From the
The structural model of AT
The octapeptide hormone Ang II
Sequential deletions of the first two residues of
the Ang II produce varying effects on AT
The
ligand binding experiments were carried out under equilibrium
conditions. Inhibitory constants were derived from competition binding
experiments using the formula K
Data for binding
studies with AT
1) The (NO
2) In the D281A receptor mutant, where
the negative charge of the Asp
3) The ratio of binding affinity of mutant receptor
to wild-type receptor for
[Sar
Collectively,
these findings suggest a direct charge-pair interaction between
Asp
We are indebted to the insightful suggestions of the
late Dr. F. Merlin Bumpus. We thank Dr. W. Greenlee of Merck Sharp and
Dohme for a generous gift of non-peptide agonist, Dr. Kunio Misono for
assistance in synthesis and characterization of peptides, Dennis Wilk
for excellent technical assistance, and Dave R. Schumick for artwork.
The assistance of Robin Lewis and Christine Kassuba in manuscript
preparation and editing is kindly acknowledged.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
angiotensin receptor
contains seven-transmembrane
-helices with three interhelical
loops on either side of the membrane. The angiotensin II binding pocket
within the receptor is not clearly defined. We showed earlier that
Lys
in transmembrane-helix-5 of the AT
receptor binds the COOH-terminal
-carboxyl group of
angiotensin II (Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P.,
Graham, R. M., Husain, A., and Karnik, S. S.(1995) J. Biol. Chem. 270, 2284-2289). We now show that His
and
Asp
, both located in the extracellular domain of the
AT
receptor, are involved in binding the
NH
-terminal Asp
and Arg
residues of
angiotensin II, respectively. The Asp
/His
interaction appears to be weak and is unlikely to be important
for agonism. But the loss of Arg
/Asp
interaction leads to partial agonism of the receptor. The action
of non-peptide agonists is not affected by Asp
mutations.
These results suggest that several independent interactions between
angiotensin II and AT
receptor are necessary for full
agonism. Since L-162,313 the non-peptide agonist of the
AT
receptor is a partial agonist that does not make contact
with Asp
, we speculate that the degree of agonism may be
increased if it is redesigned to make contacts with Asp
.
(
)
has a
variety of effects on cellular control mechanisms that influence blood
pressure regulation. Two classes of cell surface Ang II receptors,
AT
and AT
, have now been
identified
(1, 2, 3, 4, 5) . The
AT
receptor is responsible for mediating most of the known
cardiovascular effects of Ang II, including its potent vasoconstrictor
effect
(1) . The AT
receptor is a member of the
G-protein-coupled receptor
family
(2, 3, 4, 5) . In this family of
receptors the binding of small ligands occurs within the transmembrane
domain. However, for the binding of large glycoprotein hormones,
extracellular loops are employed
(6) . The nature of the Ang II
binding site is not clearly defined, but there is evidence indicating
roles for extracellular, as well as transmembrane, domains
(6) .
Previous studies have suggested that the non-peptide antagonists bind
to the transmembrane domain of AT
receptor
(7, 8, 9, 10, 11, 12, 13) .
Because they do not chemically resemble the native hormone, the
non-peptides are believed to bind to the receptor differently from
peptide ligands of the AT
receptor
(7, 8, 9, 10, 11, 12) .
We have shown that a crucial acidic pharmacophore common to both
peptide and non-peptide ligands binds to a conserved residue
Lys
in the fifth transmembrane helix of the AT
receptor and have demonstrated that the Lys
-ligand
interaction is ionic (13). Therefore, it can be postulated that the
COOH-terminal region of Ang II interacts within the transmembrane
domain of the AT
receptor. The site of binding and the
functional role of the NH
-terminal region of Ang II is,
however, unclear.
receptor binding
and agonism. Des-Asp
-Ang II (also known as Ang III) is a
full agonist analogue of Ang II which binds to the AT
receptor with a slightly reduced affinity (80% that of Ang II).
