Identification of Receptor Binding and Activation Sites in Endothelin-1 by Use of Site-Directed Mutagenesis
Abstract This study addresses the structural requirements for the intracellular processing and receptor binding properties of endothelin-1 (ET-1). Point mutants of preproendothelin-1 cDNA, with replacement of the codons for Lys9 of ET-1 by ones for Ala and Glu and of Ile20 and Trp21 by ones encoding Ala, were expressed in COS-7 cells. Competitive binding experiments on rat vascular smooth muscle cells (A-10), which were shown to be an ETA receptor–rich cell line, between [125I]ET-1 and synthetic ET-1, wild-type recombinant ET-1, and recombinant [Ala9]ET-1, [Glu9]ET-1, [Ala20]ET-1, and [Ala21]ET-1 yielded Ki values of 0.2±0.02, 0.2±0.02, 0.04±0.01, 1.4±0.2, 1.6±0.2, and >50 nmol/L, respectively. In similar experiments with ETB receptor–rich human Girardi heart cells, the corresponding values were 0.2±0.03, 0.2±0.03, 0.2±0.04, 0.2±0.06, 1.4±0.4, and >50 nmol/L. The ETA receptor–mediated contractile responses to [Glu9]ET-1 and [Ala20]ET-1, measured by using canine coronary artery rings, were decreased approximately fourfold to fivefold compared with the response produced by synthetic or wild-type recombinant ET-1, whereas [Ala9]ET-1 was found to be more potent, and [Ala21]ET-1 did not produce any contraction. These results demonstrate that Ile20 and Trp21 are involved in binding to both receptor subtypes. Of considerable interest was the observation that [Glu9]ET-1 also blunts the ETA receptor subtype–mediated contractile response to ET-1 stimulus.
Endothelins (ETs) constitute a family of peptides that have very similar structures and also share sequence homology and bioactivity with sarafotoxins, found in the venom of Atractaspis engaddensis.1 2 ET-1, a peptide with very potent vasoconstrictor,3 bronchoconstrictor,4 and mitogenic5 6 7 effects, is biosynthesized as PPET-1, an inactive precursor that is a substrate for endoproteases and exoproteases.3 This 203-amino-acid residue protein is initially cleaved by dibasic-specific endopeptidases and a carboxypeptidase to yield an intermediate peptide, big ET-1, which is further processed to ET-1 by ECE. The mature peptide exerts its biological effects by binding to two G protein–coupled receptor subtypes that have been identified in higher animals. The ETA receptor has higher affinity for ET-1 and ET-2 than ET-3,8 whereas the ETB receptor binds all three isoforms and S6c with nearly equal affinity.9
Because of its constrictor and proliferative properties, ET-1 has been suggested to be involved in a variety of diseases, including atherosclerosis, renal failure, and hypertension.7 10 To better understand the role of ET-1 in these disease states and also to develop selective receptor antagonists, structure-function studies have been performed by using ET analogues and chemical and enzymatic modifications of ET-1. These studies have focused on three highly conserved regions of the molecule, including the two disulfide bonds, the cluster of charged residues (Asp8, Lys9, and Glu10), and the hydrophobic C-terminal region.
