(Received for publication, October 17, 1994; and in revised form, November 23, 1994)
From the
Non-peptide ligands for peptide receptors of the
G-protein-coupled type are generally antagonists, except in the opiate
system. Recently, it was observed that a subset of biphenylimidazole
derivatives surprisingly possessed angiotensin-like activity in
vivo. In COS-7 cells transfected with the rat AT
receptor a prototype of these compounds, L-162,313 stimulated
phosphoinositide hydrolysis with an EC
of 33 ± 11
nM. The maximal response to the compound was 50% of that of
angiotensin II in COS-7 cells but only 3% in stably transfected Chinese
hamster ovary cells. The agonistic effect of L-162,313 was blocked by
the AT
-specific antagonist L-158,809 and was not observed
in untransfected cells. In Chinese hamster ovary cells, L-162,313 also
acted as an insurmountable antagonist of the angiotensin stimulated
phosphoinositide hydrolysis. In contrast to previously tested
non-peptide ligands, L-162,313 bound with reasonably high affinity to
the Xenopus laevis AT
receptor. In the human
receptor, the binding of L-162,313 was found to be unaffected by point
mutations in transmembrane segments III and VII, which impaired the
binding of biphenylimidazole antagonists. Substitutions in the
extracellular domains of the human and rat receptor, which impaired the
binding of angiotensin II, did not affect the binding of L-162,313. It
is concluded that a subset of biphenylimidazole compounds can act as
high affinity partial agonists on the AT
receptor. These
compounds have molecular interactions with the receptor which appear to
differ both from that of the structurally similar non-peptide
antagonists and from that of their functional counterpart, the peptide
agonist.
Non-peptide ligands have been developed in recent years for many
peptide receptors belonging to the G-protein-coupled class(1) .
In nearly all cases, these ligands are antagonists, i.e. compounds that block the function of the receptors. In the
angiotensin system, a series of non-peptide antagonists has been
described for the AT receptor, through which angiotensin II
exerts all of its important cardiovascular functions(2) .
Further chemical development of these biphenylimidazole derivatives
toward a compound with balanced affinity for the AT
and the
AT
receptor (the latter of still unknown function) led to a
series of compounds which, surprisingly, had agonistic properties in vivo. (
)Infusion of these compounds caused a
dose-dependent increase in arterial blood pressure in rats. The
increase in blood pressure in response to L-162,313, a prototype of
these compounds, could be inhibited by the non-peptide AT
antagonist L-158,809.
Thus L-162,313 represents the
first high affinity, non-peptide agonist for a non-opiate
peptide receptor.
Previously, through mutational analysis, we have
identified a series of residues in the AT receptor
important for the binding of either peptide agonist or non-peptide
antagonists(3, 4) . Angiotensin II appears to interact
with a number of discontinuously located residues in the extracellular
domains of the AT
receptor, especially in the N-terminal
extension adjacent to the first transmembrane segment (TM-I) (
)and in the C-terminal part of the last extracellular
loop(4) . These residues are believed to be in close spatial
proximity in the folded receptor structure. Mutations that affect the
non-peptide angiotensin antagonists are located in the transmembrane
segments in epitopes distinct from the binding site of the naturally
occurring peptide agonists (3, 5) in analogy with
findings in the tachykinin system (6, 7, 8, 9) . By the use of
chimeric receptors between the human and the Xenopus laevis AT
receptor, binding of a series of non-peptide
antagonists were found to be critically dependent on residues,
especially in TM-VI and TM-VII, for example Asn
in the
middle of TM-VII(3) .
In the present study we have examined
the activity of the newly developed non-peptide agonist L-162,313 in
cells stably expressing the rat AT receptor. In order to
identify residues in the AT
receptor important for the
binding of L-162,313, we have used a series of mutated AT
receptors, in which exchanges of single residues affect the
binding of either the structurally homologous non-peptide
antagonists or the functionally homologous peptide,
angiotensin II.
