Role of Aromaticity of Agonist Switches of Angiotensin II in the Activation of the AT1 Receptor*

Shin-ichiro Miura, Ying-Hong Feng, Ahsan HusainDagger , and Sadashiva S. Karnik§

From the Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195-5069

    ABSTRACT
Top
Abstract
Introduction
References

We have shown previously that the octapeptide angiotensin II (Ang II) activates the AT1 receptor through an induced-fit mechanism (Noda, K., Feng, Y. H., Liu, X. P., Saad, Y., Husain, A., and Karnik, S. S. (1996) Biochemistry 35, 16435-16442). In this activation process, interactions between Tyr4 and Phe8 of Ang II with Asn111 and His256 of the AT1 receptor, respectively, are essential for agonism. Here we show that aromaticity, primarily, and size, secondarily, of the Tyr4 side chain are important in activating the receptor. Activation analysis of AT1 receptor position 111 mutants by various Ang II position 4 analogues suggests that an amino-aromatic bonding interaction operates between the residue Asn111 of the AT1 receptor and Tyr4 of Ang II. Degree and potency of AT1 receptor activation by Ang II can be recreated by a reciprocal exchange of aromatic and amide groups between positions 4 and 111 of Ang II and the AT1 receptor, respectively. In several other bonding combinations, set up between Ang II position 4 analogues and receptor mutants, the gain of affinity is not accompanied by gain of function. Activation analysis of position 256 receptor mutants by Ang II position 8 analogues suggests that aromaticity of Phe8 and His256 side chains is crucial for receptor activation; however, a stacked rather than an amino-aromatic interaction appears to operate at this switch locus. Interaction between these residues, unlike the Tyr4:Asn111 interaction, plays an insignificant role in ligand docking.

    INTRODUCTION
Top
Abstract
Introduction
References

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 alpha -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 beta -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, beta -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 (approx 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 approx 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 approx 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 approx 2.8 kcal mol-1, whereas the hydroxyl group in Tyr4 contributed approx 0.4 kcal mol-1 and the surface area (based on comparison between Cha4 and Ile4) accounted for approx 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 Delta Delta G value for each interaction was calculated using the formula Delta Delta G = [Delta Gmut - Delta 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 (approx 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 approx 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 approx 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 approx 60% in the H256Q and approx 70% in the H256A mutant receptors. In contrast, substitution of His256 with a Tyr (H256Y) led to a approx 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 approx 40% response in the wild-type receptor, approx 52% in the H256Y mutant, approx 35% in the H256Q mutant, and approx 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 delta - pi  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 right-arrow 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 approx 160 Å (Asn111) to approx 180 Å (Gln111), and approx 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 alpha -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 alpha -COO - group will disrupt agonist potential (1, 2, 5). Such modifications include aliphatic substitutions, and aromatic groups in D-configurations (D-Phe, alpha -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.

    ACKNOWLEDGEMENTS

We thank Jingli Zhang for excellent technical assistance, Vivien Yee for assistance in modeling and surface area calculations, and Robin Lewis and Christine Kassuba for assistance in manuscript preparation.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants EY09704 and HL57470 (to S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Victor Chang Cardiac Research Institute, St. Vincent's Hospital, Darlinghurst 2010, Sydney, New South Wales, Australia.

§ To whom correspondence should be addressed: Dept. of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-1269; Fax: 216-444-9263; E-mail: karniks{at}cesmtp.ccf.org.

    ABBREVIATIONS

The abbreviations used are: Ang II, angiotensin II (DRVYIHPF); IP, inositol phosphate; Sar, sarcosine; R, inactive receptor conformation; R', unconstrained receptor conformation; R*, activated receptor conformation; Cha, beta -cyclohexylalanine; GPCR, the G-protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium.

