©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Docking of Arg of Angiotensin II with Asp of AT Receptor Is Essential for Full Agonism (*)

Ying-Hong Feng , Keita Noda , Yasser Saad , Xiao-pu Liu , Ahsan Husain , Sadashiva S. Karnik (§)

From the (1) Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195-5069

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The structural model of AT angiotensin receptor contains seven-transmembrane -helices with three interhelical loops on either side of the membrane. The angiotensin II binding pocket within the receptor is not clearly defined. We showed earlier that Lys in transmembrane-helix-5 of the AT receptor binds the COOH-terminal -carboxyl group of angiotensin II (Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P., Graham, R. M., Husain, A., and Karnik, S. S.(1995) J. Biol. Chem. 270, 2284-2289). We now show that His and Asp, both located in the extracellular domain of the AT receptor, are involved in binding the NH-terminal Asp and Arg residues of angiotensin II, respectively. The Asp/His interaction appears to be weak and is unlikely to be important for agonism. But the loss of Arg/Asp interaction leads to partial agonism of the receptor. The action of non-peptide agonists is not affected by Asp mutations. These results suggest that several independent interactions between angiotensin II and AT receptor are necessary for full agonism. Since L-162,313 the non-peptide agonist of the AT receptor is a partial agonist that does not make contact with Asp, we speculate that the degree of agonism may be increased if it is redesigned to make contacts with Asp.


INTRODUCTION

The octapeptide hormone Ang II() has a variety of effects on cellular control mechanisms that influence blood pressure regulation. Two classes of cell surface Ang II receptors, AT and AT, have now been identified (1, 2, 3, 4, 5) . The AT receptor is responsible for mediating most of the known cardiovascular effects of Ang II, including its potent vasoconstrictor effect (1) . The AT receptor is a member of the G-protein-coupled receptor family (2, 3, 4, 5) . In this family of receptors the binding of small ligands occurs within the transmembrane domain. However, for the binding of large glycoprotein hormones, extracellular loops are employed (6) . The nature of the Ang II binding site is not clearly defined, but there is evidence indicating roles for extracellular, as well as transmembrane, domains (6) . Previous studies have suggested that the non-peptide antagonists bind to the transmembrane domain of AT receptor (7, 8, 9, 10, 11, 12, 13) . Because they do not chemically resemble the native hormone, the non-peptides are believed to bind to the receptor differently from peptide ligands of the AT receptor (7, 8, 9, 10, 11, 12) . We have shown that a crucial acidic pharmacophore common to both peptide and non-peptide ligands binds to a conserved residue Lys in the fifth transmembrane helix of the AT receptor and have demonstrated that the Lys-ligand interaction is ionic (13). Therefore, it can be postulated that the COOH-terminal region of Ang II interacts within the transmembrane domain of the AT receptor. The site of binding and the functional role of the NH-terminal region of Ang II is, however, unclear.

Sequential deletions of the first two residues of the Ang II produce varying effects on AT receptor binding and agonism. Des-Asp-Ang II (also known as Ang III) is a full agonist analogue of Ang II which binds to the AT receptor with a slightly reduced affinity (80% that of Ang II). Des-Asp,des-Arg-Ang II binds poorly to the AT receptor and shows little activity, if any. Thus, Arg of Ang II is believed to provide distinctive structural requirements (1, 14, 15) . Because receptor binding and agonism is side chain-dependent in Ang II analogues modified at the second position, it is believed that hydrogen bonding or charge interactions between the second Ang II residue and the AT receptor may be important (14, 15) . The site on the AT receptor which is responsible for mediating the interaction with the Arg side chain of Ang II is as yet unknown. In this report we identify the weak and the strong docking sites of the Asp and Arg side chains of Ang II, respectively, with the AT receptor and show the importance of the Arg interaction in receptor agonism.


EXPERIMENTAL PROCEDURES

Materials

Analogues of [Sar,Ile]Ang II and Ang II were synthesized and purified by the peptide synthesis core facility of The Cleveland Clinic Foundation or obtained from Bachem. [Sar,Ile]Ang II was iodinated by the lactoperoxidase method and purified (16) . The specific activity of the I-[ Sar,Ile]Ang II was 2200 Ci/mmol. [H]Losartan (42.3 Ci/mmol) was obtained from Amersham Corp. L-162,313 was a gift from Merck Sharp and Dohme.

Mutagenesis, Expression, and Characterization of Mutant Genes

Details of rat AT receptor expression and mutagenesis were described earlier (13, 17) .

