(Received for publication, September 21, 1995; and in revised form, October 4, 1995)
From the
The acidic pharmacophores of selective ligands bind to
Lys and His
of the AT
receptor
(Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P., Graham, R. M.,
Husain, A., and Karnik, S.(1995) J. Biol. Chem. 270,
2284-2289). In this report we examine how interactions between
these residues and agonists activate inositol phosphate production in
transiently transfected COS-1 cells. [Sar
]
angiotensin (Ang II) II and [Sar
]Ang II-amide
stimulated a 5-fold inositol phosphate response from wild-type
AT
receptor. The peptide antagonist
[Sar
,Ile
]Ang II and the non-peptide
agonist L-162,313 produced a partial but saturating response.
Stimulation of wild-type receptor by [Sar
]Ang
II-amide and the mutant K199Q and K199A receptors by
[Sar
]Ang II demonstrates that AT
receptor activation is not critically dependent on the
ion-pairing of the
-COOH group of Ang II with Lys
.
The mutation of His
produced diminished inositol
phosphate response without commensurate change in binding affinity of
ligands. The His
side chain is critical for maximal
activation of the AT
receptor, although isosteric Gln
substitution is sufficient for preserving the affinity for
Phe
-substituted analogues of [Sar
]Ang
II. Therefore, AT
receptor activation requires interaction
of Phe
side chain of Ang II with His
, which
is achieved by docking the
-COOH group of Phe
to
Lys
. Furthermore, non-peptide agonists interact with
Lys
and His
in a similar fashion.
Angiotensin II (Ang II) ()is a key hormone that
influences blood pressure regulation. Two distinct classes of Ang II
receptors, AT
and AT
, mediate its
function(1, 2) . The AT
receptor is
responsible for mediating the potent vasoconstrictor effect of Ang II.
Therefore, the AT
receptor is a major target for drug
design in the treatment of hypertension, congestive heart failure, and
cardiac hypertrophy(1) . Previous structure-activity evaluation
has demonstrated that the Phe
side chain and the
-carboxyl group of Ang II are critical determinants of
angiotensin's biological potency(2) . Several non-peptide
agonists and antagonists of the AT
receptor also preserve
an acidic group attached to an aromatic function, suggesting that it is
a crucial determinant of ligand specificity(1) .
The binding
pocket of the AT receptor for various ligands is not
clearly defined. The structural model of the AT
receptor
contains seven transmembrane
-helices with three interhelical
loops on either side of the membrane. Structure-function studies of the
AT
receptor so far have indicated that the carboxyl
terminus of Ang II and the non-peptide ligands bind within the
transmembrane
domain(3, 4, 5, 6, 7, 8, 9, 10, 11) ,
but the binding of Ang II and other peptide analogues may also be
influenced by the extracellular domain of the
receptor(3, 4, 5, 6, 7, 8, 9, 10, 11) .
To identify points of interaction that are common to both peptide and
non-peptide ligands on the AT
receptor, we defined the
subsite for binding the acidic pharmacophore that is present on all
ligands(9) . We showed that the carboxyl group of Ang II,
tetrazole, and sulfonylamide groups of non-peptide antagonists bind to
the
-amino group of Lys
in the fifth transmembrane
helix of the AT
receptor. The role of this ion pair in the
activation of AT
receptor function is not clearly
established. Aumelas et al.(12) showed that the
-COOH group stabilizes the conformation of the Phe
side chain in Ang II. Therefore, the interaction of Lys
with the
-COOH group of Ang II is likely very important for
positioning the Phe
within the pocket of the AT
receptor. Molecular modelling studies indicate that Phe
of Ang II might interact with His
among other
candidate residues. (
)The functional role of His
is not demonstrated as yet. Therefore, we investigated the
following questions. Is the ion-pair interaction between Lys
and
-COOH of Ang II essential for the function of the
receptor? What is the role of His
in the AT
receptor function? The results demonstrate that the ion-pair
interaction of Ang II and Lys
is not essential for
receptor activation and that His
plays an important role
in receptor activation because it directly interacts with the Phe
side chain of the ligand.
