 |
INTRODUCTION |
A central question in characterizing the biochemistry and action
of the octapeptide hormone angiotensin II (Ang
II)1 is the mechanism by
which the Tyr4 and Phe8 residues mediate the
biological functions of Ang II. In vivo, Ang II is an
important regulator of mean arterial pressure, water-electrolyte balance, and cardiovascular homeostasis. A clear understanding of the
molecular mechanism of Ang II activation of cell surface receptors is
necessary for the development of therapeutic agents to treat disorders
such as high blood pressure and cardiac hypertrophy. A recently
proposed model, based on NMR constraints of locked Ang II analogues,
suggests that a very large pharmacophore spanning the entire structure
of Ang II is presented to the receptor. Every other Ang II residue is
involved in receptor contact. Only the Tyr4 and
Phe8 side chains are considered agonist "switches"
because analogues of Ang II function as agonists in vivo if
the position 4 residue is tyrosine and the position 8 residue is a
phenylalanine. Modifications of Tyr4 and Phe8
in Ang II give rise to antagonists in vivo that display high affinity for the Ang II receptor but at higher concentrations elicit
partial receptor agonism (1-5).
Ang II type I (AT1) and type II (AT2) receptors
belonging to the G-protein-coupled receptor (GPCR) superfamily are
mediators of Ang II effects. AT1 receptor is necessary and
sufficient for regulating blood pressure, and activates intracellular
inositol phosphate (IP) production via coupling to a pertussis
toxin-insensitive G protein (3). Ang II-binding pocket consists of
transmembrane domain and the extracellular loops. Two salt-bridge
interactions, one between the
-COO- group of
Phe8 of Ang II and Lys199 of the
AT1 receptor and the other between the Arg2 of
Ang II and the Asp281 of the AT1 receptor, have
been assigned. These salt-bridge interactions are not critical for
receptor activation. Thus, a charge-separation mechanism described for
the light activation of rhodopsin and the agonist activation of
structurally related monoamine receptors is not a valid paradigm in the
Ang II activation of the AT1 receptor. Instead, the
interactions of the Tyr4 and Phe8 residues of
Ang II initiate the AT1 receptor activation process (6-10).
We previously obtained evidence for interaction of the Tyr4
and Phe8 side chains of Ang II, respectively, with
Asn111 and His256 residues of the
AT1 receptor (10, 11). Asn111 also plays a
critical role in stabilizing the basal "inactive" conformation of
the native AT1 receptor (11-13). His256 is
important for coupling agonist occupancy to G-protein activation (10).
However, the nature of the bonds between Ang II and the receptor that
switch the AT1 receptor to its active state conformation is
not clearly defined. In this report, we examine the hypothesis that
amino-aromatic bonding between the agonist "switches"
Tyr4 and Phe8 of Ang II and the respective
agonist switch-binding residues Asn111 and
His256 of the AT1 receptor is responsible for
initiating receptor activation. The absence of amino-aromatic
interaction should primarily affect the receptor activation process.
Analogues bearing saturated unnatural amino acid analogue
-cyclohexylalanine (Cha) substituents at the
X4 and X8 positions of
Ang II were synthesized and tested with wild-type and AT1
receptor mutants with amino acid replacements at position Asn111 or His256. Cha does not have a
negatively charged planar aromatic ring but through its saturated ring
provides nearly the same size and hydrophobicity as the aromatic rings
of Tyr and Phe. We show that Cha replacements at either position 4 or
position 8 of Ang II principally hinder ligand-dependent
activation of the receptor.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Oligonucleotides were obtained from the
oligonucleotide synthesis core facility of the Lerner Research
Institute, The Cleveland Clinic Foundation.
[Sar1,Ile8]Ang II and Ang II were purchased
from Bachem. [Sar1,Ile8]Ang II was iodinated
by the lactoperoxidase method and purified, as described previously
(8). The specific activity of the
125I-[Sar1,Ile8]Ang II was 2200 Ci/mmol. Losartan was a gift from DuPont Merck Co., Wilmington, DE.
Analogues of Ang II--
Analogues of [Sar1]Ang II
were synthesized and reverse phase high performance liquid
chromatography purified by the peptide synthesis core facility at the
Lerner Research Institute. The accuracy of synthesis was confirmed by
electrospray mass spectrometry of the pure analogues using a PE-Sciex
model AP1 III spectrometer. Concentration of the peptide in stock
solutions was estimated by molarity of individual amino acids
determined against known standards on an amino acid analyzer.