Figure 1:
Inositol phosphate turnover in response
to angiotensin II and L-162,313 in transfected COS-7 and CHO cells
expressing the rat AT receptor. Panel A, IP
accumulation expressed as percentage of the maximal response during
angiotensin II stimulation in transiently transfected COS-7 cells in
response to angiotensin alone (
) or L-162,313 alone (
). Panel B, IP accumulation expressed as percentage of
accumulation of [
H]inositol phosphates after
stimulation with 10
M L-162,313 in stably
transfected CHO cells. In the CHO cells the maximal response to
L-162,313 was 3.2 ± 1.0 of the maximal angiotensin response. IP
accumulation induced by L-162,313 alone (
), and by L-162,313
after pretreatment with 10
M L-158,809
(
), or with 10
M L-158,809 (
).
The structures of L-162,313 and L-158,809 are shown in the inset. Means ± S.E. for three to five experiments
performed in triplicate are shown.
The potential antagonistic
properties of the partial agonist L-162,313 were studied in the stably
transfected CHO cells. The compound inhibited angiotensin II induced
inositol phosphate accumulation in a dose-dependent manner (Fig. 2A). Not only was the dose-response curve shifted
to the right, the maximally achievable response was also reduced. A
similar phenomenon was observed for the classical diphenylimidazole
antagonist L-158,809. Thus, both L-162,313 and L-158,809 can act as an
insurmountable antagonist on the AT receptor (Fig. 2).
Figure 2:
Inositol phosphate accumulation in
response to angiotensin II and angiotensin II plus non-peptide
compounds L-162,313 or L-158,809 in CHO cells stably transfected with
the rat AT receptor. The IP accumulation is expressed as
percentage of specific accumulation of
[
H]Inositol phosphates after stimulation with
10
M angiotensin II. Panel A,
response to angiotensin II alone (
), and response to angiotensin
II after pretreatment with L-162,313 in the following concentrations:
10
M (
), 10
M (
), or 10
M (
). Panel B, angiotensin II alone (
), or after
pretreatment with 10
M L-158,809
(
), or with 10
M L-158,809 (
).
Means ± S.E. are shown for three to five experiments (eight
experiments for angiotensin II) performed in
triplicates.
Figure 3:
Competition binding curves for the rat and X. laevis AT receptors in transiently transfected
COS-7 cells. Binding is expressed as percentage of specifically bound
I-[Sar
,Leu
]angiotensin
II in the rat (
), and Xenopus (
) AT
receptor. Upper panel, angiotensin II; middle
panel, the non-peptide antagonist L-158,809; lower
panel, the non-peptide agonist L-162,313. Arrows indicate the changes in binding affinity from the rat to the Xenopus AT
receptor.
In order to identify
interaction points for L-162,313 on the AT receptor, we
used a series of receptors with point mutations known to affect ligand
binding (Fig. 4). Although L-162,313 resembles L-158,809
chemically, the binding of the non-peptide agonist was
virtually unaffected by point substitutions of Asn residues in TM-III
([Ala
]hAT
) or in TM-VII
([Ala
]hAT
and
[Asp
]hAT
, each of which impaired
the binding of the antagonist between 9- and 48-fold (Table 1, Fig. 4). The binding of the peptide agonist angiotensin II was
also unaffected by these substitutions. Thus, the receptor interaction
of the non-peptide agonist L-162,313 appears to be rather different
from that of the structurally homologous non-peptide antagonists.
Figure 4:
A serpentine diagram of the rat AT angiotensin receptor. Residues mutated in the present study are
indicated in white and black (see Table 1). Angiotensin II
binding was affected by the substitutions in the exterior part of the
receptor(4) , whereas the binding of diphenylimidazole
non-peptide antagonists was selectively affected by the substitutions
in TM-III and -VII(3) . Residues conserved among mammalian
AT
, X. laevis AT
, and rat AT
receptors are indicated in black on gray.
Residues Asn
, Asp
, and Asn
are conserved residues, which have been mutated in the present
study. Nonconserved residues are indicated in black on white.
