors to SQ29,548 was shown in Fig. 5. Only one
recombinant TP receptor with a V176L mutation retained the binding
activity to SQ29,548 (Fig. 5B). All other mutants showed
significantly decreased or lost binding activity (Fig. 5,
B-E). In contrast, the control mutants of the TP receptor,
Y178W and S181T, which are highly conserved in all the prostanoid
receptors (Fig. 6), remained full binding
activities to SQ29,548 as compared with the wild-type TP receptor (Fig.
7). These results indicate that the
hydrophobic side chain of Val176 is important for the
interaction with SQ29,548. For the residues Leu185,
Thr186, and Leu187, any structural changes to
the side chain will affect TP receptor binding to its antagonist.

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Fig. 4.
The amino acid sequence of wild-type and the
type of mutated residues (bold) of the recombinant TP
receptors are indicated.
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Fig. 5.
A, Western blot of the wild-type and
mutant TP receptors on COS-7 cells. The procedure was the same as
described in the legend to Fig. 3. B-E, binding activity of
mutant TP receptors with replacement of the residues under different
consideration as shown in Fig. 4.
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Fig. 6.
Sequence alignment of the eLP2 of different
prostanoid receptors. The highlighted letters are conserved among
all of the receptors. The mutation of Y178 and S181 to W and T, which
were conserved in other prostanoid receptors, were used as control
mutants.
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Fig. 7.
Comparison of the binding activities for the
control mutants of the TP receptors. As compared with the
wild-type TP receptor, Y178W and S181T retained all the binding
activity to its ligand, SQ29,548.
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 |
DISCUSSION |
Several alignment-based mutageneses supported the proposition that
the third and seventh transmembrane domains of TP receptor were
involved in ligand binding for the prostanoid receptors (18, 23).
However, most of the residues located on the transmembrane domain are
covered by the extrarcellular domains of the receptor and the cell
membrane molecules. The initial specific ligand docking residues shall
be on the surface of the receptor molecule. Based on our molecular
modeling studies, the TP receptor ligand, SQ29,548 (about 20 Å long),
must contact the extracellular domains of the receptor before binding
to the residues on the third and seventh transmembrane domains. It is
also believed that the initial contact residues on the molecular
surface shall be specific. This hypothesis comes from our previous
study (1). The synthetic constrained eLP2 peptide of the TP receptor
has been shown to change the conformation upon the addition of the
receptor antagonist SQ29,548 using fluorescence spectroscopic studies
(1). The interaction was also supported by the separated CD
spectroscopic studies (1). These results provide the evidences that the
second extracellular loop of the TP receptor is involved in the
receptor ligand recognition. Studies from other groups also provided
evidence to support the hypothesis of the second extracellular loop
involving ligand recognition. Tai's group and Dorn II's group
suggested that extracellular loops one and two were involved in ligand
binding (25, 27), with point mutations of several cysteine residues at
these loops exhibiting no binding activity. Residues 198-205 in the
second extracellular loop of the EP3 receptor have been reported as an
essential determinant of ligand selectivity (19). However, the residues
responsible for the ligand recognition have not been thoroughly
examined. The mutations based on alignment only are controversial and
need structural information to support them. But, x-ray structure is not yet available for any mammalian G-protein-coupled receptor due to
the difficulties in crystallizing the membrane-bound proteins. The
structural bases of the ligand-specific recognition with the extracellular parts of the prostanoid receptors are not well known. So,
characterization of the ligand recognition sites on any prostanoid receptor at the three-dimensional structural level represents a key
step to reveal the specific recognition of the different prostaglandins
and thromboxane by their receptors.
In this paper, to further identify the interaction and localize the
residues within the eLP2 region responsible for the important ligand
recognition, two-dimensional NMR spectroscopy was successfully carried
out and the results showed the detail contacts between the eLP2 peptide
and the receptor antagonist (Fig. 2C). The observed ligand
recognition residues on the eLP2 peptide were further confirmed by the
site-directed mutagenesis approach for the native TP receptor (Figs. 3
and 5). The combination of the two-dimensional NMR experiments and the
NMR experiment-guided mutagenesis methods provided a quicker way to
identify the important ligand recognition site of the TP receptor. This
approach can be used to characterize the ligand binding to other
domains of the receptor.
During our preparation of this manuscript, Le Breton's group reported
a mapping of the ligand-binding site of the human TP receptor using
photoaffinity labeling and site-specific antibody probes (28). The
antibody screening revealed that inhibition of the amino acid region
Cys183-Asp193 was critical for radioligand
binding and platelet aggregation. The studies provided evidences that
the ligand interacts with amino acids within the second extracellular
loop of the TP receptor (28). It further supported our conclusion
described in this paper in which the four residues Val176,
Leu185, Thr186, and Leu187 within
the second extracellular loop are identified as important residues for
the receptor ligand recognition. In comparison, the combination of the
two-dimensional NMR experiments and the NMR experiment-guided
mutagenesis approach could give detailed structural information about
the interaction of the receptor and ligand, which could not be achieved
by other approaches, including general mutation approach, photoaffinity
labeling, and site-specific antibody screening. The agreement among our
conclusion with the photoaffinity labeling and site-specific antibody
investigation has further supported the reliability of the NMR
experiment-based mutagenisis approach used for the identification of
the ligand recognition site of the TP receptor. One of the key factors
in these studies is to design a synthetic peptide with biological
function. By using a constrained peptide to mimic the extramembrane
loops of TP receptor, we successfully identified the ligand recognition site for the receptor.
Our identification of the important residues of TP eLP2 responsible for
the contact with TP receptor ligand reported here does not exclude the
other possible ligand-binding sites reported by other groups. We
suspected that the ligand-docking site might differ from the final
ligand-binding site, because the residues important to TP receptor
ligand binding located within the transmembrane domains are conserved.
The initial docking residues of the prostanoid receptors with their
ligand shall be specific. The traditional alignment-based mutagenesis
approach may pick up some residues, which may not be involved in direct
ligand contact, but which indirectly affect the protein activity
through the change of protein conformation distantly. Nevertheless, our
proton level information for identification of the TP receptor ligand
recognition site on the extracellular domain will serve as a very
valuable tool to characterize the structure of the TP receptor ligand
docking site and understand the biological mechanism of
TXA2 binding to its receptor. In addition, it also provides
great reference information to determine the ligand-docking sites for
other prostanoid receptors, and understand the specific recognition
among the eight different prostanoid receptors. In general, the NMR
experiment-based mutagenesis approach is also suitable for
identification of the ligand recognition sites for other
G-protein-coupled receptors.
We thank Dr. Xiaolian Gao in the Chemistry
Department, University of Houston, for access to the NMR facility and
providing valuable advice on taking the NMR Spectra. We also thank
Susan Mitterling for the manuscript editing assistance.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M209337200
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