Des-Asp
,des-Arg
-Ang II binds poorly to the
AT
receptor and shows little activity, if any. Thus,
Arg
of Ang II is believed to provide distinctive structural
requirements
(1, 14, 15) . Because receptor
binding and agonism is side chain-dependent in Ang II analogues
modified at the second position, it is believed that hydrogen bonding
or charge interactions between the second Ang II residue and the
AT
receptor may be important
(14, 15) . The
site on the AT
receptor which is responsible for mediating
the interaction with the Arg
side chain of Ang II is as yet
unknown. In this report we identify the weak and the strong docking
sites of the Asp
and Arg
side chains of Ang II,
respectively, with the AT
receptor and show the importance
of the Arg
interaction in receptor agonism.
Materials
Analogues of
[Sar,Ile
]Ang II and Ang II were
synthesized and purified by the peptide synthesis core facility of The
Cleveland Clinic Foundation or obtained from Bachem.
[Sar
,Ile
]Ang II was iodinated by the
lactoperoxidase method and purified
(16) . The specific activity
of the
I-[ Sar
,Ile
]Ang
II was 2200 Ci/mmol. [
H]Losartan (42.3 Ci/mmol)
was obtained from Amersham Corp. L-162,313 was a gift from
Merck Sharp and Dohme.
Mutagenesis, Expression, and Characterization of Mutant
Genes
Details of rat AT receptor expression and
mutagenesis were described earlier
(13, 17) .
=
IC
/(1 -
[L]/K
), where IC
is the concentration of unlabeled analogue that displaces 50% of
specifically bound radioligand, [L] is the concentration of
radioligand, and K
is the equilibrium
dissociation constant of AT
receptor for
I-[Sar
,Ile
]Ang II or
[
H]losartan
(18) . The
K
values represent mean ± S.E. of
three to five independent determinations.
Inositol Phosphate Formation Studies
Transfected
COS-1 cells were labeled for 24 h at 37 °C in CO incubator with [
H]myo-inositol
(Amersham) in Dulbecco's modified Eagle's medium containing
10% bovine calf serum. After labeling, cells were washed with HBSS and
exposed with 10 mM LiCl in HBSS for 30 min. Cells were treated
with or without various concentrations of the ligand in HBSS containing
20 mM sodium phosphate, pH 7.4, for 45 min. Cells were lysed
with 0.4 M perchloric acid, and total inositol phosphate
production was measured as described previously (19). Total inositol
phosphate production is expressed as a percentage compared with the
maximum stimulation of transfected wild-type AT
receptor by
[Sar
]Ang II.
Interaction of Arg
[SarSide Chain of
,Ile
]Ang II with
the AT
Receptor Identification of Candidate Residues in the AT
Receptor Responsible for Interactions with Arg
of
[Sar
,Ile
]Ang
II-The Arg
side chain of Ang II is critical for
binding to both the AT
and AT
Ang II receptor
subtypes. Therefore, acidic residues residing in the extracellular and
trans-membrane domain that are conserved in both AT
and
AT
receptors were identified as potential candidates for
binding the Arg
side chain (). These conserved
residues are Asp
, Glu
, Asp
,
Asp
, and Asp
. These residues in the rat
AT
receptor were substituted with neutral (Ala or Gln) or
positively charged (Lys) side chains or side chains of different length
(Glu or Asp) by mutagenesis to examine the contribution of charge,
hydrogen bonding, or side chain length on Ang II binding. The wild-type
and mutant genes were transiently expressed in COS cells. Immunoblot
analysis indicated that these mutant receptor proteins were expressed
within a 20-100% range of expression of the wild-type AT
receptor. The ability of expressed receptor proteins to bind to
the peptide antagonist of
[Sar
,Ile
]Ang II and its analogues
modified at the Arg
position were analyzed by competition
binding studies. The binding affinity of the AT
receptor-selective non-peptide antagonist losartan was also
measured. Losartan is believed to mimic the COOH terminus of Ang II,
based on pharmacophore overlay studies
(1) .