To evaluate the role of the aromaticity of Tyr4 and
Phe8 rings without a change of residue size,
-cyclohexylalanine, an unnatural analogue of Phe, was substituted at
these positions to obtain [Sar1,Cha4]Ang II
and [Sar1,Cha8]Ang II. In addition, we
synthesized three analogues of [Sar1]Ang II containing
position 4 side chain size modifications to evaluate the influence of
size; [Sar1,Phe4]Ang II serves as a control
for the size difference between Tyr and Cha.
[Sar1,di-I-Tyr4]Ang II analogue retains
the aromatic ring but bulkier than Tyr. The smaller side chain
substituted Ang II analogues, [Sar1,Ala4]Ang
II and [Sar1,Ile4]Ang II both lack an
aromatic ring. The surface area accessible for interaction with the
receptor, defined as the area accessible to a sphere of water molecule
of 1.4 Å in diameter (14), was estimated in each case as described
below. The pharmacological studies on the wild-type and mutant
receptors using various position 4 analogues is shown in Figs.
1-6.
Molecular Graphics and Estimation of Side Chain Surface
Area--
To generate the surface area values of Cha, mono-I-Tyr, and
di-I-Tyr, we modeled the different molecules using bond lengths and
angles extracted from a small molecule structure data base in an
interactive graphics program called "O" (15). We then used the
program "GRASP" to calculate the surface areas (16). The surface
area values for the natural amino acid residues are from Ref. 14.
Mutagenesis and Expression of the AT1
Receptor--
The synthetic rat AT1 receptor gene, cloned
in the shuttle expression vector pMT-2, was used for expression and
mutagenesis, as described previously (8-11, 17). Mutants were prepared
either by the restriction fragment replacement method or by the
polymerase chain reaction method, and DNA sequence analysis was done to
confirm the mutations. To express the AT1 receptor protein,
10 µg of purified plasmid DNA/107 cells was used in
transfection. COS1 cells (American Type Culture Collection, Rockville,
MD), cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum, were transfected by the
DEAE-dextran method. Transfected cells cultured for 72 h were
harvested and cell membranes were prepared by the nitrogen parbomb
disruption method. The receptor expression was assessed in each case by
immunoblot analysis (data not shown) and by
125I-[Sar1,Ile8]Ang II saturation
binding analysis.
Radioligand Binding
Studies--
125I-[Sar1,Ile8]Ang
II-binding experiments were carried out under equilibrium conditions,
as described previously (8-11, 17). For competition binding studies,
membranes expressing the wild-type receptor or the mutants were
incubated at room temperature for 1 h with 300 pM
125I-[Sar1,Ile8]Ang II and
various concentrations of the agonist or antagonist. All binding
experiments were carried out at 25 °C in a 250-µl volume.
Nonspecific binding of the radioligand measured in the presence of 10 µM
127I-[Sar1,Ile8]Ang II was
<3-5% of the total binding. After equilibrium was reached, the
binding experiments were stopped by filtering the binding mixture
through Whatman GF/C glass fiber filters, which were extensively washed
further with binding buffer to wash the free radioligand. The bound
ligand fraction was determined from the counts per minute (cpm)
remaining on the membrane. Equilibrium binding kinetics were determined
using the computer program LigandTM. The
Kd values represent the mean ± S.E. of three
to five independent determinations.
Inositol Phosphate Formation Studies--
COS1 cells (cultured
in 60-mm Petri dishes), 24 h after transfection, were labeled for
24 h with [3H]myoinositol (1.5 µCi/Petri dish),
specific activity 22 Ci/mmol (Amersham), at 37 °C in DMEM containing
10% bovine calf serum. On the day of the functional assay
(i.e. 48 h after transfection), the labeled cells were
washed with serum-free medium three times and incubated with DMEM
containing 10 mM LiCl for 20 min; agonists were added and
incubation continued for another 45 min at 37 °C. At the end of
incubation, the medium was removed, and total soluble IP was extracted
from the cells by the perchloric acid extraction method, as described
previously (8-11). The amount of [3H]IP eluted from the
column was counted and a concentration-response curve generated using
iterative nonlinear regression analysis (see Refs. 8-11 and 17 for
additional details).
 |
RESULTS |
The Experimental System--
Transiently transfected COS1 cell
model system was used for analysis as described previously (8-11, 17).