    REFERENCES
Top
Abstract
Introduction
References
  1. Marshall, G. R., Bosshard, H. E., Vine, W. H., Glickson, J. D., and Needleman, P. (1974) in Recent Advances in Renal Physiology and Pharmacology (Wesson, L. G., and Fanelli, G. M., Jr., eds), pp. 215-256, University Park Press, Baltimore
  2. Bumpus, F. M., and Khosla, M. C. (1977) in Hypertension: Physiology and Treatment (Genest, J., Koiw, E., and Kuchel, O., eds), pp. 183-201, McGraw-Hill, New York
  3. Timmermans, P. B. M. W. M., Wong, P. C., Chiu, A. T., Herblin, W. F., Benefield, P., Carini, D. J., Lee, R. J., Wexler, R. R., Saye, J. M., and Smith, R. D. (1993) Pharmacol. Rev. 45, 205-251[Medline] [Order article via Infotrieve]
  4. Nikiforovich, G. V., Kao, J. L.-F., Pluchinska, K., Zhang, W. J., and Marshall, G. R. (1994) Biochemistry 33, 3591-3598[Medline] [Order article via Infotrieve]
  5. Samanen, J., and Regoli, D. (1994) in Angiotensin II Receptors: Medicinal Chemistry (Rufolo, R., ed), Vol. 2, pp. 11-97, CRC Press, Boca Raton, FL
  6. Karnik, S. S., Husain, A., and Graham, R. M. (1996) Clin. Exp. Pharm. Physiol. Suppl. 3, S58-S66
  7. Yamano, Y., Ohyama, K., Chaki, S., Guo, D.-F., and Inagami, T. (1992) Biochem. Biophys. Res. Commun. 187, 1426-1431[Medline] [Order article via Infotrieve]
  8. 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[Abstract/Free Full Text]
  9. Feng, Y.-H., Noda, K., Saad, Y., Liu, X., Husain, A., and Karnik, S. S. (1995) J. Biol. Chem. 270, 12846-12850[Abstract/Free Full Text]
  10. Noda, K., Saad, Y., and Karnik, S. S. (1995) J. Biol. Chem. 270, 28511-28514[Abstract/Free Full Text]
  11. Noda, K., Feng, Y. H., Liu, X. P., Saad, Y., Husain, A., and Karnik, S. S. (1996) Biochemistry 35, 16435-16442[CrossRef][Medline] [Order article via Infotrieve]
  12. Balmforth, A, J., Lee, A. J., Warburton, P., Donnelly, D., and Ball, S. C. (1997) J. Biol. Chem. 272, 4245-4251[Abstract/Free Full Text]
  13. Groblewski, T., Maigret, B., Larguier, R., Lombard, C., Bonnafous, J.-C., and Marie, J. (1997) J. Biol. Chem. 272, 1822-2836[Abstract/Free Full Text]
  14. Creighton, T. E. (1984) Proteins: Structural and Molecular Principles, pp. 2-60, Freeman and Co., New York
  15. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
  16. Nicholls, A. J. (1993) GRASP Manual, Columbia University, New York
  17. Noda, K., Saad, Y., Graham, R. M., and Karnik, S. S. (1994) J. Biol. Chem. 269, 6743-6752[Abstract/Free Full Text]
  18. Samanen, J., Cash, T., Narindray, D., Brandies, E., Yellin, T., and Regoli, D. (1989) J. Med. Chem. 32, 1366-1370[Medline] [Order article via Infotrieve]
  19. Guillemette, G., Bernier, M., Parent, P., Leduc, R., and Escher, E. (1984) J. Med. Chem. 27, 315-320[Medline] [Order article via Infotrieve]
  20. Bihoreau, C., Monnot, C., Davis, E., Teutsch, B., Bernstein, K. E., Corvol, P., and Clauser, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5133-5137[Abstract]
  21. Marie, J., Maigret, B., Joseph, M. P., Larguier, R., Nouet, S., Lombard, C., and Bonnafous, J. C. (1994) J. Biol. Chem. 269, 20815-20818[Abstract/Free Full Text]
  22. Monnot, C., Bihoreau, C., Conchon, S., Curnow, K. M., Corvol, P., and Clauser, E. (1996) J. Biol. Chem. 271, 1507-1513[Abstract/Free Full Text]
  23. Armstrong, K. M., Fairman, R., and Baldwin, R. L (1993) J. Mol. Biol. 230, 284-291[CrossRef][Medline] [Order article via Infotrieve]
  24. Walksman, G., Kominos, D., Robertson, S. C., Pant, N., Baltimore, D., Birg, R. B., Cowburn, D., Hanafusa, H., Mayer, B. J., Overduin, M., Resh, M., Rios, C. B., Silverman, L., and Kurian, J. (1992) Nature 358, 646-653[CrossRef][Medline] [Order article via Infotrieve]
  25. Strader, C. D., Fong, T. M., Tota, M. R., and Underwood, D. (1994) Annu. Rev. Biochem. 63, 101-132[CrossRef][Medline] [Order article via Infotrieve]
  26. Levitt, M., and Perutz, M. F. (1988) J. Mol. Biol. 201, 751-754[Medline] [Order article via Infotrieve]
  27. Dougherty, D. A., and Stauffer, D. A. (1990) Science 250, 1558-1560[Medline] [Order article via Infotrieve]
  28. Gether, U., and Kobilka, B. K. (1998) J. Biol. Chem. 273, 17979-17982[Free Full Text]
  29. Mitchell, J. B. O., Nandi, C. L., McDonald, I. K., and Thornton, J. M. (1994) J. Mol. Biol. 239, 315-331[CrossRef][Medline] [Order article via Infotrieve]
  30. Underwood, D. J., Strader, C. D., Rivero, R., Patchett, A. A., Greenlee, W., and Prendergast, K. (1994) Chem. Biol. 1, 211-221[Medline] [Order article via Infotrieve]
  31. Perlman, S., Schambye, H. T., Rivero, R. A., Greenlee, W. J., Hjorth, S. A., and Schwartz, T. W. (1995) J. Biol. Chem. 270, 1493-1496[Abstract/Free Full Text]
  32. Feng, Y.-H., Miura, S.-I., Husain, A., and Karnik, S. (1998) Biochemistry 45, 15791-15798[CrossRef]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.