receptor mutants, where the conserved
acidic residues (Asp
, Glu
,
Asp
, Asp
, and Asp
) were
individually mutated to neutral or basic residues, are summarized in
. With the exception of the D74K mutant, these neutral or
basic mutations did not significantly decrease losartan binding to the
receptor, indicating that a global change in receptor structure had not
occurred. However, because both
[Sar
,Ile
]Ang II and losartan did not
bind to D74K, it is likely that this mutation produced a global defect
in receptor folding. E185Q, E185K, D263A, and D263K mutations in the
AT
receptor had no effect on
[Sar
,Ile
]Ang II binding affinity.
Previously, Bihareau et al.(7) have shown a lack of
effect of a neutral mutation at the Asp
locus on
[Sar
,Ile
]Ang II binding affinity.
These findings suggest that Asp
, Glu
, and
Asp
do not play an important role in binding
[Sar
,Ile
]Ang II and losartan. D278A
and D281A mutations in the AT
receptor produced a 13- and
33-fold decrease in [Sar
,Ile
]Ang II
binding affinity. This disruption in
[Sar
,Ile
]Ang II binding affinity was
exacerbated in the charge reversal mutants D278K (by 7-fold compared
with D278A) and D281K (by 1150-fold compared with D281A; see
). Since charge reversal at the Arg
docking
site is expected to produce a greater decrease in
[Sar
,Ile
]Ang II binding affinity than
that produced by a neutral mutation, both Asp
and
Asp
were considered as candidate docking sites for the
Arg
side chain of
[Sar
,Ile
]Ang II. Arg
Side Chain of
[Sar
,Ile
]Ang II Binds to
Asp
of the AT
Receptor-In
order to determine if the Asp
or the Asp
side chain was the docking site for the positively charged
guanidinium group of Arg
in
[Sar
,Ile
]Ang II, the following
additional criteria were used. First, the change in binding affinity
attributed to the interaction between the positive charge of the
Arg
side chain of
[Sar
,Ile
]Ang II and the potential
counterion residue on the AT
receptor should be similar to
the decrease in [Sar
,Ile
]Ang II
binding affinity observed when the negatively charged counterion
residue is replaced by a neutral residue. Second, using an Ang II
analogue as ligand in which the positively charged Arg
is
neutralized, differences in binding affinity between the wild-type
receptor and the neutral residue mutant receptor should be minimal.
Finally, differences in binding affinity between the wild-type receptor
and the mutant receptor where a charge reversal has occurred at the
counterion site should be high using
[Sar
,Ile
]Ang II as ligand and low
using an Ang II analogue in which the positively charged Arg
is neutralized.
)Arg side chain in the
Ang II analog [Sar
,(NO
)Arg
,
Ile
]Ang II retains the hydrogen bonding ability of
Arg, but lacks the positive charge. A 37-fold decrease in affinity of
[Sar
,(NO
)Arg
,Ile
]Ang
II binding to the wild-type receptor compared with
[Sar
,Ile
]Ang II binding to the
wild-type receptor reflects a loss of binding energy which is due to
the lack of the Arg positive charge. The magnitude of this change in
affinity is commensurate with the decrease in
[Sar
,Ile
]Ang II binding affinity
between the wild-type AT
receptor and its D281A mutant
(33-fold decrease). The change for the D278A mutant was relatively
smaller (13-fold decrease).
side chain has been
eliminated, the affinity of
[Sar
,(NO
)Arg
,Ile
]Ang
II binding was only marginally different (by 3-fold) from that of
[Sar
,Ile
]Ang II binding. If
Asp
is the counterion site, this effect would be
expected, since the attractive force of the counterion would not be
present in either situation. In distinct contrast to the observations
with the Asp
mutant, the affinity of
[Sar
,(NO
)Arg
,Ile
]Ang
II binding to the D278A mutant was 162-fold lower than that of
[Sar
,Ile
]Ang II binding
().
