Residues Val254, His256, and Phe259 of the Angiotensin II AT1 Receptor Are Not Involved in Ligand Binding but Participate in Signal Transduction
Heliana M. C. B. Han,
Suma I. Shimuta,
Célia A. Kanashiro,
Laerte Oliveira,
Sang W. Han and
Antonio C. M. Paiva
Department of Biophysics Escola Paulista de Medicina
Federal University of São Paulo 04023062 São Paulo,
SP, Brazil
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ABSTRACT
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The role of the external third of helix VI of the
angiotensin II (AII) AT1 receptor for the
interaction with its ligand and for the subsequent signal transduction
was investigated by individually replacing residues 252256 by Ala,
and residues 259 or 261 by Tyr, and permanently transfecting the
resulting mutants to Chinese hamster ovary (CHO) cells. Binding
experiments showed no great changes in affinity of any of the mutants
for AII, [Sar1]-AII, or
[Sar1, Leu8]-AII, but
the affinity for the nonpeptide antagonist DuP753 was significantly
decreased. The inositol phosphate response to AII was remarkably
decreased in mutants V254A, H256A, and F259Y. These results indicate
that AT1 residues
Val254, His256, and
Phe259 are not involved in ligand binding but
participate in signal transduction. Based in these results and in
others from the literature, it is suggested that, in addition to the
His256 imidazole ring, the
Phe259 aromatic ring interacts with the AIIs
Phe8, thus contributing to the
signal-triggering mechanism.
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INTRODUCTION
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Over the years, a great deal of data have been collected on the
structural requisites for the potent effects of the octapeptide hormone
angiotensin II (AII: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) upon different
biological systems (for a review see Ref.1). These findings showed
that, among other features, the agonist molecule must have a Phe
residue at the C-terminal position and a free C-terminal carboxylate to
exert its biological activity. In particular, the Phe8
side-chain, although not needed for binding to the receptor, is very
important for triggering the cellular response. Thus, when
Phe8 is replaced by alanine or leucine, for instance,
high-affinity analogs with low activity are obtained, which act as
competitive antagonists of AII (2, 3).
More recently, two AII receptor subtypes (AT1 and
AT2) have been cloned and sequenced and shown to belong to
the family of rhodopsin-like G protein-coupled receptors (GPCRs),
characterized by having seven transmembrane
-helices (TM-ITM-VII)
linked by three extracellular and three intracellular loops (4). The
AT1 receptor has been shown to be responsible for most of
the known biological effects of AII, and a great deal of information
was recently reported about the importance of its amino acid residues
for ligand binding (5, 6, 7, 8, 9, 10, 11, 12, 13). As a result, some interactions have been
proposed between the AII molecule and different portions of the
AT1 receptor, as modeled with basis on the rhodopsin
seven-helix configuration (6, 14, 15). Among these interactions, it was
proposed that the
-ammonium group of Lys199, at the
external third of AT1s TM-V, is the counterion for the
C-terminal carboxylate of AII (5, 6, 7). This interaction would pull the
C-terminal His-Pro-Phe sequence of AII to a deeper position in the
locus of the receptor central cavity surrounded by helices IIIVI (6, 11). The possibility therefore arises that other residues placed at the
external third of helices IVVI, and pointing to the receptor central
cavity, might also be crucial for AII binding. Previous attempts to
confirm this hypothesis experimentally have been unsuccessful. Thus,
whereas the K199A mutant presented a remarkable loss of affinity
(increase in binding constant by at least 2 orders of magnitude)
(5, 6, 7), not more than 30-fold losses were observed in mutants modified
in residues of helices IIIVI, which are thought to be close to
Lys199 in the seven-helix bundle configuration (6, 7, 9, 10, 11). However, a single but quite significant finding was that the
H256A mutation does not affect ligand binding but impairs the signal
transduction triggered by AII through the activation of phospholipase C
(11). This finding was interpreted as indication that the
His256 side chain might be interacting with AIIs
Phe8 aromatic ring, thus stabilizing a productive
peptide-receptor complex. According to this model, the cavity
surrounded by helices IIIVI might be the receptor locus at which the
signal transduction is originated after agonist binding.
In the present work we have investigated this hypothesis by studying
the effect of point mutations in different residues of the
AT1 receptors TM-VI upon the phospholipase C response of
permanently transfected Chinese hamster ovary (CHO) cells. Our results
indicate that mutations at residues Val254 and
Phe259 of the AT1 receptor also cause
impairment of signal transduction without remarkable loss of affinity
for the agonist. These results led us to suggest that
AT1s Phe259 side chain might also be
interacting with the AIIs Phe8 aromatic ring, playing a
role that does not depend on His256 but alone is able to
elicit the receptors physiological response.