Figure 1:
Inositol phosphate production in
COS-1 cells expressing wild-type and various mutants of the rat
AT receptor gene. Basal, shown as an open box, is
the IP production in transfected cells without stimulation by
[Sar
]Ang II and the filled box is with
stimulation by 10
M
[Sar
]Ang II. The concentration of other ligands
are 10
M Ang II, 10
ML-162,313, 10
M DUP753. The values represent mean ± S.E. of three or more
independent transfection experiments performed in duplicate for each
mutant. The level of expression of each of the mutant receptor proteins
was within 3-fold of the wild-type receptor expression. The B
values estimated per mg of total membrane
protein are as follows: wild type, 5.2 ± 0.2 pmol; K199Q, 2.8
± 0.4; K199A, 2.4 ± 0.2; H256Q, 2.1 ± 0.5; H256A,
4.3 ± 0.2.
The concentration dependence (EC = 2 ± 0.1 nM) and level of stimulation (3-
to 5-fold over the basal) of IP formation were similar to the
previously reported characteristics of AT
receptor in the
transfected COS-1 cells (Fig. 2; (8) and (10) ). [Sar
]Ang II-amide
(EC
of 73 ± 4 nM) produced nearly the same
maximal level of response, although its affinity for the wild-type
AT
receptor is 10-fold lower. Since
[Sar
]Ang II-amide could be converted to
[Sar
]Ang II because of spontaneous deamidation,
fresh stock solutions were used each time. The molecular weight of the
peptide in the assayed stock solution was confirmed by mass
spectrometry. The antagonist
[Sar
,Ile
]Ang II (EC
= 1.5 ± 0.3 nM) produced <20% of the
maximal [Sar
]Ang II response. The maximal
response to stimulation with the non-peptide agonist L-162,313
was 40 ± 5% of the [Sar
]Ang II response
with an EC
of 98 ± 26 nM, as reported
earlier(8, 10) . L-162,313 bound to wild-type
receptors with an affinity of 14 ± 3 nM (Fig. 2).
Figure 2:
Relative potency of agonists to activate
the inositol phosphate production in COS-1 cells transfected with
wild-type AT receptor gene. IP accumulation (mean ±
S.E.) is expressed as percentage of specific IP accumulation after
stimulation with 10
M
[Sar
]Ang II. The ligand concentration stimulating
50% of the maximal response calculated are as follows:
[Sar
]Ang II, 2 ± 0.1 nM;
[Sar
,Ile
], 1.5 ± 0.3
nM; [Sar
]Ang II-amide, 73 ± 4
nM; and the non-peptide L-162,313, 98 ± 26
nM. 10
M DUP does not inhibit
basal IP production in the untransfected COS-1
cells.
Figure 3:
The
agonist stimulation of the mutant AT receptors by
[Sar
]Ang II (A-C) and the
non-peptide, L-162,313 (D). The IP produced in each
case is represented as the percentage of maximum response for the
wild-type AT
receptor in parallel experiments under
identical conditions. The values (mean ± S.E.) are from three
independent experiments.
The receptor activation seems to be influenced by the
kind of side chain at position 256. In the H256A and H256Q mutants, the
maximal response produced is 20-40% of that for the wild-type (Fig. 3B). The EC for this diminished
response is 1.8 nM for H256Q and 5.4 nM for H256A.
The affinity of [Sar
]Ang II to both the mutants
(0.6 nM and 0.8 nM) is nearly identical with that of
the wild-type AT
receptor (see (9) ). The mutant
H256R, K199A/H256A, and K199A/H256R receptors were completely
defective, although they bound [Sar
]Ang II with
high affinity (K
= 0.8 nM, 3.9
nM, and 1.6 nM, respectively) (Fig. 3C and (9) ).
As shown in Fig. 3D, the
K199Q mutant is not stimulated by the non-peptide agonist L-162,313. This mutation caused an approximate 7-fold decrease
of L-162,313 binding affinity (103 ± 8 nM).
The stimulation of H256Q and the H256A mutants was diminished,
respectively, to 10 ± 5% and 18 ± 10% of the wild-type
receptor stimulation. The K of L-162,313
to mutants is 110 ± 11 nM for H256Q and 191 ± 14
nM for H256A mutants.
Figure 4:
Influence of modification of
Phe side chain of Ang II on interaction with wild-type and
His
mutants. A, relative changes in the affinity
of wild-type and the mutant H256A AT
receptor toward
analogues of Ang II carrying modification of the Phe
side
chain. The values represent (mean ± S.E.) K
/K
of two to four independent determinations. B,
inositol phosphate production in response to various analogues in
transfected COS-1 cells containing matched receptor
density.
In the H256A
mutant, the affinity loss is larger for Ala and Thr side chains at
position 8 of the Ang II than with wild-type AT receptor.
