©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Non-peptide Angiotensin Agonist
FUNCTIONAL AND MOLECULAR INTERACTION WITH THE AT(1) RECEPTOR (*)

(Received for publication, October 17, 1994; and in revised form, November 23, 1994)

Signe Perlman (1) Hans T. Schambye (1) Ralph A. Rivero (2) William J. Greenlee (2) Siv A. Hjorth (1) Thue W. Schwartz (1)(§)

From the  (1)Laboratory for Molecular Endocrinology, University Department of Clinical Biochemistry, Rigshospitalet 6321, Blegdamsvej 9, DK-2100 Copenhagen, Denmark and the (2)Department of Exploratory Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(1) 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(1)-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(1) 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(1) 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.


INTRODUCTION

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(1) 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(1) and the AT(2) receptor (the latter of still unknown function) led to a series of compounds which, surprisingly, had agonistic properties in vivo. (^1)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(1) antagonist L-158,809.^1 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(1) 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(1) receptor, especially in the N-terminal extension adjacent to the first transmembrane segment (TM-I) (^2)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(1) 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(1) receptor. In order to identify residues in the AT(1) receptor important for the binding of L-162,313, we have used a series of mutated AT(1) 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.


EXPERIMENTAL PROCEDURES

Peptide and Non-peptide Ligands

Angiotensin II and [Sar^1, Leu^8]angiotensin II were purchased from Peninsula Laboratories. The non-peptide compounds L-162,313 and L-158,809 were synthesized as described(10, 11) . The radiolabeled non-peptide antagonist I-L-735,286 (12) was kindly provided by Drs. N. J. Brenner and H. D. Burns (Merck Research Laboratories, West Point, PA).

Receptor Mutagenesis

The rat AT(1) receptor cDNA (13) was generously provided by Dr. T. J. Murphy (Emory University, Atlanta, GA) and the human AT(1) receptor cDNA (14) by Dr. D. J. Bergsma (SmithKline Beecham, King of Prussia, PA). A ``cassette'' gene was initially generated from the rat receptor cDNA(4) . Mutations were introduced by the PCR overlap extension technique as described previously(4, 15) . The full-length PCR fragments were digested with appropriate restriction enzymes and subsequently inserted into the likewise digested expression vector pTEJ8(16) . PCR reactions employed Pfu polymerase (Stratagene), using the reaction conditions recommended by the manufacturer. Temperature cycling consisted of 30-35 cycles at 94 °C for 1 min, 45-50 °C for 1 min, and 72 °C for 1 min. All receptor constructs were initially identified by the presence of a diagnostic restriction site, and subsequently verified by dideoxynucleotide sequencing (Sequenase kit, U. S. Biochemical Corp.).

Cell Culture and Transfections

Expression plasmids containing wild type and mutated AT(1) receptors were transiently transfected into COS-7 cells by the calcium phosphate precipitation method(17) . CHO (Chinese hamster ovary) cell clones stably expressing the AT(1) receptor or the substance P (NK(1)) receptor were established and the cells grown as described(17) .

Phosphoinositide Turnover

1-2 times 10^6 CHO cells or 0.5 times 10^6 COS-7 cells expressing the rat AT(1) receptor were cultivated for 24 h in inositol-free media (RPMI 1640, supplemented with 10% fetal calf serum, 2 mM glutamine, and 0.1 mg/ml gentamicin) in six-well plates, each well containing 5 µCi of myo-[^3H]inositol (Amersham Corp.), as described(8) . The cells were subsequently washed twice with buffer (20 mM Hepes, 140 mM NaCl, 5 mM KCl, 10 mM MgSO(4), 1 mM CaCl(2), 10 mM glucose, pH 7.4) and incubated for 30 min at 37 °C with the same buffer including 10 mM LiCl. In experiments with antagonists, these were added 30 min prior to stimulation with the agonists. After incubating for 1 h, the reaction was terminated by adding 0.5 ml of 10% perchloric acid and precipitated protein was removed by centrifugation. The supernatants were neutralized using a 50/50 (v/v) mixture of Freon and tri-n-octylamine(18) , after which 2 ml of water was added, and incubated for 30 min with 0.5 ml of an anion exchanger, Bio-Rad AG 1-X8 resin(19) . The resin was washed three times with 5 mMmyo-inositol, and the generated [^3H]inositol phosphates were eluted by adding 1 ml of 1.0 M ammonium formate in 0.1 M formic acid.

Binding Experiments

Monoiodinated I-[Sar^1,Leu^8]angiotensin II was prepared by the IODO-GEN method and purified by reversed-phase high performance liquid chromatography, using a gradient from 17 to 29% acetonitrile as described(3) . One day after transfection and 24 h prior to the binding experiments, the transfected cells were transferred to 6-, 12-, or 24-well culture plates, with 0.1-3.5 times 10^5 cells/well, aiming at a total binding of 5-10% of the radiolabeled peptide or non-peptide. The cells were washed twice with buffer (25 mM Tris, 5 mM MgCl(2), 140 mM NaCl, pH 7.4) before and after the binding. The binding was carried out for 24 h at 4 °C with 50 pM either I-L-735,286 or I-[Sar^1,Leu^8]angiotensin II and variable amounts of unlabeled non-peptide or peptide ligands in 0.5-1 ml of binding buffer without NaCl. Data were analyzed by computerized nonlinear regression analysis using INPLOT 4.0 (Graph-Pad Software, San Diego).