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RESULTS
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AT1 Mutants
The mutants were obtained from point mutations carried out on the
amino acid sequence S252 W253 V254
P255 H256... . F259...
F261, corresponding to the extracellular third of TM-VI of
the AT1 receptor. AT1 residues 252256 were
mutated to Ala, residues 259 and 261 were mutated to Tyr, and the
resulting mutants were permanently transfected in CHO cells. The
transfected cells were assayed for their affinity toward different
ligands and for receptor-mediated phospholipase C activation.
Binding Experiments
IC50 values were estimated from binding experiments in
which competition curves were obtained for the displacement of the
respective iodinated peptide by AII as well as by the agonist peptide
analog [Sar1]-AII, the antagonist peptide
[Sar1, Leu8]-AII, and for the displacement of
the nonpeptide antagonist DuP753 by its 3H-labeled analog.
With the exception of an increased affinity observed in the S252A
mutant, the IC50 values obtained for AII with all the
mutants were not much different from those obtained with the wild-type
receptor (Table 1
). This also occurred
with [Sar1]-AII and [Sar1,
Leu8]-AII, with the exception of the mutant F261Y, which
did not bind these two ligands. This intriguing but reproducible
finding may reflect a difference in the interaction of the
Sar1 analog with AT1s helix VI and will
require further investigation. With the nonpeptide antagonist DuP753,
significant binding impairment was observed with most of the receptor
mutants. Thus, with the exception of the P255A mutant, which showed
only an 8-fold decrease in affinity, the other mutants showed a 20- to
30-fold loss. These changes in affinity are in agreement with the idea
that the binding of nonpeptide ligands involves sites on transmembrane
helices VI and VII (16).
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Table 1. IC50 Values (nM) for
the Binding of Different Ligands to AT1 Receptors with
Single-Site Mutations in the External Third of
TM-VI
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Inositol Phosphate Responses
The cells transfected with the wild-type and mutant receptors were
stimulated with AII and their response, in terms of inositol phosphate
accumulation, was measured. Table 2
shows
that, except for V254A and H256A, which presented no detectable
response, the ED50 values obtained for the other mutants
were not significantly different from that of the wild-type receptor.
The values for the maximum effects (Emax), however, show
that, in addition to V254A and H256A, other mutants showed significant
losses in their ability to activate the inositol phosphate response.
Thus, the S252A, F259Y, and F261Y mutants had Emax values
that were, respectively, 38%, 13%, and 54% of that of the wild-type
receptor. To determine whether this diminished response could be due to
a lower receptor concentration, we used the same cell batches used for
the inositol phosphate measurements to perform saturation binding
curves with 125I-labeled AII. The maximum binding
(Bmax) and specific activity
(Emax/Bmax) values obtained in mutant and
wild-type receptors are shown in the last two columns of Table 2
. These
values indicate that the smaller capacity of the S252A and F261Y
mutants to induce response (38% and 54% of the wild type,
respectively) may be attributed, at least in part, to the smaller
receptor concentrations (
50% relative to wild type) as denoted from
the wild-type levels of specific activity. Mutants V254A, H256A, and
F259Y, however, induced wild-type-like Bmax values but
still presented a low response to AII, indicating that the activity of
these receptor species was really affected by the corresponding
mutations.
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DISCUSSION
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Our results show that the amino acid residues Val254,
His256, and Phe259, located on the external
third of TM-VI of the AT1 receptor, which are not important
for ligand binding, are involved in receptor-mediated G protein
activation. In the case of His256, this agrees with the
previous finding (11) that mutation in this residue causes diminished
inositol phosphate response without commensurate change in binding
affinity of ligands. Mutations at the 254 position have not yet been
reported, whereas at position 259 only binding studies were reported
for the F259A mutant, which showed approximately 15-fold decreased
affinity for AII (6). This is significantly larger than the 2-fold loss
of affinity found for F259Y (Table 1
), suggesting that the benzene ring
of the Phe259 side chain has contributed in part to the
free energy of receptor binding.
Noda et al. (11) attributed an important role to
His256 in the signal-triggering mechanism of the
AT1 receptor, proposing that the imidazole ring of this
residue interacts with AIIs Phe8 aromatic ring while the
peptides terminal carboxyl is salt-bridged to the receptors
Lys199 ammonium group. In addition, experimental findings
obtained with Lys199 receptor mutants show that this
residue can modulate the effect of His256 side chain on
receptor binding. Whereas the H256A mutation does not affect binding,
the loss of affinity of the [K199A;H256A] double mutant is
significantly larger than that of the K199A mutant (7), suggesting
that, in the AT1 structure, the Lys199 ammonium
group is able to keep the His256 imidazole ring at a
specific position, perhaps by forming an intramolecular hydrogen bond.