Immunoblotting experiments (data not shown) indicated that the
expression of the mutant AT1 receptors described in this
report were ±20% of the level of the wild-type receptor expression.
This level of variability in receptor polypeptide expression did not
cause significant variation in cell surface receptor numbers
(
1.4-1.6 × 105 sites/cell), which was determined
from acid-labile
125I-[Sar1,Ile8]Ang II binding in
intact cells. The Bmax estimated varied
2-fold (see Table I). Statistical
analysis of 125I-[Sar1,Ile8]Ang
II binding kinetics was best fit to a one-site model, which indicated
that a homogeneous population of wild-type and mutant receptors were
produced in COS1 cells. Affinity of the wild-type AT1
receptor to the radioligand was 0.37 ± 0.02 nM. The
Kd values for the agonist [Sar1]Ang II
and the native hormone Ang II were 0.33 ± 0.02 and 1.48 ± 0.05 nM, respectively. The measured affinities did not
change significantly in the presence of analogues of GTP, since our
membrane preparations have been EDTA washed to uncouple G-proteins.
Hence, the Kd values in Table I represent the
intrinsic affinity of the receptor in the absence of G-protein
coupling. The expressed AT1 receptor bound the nonpeptide
antagonist losartan with high affinity (Kd,
10 ± 2 nM) and did not bind the AT2
receptor-selective antagonist PD123319 (Kd
>10
5 M). The ability of the AT1
receptor to activate IP production in COS1 cells is shown in Fig.
1.
View this table:
[in this window]
[in a new window]
|
Table I
Ligand affinity of Asn111 and His256 substitution
mutants
The Kd and Bmax values represent
the mean ± S.E. obtained from three to five independent
transfection experiments performed in duplicate.
Bmax values represent total receptors present in
plasma and endoplasmic reticulum membranes. ND, not determined.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Activation of the function of the
AT1 receptor by analogues of angiotensin II. IP
production stimulated by the AT1 receptor, expressed
(A) in response to varying concentration of expression
plasmid DNA or (B) in response to the addition of varying
concentrations of [Sar1]Ang II,
[Sar1,Cha4]Ang II, and
[Sar1,Cha8]Ang II. COS1 cells were
transfected with 10 µg of expression vector in B. Three to
five dose-response experiments were performed to determine each
response curve. IP production in mock-transfected cells and in
transfected cells in the absence of ligand are significantly different,
as indicated by arrows.
|
|
[Sar1]Ang II-stimulated IP production in COS1 cells
varied with the plasmid DNA transfected (see Fig. 1A).
Expression plasmid concentrations, >4 µg of DNA/dish, did not
further increase the maximal IP produced, as well as cell surface
receptor number (see above). All IP measurements shown in this report
were carried out at 10 µg of expression plasmid DNA/dish. Therefore,
the maximal IP values given in each case truly represent the magnitude
of signal transduction and not the differences in cell surface receptor numbers. The basal IP production without [Sar1]Ang II
treatment is <5 ± 0.3% when compared with the maximal IP
response (taken as 100%) elicited by [Sar1]Ang II
concentrations >10
7 M in transfected COS1 cells.
Pharmacological Characterization of Tyr4-modified
[Sar1]Ang II Analogues--
Interaction of
[X4]Ang II analogues with the wild-type
AT1 suggests that aromaticity and the size provided by
Tyr4 in Ang II are critical for function. The affinity of
the AT1 receptor for
[Sar1,Cha4]Ang II and
[Sar1,Phe4]Ang II (the size of Phe is nearly
the same as Cha) was 230- and 2-fold lower, respectively, than the
affinity of [Sar1]Ang II (see Fig.