The presence of disulfide bonds is required for receptor binding and bioactivity mediated by the ETA receptor11 but not the ETB receptor12 subtype. The replacement of the charged groups of Asp8 and Glu10 by Asn and Gln, respectively, results in the complete loss of vasoconstrictor activity.11 Additionally, the vasoconstrictive response to Lys9-nicked ET-1, in which the peptide bond between Lys9 and Glu10 is cleaved, develops and decays much more rapidly.13 This observation suggests that the interaction between Lys9 and the surrounding charged residues is important in association and dissociation of ET-1 to and from the receptor.13 Furthermore, a change in the kinetics of receptor binding may be important in influencing biological activities. Since S6c, in which Lys9 is replaced by Glu, is an ETB-selective agonist, it was also proposed that variations in the net charge in the loop region of the molecule, ie, including Ser4 to Glu10, are important for receptor subtype specificity and affinity.14 Several groups also demonstrated that the C-terminal Trp is involved in receptor binding.13 15
These reports from different research groups involve either receptor binding experiments or contractility assays. However, a comprehensive study including both measurements under the same conditions has not been reported. Furthermore, these investigations were conducted only on vascular smooth muscle, where the ETA receptor subtype is predominant. Although it was demonstrated that the C-terminal fragment of ET-1 (residues 16 to 21) acts as an agonist in the guinea pig bronchus,16 which possesses predominantly ETB receptors, our knowledge of the contribution of the residues in this region on binding to ETB receptors and subsequent bioactivity is limited.
To develop a working model and investigate the contributions of the charge interactions in the loop region and of the hydrophobic residues in the C-terminal region of the peptide to the binding to both receptor subtypes and contractility mediated by the ETA receptor subtype, we substituted Lys9 with Ala and Glu and substituted Ile20 and Trp21 with Ala in the PPET-1 cDNA by using site-directed mutagenesis. This approach allowed us to study the contribution of these residues to the biosynthesis and posttranslational modifications of these peptides as well. The results of the present study demonstrate that Ile20 and Trp21 are important for binding to both receptor subtypes and that replacement of Lys9 by Glu yields a peptide that has partial agonist and inhibitory properties for the ETA receptor.
Materials and Methods
BQ-123, S6c, [Ala1,3,11,15]ET-1, big ET-1, and ET-1 synthetic peptides and RIA kits were obtained from Peninsula Laboratories, Inc. A Transformer Mutagenesis kit was purchased from Clontech. AimV serum-free medium was a product of GIBCO BRL. Transfectam and the pSVL expression vector were obtained from Promega and Pharmacia, respectively. The C-18 column (4.6×250 mm) and [125I]ET-1 (2200 Ci/mmol) were purchased from Alltech and New England Nuclear, respectively. The PPET-1 cDNA was kindly provided by Dr Carla Zoja, Mario Negri Institute for Pharmacological Research, Bergamo, Italy. The adult female dogs for the contractility studies were obtained from the Athens/Clarke County Humane Society (Ga).
Rat vascular smooth muscle cells (A-10) and African green monkey kidney epithelial cells (COS-7) were obtained from American Type Culture Collection and maintained as recommended. Girardi heart cells were kindly provided by Dr M. Fujimoto, Shionogi Research Laboratories, Osaka, Japan, and maintained as described previously.17 Briefly, all three cell lines were grown in DMEM containing 10% fetal bovine serum, 50 U/mL penicillin, 50 μg/mL streptomycin, and 125 ng/mL amphotericin B and maintained at 37°C under an atmosphere of 5% CO2.
Preparation of Mutant cDNAs and Transient Transfection
The pSVL–PPET-1 construct was used for site-directed mutagenesis with the Transformer Mutagenesis kit, which allows the introduction of specific base changes into double-stranded plasmid. The primers were designed to replace the codons in the PPET-1 cDNA for amino acid residues Lys9, Ile20, or Trp21 by ones that encode Ala. Lys9 was also replaced by an oppositely charged residue, Glu (Fig 1⇓). The primer sequences are as follows: [Ala9]ET-1, CTG ATG GAT GCA GAG TGT GTC; [Glu9]ET-1, CTG ATG GAT GAA GAG TGT GTC; [Ala20]ET-1, CTG GAC ATC GCT TGG GTC AAC; and [Ala21]ET-1, GAC ATC ATT GCG GTC AAC ACT. The mutated plasmids were verified by sequencing and mutant clones were termed [Ala9-ET-1]PPET-1, [Glu9-ET-1]PPET-1, [Ala20-ET-1]PPET-1, and [Ala21-ET-1]PPET-1, respectively.
