©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenesis and the Molecular Modeling of the Rat Angiotensin II Receptor (AT) (*)

Yoshiaki Yamano , Kenji Ohyama (1), Mitsuhiro Kikyo , Tomoaki Sano (1), Yoshiko Nakagomi (1), Yoshihisa Inoue (2), Norifumi Nakamura (2), Isao Morishima , Deng-Fu Guo (3), Takao Hamakubo (3), Tadashi Inagami (3)(§)

From the (1) Laboratory of Metabolic Biochemistry, Faculty of Agriculture, Tottori University, Tottori 680, Japan, the Department of Pediatrics, Yamanashi Medical School, Yamanashi 409-38, Japan, the (2) Green Cross Corp., Shodai-Ohtani, 2-25-1, Hirakata, Osaka 573, Japan, and the (3) Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the 2 adrenergic receptor model built on the template of the bacteriorhodopsin structure. These data indicated that there are conserved residues that are actively involved in the receptor-ligand interaction. Eleven conserved residues in AT, His, Arg, Glu, His, Glu, Lys, Trp, His, Phe, Thr, and Asp, were targeted individually for site-directed mutation to Ala. Using COS-7 cells transiently expressing these mutated receptors, we found that the binding of angiotensin II was not affected in three of the mutations in the second extracellular loop, whereas the ligand binding affinity was greatly reduced in mutants Lys Ala, Trp Ala, Phe Ala, Asp Ala, and Arg Ala. These amino acid residues appeared to provide binding sites for Ang II. The molecular modeling provided useful structural information for the peptide hormone receptor AT. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg but not Lys.


INTRODUCTION

The octapeptide angiotensin II (Ang II)() is well known for its important roles in the regulation of cardiovascular functions and electrolyte homeostasis (1) . Its structure-function relationship has been extensively studied (2) , and it has been postulated that its receptor-bound form assumes a conformation with a twisted U-shaped bend (3). The Ang II-binding sites for which a specific ligand is displaced by nanomolar concentrations of losartan are now referred to as the angiotensin type 1 (AT) receptors, and those displaced by CGP42112A or PD123319 are designated as the type 2 (AT) receptors (4) . The primary sequences of AT receptors of various mammalian species have been determined (5, 6, 7, 8, 9, 10, 11, 12, 13) in recent years, and AT receptors have also been cloned (14, 15) . They were found to belong to the G-protein coupled receptor (GPCR) family. In general, AT receptors mediate most of the responses commonly associated with Ang II, are dithiothreitol sensitive, linked to G-protein, and not displaced by PD123319. AT receptors are for the most part losartan insensitive, not inactivated by dithiothreitol, and do not mediate physiological responses involving the second messenger systems commonly associated with AT receptors. AT receptors are further divided into subtypes which are referred to as ``A'' and ``B'' subtypes. AT and AT show an approximately 95% sequence homology (16-23). The multiple amino acid sequence alignment of several AT receptors is shown in Fig. 1.


Figure 1: Alignment of amino acid sequences of angiotensin II receptors (AT). Positions of the putative transmembrane domains I-VII are indicated by solid lines. The sequences of rAT and rAT are aligned in parallel. Skipped sequences as indicated by hyphens are introduced for maximizing sequence homology, and residues identical with those of rAT are indicated by asterisks. rAT2, rat AT (14, 15); rAT1a, rat AT (6, 16); rAT1b, rat AT (18); hAT1, human AT (7); bAT1, bovine AT (5); tAT1, turkey AT (26); xAT, XenopusAT (25).



Recently, Ang II receptors have been cloned from an amphibian (24, 25) and avian species (26) , and they were found functionally similar to mammalian AT receptors. However, these receptors do not recognize AT- or AT-specific nonpeptide antagonists (8, 9, 10) . The binding site for the AT-specific antagonist losartan was explored by replacing nonconserved amino acid residues of the rat AT receptor with amino acid species at the corresponding positions in the amphibian receptor (27) . The losartan-binding site was defined in several membrane spanning domains of the rat AT receptor. A recent study of chimeric human-amphibian Ang II receptors showed that the binding mode for peptide and nonpeptide ligands is rather different and that competitive and insurmountable (noncompetitive) antagonists presumably bind to overlapping but distinct sites located in the sixth and seventh transmembrane (TM) domain (28) . The objective of the present studies is to determine the binding sites for Ang II utilizing site-directed mutagenesis and incorporating a computer-assisted receptor modeling approach.