The ligand binding experiments were carried out under equilibrium conditions. Inhibitory constants were derived from competition binding experiments using the formula K= IC/(1 - [L]/K ), where IC is the concentration of unlabeled analogue that displaces 50% of specifically bound radioligand, [L] is the concentration of radioligand, and K is the equilibrium dissociation constant of AT receptor for I-[Sar,Ile]Ang II or [H]losartan (18) . The K values represent mean ± S.E. of three to five independent determinations.

Inositol Phosphate Formation Studies

Transfected COS-1 cells were labeled for 24 h at 37 °C in CO incubator with [H]myo-inositol (Amersham) in Dulbecco's modified Eagle's medium containing 10% bovine calf serum. After labeling, cells were washed with HBSS and exposed with 10 mM LiCl in HBSS for 30 min. Cells were treated with or without various concentrations of the ligand in HBSS containing 20 mM sodium phosphate, pH 7.4, for 45 min. Cells were lysed with 0.4 M perchloric acid, and total inositol phosphate production was measured as described previously (19). Total inositol phosphate production is expressed as a percentage compared with the maximum stimulation of transfected wild-type AT receptor by [Sar]Ang II.


RESULTS AND DISCUSSION

Interaction of ArgSide Chain of

[Sar,Ile]Ang II with the ATReceptor Identification of Candidate Residues in the ATReceptor Responsible for Interactions with Argof [Sar,Ile]Ang II-The Arg side chain of Ang II is critical for binding to both the AT and AT Ang II receptor subtypes. Therefore, acidic residues residing in the extracellular and trans-membrane domain that are conserved in both AT and AT receptors were identified as potential candidates for binding the Arg side chain (). These conserved residues are Asp, Glu, Asp, Asp, and Asp. These residues in the rat AT receptor were substituted with neutral (Ala or Gln) or positively charged (Lys) side chains or side chains of different length (Glu or Asp) by mutagenesis to examine the contribution of charge, hydrogen bonding, or side chain length on Ang II binding. The wild-type and mutant genes were transiently expressed in COS cells. Immunoblot analysis indicated that these mutant receptor proteins were expressed within a 20-100% range of expression of the wild-type AT receptor. The ability of expressed receptor proteins to bind to the peptide antagonist of [Sar,Ile]Ang II and its analogues modified at the Arg position were analyzed by competition binding studies. The binding affinity of the AT receptor-selective non-peptide antagonist losartan was also measured. Losartan is believed to mimic the COOH terminus of Ang II, based on pharmacophore overlay studies (1) .

Data for binding studies with AT receptor mutants, where the conserved acidic residues (Asp, Glu, Asp, Asp, and Asp) were individually mutated to neutral or basic residues, are summarized in . With the exception of the D74K mutant, these neutral or basic mutations did not significantly decrease losartan binding to the receptor, indicating that a global change in receptor structure had not occurred. However, because both [Sar,Ile]Ang II and losartan did not bind to D74K, it is likely that this mutation produced a global defect in receptor folding. E185Q, E185K, D263A, and D263K mutations in the AT receptor had no effect on [Sar,Ile]Ang II binding affinity. Previously, Bihareau et al.(7) have shown a lack of effect of a neutral mutation at the Asp locus on [Sar,Ile]Ang II binding affinity. These findings suggest that Asp, Glu, and Asp do not play an important role in binding [Sar,Ile]Ang II and losartan. D278A and D281A mutations in the AT receptor produced a 13- and 33-fold decrease in [Sar,Ile]Ang II binding affinity. This disruption in [Sar,Ile]Ang II binding affinity was exacerbated in the charge reversal mutants D278K (by 7-fold compared with D278A) and D281K (by 1150-fold compared with D281A; see ). Since charge reversal at the Arg docking site is expected to produce a greater decrease in [Sar,Ile]Ang II binding affinity than that produced by a neutral mutation, both Asp and Asp were considered as candidate docking sites for the Arg side chain of [Sar,Ile]Ang II. ArgSide Chain of [Sar,Ile]Ang II Binds to Aspof the ATReceptor-In order to determine if the Asp or the Asp side chain was the docking site for the positively charged guanidinium group of Arg in [Sar,Ile]Ang II, the following additional criteria were used. First, the change in binding affinity attributed to the interaction between the positive charge of the Arg side chain of [Sar,Ile]Ang II and the potential counterion residue on the AT receptor should be similar to the decrease in [Sar,Ile]Ang II binding affinity observed when the negatively charged counterion residue is replaced by a neutral residue. Second, using an Ang II analogue as ligand in which the positively charged Arg is neutralized, differences in binding affinity between the wild-type receptor and the neutral residue mutant receptor should be minimal. Finally, differences in binding affinity between the wild-type receptor and the mutant receptor where a charge reversal has occurred at the counterion site should be high using [Sar,Ile]Ang II as ligand and low using an Ang II analogue in which the positively charged Arg is neutralized.