2A), indicating that the
aromatic ring is an important determinant of affinity. Size restriction
in the interaction of aromatic group with the receptor is evident from
328-fold reduction of affinity for
[Sar1,di-I-Tyr4]Ang II (with an
40-Å
increase of accessible area). It is known that the addition of iodine
atom on the Tyr4 ring alters the interaction with the
receptor. [Sar1,mono-I-Tyr4]Ang II is a full
agonist with slightly improved affinity, while [Sar1,di-I-Tyr4]Ang II is a partial agonist
with lower affinity. Samanen et al. (18) and Guillemette
et al. (19) also reported that different halide
modifications of the position 4 aromatic ring of Ang II increase the
electronegativity associated with a reduction of affinity for the
receptor. The 458- and 710-fold reduced affinity, respectively, for
aliphatic amino acid-substituted analogues
[Sar1,Ala4]Ang II and
[Sar1,Ile4]Ang II is also consistent with
size and aromaticity constraints.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
A, effect of accessible surface area of
residues substituted for Tyr4 in [Sar1]Ang II
on binding to the AT1 receptor. The affinity values for
125I-labeled [Sar1,Ile8]Ang II
and the position 4-modified analogues of [Sar1]Ang II are
calculated from saturation and competition binding curves,
respectively. The accessible surface area of natural amino acid side
chains are as defined and derived from Creighton (14). The surface area
values of unnatural analogues were calculated as described under
"Experimental Procedures." The values given are an average of three
to five determinations. The S.E. (<8%) is not shown. B,
effect of affinity changes on maximal IP response from the
AT1 receptor.The maximal IP response values elicited by
[Sar1]Ang II and Ang II are identical; this was used as
100%. The values shown are average ± S.E. of three to five
independent determinations in each case.
|
|
The binding energy contributions for the interactions were calculated
as described in legend to Table II. The
energy of interactions indicates that the aromatic ring in
Tyr4 contributed
2.8 kcal mol
1, whereas
the hydroxyl group in Tyr4 contributed
0.4 kcal
mol
1 and the surface area (based on comparison between
Cha4 and Ile4) accounted for
0.6-1.0 kcal
mol
1 (see Table II).
View this table:
[in this window]
[in a new window]
|
Table II
Activation of AT1 receptor by different types of interactions
between [X4] of ligand and [X111] of the receptor
The  G value for each interaction was calculated using
the formula  G = [ Gmut GWT] = [ RT ln
(Kd(mut)] [ RT ln
(Kd(WT)] as described previously (8).
|
|
Data in Table II indicate that AT1 receptor activation is
specifically hindered by Cha4 substitution in Ang II. The
[Sar1,Cha4]Ang II analogue partially
activated (48 ± 5%) the receptor function at concentration
1320-fold > Kd (see Fig. 1). In contrast, the
maximum IP production stimulated by
[Sar1,di-I-Tyr4]Ang II,
[Sar1,Ala4]Ang II, and
[Sar1,Ile4]Ang II was >80 ± 8% of
that produced by the [Sar1]Ang II. The maximal IP
stimulation by [Sar1,Phe4]Ang II was 81 ± 8%, but the potency was 100-fold greater. The reduction (
20%)
of maximum IP displayed [Sar1,Phe4]Ang II
could not be recovered in any other combination, implying that the
hydroxyl group of Tyr4 in Ang II is crucial for activation
(see below). Taken together, these observations indicate that
activation of receptor by [X4]Ang II analogues
is not directly dependent on their binding to the AT1
receptor (Fig. 2B).
Effect of Size Substitution of Asn111 on Interaction
with Different [X4]Ang II Analogues--
Interaction of
the [X4]Ang II analogues with the residue 111 mutant receptors suggests that the interaction between the
Asn111 of the AT1 receptor and the
Tyr4 of Ang II is a side chain size-dependent
amino-aromatic bonding, which is essential for effective
hormone-receptor coupling (Fig. 3). Amide
group bearing substitution mutants N111Q and N111H bound [Sar1]Ang II and [Sar1,Phe4]Ang
II with 1.3-fold better affinity when compared with the wild-type AT1 receptor. These mutants also bound
[Sar1,Cha4]Ang II and
[Sar1,Ile4]Ang II analogues with slightly
improved affinity over that of the wild-type receptor. The N111K mutant
affinity for these analogues is comparable to that exhibited by the
wild-type receptor (Fig. 3).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Influence of different position 111 mutations
on the affinity and activation by various
[X4]Ang II analogues. The side
chain size in each case is drawn to scale using Chem
Office®. The values shown in each case are:
Kd (nM; top row),
relative activation (% IP; middle row) and the
EC50 (nM, bottom row).
The values are derived from concentration-response curves in which
ligand concentrations were varied from 10 10 M
to 10 4 M. The IP response produced by the
wild-type receptor by 10 µM [Sar1]Ang II
(see Fig. 1 for cpm values) was taken as 100%. In each experiment,
this control was included for comparison and values are normalized for
variations in IP formation in basal and mock-transfected cells.