The three-dimensional structure of bacteriorhodopsin (bRh), a seven transmembrane domain protein, has been obtained by high-resolution electron cryomicroscopy (28) . It provided a modeling template for the overall structure of GPCRs (29, 30, 31, 32, 33, 34, 35) . Although, the sequence homology between GPCRs and bRh is low, several relevant features of the x-ray crystallographic structure of photosynthetic reaction center (36) were incorporated in modeling many GPCRs including the -adrenergic receptor (2-AR).

Amino acid sequences of a wide variety of GPCRs have been accumulated (37-44). By multiple amino acid sequence alignment, highly conserved domains and residues as well as unique sequences for specific ligand-binding sites can be identified. These data permit modeling of the AT receptor on the template of an existing model such as that of 2-AR (45, 46) .

In prior studies, site-directed mutagenesis in AT of a limited number of residues in the transmembrane domains indicated that Lys in the fifth transmembrane domain (TM 5th) as a key residue for ligand binding (47) . In order to analyze ligand binding, a model for the docking of Ang II was constructed on the basis of positional and possible functional interactions of a limited number of residues in the receptor model. A hypothesis for the docking mode was then tested on the basis of mutagenesis of several highly conserved amino acid residues considered critical for Ang II binding.

We present the homology-based model of the rat AT receptor constructed after the model of 2-AR which was used in the present study as a template. We propose a mechanism for docking of a ligand to the AT receptor by determining the effects of mutagenesis of critical receptor amino acid residues on the binding affinity.


MATERIALS AND METHODS

Molecular Modeling of the AT Receptor

The multiple amino acid sequence alignment of several Ang II receptors is shown in Fig. 1(5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23) . Information on conserved and non-conserved amino acid sequences were used for homology-based modeling of AT on the template of 2-AR model. The modeling was performed using the molecular modeling software SYBYL 5.5 on an UNIX workstation model IRIS/indigo (Silicon Graphics Inc., Mt. View, CA). The extracellular and intracellular loops were constructed, and the restraint minimization was performed as described above for 2-AR. A restrained and full conjugate gradient geometry optimization was then carried out using Discover 2.9. Following this step, loop regions were removed.

Molecular Modeling of Ang-II and a Docking Model

The full conjugate gradient geometry optimization of the Ang II derivative, (Pen, Pen)Ang II (48, 49) was performed using Discover 2.9 so that this derivative forms a twisted U-shape conformation (3) (Pen is penicillamine). The conformation of the -carboxyl and -amino groups of Asp of Ang II was fixed so as to form an intramolecular salt. Docking studies were carried out using SYBYL 5.5 by placing Phe of Ang II on Trp, His on Asp, Arg on Asp, Tyr on Arg (see ``Results and Discussion''). Following the docking of Ang II to the AT receptor model, and modifying the backbone and side chain conformation, 100 cycles of the steepest descent optimization were performed followed by about 1000 cycles of the conjugate gradient optimization with strong backbone restraints (1000 iterations) for the AT receptor as a routine. Later, the backbone conformation of Ang II reported by Nikiforovich et al.(50, 51) was adopted. Further details of the modeling procedures involving a -AR model as an intermediate step are provided under the ``Appendix.''

Site-directed Mutagenesis of Rat Angiotensin II Receptor

A 2-kb KpnI-EcoRI fragment of the rat AT cDNA (16) was subcloned into the polylinker site of the plasmid vector pBluescript II KS, and a single-stranded DNA was prepared using the helper phage R408. Site-directed mutagenesis was performed following the procedure of Kunkel (52) . Sites of mutations were confirmed by Sanger's dideoxynucleotide sequencing method (53) . The mutant DNA insert was excised from the plasmid vector and introduced into the mammalian expression vector pcDNAI. Eleven conserved residues in AT were targeted individually for site-directed mutation to alanine.