1) The (NO)Arg side chain in the Ang II analog [Sar,(NO)Arg, Ile]Ang II retains the hydrogen bonding ability of Arg, but lacks the positive charge. A 37-fold decrease in affinity of [Sar,(NO)Arg,Ile]Ang II binding to the wild-type receptor compared with [Sar,Ile]Ang II binding to the wild-type receptor reflects a loss of binding energy which is due to the lack of the Arg positive charge. The magnitude of this change in affinity is commensurate with the decrease in [Sar,Ile]Ang II binding affinity between the wild-type AT receptor and its D281A mutant (33-fold decrease). The change for the D278A mutant was relatively smaller (13-fold decrease).

2) In the D281A receptor mutant, where the negative charge of the Asp side chain has been eliminated, the affinity of [Sar,(NO)Arg,Ile]Ang II binding was only marginally different (by 3-fold) from that of [Sar,Ile]Ang II binding. If Asp is the counterion site, this effect would be expected, since the attractive force of the counterion would not be present in either situation. In distinct contrast to the observations with the Asp mutant, the affinity of [Sar,(NO)Arg,Ile]Ang II binding to the D278A mutant was 162-fold lower than that of [Sar,Ile]Ang II binding ().

3) The ratio of binding affinity of mutant receptor to wild-type receptor for [Sar,Ile]Ang II was 90:1 for the D278K mutant and 38,300:1 for the D281K mutant. This ratio for [Sar,(NO)Arg,Ile]Ang II was 77:1 for the D278K mutant and 354:1 for the D281K mutant (). These findings indicate that a charge reversal of the Asp residue has no selective effect on the modification of the Arg side chain of the ligand. But, a substantial improvement in ligand affinity was observed when the repulsive force between Lys and Arg side chain of the ligand was replaced by a neutral hydrogen bond interaction.

Collectively, these findings suggest a direct charge-pair interaction between Asp of the native AT receptor and the Arg side chain of the ligand. In contrast, the mutants D278A and D278K bound [Sar,(NO)Arg,Ile]Ang II with 58- and 77-fold lower affinities, respectively, compared with the wild-type receptor. A general loss of binding affinity toward two changes tested at the Asp position (data not shown) suggests an indirect role for this residue in ligand binding. It has been suggested that an Arg side chain can participate in as many as five hydrogen bonds (20) . Therefore, a potentially weak role for Asp in stabilization of the Asp-Arg salt bridge is possible because of its close proximity to Asp. Properties of the Ion-Pair Interaction between Aspand ArgSide Chain of [Sar,Ile]Ang II-In order to further define the flexibility of the Asp-Arg interaction in terms of side chain size and hydrophobicity, we examined two additional [Sar,Ile]Ang II analogues, [Sar,(homo)Arg,Ile]Ang II and [Sar,Gln,Ile]Ang II. A (homo)Arg substitution at the Ang II Arg position leads to an increase in side chain length by a single methylene unit. The binding affinity of the wild-type AT receptor for [Sar,(homo)Arg,Ile]Ang II is 7-fold lower than for [Sar,Ile]Ang II. A methylene unit increase in the side chain by the replacement of Asp with Glu also leads to a mutant receptor with a 23-fold reduction of [Sar,Ile]Ang II affinity. The reduction of affinity of Lys mutant receptor was maximal with all Ang II analogues examined. The Gln-substituted analogue [Sar,Gln,Ile]Ang II can interact through hydrogen bonding, but through a two-methylene unit shorter side chain than the Arg side chain. The binding affinity of this analogue to the wild-type receptor was 196-fold lower than the affinity of [Sar,Ile]Ang II and 4-fold lower than [Sar,(NO)Arg,Ile]Ang II. The ratio of affinity of [Sar,Gln,Ile]Ang II toward the wild-type and the D281K mutant receptors is 1:550. The ratio of affinity of [Sar,Gln,Ile]Ang II toward the D281A and D281K mutants is 1:180. The ratio of [Sar,Ile]Ang II affinity for these mutants is 1:38,300. This indicates that the longer Lys side chain of the receptor is better accommodated by the shorter Gln side chain than the Arg side chain of [Sar,Ile]Ang II. Thus, the length of interacting side chains is important. The binding specificity is produced by hydrogen bonding, but appears to be best mediated by a charge-pair interaction between Asp and Arg of Ang II. The change in the structure of Lys mutant receptor seems to be limited to the site of interaction of Arg of Ang II. For example, the defect does not propagate to the binding site of losartan. Therefore, the huge loss of binding affinity may reflect a strong repulsive interaction in a restricted pocket (see Ref. 21 for other examples).