However, the affinity of the H256A mutant was increased toward the
[Trp
]Ang II analogue in contrast to a decrease of
affinity of wild-type receptor (Fig. 4A). However, the
[Trp
]Ang II-stimulated IP responses from H256Q
and H256A mutants were approximately 40% of that from the wild-type
AT
receptor (Fig. 4B).
The Phe side chain of Ang II plays a crucial role
in the activation of the AT
receptor(1, 2) . Because several weak interactions may
collectively bind the Phe
side chain, mutation-induced loss
of affinity may be difficult to measure. Thus, it was anticipated that
the Phe
binding site would be difficult to locate. We
approached this problem by initially identifying the docking residues
for the Arg
and the
-COOH group of Ang
II(9, 10) . A molecular model based on Ang II docked
to Lys
and Asp
predicted several potential
sites for the interaction of the Phe
side chain. Since
replacement of the Phe
side chain of Ang II with aliphatic
side chains such as Ile
, Ala
, and Thr
produces poor agonists without substantial change of affinity,
the replacement of the complementary interacting residue must also
produce a functionally defective receptor with no change in affinity
for Ang II. Furthermore, the Phe
side chain docking site is
likely to be proximal to Lys
because it binds the
-COOH group of Phe
. Topological location and the
functional defect caused by the His
mutations are
consistent with this expectation.
The H256Q and H256A mutants cause
only small changes in agonist affinity, but a substantial defect in IP
response (Fig. 3B). As shown in Fig. 4A, reduction of the size of the His side chain correlates with a change of binding affinity for
position 8 analogues of Ang II. For example, the bulkier side chain of
[Trp
]Ang II binds better to the His
Ala mutant receptor than to the His
Gln mutant receptor or the wild-type receptor. The Ala
,
Thr
, and Ile
analogues of Ang II lead to weaker
binding to Ala
receptor than to the His
and
Gln
receptors. Schambye et al.(11) independently observed that increase of side chain
size in a H256F mutant AT
receptor improves affinity for
[Sar
,Leu
]Ang II. The van der Waals
contacts between His
and the angiotensin position 8 side
chain appears to be a critical factor for the differences in affinity
of analogues shown in Fig. 4A. This presupposes that
direct contact of His
with the Phe
side chain
is responsible for ``transmitting'' the agonist occupancy of
the ligand pocket as a signal for receptor activation. Then one would
expect that activation of the His
Ala mutant by
Ang II should be similar to the level of activation of wild-type
receptor by Ala
, Thr
, and Ile
analogues of Ang II. Inconsistency in the observed response (Fig. 3B and Fig. 4B) is most likely
due to the involvement of more than one residue making contact with the
Phe
side chain. It is possible that several residues are
involved in stabilizing the bulkier Phe
side chain, as has
been commonly observed in protein structures(17) . However, the
interaction of Phe
of Ang II with the His
plays an important role in receptor activation. This cannot be
explained by the simple contact between them, because substituting a
Gln for His
to provide isosteric hydrogen bonding
properties also produced a 60% reduction of IP response. The histidine
side chain has the unique protonation-tautomerism enabling it to act as
a crucial bridging residue in a hydrogen-bonded network in the
activated state(18) . The Gln
side chain may be
very inefficient in this process because it lacks tautomerism. However,
the ultimate chemical basis for the function of His
needs
high resolution structural evidence, which is currently not available
for this receptor. The most significant conclusion from the present
results, therefore, is that His
is a point of contact
between agonists and the AT
receptor where the process of
receptor activation is initiated.
Because the interaction of
His with Ang II provides nearly insignificant binding
energy, it is important to understand how this crucial interaction is
achieved. Docking the
-COOH group of Phe
to the
Lys
side chain is very important for positioning the
Phe
side chain of [Sar
]Ang II. The
modification of the Phe
side chain to Ile
, for
example, reduces the affinity 2-fold, but modification of the
-COOH group reduces the affinity of both
[Sar
]Ang II-amide and
[Sar
,Ile
] Ang II-amide by
20-fold(9) . It is very likely that loss of the docking
interaction will affect positioning of the Phe
side chain.
Therefore, the effect of Lys
mutation on receptor
activation may be a direct consequence of the changes in positioning
the Phe
side chain of [Sar
]Ang II in
the mutant receptors. The effects of ligand modification and
complementary changes in the receptor confirm this.