Cell Culture

COS-7 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) penicillin and streptomycin under 5% CO, 95% air at 37 °C. Cells were seeded at 3 10 cells in 9-cm dishes and split every 3 days.

Expression of the Mutant Angiotensin II Receptors and Ligand Binding Assay

COS-7 cells were transfected with mutated AT cDNAs by electroporation as described elsewhere (54) . These cells were cultured for 3 days, and the mutated receptors were allowed to express transiently. Ligand binding studies were performed at 4 °C following the procedure reported using I-Ang II as an agonist ligand, I-EXP985 as an antagonist ligand, and results were analyzed by Scatchard analysis (55) .

Flow Cytometric Confirmation of Receptor Expression in Transfected COS-7 Cells

COS-7 cells transfected with the receptor cDNAs used in the binding study were collected and washed once with Hanks' balanced salt solution. Cells (1 10 cells) were incubated in Dulbecco's modified Eagle's medium containing 2% fetal calf serum and -globulin for 30 min at 4 °C. Rabbit polyclonal anti-AT antibodies against a 15-residue epitope (Lys-Tyr) in the N-terminal region of rat AT were produced by immunization with the peptide coupled to thyroglobulin. Cells were incubated with the anti-rat AT antibodies at 4 °C for 60 min, washed three times with phosphate-buffered saline, then incubated with the fluorescence-labeled goat anti-rabbit IgG F(ab`)2 fragment at a 1:50 dilution for 30 min at 4 °C, and subjected to an Epics Profile flow cytometer equipped with a 488 nm laser beam (Counter Electronics Co., Ltd.). Cells transfected with pcDNAI containing a mutated AT cDNA insert expressed immunoreactive rat AT as shown in Fig. 2. Expression levels of mutated AT receptors were comparable with that of the unmutated AT


Figure 2: Flow cytometric analysis of mutated rat AT expressed on the surface of COS-7 cells. Distribution of fluorescence positive cells with fluorescence intensity higher than the basal level (the dotted line) is indicated by shaded peaks. Percentage of positive cells is also indicated. pcDNAI, pcDNAI vector transfected; wild AT, nonmutated rat AT transfected; Mut-HR166,167AA, mutant HisArg AlaAla, Mut-W253A, mutant Trp Ala; Mut-F259A, mutant Phe Ala; Mut-D263A, mutant Asp Ala. Other mutants used in this experiment showed similar results.




RESULTS AND DISCUSSION

Molecular Modeling of the AT Receptor

The multiple amino acid sequence alignment of several Ang II receptors in Fig. 1A(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) was used to build an AT receptor model based on the -AR model as the template. A number of amino acids in the putative TMs are conserved in a large number of GPCRs and probably are of functional or structural importance. The existence and function of conserved residues are best explained if they are situated on the inside of the TM bundle or in an area which is facing other helices. Residues facing the membrane environment can be mutated more easily without affecting the function of the receptor and should be less conserved. The AT receptor has two relatively short loops between TM1 and TM2 and also between TM6 and TM7. This was helpful in determining the exact position of the TMs connected to these loops.

Additional bent helices by prolines were introduced into TM1 and TM7. Finally, the model of the AT receptor TM bundle thus obtained was energy minimized (Discover Force Field) with the backbone fixed with the aim of releasing side chain repulsion. Further minimization with no restriction of the helix bundle (1000 iterations) was performed. These minimizations did not significantly change the relative positions of the TMs.