This interaction might not contribute to ligand-receptor affinity, as
shown experimentally (6, 7), but could be important to form a more
productive receptor-agonist binding mode. This might involve a
relay-like structure (17) consisting of AIIs C-terminal carboxylate,
the receptors Lys199 ammonium group, and the
His256 imidazole ring.
Table 2
shows that the F259Y mutation significantly impairs the
signaling in AT1 receptors, allowing us to suggest, in
addition to a previously drawn picture (11), that the receptors
Phe259 side chain also interacts with AIIs
Phe8 aromatic ring, but places it at a different position
than that of His256. In a schematic representation of the
AII-AT1 complex (Fig. 1
), the
His256 and Phe259 side chains are shown at the
same side of helix VI, pointing at different levels toward AIIs C
terminus and flanking the agonists Phe8 benzene ring. In
this configuration one of the His256 imidazole nitrogens
may point toward the salt bridge between the Lys199
ammonium and the AII carboxyl groups, a more favorable position for the
histidine side chain, which is more commonly found forming bridges
between hydrogen-donor and -acceptor groups than in hydrophobic
clusters (17). Inoue et al. (13) propose a model showing
also a clustering of His256, Phe259, and AIIs
Phe8 side chains. This role of Phe259 has not
yet been experimentally verified by observing the effect of nonaromatic
replacement of this residue on AT1 activation.
Nevertheless, a plausible explanation for the deleterious effect of the
F259Y mutation on signaling would be a possible interaction of the
Tyr259 phenoxyl with some other side chain which would
shift its aromatic ring to a position removed from AIIs
Phe8 and the receptors His256.

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Figure 1. A Computer-Aided Representation of the Interaction
of AIIs C Terminus with the External Third of TM-V and TM-VI of the
AT1 Receptor
Using the program WHAT IF (23), the side chains corresponding to the
AT1 receptor sequences were added to a backbone template
taken from the corresponding regions of the bacteriorhodopsin 3D
structure (24). Based on previous models (6, 7, 13), the
Lys199 (TM-V) and His256 (TM-VI) side chains
were set close to each other and at bond distances of the AIIs
Phe8 (F8) side chain and the C-terminal carboxylate. The
aromatic rings of the receptors Phe259 and the peptides
Phe8 are in close proximity, allowing a double aromatic
ring side-coupling to be admitted in the structure.
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MATERIALS AND METHODS
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Materials
125I-labeled and 3H-labeled DuP753 were
purchased from New England Nuclear (Boston, MA), and DuP753 was kindly
provided by the DuPont/Merck Pharmaceutical Co. (Wilmington, DE). The
peptides AII, [Sar1]-AII, and
[Sar1,Leu8]-AII were synthesized and labeled
with 125I (18) using the iodo-beads method (Pierce Chemical
Co., Rockford, IL) in our laboratory. Myo-[2-3H]inositol
was purchased from Amersham International (Little Chalfont,
Buckinghamshire, UK) and Dowex-AG-1 x 8 resin (100200 mesh in
formate form) from Bio-Rad (Richmond, CA).
Site-Directed Mutagenesis of AT1
Receptor
A new AT1 receptor expression vector (pAT1R-NF),
containing an epitope ("Flag") with the hydrophilic amino acid
sequence Asp-Tyr-Leu-Asp-Asp-Asp-Asp-Leu at the N terminus (19), was
generated using the expression vector pTEJ8 (20), carrying the rat
AT1A receptor cDNA and the neoR resistance
marker, which was kindly provided by Dr T. W. Schwartz. The
construction of mutants with pAT1R-NF was done with the PCR overlap
extension technique using Vent polymerase (New England Biolabs, Inc.
Beverly, MA) for amplification. For identification of mutant receptor
cDNA, the silent restriction sites introduced during the
synthesis of oligonucleotides were first verified. Later, the region of
DNA corresponding to the PCR-amplified cassette was sequenced by
Sangers dideoxynucleotide sequencing method (20).