Residues important for the binding of the peptide agonist
angiotensin II have previously been identified in the extracellular
domain of the AT receptor, discontinuously located in the
N-terminal extension (Ile
, His
,
Tyr
, and Ile
), in the first extracellular
loop (Tyr
), and in the third extracellular loop
(Asp
and Asp
)(4) . As shown in Table 1, exchange of these residues did not affect the binding of
neither the non-peptide agonist L-162,313, F
= 0.8-1.9, nor the binding of the non-peptide
antagonist L-158,809, F
= 0.5-2.8,
whereas the affinity for angiotensin II was decreased 11 to more than
1000-fold in agreement with previous results(4) . Thus, the
binding of the non-peptide agonist is apparently independent
of residues presently known to be important for the binding of the peptide agonist angiotensin II.
In the present study we have examined the activity of
L-162,313, the first non-peptide agonist outside the opiate
system, on the cloned AT receptor expressed in transfected
cells. The compound is a high affinity ligand of the AT
receptor and is a partial agonist with both agonistic and
antagonistic prop-erties. The binding of the compound is unaffected by
a series of receptor point mutations, which selectively affect the
binding of either the peptide agonists or the non-peptide antagonists.
This indicates that the three types of ligands may bind in rather
distinct ways to the AT
receptor. Apparently, the small
structural differences that change a non-peptide compound from being an
antagonist to an agonist rather significantly changes its interaction
with the receptor at the molecular level.
On the rat AT receptor expressed in CHO or COS-7 cells, L-162,313 is a partial
agonist with variable efficacy. Interestingly, in vivo the
compound was found to be a full agonist, i.e. it increased
blood pressure to the same degree as angiotensin II.
However, the response to angiotensin was rapid and of short
duration, whereas blood pressure increased rather slowly after infusion
of L-162,313 and the response was more sustained. The increase in blood
pressure caused by infusion of a hypertensive agent is a net result of
the agent itself and of counter-regulatory mechanisms. A full and a
partial hypertensive agent may thus lead to the same increase in blood
pressure, depending on the magnitude of the counter-regulatory
mechanisms. In dipsogenic assays L-162,313 is only a partial
agonist
. It is therefore likely that L-162,313 is a partial
agonist in vivo as it is in vitro, inducing only a
partial stimulation of the AT
receptors, but resulting in a
full response in blood pressure.
Mutations that affect the binding
of the non-peptide antagonists in the AT receptor are
located in a pocket rather deeply in between TM-III, TM-VI, and
TM-VII(3, 5) , whereas those affecting the peptide
agonist are located in the extracellular domains of the
receptor(4) . Surprisingly, the non-peptide agonist L-162,313
does not fit into any of these pictures. The difference in
susceptibility to receptor mutations between L-162,313 and the group of
structurally related biphenylimidazole antagonists is even more
surprising, as L-162,313 not only is an agonist but, like the
homologous compounds, can act as an antagonist on the AT
receptor too. L-162,313 is also unique among non-peptide ligands
for the AT
receptor in respect of its relatively high
affinity for the X. laevis AT
receptor (Fig. 3C). Interestingly, L-162,313 also binds with
high affinity to the AT
type angiotensin receptor. The
sequence identity between the mammalian and the Xenopus AT
receptors is 63-65% and between the human
AT
and AT
receptors,
32%(13, 14, 20, 21) . In this
respect L-162,313 is more similar to the peptide agonist angiotensin
II, which binds equally well to the AT
and the AT
receptors and with even higher affinity to the Xenopus AT
receptor. Nevertheless, L-162,313 does not appear
to bind to any of the presumed interaction points for angiotensin II
currently identified in the exterior domain of the AT
receptor (Table 1). Thus, either the three types of ligands
(peptide agonist, non-peptide agonist, and non-peptide antagonist) bind
in three rather separate fashions to their common target receptor, or
some crucial common binding epitope(s) has not yet been identified. In
this connection, we are currently systematically probing the outer
parts of the other ligand binding pockets in the AT
receptor(9) , with special focus on residues that are
common to both the AT
, the AT
, and the X.
laevis AT
receptor (Fig. 4). Identification of
the binding site for the partial agonist L-162,313 may thus provide
important knowledge on the function of the angiotensin II receptor.