Amine-Aromatic (NH-) Interaction

The amine-aromatic interaction, discovered recently in hemoglobin (56) and repeatedly confirmed in x-ray crystallographic structures of a number of proteins (57), were incorporated in the modeling of AT. The interaction of amine, amide, or guanido nitrogen with aromatic electrons within 3-4 Å of the flat face of aromatic ring was computed by molecular orbital calculation according to the Density Functional Theory.()

Since the side chain of a lysyl residue is long, the -amino group of Lys (TM5), which is important for ligand binding (), might not be located near the outer surface. Frequently, there are aromatic amino acid residues surrounding the ionic bridge formed between GPCRs and their ligands (30) as shown in Fig. 3. Tryptophyl residues are found in TM4, 6, and 7 in most GPCRs (37, 38, 39, 40, 41, 42, 43, 44, 59) . It is possible that many of them stabilize an ammonium salt by an aromatic-amine interaction. In the x-ray crystallographic structure of the phosphotyrosine recognition domain, SH2, of v-src complexed with a tyrosine-phosphorylated peptide, one of the guanidine nitrogens of an arginyl residue is situated immediately above an aromatic ring, and the -amino group of a lysyl residue is immediately below the phosphorylated tyrosine phonemic ring (60) . A similar ammonium-aromatic interaction has been observed by other investigators (61, 62) . Our molecular orbital calculation showed that the interaction produces a stabilization of about 5.3 kcal/mol, virtually indistinguishable from a hydrogen bond which averages 6.1 kcal/mol (63) . Thus, it is likely that the -amino group of Lys may be located on Trp in TM6 or Trp in TM4. From the docking model shown below, Trp looked more likely (Fig. 3).


Figure 3: Schematic representation of the interaction between rat AT in the sixth transmembrane region and angiotensin II.



The mutation of Lys to Ala resulted in a greater than 50-fold increase in K for Ang to from 1.7 nM to >100 nM; Trp to Ala increased K 7-fold to 12.5 nM (). These results led to the plausible postulate that the C-terminal carboxylate anion of Phe of Ang-II and the -amino group of Lys which is located on top of the indole ring of Trp form an ionic link and that Trp stabilizes the salt bridge.

Phe in TM6 of 2-AR is very important for ligand binding. His of AT is considered to be an equivalent of Phe of 2-AR because of its position. We mutated His to Ala. The K value for Ang II increased to 4.5 nM from 1.7 nM of the native receptor. The mutation of neighboring residues also affects the ligand binding (). In mutagenesis studies of nonconserved residues in AT, H. Ji et al.(27) found that Ser Cys resulted in a considerable reduction in the binding affinity of saralasin.

Binding Site for His of Angiotensin II

We studied the binding of an imidazole group to various receptors and enzymes in search of a general pattern for imidazole binding. The imidazole ring of histamine is known to bind to Thr, Asn, and Phe in TM5 of the histamine H1 receptor (64) , and to Asp, Thr, and Phe in TM5 of the histamine H2 receptor (65) . The protonation-tautomerism of an imidazole sandwiched between two hydrogen-bonding ligands seems to be stabilized by an adjacent aromatic ring.

An analogous triad Phe Thr Asp is present in AT in TM6. We established a model illustrated in Fig. 4, in which the imidazole of His and the C-terminal carboxylate of Phe of Ang II are accommodated by TM6. The imidazole group was placed between the -carboxyl group of Thr and -carboxyl group of Asp, and the C-terminal carboxylate anion on the -amino group located on the indole ring of Trp. The model accommodated this configuration well. The interactive centers of Trp and Asp are 16 Å apart. This distance is compatible with that between the C-terminal carboxylate and imidazole groups of Ang II plus the calculated distances of interaction between Phe of Ang II and Trp (4.7 Å), and that between His of Ang II and Asp (3.9 Å). Significant increases in K accompanying mutations of individual amino acid residues in this triad, except for Thr, to an alanyl residue are compatible with probable roles of these residues in ligand binding. His and Phe are 2 of the functionally important residues for the agonistic activity of Ang II (66) . Although a small effect on K of mutation Thr Ala is somewhat different from a larger change due to a similar mutation in the histamine H2-receptor, it is significant compared with the mutation of the next residue Ile, which had no effect on saralasin binding as reported by H. Ji et al.(27) . The small effects of Asp Ala and Thr Ala are also compatible with a relatively small contribution of His to the binding of Ang II (67) . It looks as if AT binds its ligand with a somewhat different manner from H2-R. The hydrogen bonding of the imidazole of His to Thr is weak or not essential in case of AT ().