Relative activation value close to 100% indicates that activation of
the mutant by the analogue and [Sar1]Ang II are nearly
identical. Note that substitution with smaller residues, such as Gly.
Ala, Ser, and Cys for Asn111, gives rise to a partially
activated receptor that binds almost all Ang II analogues with high
affinity, and maximal stimulation of these mutant receptors requires
Ang II, but not its agonist side chains. Hence, those mutations are not
included in the current analysis (32).
|
|
In contrast, the affinity of [Sar1]Ang II was decreased
1.6-, 5.1-, and 3.3- fold, respectively, in the N111I, N111F, and N111Y mutants. Substitution of a larger residue at position 111 that did not
provide an amide group cost 0.2-1.2 kcal mol
1 more
energy in binding Tyr4. The
[Sar1,Cha4]Ang II and
[Sar1,Ile4]Ang II analogues interacted with
these three mutants with significantly higher affinity, perhaps through
hydrophobic interaction restoring specificity. Most importantly,
magnitude of increased affinity of the N111I mutant was consistent with
an interaction dependent on size and hydrophobicity. These results
further substantiate our previous proposal that Asn111 in
the middle of transmembrane helix III of the AT1 receptor interacts with Tyr4 of Ang II (11).
The gain of affinity, however, is not accompanied by gain of function
in most bonding combinations set up between Ang II position 4 analogues
and receptor mutants (see Fig. 3) to evaluate the critical requirement
for amino-aromatic interaction in signal transduction. The maximal IP
production stimulated by [Sar1]Ang II and
[Sar1,Xaa4]Ang II in most of the mutants
shown in Fig. 3 was
50% reduced in comparison to the wild-type
AT1 receptor. In several combinations the binding
interaction was preserved, for instance
Tyr4-Gln111,
Tyr4-His111,
Tyr4-Lys111,
Tyr4-Ile111,
Phe4-Gln111,
Phe4-His111, and
Phe4-Ile111, but the activation was inadequate
(see Fig. 3). The Kd and EC50 in these
ligand-receptor combinations were close to (<2-fold) that of a
wild-type situation, but the maximal IP response was impaired. Thus,
the increased size of the residue 111 affected receptor activation much
more than the binding affinity (Kd) and potency
(EC50). An exception is the
Phe4-Tyr111 interaction, in which the measured
Kd, EC50, and maximal IP values are
comparable to that of Phe4-Asn111 interaction.
The reasons for this are unknown.
Reversal of Amino-aromatic Interaction--
An efficient
functional interaction is reproduced by substitution of an Asn residue
at position 4 in [Sar1]Ang II and a Phe residue at
position 111 in the receptor (see Fig.
4, also see Table II). The binding
affinity of [Sar1,Asn4]Ang II for the
wild-type AT1 receptor is 230-fold reduced. High affinity
for [Sar1,Asn4]Ang II was regained in the
N111F (Kd = 0.49 ± 0.05 nM) and
N111Y (Kd = 1.8 ± 0.2 nM) mutant
AT1 receptors. The most impressive effect of the reversal
of the bonding was gain of function: the IP production stimulated by
[Sar1,Asn4]Ang II in the N111F receptor was
identical (maximum = 79 ± 6% and EC50 = 3.8 ± 0.7 nM) to that elicited by
[Sar1,Phe4]Ang II in the wild-type receptor.
Although the maximum IP production stimulated by
[Sar1,Phe4]Ang II in N111F mutant receptor
was nearly the same, the EC50 was distinct (see Fig.
4C). Rather surprisingly, in the N111Y mutant, the IP
stimulation by [Sar1,Asn4]Ang II did not
reach the wild-type level. Activation of function was impaired in this
mutant in response to several other Ang II analogues as well. Reasons
for this discrepancy are not known. Molecular dynamic simulations
indicate that Tyr111 hydroxyl group is oriented toward
transmembrane domain II (Asn69 is 2.1 Å from
Tyr111 hydroxyl group), and may have interfered with
interactions between transmembrane helices II, III, and VII. N111F does
not have this effect, and with regard to the aromaticity Phe and Tyr
are equivalent. This might partly explain lower IP stimulation in the
N111Y mutant.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 4.
Reversal of amino-aromatic interaction.