,Ile
]Ang II was 90:1 for the
D278K mutant and 38,300:1 for the D281K mutant. This ratio for
[Sar
,(NO
)Arg
,Ile
]Ang
II was 77:1 for the D278K mutant and 354:1 for the D281K mutant
(). These findings indicate that a charge reversal of the
Asp
residue has no selective effect on the modification
of the Arg
side chain of the ligand. But, a substantial
improvement in ligand affinity was observed when the repulsive force
between Lys
and Arg
side chain of the ligand
was replaced by a neutral hydrogen bond interaction.
of the native AT
receptor and the
Arg
side chain of the ligand. In contrast, the mutants
D278A and D278K bound
[Sar
,(NO
)Arg
,Ile
]Ang
II with 58- and 77-fold lower affinities, respectively, compared with
the wild-type receptor. A general loss of binding affinity toward two
changes tested at the Asp
position (data not shown)
suggests an indirect role for this residue in ligand binding. It has
been suggested that an Arg side chain can participate in as many as
five hydrogen bonds
(20) . Therefore, a potentially weak role for
Asp
in stabilization of the Asp
-Arg
salt bridge is possible because of its close proximity to
Asp
. Properties of the Ion-Pair Interaction between Asp
and
Arg
Side Chain of
[Sar
,Ile
]Ang II-In
order to further define the flexibility of the
Asp
-Arg
interaction in terms of side chain
size and hydrophobicity, we examined two additional
[Sar
,Ile
]Ang II analogues,
[Sar
,(homo)Arg
,Ile
]Ang II
and [Sar
,Gln
,Ile
]Ang II.
A (homo)Arg
substitution at the Ang II Arg
position leads to an increase in side chain length by a single
methylene unit. The binding affinity of the wild-type AT
receptor for
[Sar
,(homo)Arg
,Ile
]Ang II
is 7-fold lower than for [Sar
,Ile
]Ang
II. A methylene unit increase in the side chain by the replacement of
Asp
with Glu
also leads to a mutant
receptor with a 23-fold reduction of
[Sar
,Ile
]Ang II affinity. The
reduction of affinity of Lys
mutant receptor was maximal
with all Ang II analogues examined. The Gln
-substituted
analogue [Sar
,Gln
,Ile
]Ang
II can interact through hydrogen bonding, but through a two-methylene
unit shorter side chain than the Arg
side chain. The
binding affinity of this analogue to the wild-type receptor was
196-fold lower than the affinity of
[Sar
,Ile
]Ang II and 4-fold lower than
[Sar
,(NO
)Arg
,Ile
]Ang
II. The ratio of affinity of
[Sar
,Gln
,Ile
]Ang II
toward the wild-type and the D281K mutant receptors is 1:550. The ratio
of affinity of
[Sar
,Gln
,Ile
]Ang II
toward the D281A and D281K mutants is 1:180. The ratio of
[Sar
,Ile
]Ang II affinity for these
mutants is 1:38,300. This indicates that the longer Lys
side chain of the receptor is better accommodated by the shorter
Gln
side chain than the Arg
side chain of
[Sar
,Ile
]Ang II. Thus, the length of
interacting side chains is important. The binding specificity is
produced by hydrogen bonding, but appears to be best mediated by a
charge-pair interaction between Asp
and Arg
of Ang II. The change in the structure of Lys
mutant receptor seems to be limited to the site of interaction of
Arg
of Ang II. For example, the defect does not propagate
to the binding site of losartan. Therefore, the huge loss of binding
affinity may reflect a strong repulsive interaction in a restricted
pocket (see Ref. 21 for other examples).
Role of Asp
[SarSide Chain in Signal
Transduction
]Ang II produced an
increase in inositol phosphate accumulation in a dose-dependent manner
with an EC
of 50 nM in COS cells transfected with
the wild-type AT
receptor gene (Fig. 1).