Figure 4: View of rat AT receptor model from the side (A) and the top of receptor well (B). The transmembrane helices are represented by the solid ribbon. The oxygen atom is shown in red, and the nitrogen atom in purple. The extracellular space is in the top of the figure.



Contribution of Arg to the Binding of Angiotensin II

In the Ang II molecule, the phenolic side chain of Tyr has a very strong interaction with the receptor (67, 68) . It is likely that phenolic group interacts with guanido group as reported in various x-ray crystallographic structures. In our model with Phe and His fixed, the side chain of Arg extends toward the phenolic group of Tyr of Ang II. Thus we propose that Arg in TM4 is important for Ang II binding. This notion is supported since Arg is conserved throughout Ang II receptor isoforms.

The Binding Mode of Nonpeptide Antagonist EXP985

Finally, the present approach brought out an interesting contrast between the binding of the peptide agonist Ang II and the non-peptide antagonist EXP985, an analog of losartan. As shown in , mutations of many residues had comparable effects on the binding of the agonist Ang II and the nonpeptidic antagonist. However, mutations, Lys Ala and Trp Ala had hardly any effect on the binding of EXP985, whereas these individual mutations drastically weakened Ang II binding. As discussed above, Lys and Trp are most likely hydrogen bonded in the ligand-free state. Because of mutual proximity and close interaction, the mutation of either one of these residues will disrupt the ligand binding function of the two associated side chain groups in parallel. Thus, the specific and parallel change of Lys Ala and Trp Ala on the binding of Ang II, and a lack of effect of either of these mutations on the binding of the nonspecific antagonist EXP985 gave further support to the present hypothesis that Lys and Trp function as a unit in association with each other. These observations indicate that the anionic tetrazolium widely used in many nonpeptidic antagonists binds selectively to the cationic side chain of Arg rather than Lys. Further, contribution of Asp, presumably in the binding of the imidazole ring of His of Ang II, seems to play a relatively minor role for the binding of nonpeptidic antagonist.

These results showed that the nonpeptide agonist binds near the surface area. It showed that the binding mode of Ang II and the nonpeptide antagonist is different, which agrees with the results of Schambye et al.(69) . Of 11 residues mutated, ionic groups in the second extracellular loop (Glu, His, Gln) seem to play only minor, if any, roles in the binding for both the agonist and antagonist.

In summary, the present AT receptor modeling and mutagenesis presented the following hypotheses.

1) The aromatic amino acid residue, Trp stabilizes the ionic bridge formed between Lys of AT and the carboxylate anion of Phe of Ang II.

2) Phe and Asp in TM6 provide the binding site for His of Ang II.

3) Ang II penetrates almost one-third of the way into the membrane to bind the receptor.

The result indicates that the anionic tetrazolium of EXP985 binds selectively to the cationic side chain of Arg of AT. In AT, mutants, Val Ile, Ala Ser, and Ser Cys had a marked effect on losartan binding (27) . It shows that the mutation of neighboring amino acids also reduces ligand binding ability, and our present results are in agreement with these observations.