A, relative affinity of various mutants to select
[X4]Ang II analogues. The affinity of
[Sar1]Ang II for the wild-type receptor (0.33 nM) is used as a common denominator to calculate -fold
affinity. B, activation by the same analogues. Note that the
maximal IP production in the Phe4-Phe111
combination (79%) is comparable to that obtained with
Phe4-Asn111 and
Asn4-Phe111 combinations (81%). However, the
Kd value for Phe4-Phe111
combination is 4-fold higher than that for the
Phe4-Asn111 and
Asn4-Phe111 combinations. C, shift
in the potency of [Sar1,Asn4]Ang II at the
Phe111 mutant compared with that of the wild-type
receptor.
|
|
In addition we examined several additional analogues. Binding of
[Sar1,Gln4]Ang II to the N111F
(Kd = 24 nM) mutant was not a high affinity interaction and was not associated with a gain of function. The affinity of N111F, N111Y, and N111K mutants toward
[Sar1,Asp4]Ang II was 91, 20, and 468 nM and toward [Sar1,Glu4]Ang II
was 290, 198, and 2980 nM, respectively. These observations suggest that providing an electronegative group at the
X4 position is insufficient. The fact that
Asn111/Phe4 and
Phe111/Asn4 combinations produced identical
affinity and potency demonstrates that an amino-aromatic interaction is
essential for the fidelity of AT1 receptor-Ang II coupling.
The wild-type receptor interaction with Ang II yields
20% more IP,
suggesting that the critical interaction provided by the
Tyr4 hydroxyl group is not mimicked in
Asn111/Phe4 and
Phe111/Asn4 combinations. If conventional
electrostatic/hydrogen bonding occurred between Asn111 and
Tyr4, our analysis would have been expected to restore
affinity and potency (equal to that of the wild-type receptor) in
several different combinations.
Pharmacological Characterization of Phe8-modified
[Sar1]Ang II Analogues--
Substitution of
Cha8 for Phe8 did not significantly affect the
binding affinity, but reduced the level of AT1 receptor
activation (Table I, Fig. 5). The maximal
IP response elicited by [Sar1,Cha8]Ang II was
reduced by 60 ± 5%, which appears to be caused by diminished
AT1 receptor activation by the bound analogue. Size alteration at the Phe8 position has a nearly insignificant
effect on binding. The change of Kd resulting from
the substitution of Phe8 with Gly8,
Ala8, Thr8, and Ile8 was within
6-fold. Replacement with Glu8 and Trp8 produced
lower affinity, 89 ± 9 and 13.4 ± 2 nM,
respectively, indicating that charged groups and large hydrophobic
groups are not accommodated at the putative Phe8 binding
site on the receptor. Preservation of the aromatic character, as in
[Sar1,Trp8]Ang II, yielded nearly full
receptor activation, suggesting that the reduction of binding affinity
at the Phe8 binding pocket did not affect the ability to
activate. Thus, the aromaticity of Phe8 is required for
receptor activation, but essentially plays no role in the
receptor-binding step.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
AT1 receptor binding and
activation by Phe8-modified analogues of Ang II. The
IP response values shown are obtained at identical levels of
receptor/cell. The maximal response (100%) shown is at 10 µM concentration of each ligand is unaffected by
increases in ligand concentration. Loss of aromaticity leads to
diminished stimulation. An apparent linear correlation between the
residual IP stimulation and the surface area of the side chain at
position 8 of Ang II was observed. The Kd value of
[Sar1,Glu8]Ang II was 89 ± 8 nM is not shown in the figure because it is outside of the
scale.
|
|
Effect of Different Substitutions of His256 on
Activation by Ang II--
Ang II-Phe8-mediated functional
activation is specifically and uniquely dependent on His256
(10). Substitution of His256 did not affect Ang II binding
affinity, but caused receptor to lose the ability to be activated by
Ang II. We examined which residue combination would mimic the type of
functional interaction between Phe8 and His256,
employing three substitution mutants of His256 (Fig.
6). The binding affinity of H256A, H256Q,
and H256Y mutants for [Sar1]Ang II was within 2-fold of
the binding affinity for the wild-type. The receptor activation was
reduced
60% in the H256Q and
70% in the H256A mutant receptors.