Cell Culture and Transfection
Chinese hamster ovary cells were cultured in DMEM containing
10% FBS, penicillin, and streptomycin in a humidified atmosphere of
5% CO2 and 95% air. Cell transfection was performed by
calcium phosphate coprecipitation with plasmid DNA purified on Qiagen
columns (QIAGEN Inc., Chatsworth, CA). Resistant cells were selected
with 0.8 mg/ml geneticin (GIBCO/BRL, Gaithersburg, MD) for about 2
weeks, until antibiotic-resistant clones were obtained. The expressed
receptors were visualized by immunocytochemistry using anti-Flag M2
(Eastman Kodak Co., Rochester, NY) as primary antibody and rabbit
antimouse IgG labeled with fluorescein isothiocyanate (Sigma Chemical
Co., St. Louis, MO) as second antibody, as described elsewhere (19).
Northern blot analysis of nontransfected CHO cells showed no detectable
signal for AT1 receptor expression. After the cells were grown in the
selective medium, they were frozen in liquid nitrogen still in the
presence of geneticin. For each experiment, only one vial of the cell
stock was used, and cell amplification was carried out, in most cases
with less than eight passages. Binding experiments done with and
without geneticin in the medium showed no significant differences.
Binding Experiments
The cells were seeded at 5 x 104 cells per
well in 24-well plates, and left for 24 h at 37 C in a humidified
incubator with 5% CO2, 95% air. The culture medium was
siphoned off, and the cells were washed twice with cold PBS. They were
then suspended in cold binding buffer (50 mM Tris-HCl, pH
7.5, 120 mM NaCl, 4 mM KCl, 5 mM
MgCl2, 1 mM CaCl2, 10 µg/ml
bacitracin, and 2 mg/ml glucose) in the presence of a fixed
concentration of the radioligands [125I]AII, or
[125I-Sar1]AII, or
[125I-Sar1,Leu8]AII, or
[3H]DuP753 and of different concentrations of the
respective unlabeled compounds (1 pM to 1
µM). The plates were incubated overnight at 4 C with
gentle shaking. The binding buffer was siphoned off and the cells were
lysed with 2% NP-40 solution containing 8 M urea and 3
M acetic acid. Receptor-bound radiolabels were counted on a
-counter (Packard Instrument Co., Downers Grove, IL), and the
results were treated by nonlinear regression analysis using the Inplot
software (Graph-Pad Software, San Diego, CA) to determine kinetic
constants.
No evidence of the presence of AT1 receptors was found in
the untransfected CHO cells, since no signal was detected in Northern
blots, and no AII binding was seen in control experiments using
untransfected cells.
Inositol Phosphate Turnover
Confluent cells (12 x 106 cells) expressing
the wild-type rat AT1 receptor or mutants were cultivated
for 18 h in inositol-free medium (199 Dulbecco with
NaHCO3 supplemented with 10% FCS, 2 mM
glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin) in 3.5-ml
culture plates containing 10 µCi [3H]myoinositol
(Amersham). The cells were washed twice with Tyrode solution (137
mM NaCl, 2.68 mM KCl, 1.36 mM
CaCl2, 0.49 mM MgCl2, 12
mM NaHCO3, 0.36 mM
NaH2PO4, 5.6 mM
D-glucose) and subsequently incubated for 30 min with the
same solution containing 10 mM LiCl. AII dose-response
experiments were performed with 30-min incubation time, and the
reaction was terminated by siphoning off the medium and adding a
mixture of 1 ml 0.1 M NaOH and 1 ml of chloroform-methanol
(1:1, vol/vol). After centrifugation, 0.5 ml H2O and 0.5 ml
chloroform were added to the aqueous phase containing the inositol
phosphates, the mixture was centrifuged, and the aqueous phase was
applied to a column containing 0.5 ml of AG1-X8 anion-exchanger resin
(Bio-Rad, Richmond, CA) (21). The resin was washed three times with 5
mM myoinositol and the [3H]inositol
phosphates were eluted by adding 1 ml of 1.0 M ammonium
formate in 0.1 M formic acid. The data were analyzed by
Wilkinsons treatment (22). Maximum binding values were determined on
the same batch of the cells used in the inositol phosphate assays, by
Scatchard analyses of saturation binding curves with
[125I]AII (specific activity, 2,000 Ci/mmol).
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ACKNOWLEDGMENTS
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We are grateful to Professor Thue W. Schwartz for kindly
providing plasmids for the pTEJ8 vector.
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FOOTNOTES
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Address requests for reprints to: Antonio C. M. Paiva, Department of Biophysics, Escola Paulista de Medicina, Rua Botucatú, 862, 04023062 São Paulo, SP, Brazil. E-mail:
acmpaiva{at}biofis.epm.br
This work was supported by grants and Fellowships from the Brazilian
National Research Council (CNPq) and the São Paulo State Research
Foundation (FAPESP).
Received for publication November 17, 1997.
Revision received February 23, 1998.
Accepted for publication March 11, 1998.
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