APPENDIX

Molecular Modeling of 2-ARM

For the molecular modeling of 2-AR, we used the modeling program SYBYL 5.4 (Tripos Associates, St. Louis) on a micro VAX-II (Degital Equipment Corp., Boston) and PS 390 (Evans & Sutherland, Salt Lake City), and Discover (Biosym. Technologies Inc., San Diego) version 2.1 was used for optimization. The result of the multiple sequence alignment of several GPCRs was used to build the model of 2-AR on the basis of the three-dimensional structure of bRh (28) . The angle of a kink in a helical column due to a proline residue in a transmembrane helical structure was cited from the x-ray crystallographic structure of photosynthetic reaction center (36). Results of mutation studies of 2-AR (45, 46) were also incorporated. The extracellular and intracellular loops were generated using the ``Loop Search'' in SYBYL, then 100 cycles of a steepest descent optimization were performed with strong backbone restraints (1000 iterations) as usual. These ``Loop Search'' and restrained minimization processes were repeated until this protein satisfied acceptable - angle values for the peptide backbone. The docking model of 2-AR was constructed by fitting each helix of 2-AR to the corresponding bRh in order to minimize the root-mean-square (rms) value (32) . A full conjugate gradient optimization of the model was performed without the backbone restraints until the rms change or gradient reached their minimum levels.

Molecular Modeling of the 2-Adrenergic Receptor as a Template for AT

Various GPCRs were cloned and sequenced, and their molecular models were constructed based on the overall structure of bRh alignment to 2-AR (29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44) . A mumber of residues are highly conserved throughout the GPCR family, and other residues are conserved among Ang II receptors shown in Fig. 1 . The effect of the mutation of the highly conserved aspartyl residue (Asp of 2-AR and Asp of AT) in TM2 on the receptor structure and function has been experimentally demonstrated (58, 71, 72). Many investigators speculated that the residues conserved throughout the GPCRs are probably involved in their general structural and functional roles for transmission of signals from a bound ligand, whereas those conserved only within Ang II receptors will play important roles in the specific ligand binding.

The 2-AR model, constructed after the three-dimensional structure of bRh (28) , was used as a template for the AT receptor model in the present study according to a similar method used for other receptors (29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44) . This two-step strategy was used because (i) the bRh is the only seven membrane spanning domain protein for which atomic coordinates are available; (ii) homology between bRh and AT is very low; (iii) a reasonable homology is seen between 2-AR and AT in their transmembrane domains; (iv) the interaction between 2-AR and its ligands has been well studied (45, 46) ; (v) 2-AR and AT have large numbers of proline residues compared with bRh; and (vi) bRh binds retinal, whereas GPCRs do not. Thus, we proceeded to use only the topology of the seven transmembrane spanning helices of bRh and refined the 2-AR model in reference to the results of site-directed mutagenesis studies (45, 46). Because 2-AR has more proline residues than bRh and a proline residue introduces a kink to the transmembrane conformation of each helix, we had to modify the backbone conformation of each helix according to the published method (29, 30, 31, 32, 33, 34, 35) . Since the bRh structure is of a low resolution, we adopted the x-ray crystallographic structure of the membrane photosynthetic reaction center (37, 38, 39, 40, 41, 42, 43, 44) , for introducing proline kinks to the helical columns of 2-AR.

After the initial 2-AR model was constructed, we docked a 2 agonist and antagonists and rotated or translationally shifted the position of each helix.

  
Table: Binding affinity of peptide and nonpeptide ligands for rat AT wild type receptor and mutant receptors

Data represent results of three identical series of binding isotherms followed by Scatchard analysis. Results are presented as means ± S.E.



FOOTNOTES

*
This work was supported by a scientific grant from the Ministry of Education, Science and Culture, the Uehara Memorial Foundation, Japan, and by United States Public Health Service Research Grants HL-14192 and HL-35323 from the National Institute of Health. 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.: 615-322-4347; Fax: 615-343-5244.

The abbreviations used are: Ang II, angiotensin II; AT, angiotensin type 1 receptor; AT, angiotensin type 2 receptor; GPCR, G-protein coupled receptor; TM, transmembrane domain; bRh, bacteriorhodopsin; 2-AR, 2-adrenergic receptor; Pen, penicillamine; SH2, src homology-2; kb, kilobase(s).

Y. Inoue, Y. Yamamura, N. Nakamura, Y. Yamano, K. Ohyama, T. Inagami, A. Scheiner, M. Wrinn, and J. Andzelm, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. K. Prendergast, H. T. Schambye, and J.-C. Bonnafous for useful discussions and comments and Dr. Erwin J. Landon for critical review of the manuscript.


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