RESULTS

Phosphoinositide Turnover

L-162,313 increased inositol phosphate accumulation in a dose-dependent manner with an EC of 33 ± 11 nM and 13 ± 6 nM in, respectively, CHO and COS-7 cells transfected with the rat AT(1) receptor (Fig. 1). In the COS-7 cells, the maximal response to stimulation with L-162,313 was 50 ± 7%, and in the CHO cells 3.2 ± 1.0% of the maximal angiotensin II response. In both cell systems a tendency to a bell shaped dose-response curve for L-162,313 was observed with a maximal response around 100 nM. The AT(1)-specific non-peptide antagonist L-158,809 was able to block the L-162,313 response, indicating that the observed inositol phosphate accumulation was mediated specifically through the AT(1) receptor (Fig. 1). This was further substantiated by the lack of response to L-162,313 in untransfected CHO cells and in CHO cells transfected with the unrelated tachykinin NK-1 receptor (data not shown). High concentrations of L-162,313 induced an unspecific increase in phosphoinositide turnover which could not be blocked by L-158,809 and which was also observed in untransfected CHO cells, as well as in the NK-1-transfected cells (data not shown).


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(1) 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 (bullet). Panel B, IP accumulation expressed as percentage of accumulation of [^3H]inositol phosphates after stimulation with 10M 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 (bullet), and by L-162,313 after pretreatment with 10M L-158,809 (), or with 10M L-158,809 (circle). 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(1) 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(1) receptor. The IP accumulation is expressed as percentage of specific accumulation of [^3H]Inositol phosphates after stimulation with 10M angiotensin II. Panel A, response to angiotensin II alone (circle), and response to angiotensin II after pretreatment with L-162,313 in the following concentrations: 10M (up triangle), 10M (), or 10M (bullet). Panel B, angiotensin II alone (circle), or after pretreatment with 10M L-158,809 (box), or with 10M L-158,809 (circle). Means ± S.E. are shown for three to five experiments (eight experiments for angiotensin II) performed in triplicates.



Binding Experiments and Receptor Mutagenesis

L-162,313 displaced radiolabeled peptide from the rat and human AT(1) receptor in transfected COS-7 cells with an IC value of 14.9 ± 2.9 and 11.9 ± 0.3 nM, respectively (Table 1). Surprisingly, the X. laevis AT(1) receptor bound L-162,313 with an affinity only 10-fold lower than the affinity of the rat AT(1) receptor ( Table 1and Fig. 3C). In contrast, the affinity of the structurally homologous non-peptide antagonist L-158,809 was 2000-fold lower in the X. laevis AT(1) receptor than in the rat receptor ( Table 1and Fig. 3B), as previously shown for this and 10 other diphenylimidazole antagonists(3) . In fact, L-158,809 was even the one that bound with highest affinity to the Xenopus receptor among the non-peptide antagonists(3) .




Figure 3: Competition binding curves for the rat and X. laevis AT(1) receptors in transiently transfected COS-7 cells. Binding is expressed as percentage of specifically bound I-[Sar^1,Leu^8]angiotensin II in the rat (box), and Xenopus () AT(1) 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(1) receptor.



In order to identify interaction points for L-162,313 on the AT(1) 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(1)) or in TM-VII ([Ala]hAT(1) and [Asp]hAT(1), 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(1), X. laevis AT(1), and rat AT(2) 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(1) receptor, discontinuously located in the N-terminal extension (Ile^14, 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.


DISCUSSION

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(1) receptor expressed in transfected cells. The compound is a high affinity ligand of the AT(1) 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(1) 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(1) 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.^1 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^1. 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(1) receptors, but resulting in a full response in blood pressure.

Mutations that affect the binding of the non-peptide antagonists in the AT(1) 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(1) receptor too. L-162,313 is also unique among non-peptide ligands for the AT(1) receptor in respect of its relatively high affinity for the X. laevis AT(1) receptor (Fig. 3C). Interestingly, L-162,313 also binds with high affinity to the AT(2) type angiotensin receptor. The sequence identity between the mammalian and the Xenopus AT(1) receptors is 63-65% and between the human AT(1) and AT(2) 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(1) and the AT(2) receptors and with even higher affinity to the Xenopus AT(1) 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(1) 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(1) receptor(9) , with special focus on residues that are common to both the AT(1), the AT(2), and the X. laevis AT(1) 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.


FOOTNOTES

*
This work was supported by grants from the Danish Medical Research Council, the Danish Heart Association, the Novo Nordisk Foundation, and the Danish Biotechnology Center for Signal Peptide Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 45-3545-6321; Fax: 45-3135-2995.

(^1)
S. D. Kivlighn, G. J. Zingaro, R. A. Rivero, W. R. Hockle, V. J. Lotti, R. S. J. Chang, T. W. Schorn, N. Kevin, R. G. Johnson, W. J. Greenlee, and P. K. S. Siegl, manuscript in preparation.

(^2)
The abbreviations used are: TM, transmembrane; PCR, polymerase chain reaction; IP, inositol phosphate; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We are very grateful for devoted technical assistance by Dorte Frederiksen and Lisa Zeh.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.