[Sar
]Ang II-amide activated the wild-type
receptor with a rightward shift of the dose-response curve with nearly
the same maximal IP response as did the [Sar
]Ang
II. This observation is consistent with earlier bioassay results where
Ang II-amide demonstrated full potency, but at a higher concentration
relative to Ang II(2, 18) . The distance of
interaction between Lys
and the Ang II-amide compared to
Ang II must remain the same, but the modification replaces an ion-pair
interaction by a neutral hydrogen bond interaction. Therefore,
stimulation by [Sar
]Ang II does not require the
negative charge of the
-COOH group. When Lys
is
mutated to Gln
, the effect is consistent with loss of
charge-pair interaction and reduction of binding affinity (see (9) ). The decrease of maximal response (Fig. 3)
correlates with a decrease of side chain length(17) . If the
Gln
side chain forms a hydrogen bond with the
-COOH-group of Ang II, then one would expect that the Ala
mutant receptor should be poorly activated by
[Sar
]Ang II. The 60% decrease in stimulation by
the K199A mutant confirmed this. Basis for the defect is consistent
with reduction in the volume of side chain combined with loss of
hydrogen bonding ability (87 Å
versus 169
Å
)(17) . Therefore, we conclude that the loss
of van der Waals interaction in the Gln
or Ala
receptors leads to the partial agonism with
[Sar
]Ang II stimulation, presumably due to
problems with positioning the Phe
side chain.
To explain
the putative function of Lys and His
, we
propose that His
interacts directly with the Phe
side chain of [Sar
]Ang II when the
-COOH group of Phe
is bound to Lys
. It
has been demonstrated that the rotational entropy of the Phe
side chain of Ang II is restricted by its interaction with the
-COOH group(12, 19) . Therefore, interaction with
Lys
contributes to most of the binding energy without
requiring an additional contribution from the His
interaction. A concerted interaction of
-COOH and the side
chain of Phe
with the receptor may be essential for potent
activation of the receptor. This suggestion needs further confirmation,
although it provides an explanation for the defect in double mutants
where the Arg
functions as a counterion for docking the
-COOH group of Ang II(9) . Because Arg
is
utilized only for binding the
-COOH group of Ang II in the double
mutant K199A/H256R, the Phe
side chain cannot be positioned
properly, resulting in an inactive phenotype.
The poor IP response
of the two AT receptor mutants (Fig. 3) stimulated
by the non-peptide agonist L-162,313 is also associated with
no significant reduction of binding affinity. Since L-162,313
contains a sulfonylamide pharmacophore, it is likely to utilize
Lys
for docking, and His
may stabilize its
binding (see (9) ). Because L-162,313 has fewer
contacts with the receptor, loss of any one contact may significantly
affect its agonist function. Most likely, His
functions
as a counterion for the sulfonylamide moiety of L-162,313 in
the K199A and K199Q mutants. In that configuration, L-162,313
is likely to function as an antagonist, thus explaining the complete
loss of activity (see discussion on H256R mutant above, for example).
The partial defect in the H256Q and H256A mutants also suggests that
there might be direct interaction of His
with L-162,313 that is critical for receptor activation in a
fashion similar to the interaction of Ang II with His
.
Perlman et al.(8) have suggested that the molecular
interactions of the L-162,313 may differ from both peptide and
non-peptides that selectively bind to AT
receptor. The
results presented here suggest an overlap in the binding pocket for
these two agonists, at least with regard to Lys
and
His
, which also form the subsite for the
carboxyl-terminal end of Ang II. Therefore, L-162,313 may
truly be considered an analogue of the carboxyl-terminal fragment of
Ang II.
It can now be argued that Lys and His
make direct contacts with all classes of AT
receptor-specific ligands (also discussed in (9) ). The
type of interaction with these residues distinguishes agonists from
antagonists. The position of His
might be perturbed by
antagonists and agonists differently. For example, both Lys
and His
interact with the tetrazole group of
biphenyl antagonists, but only His
may interact with the
carboxyl group of imidazolyl-acrylic acid antagonists (9, 11) . Presumably, these interactions stabilize an
inactive conformation of the AT
receptor. The activating
conformation of the receptor might require specific interaction of the
acidic group with Lys
and weak electrostatic interaction
with His
, each with considerably stringent
stereospecificity. Both of these residues are conserved among all
angiotensin receptors, and His
corresponds to a
well-defined ligand-binding residue in opsins and the amine
receptors(20) . Hence, we conclude that Lys
and
His
constitute the critical components of the ligand
pocket of the AT
receptor that undergoes
stabilization-destabilization to initiate intramolecular events that
are ultimately responsible for signal transduction by AT
receptors.