Role of AspSide Chain in Signal Transduction

[Sar]Ang II produced an increase in inositol phosphate accumulation in a dose-dependent manner with an EC of 50 nM in COS cells transfected with the wild-type AT receptor gene (Fig. 1). [Sar,Gln]Ang II also produced full agonism but with an EC of 300 nM. Because the Gln side chain retains the hydrogen bonding potential of Arg, but lacks the positive charge, an ion-pair interaction with the Asp residue does not appear to be a critical requirement for the activation of the AT receptor. To determine if the hydrogen bonding interaction between the Arg side chain of Ang II and Asp side chain of the AT receptor is important for receptor agonism, we examined an AT receptor mutant where the Asp was mutated to the non-hydrogen-bonding residue Ala. The maximal inositol phosphate response elicited by [Sar]Ang II in COS cells expressing the AT receptor mutant, D281A, was 40% of that produced in COS cells expressing the wild-type receptor. These responses were completely inhibited by losartan. Because the expression levels of the D281A and the wild-type receptor were similar (B estimated for the wild-type receptor was 3-5 pmol/mg and for the D281A mutant was 1-3 pmol/mg) the differences in the cell surface expression of the receptor is not the basis of diminished signaling in the D281A mutant. The decreased response likely indicates an intrinsic loss of efficiency in ligand-mediated activation. Since the Ala side chain lacks hydrogen bonding ability, a critical contact between the D281A mutant receptor and the native ligand might be lacking. The consequence of this loss might be that the native ligand is recognized as a partial agonist by the mutant receptor.


Figure 1: Inositol phosphate formation by transfected wild-type AT receptor and the D281A mutant. Inositol phosphate formed in response to [Sar]Ang II,[Sar,Gln]Ang II and the non-peptide agonist, L-162,313, in COS 1 cells transfected with wild-type and the D281A mutant. The affinity constant (K) of wild-type and mutant D281A receptors, respectively, for [Sar]Ang II is 0.32 nM and 83.2 nM, for [Sar,Gln]Ang II is 52.7 nM and 304 nM, and for L-162,313 is 56.2 nM and 50.4 nM.



It has been proposed that losartan and the non-peptide AT receptor agonist L-162,313 mimic the COOH terminus of Ang II in their binding to the AT receptor (12, 22). This implies that L-162,313 may not make contacts with the AT receptor at the Arg docking site of Ang II. Consistent with this contention, binding of losartan and L-162,313 is not decreased in the D281A receptor mutant compared with the wild-type receptor (see Fig. 1legend). As has been described by others (12, 22) , the maximal response to stimulation by the non-peptide agonist L-162,313 was 25% of the maximum response elicited by [Sar]Ang II. Also, the maximal inositol phosphate response to stimulation by L-162,313 was identical between the wild-type receptor and its D281A mutant (Fig. 1). Furthermore, all of these responses were completely inhibited by losartan. Since functional responses to L-162,313 are not affected by the D281A mutation, the lack of interaction of L-162,313 with the AT receptor at the Arg docking site may limit its ability to fully activate the receptor.

The -Carboxylate of Aspof Ang II Interacts with His

We have previously shown that the conserved His residues in the extracellular and transmembrane domain of AT receptor are not involved in binding the COOH-terminal carboxylate of Ang II (13) . We now find that a Glu substitution of the conserved His specifically reduced the binding affinity of Ang II by about 56-fold (K= 114 ± 26 nM). The same receptor mutant bound [Ala]Ang II (K= 6.4 nM), [Asn]Ang II (K= 4.6 nM), and [Sar,Ile]Ang II (K= 0.6 nM) with small changes in affinity. The selective decrease of affinity for Ang II in Glu substitution can be accounted for by potential electrostatic repulsion. Therefore, His appears to be directly involved in binding the -carboxyl group of Asp of Ang II. The function of the receptor is not affected in any of the His mutants (data not shown). The His interaction with Asp of Ang II presumably contributes toward stabilization of receptor-bound conformation of Ang II, but it is unlikely to be a key interaction governing receptor function. This is consistent with the conclusions that Asp Ang II analogues (e.g. [Sar]Ang II) may improve affinity without altering agonistic properties (14, 15) .