[Sar
,Gln
]Ang II also produced full
agonism but with an EC
of 300 nM. Because the Gln
side chain retains the hydrogen bonding potential of Arg, but lacks the
positive charge, an ion-pair interaction with the Asp
residue does not appear to be a critical requirement for the
activation of the AT
receptor. To determine if the hydrogen
bonding interaction between the Arg
side chain of Ang II
and Asp
side chain of the AT
receptor is
important for receptor agonism, we examined an AT
receptor
mutant where the Asp
was mutated to the
non-hydrogen-bonding residue Ala. The maximal inositol phosphate
response elicited by [Sar
]Ang II in COS cells
expressing the AT
receptor mutant, D281A, was 40% of that
produced in COS cells expressing the wild-type receptor. These
responses were completely inhibited by losartan. Because the expression
levels of the D281A and the wild-type receptor were similar (B
estimated for the wild-type receptor was 3-5 pmol/mg and
for the D281A mutant was 1-3 pmol/mg) the differences in the cell
surface expression of the receptor is not the basis of diminished
signaling in the D281A mutant. The decreased response likely indicates
an intrinsic loss of efficiency in ligand-mediated activation. Since
the Ala
side chain lacks hydrogen bonding ability, a
critical contact between the D281A mutant receptor and the native
ligand might be lacking. The consequence of this loss might be that the
native ligand is recognized as a partial agonist by the mutant
receptor.
Figure 1:
Inositol phosphate
formation by transfected wild-type AT receptor and the
D281A mutant. Inositol phosphate formed in response to
[Sar
]Ang
II,[Sar
,Gln
]Ang II and the
non-peptide agonist, L-162,313, in COS 1 cells transfected
with wild-type and the D281A mutant. The affinity constant (K)
of wild-type and mutant D281A receptors, respectively, for
[Sar
]Ang II is 0.32 nM and 83.2
nM, for [Sar
,Gln
]Ang II is
52.7 nM and 304 nM, and for L-162,313 is
56.2 nM and 50.4 nM.
It has been proposed that losartan and the non-peptide
AT receptor agonist L-162,313 mimic the COOH
terminus of Ang II in their binding to the AT
receptor (12,
22). This implies that L-162,313 may not make contacts with
the AT
receptor at the Arg
docking site of Ang
II. Consistent with this contention, binding of losartan and
L-162,313 is not decreased in the D281A receptor mutant
compared with the wild-type receptor (see Fig. 1legend). As has
been described by others
(12, 22) , the maximal response
to stimulation by the non-peptide agonist L-162,313 was 25% of
the maximum response elicited by [Sar
]Ang II.
Also, the maximal inositol phosphate response to stimulation by
L-162,313 was identical between the wild-type receptor and its
D281A mutant (Fig. 1). Furthermore, all of these responses were
completely inhibited by losartan. Since functional responses to
L-162,313 are not affected by the D281A mutation, the lack of
interaction of L-162,313 with the AT
receptor at
the Arg
docking site may limit its ability to fully
activate the receptor.
The
We have previously shown that
the conserved His residues in the extracellular and transmembrane
domain of AT-Carboxylate of Asp
of Ang II
Interacts with His
receptor are not involved in binding the
COOH-terminal carboxylate of Ang II
(13) . We now find that a Glu
substitution of the conserved His
specifically reduced
the binding affinity of Ang II by about 56-fold
(K
= 114 ± 26 nM).