In contrast, substitution of His256 with a Tyr (H256Y) led
to a
6% reduction of maximal IP response. Activation by the
[Sar1Cha8]Ang II was relatively less affected
by various mutations (see Fig. 6). The
[Sar1Cha8]Ang II evoked
40% response in
the wild-type receptor,
52% in the H256Y mutant,
35% in the
H256Q mutant, and
28% in the H256A mutant receptor. The most
surprising outcome in this analysis was the ability of a tyrosine
substituted for His256 to display full activation, which
suggests that a stacking interaction involving the aromatic rings of
His256 and Phe8 might be responsible for
AT1 receptor activation.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of different substitutions of
His256 on binding and activation by [Sar1]Ang
II and [Sar1,Cha8]Ang II.
|
|
 |
DISCUSSION |
Like a large variety of hormone GPCRs, the molecular mechanism
involved in Ang II-mediated AT1 receptor activation remains unclear. Some insights into the mechanism have come from
structure-function studies and the discovery of mutations that uncouple
receptor activation from agonist binding (8-13, 20-22). Two most
important such mutations in AT1 receptor affect amide group
bearing residues, Asn111 and His256, both
interact with the agonist switches, Tyr4 and
Phe8 of Ang II. Since aromatic rings are able to
participate in amino-aromatic bonding (23-25) in addition to the
obvious hydrophobic, hydrogen bonding and van der Waals interactions
because of an accessible center of negative charge (from the
electrons), our goal was to distinguish potential bonding
interactions of Tyr4 and Phe8 in Ang II.
Because the information available regarding Ang II function was
obtained from in vivo functional studies prior to the
cloning of the receptor, and also before radioligand binding assay came
into routine use (5), our study is justified. Primary comparison
involved Cha-substituted Ang II analogues but the aliphatic-substituted analogues served mostly to calibrate size effects. Cha lacks a negative
charge, lacks hydrogen bonding potential, and provides hydrophobicity.
The volume effect from Cha modification is not significant because the
space-filling models indicate that the overall sizes of chair and boat
configurations of the cyclohexyl ring and the planar configuration of
aromatic ring are substantially similar (23). Since the functional
significance of the highly directional and significantly attractive
interaction between amide groups of Asn, Gln, His, and Lys and the
aromatic rings of Tyr, Phe, and Trp has been questioned in several
proteins (23-25), we evaluated whether the amino-aromatic bonding
interactions are essential in Ang II-AT1 receptor coupling.
Our results indicate that the aromaticity of both Tyr4 and
Phe8 is crucial, but an amino-aromatic bonding operates at
the Tyr4 switch of Ang II and a stacked rather than an
amino-aromatic interaction appears to operate at the Phe8
switch locus.
The complex role of Tyr4-Asn111 amino-aromatic
bonding in Ang II- AT1 receptor coupling is suggested by
the observation that both affinity and potency of receptor activation
is recreated by reversal of interaction (Fig. 4), but not in several
other combinations that restore binding affinity. Functional equivalent
of this bonding is recapitulated in Phe4-Phe111
Phe4-Tyr111, and
Tyr4-Tyr111 interactions. An edge-to-face
aromatic-aromatic interaction that might be operating in all three
instances would be similar to an amino-aromatic interaction based on
past examples (23, 25-27). The enthalpic contribution of this
interaction to Ang II binding is estimated at 3.2 kcal/mol (see Table
II) which is comparable to the estimated energy (3.3 kcal/mol) of
amino-aromatic hydrogen bonds in the protein structure data base (26).
This interaction is superimposed with size and hydrophobicity
constraints. For instance, the reduction in binding affinity resulting
from Tyr4
Ile4 change in
[Sar1]Ang II could be overcome by substitution of
Asn111 in the receptor with larger hydrophobic residues
Ile, Phe, and Tyr but not by larger hydrophilic residues Lys, Gln, and
His (Fig. 3). Increasing the accessible surface area from
160 Å (Asn111) to
180 Å (Gln111), and
195 Å (His111) appeared to increase affinity for
[Sar1]Ang II, and
[Sar1,Phe4]Ang II. The gain of affinity in
both instances, however, is without gain in receptor activation,
indicating that Tyr4 is the most complex switch in Ang II
because the receptor-binding and agonism-specifying elements are
structurally integrated. The size chiefly influences the
Kd, but receptor activation requires stringent
conservation of size and aromaticity.
The binding and agonism-specifying elements are structurally separate
in the Phe8 switch. The
-COO
in
Phe8 is the docking group and the benzyl-alanyl moiety is
the agonist switch, modifications of which generated potent antagonists
without compromising the binding affinity (also seen in Fig. 5).