Summary and Conclusions

The most important finding described in this paper is the interaction between Asp of AT receptor and Arg of Ang II. Salt bridge and hydrogen bond interactions involving these two side chains play an important role in receptor agonism. However, a relatively less significant interaction between His of AT receptor and Asp of Ang II was identified based on repulsive interaction (Fig. 2). Assignment of these two contacts, in addition to the docking of COOH-terminal carboxylate to Lys(13, 23) , now provide definitive attachment points for modeling AT receptor-Ang II structure. These studies are in progress. These two points of contact between the NH terminus of Ang II and the extracellular domain of the AT receptor are not utilized by the non-peptide agonists and antagonists in binding to this receptor. COOH terminus of Ang II and the non-peptides evidently bind within the transmembrane domain through Lys residue (13, 23) . Only partial activation of the receptor is achieved when the interaction between the Asp side chain located in the extracellular domain and ligand is disrupted. Detailed understanding of the mechanism of receptor activation requires more information than is available at this time. Nevertheless, some qualitative conclusions are justified. For example, the non-peptide agonist L-162,313 may be a partial agonist, because it does not interact with Asp side chain. Redesigning L-162,313 structure such that it can interact with Asp side chain might improve its potency comparable with that of native hormone. The potency of non-peptide antagonists may also be improved by additional contacts with the Asp and His side chains. The extracellular loops of the G-protein-coupled receptors are generally thought to be disordered and hence are unattractive targets for rational drug design. An entirely different possibility that is emerging for the AT receptor is that the extracellular loops and the NH-terminal tail are an integral part of the ligand binding pocket that is responsible for binding at least two NH-terminal residues of Ang II. This structure is probably achieved through a combination of extracellular disulfide bonds (23) , hydrogen bonding, and hydrophobic interaction (11) in order to shield the extracellular hormone-receptor contacts from bulk water. These findings have important implications for model building studies, which until now have lacked experimental data on the docking of the Ang II to the AT receptor (24) .


Figure 2: A current model depicting the interactions between AT angiotensin receptor and Ang II. The order of helices is based on the bacteriorhodopsin structure. The orientation of Asp and Arg side chains of Ang II is based on this report. The interaction of the Ang II COOH-terminal carboxylate and Lys is based on previous studies (13, 23).



  
Table: Binding affinity of wild-type and mutant receptors for peptide and non-peptide antagonists

I-[Sar,Ile]Ang II, a peptide antagonist of the AT receptor, was used in binding studies with all AT receptor mutants where the affinity for [Sar,Ile]Ang II was <0.1 µM. The non-peptide antagonist, [H]losartan, was used as the radioligand in the mutants where the affinity for [Sar,Ile]Ang II was >0.1 µM. Values are means ± S.E. of two to five independent measurements. The residue Asp is conserved in all AT receptors, but not in AT receptors.


  
Table: 550


FOOTNOTES

*
This work was supported in part by Specialized Center for Research in Hypertension Grant HL33713 from the National Institutes of Health, a grant-in-aid from the North East Ohio Affiliate of the American Heart Association, and a fellowship from the American Heart Association, Ohio Chapter (to K. N.). 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: Dept. of Molecular Cardiology, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-1269; Fax: 216-444-9263.

The abbreviations used are: Ang II, angiotensin II (NH-D-R-V-Y-I-H-P-F-COOH); L-162,313, [5,7-dimethyl-2-ethyl-3-[(4-[2(n-butyloxycarbonylsulfonamido)-isobutyl-thienyl]]phenyl]methyl imidazo[4,5,6]pyridine (22); HBSS, Hank' balanced salt solution.


ACKNOWLEDGEMENTS

We are indebted to the insightful suggestions of the late Dr. F. Merlin Bumpus. We thank Dr. W. Greenlee of Merck Sharp and Dohme for a generous gift of non-peptide agonist, Dr. Kunio Misono for assistance in synthesis and characterization of peptides, Dennis Wilk for excellent technical assistance, and Dave R. Schumick for artwork. The assistance of Robin Lewis and Christine Kassuba in manuscript preparation and editing is kindly acknowledged.


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