The same receptor mutant bound [Ala
]Ang II
(K
= 6.4 nM),
[Asn
]Ang II (K
= 4.6 nM), and
[Sar
,Ile
]Ang II
(K
= 0.6 nM) with small
changes in affinity. The selective decrease of affinity for Ang II in
Glu
substitution can be accounted for by potential
electrostatic repulsion. Therefore, His
appears to be
directly involved in binding the
-carboxyl group of Asp
of Ang II. The function of the receptor is not affected in any of
the His
mutants (data not shown). The His
interaction with Asp
of Ang II presumably contributes
toward stabilization of receptor-bound conformation of Ang II, but it
is unlikely to be a key interaction governing receptor function. This
is consistent with the conclusions that Asp
Ang II
analogues (e.g. [Sar
]Ang II) may improve
affinity without altering agonistic properties
(14, 15) .
Summary and Conclusions
The most important finding described in this paper is the
interaction between Asp of AT
receptor and
Arg
of Ang II. Salt bridge and hydrogen bond interactions
involving these two side chains play an important role in receptor
agonism. However, a relatively less significant interaction between
His
of AT
receptor and Asp
of Ang
II was identified based on repulsive interaction (Fig. 2).
Assignment of these two contacts, in addition to the docking of
COOH-terminal carboxylate to Lys
(13, 23) ,
now provide definitive attachment points for modeling AT
receptor-Ang II structure. These studies are in progress. These
two points of contact between the NH
terminus of Ang II and
the extracellular domain of the AT
receptor are not
utilized by the non-peptide agonists and antagonists in binding to this
receptor. COOH terminus of Ang II and the non-peptides evidently bind
within the transmembrane domain through Lys
residue
(13, 23) . Only partial activation of the
receptor is achieved when the interaction between the Asp
side chain located in the extracellular domain and ligand is
disrupted. Detailed understanding of the mechanism of receptor
activation requires more information than is available at this time.
Nevertheless, some qualitative conclusions are justified. For example,
the non-peptide agonist L-162,313 may be a partial agonist,
because it does not interact with Asp
side chain.
Redesigning L-162,313 structure such that it can interact with
Asp
side chain might improve its potency comparable with
that of native hormone. The potency of non-peptide antagonists may also
be improved by additional contacts with the Asp
and
His
side chains. The extracellular loops of the
G-protein-coupled receptors are generally thought to be disordered and
hence are unattractive targets for rational drug design. An entirely
different possibility that is emerging for the AT
receptor
is that the extracellular loops and the NH
-terminal tail
are an integral part of the ligand binding pocket that is responsible
for binding at least two NH
-terminal residues of Ang II.
This structure is probably achieved through a combination of
extracellular disulfide bonds
(23) , hydrogen bonding, and
hydrophobic interaction
(11) in order to shield the
extracellular hormone-receptor contacts from bulk water. These findings
have important implications for model building studies, which until now
have lacked experimental data on the docking of the Ang II to the
AT
receptor
(24) .
Figure 2:
A current model depicting the interactions
between AT angiotensin receptor and Ang II. The order of
helices is based on the bacteriorhodopsin structure. The orientation of
Asp
and Arg
side chains of Ang II is based on
this report. The interaction of the Ang II COOH-terminal carboxylate
and Lys
is based on previous studies (13,
23).
Table:
Binding
affinity of wild-type and mutant receptors for peptide and non-peptide
antagonists
I-[Sar
,Ile
]Ang II, a
peptide antagonist of the AT
receptor, was used in binding
studies with all AT
receptor mutants where the affinity for
[Sar
,Ile
]Ang II was <0.1
µM. The non-peptide antagonist,
[
H]losartan, was used as the radioligand in the
mutants where the affinity for
[Sar
,Ile
]Ang II was >0.1
µM. Values are means ± S.E. of two to five
independent measurements. The residue Asp
is conserved in
all AT
receptors, but not in AT
receptors.
-D-R-V-Y-I-H-P-F-COOH);
L-162,313,
[5,7-dimethyl-2-ethyl-3-[(4-[2(n-butyloxycarbonylsulfonamido)-isobutyl-thienyl]]phenyl]methyl
imidazo[4,5,6]pyridine (22); HBSS, Hank' balanced salt
solution.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.