Classical structure-activity relationship studies portrayed that any
modification of the Phe8 that prevents the planar
arrangement of benzyl side chain over the
Pro7-Phe8 amide bond and the
-COO
group will disrupt agonist potential (1, 2, 5). Such modifications include aliphatic substitutions, and aromatic groups in
D-configurations (D-Phe,
-MePhe) or with
bulky ring substitutions (Trp, Ind) (1-6). These evidences indicate
that rigid planar configuration is a restriction in the interactions of
Phe8 switch with the receptor. Proposed stacking
interaction between the aromatic rings of His256 and
Phe8 is consistent with this requirement. When
His256 is replaced with isosteric Gln or of
Phe8 with isosteric Cha, an inefficient hormone-receptor
coupling occurs because Gln and Cha do not participate in the planar
stacking interaction with an aromatic ring. Since the
Tyr256 mutant is fully active, this suggests that the
His256-Phe8 interaction is not an
ion-quadripole type of interaction. In the protein structure data bank,
stacked geometry is far more abundant for His-Phe interactions than
typical amino-aromatic interactions (29). A true stacking interaction
(with 1/r6 dispersion force between two
strategically placed planar rings nearly parallel) is a weaker
interaction than an amino-aromatic interaction, consistent with the
insignificant contribution of Phe8 to the binding affinity
of Ang II. Underwood et al. have previously suggested an
aromatic stacking interaction model for AT1 receptor agonism involving the nonpeptide agonist L-162,313 (30).
Evidences suggest that His256 makes contact with
AT1 receptor specific non peptide antagonists also (10,
31). Therefore, we propose that actuation of the His256
switch of the receptor is a common step for AT1 receptor
activation by both peptide and nonpeptide agonists and the site for
antagonist action. Preservation of planar benzyl imidazole or benzyl
acrylic acid ring structure is crucial in these ligands, suggesting
that a stacking interaction with His256 is likely in the
mechanism of receptor antagonism as well.
Based on these results, we speculate that the functional coupling of
Ang II binding to receptor activation requires a structural coupling
through stringent bonding between the agonist switches and respective
switch-binding residues. In the wild-type receptor, because
Asn111 is smaller, a conformational change might be
required to facilitate the amino aromatic
Tyr4-Asn111 interaction. The proposed
conformational change is the basis for efficient activation of
function. Ang II binding, in all likelihood, does not trigger a similar
conformational change in the N111Q, N111H, N111K, N111I, and N111F
mutants, which forms the basis for defect. Although an amino-aromatic
bonding is expected in N111Q mutant (also N111H), the defect in IP
formation suggests that one methylene unit increase in size is
detrimental and perhaps uncouples binding and activation. In contrast,
the stacking Phe8-His256 interaction is not
accompanied by a large conformational change, but likely exposes sites
within the receptor for the crucial interactions. Numerous studies have
described partial agonist effects of modifying Tyr4 and
Phe8 in Ang II. For the most part these reports indicated
that conservation of these two side chains is critical for Ang II
function. Our results extend these observations in that highly
efficient receptor activation of Ang II analogues is lost, leaving high
affinity binding intact, suggesting the analogue-receptor docking
occurs, but due to lack of the required bonding interactions,
functional response of the receptor is incomplete. This finding has
important implications for drug discovery.
 |
CONCLUSIONS |
Different roles played by the agonist switch residues
Tyr4 and Phe8 of Ang II in the AT1
receptor activation are pointed out. The conclusion that two critical
coupling contacts between Ang II and the AT1 receptor
involve the transmembrane helices III and VI of the receptor suggests
that the receptor activation by Ang II may involve motion of these
helices, a phenomenon also observed in other GPCRs (28). Conformational
changes induced by the two distinctly different types of bonding
interactions could orient helices and loops on the receptor so as to
enhance the efficiency of receptor-G-protein coupling. Such
conformational switching could also serve as the basis for agonist
potency because the ability of a full agonist analogue to properly
align helices far exceeds that of partial agonist analogues. The
working model suggested is based on the assumption that different
peptide analogues induce non-identical conformation of the activated
receptor that lead to differences in the kinetics and the magnitudes of
responses. Whether this induced-fit mechanism is unique to the
subfamily of peptide hormone GPCRs or is a more general mechanism that
has not been considered in prototypical GPCRs is unclear at present.