To express the recombinant proteins, COS-7 cells were seeded at a density of 3×106 cells per 175-cm2 flask the day before the transfection. The following day, the medium was aspirated, and the cells were washed with serum-free medium. Then, the expression vector carrying the mutant or the wild-type PPET-1 cDNAs was introduced by incubating cells with Transfectam for 4 hours at 37°C. After transfection, the cells were incubated in DMEM containing 10% fetal bovine serum for 18 hours, and then the medium was replaced by AimV serum-free medium (50 mL per 175-cm2 flask). After 48 hours, the medium was collected and stored at −20°C for subsequent RIA. For HPLC analysis, samples of the expression medium were extracted by using Sep-Pak C18 cartridges. Briefly, 1 mL of medium was acidified with 1 mL of 4% acetic acid and then applied to a cartridge that had been sequentially pretreated with 5 mL each of 4% acetic acid in 86% ethanol, methanol, distilled water, and 4% acetic acid. The column was rinsed twice with 3 mL of water, and the sample was eluted with 3 mL of 4% acetic acid in 86% ethanol. The recovery of [125I]ET-1 was 80% with this procedure. The fractions were dried in a SpeedVac and reconstituted in 0.1% acetic acid in distilled water and analyzed by HPLC. Transfection experiments were performed at least six independent times.
RIA of ET-1 and Big ET-1
The concentrations of ir-ET-1 and ir-big ET-1 in the transfection media were determined with RIA kits. The cross-reactivity of the antibody used in the ET-1 RIA was stated to be 35% with big ET-1 and 7% each with ET-2 and ET-3. The antibody used in the big ET-1 RIA was claimed to be 100% specific for big ET-1 with no cross-reactivity with the ETs. The sensitivity of both assays was 10 to 1280 pg/mL. The media collected from six independent transfection experiments (n=6) were assayed in three separate RIA runs, all performed in duplicate to obtain n=18 for statistical analysis. The media were lyophilized and reconstituted in a smaller volume to concentrate the samples for receptor binding experiments and contractility assays as described below. The different dilutions of the concentrated samples were also analyzed by RIA to quantify the amount of ir-ET-1 and ir-big ET-1. To ensure that the mutant ET-1s reacted with the antibody in the ET-1 RIA as did wild-type and synthetic ET-1, dilution curves were prepared, and log-logit plots were analyzed. For these, eight concentrations of each recombinant peptide were compared with those of synthetic ET-1 over the concentration range of 10 to 1280 pg/mL.
The transfection media were separated on a reverse-phase column by using a Perkin-Elmer 1020 LC Plus system. After injection of each sample (150 μL), the column was developed by using a gradient of solvent A (water in 0.1% [vol/vol] trifluoroacetic acid) and solvent B (100% acetonitrile in 0.1% [vol/vol] trifluoroacetic acid) applied at a flow rate of 1 mL/min. The discontinuous gradient was established in six steps, half of which involved linear gradients, as follows: step 1, 100% A for 5 minutes; step 2, 80% A and 20% B for 5 minutes; step 3, 50% A and 50% B for 60 minutes; step 4, 30% A and 70% B for 5 minutes; step 5, 30% A and 70% B for 5 minutes; and step 6, 100% A for 1 minute. Synthetic ET-1 and big ET-1 were analyzed under the same conditions. The HPLC fractions (1 mL) were collected, and 100 μL of each was assayed by RIA. HPLC separation was performed at least twice by using transfection media collected from two separate experiments.
Adult female dogs were euthanatized with sodium pentobarbital. The hearts were excised and placed in chilled oxygenated Krebs’ buffer of the following composition (mmol/L): KCl 4.6, CaCl2 2.5, KH2PO4 1.2, NaCl 118, NaHCO3 2.5, and dextrose 11. The left anterior descending coronary artery was carefully isolated, cut into 2- to 4-mm rings, and mounted at the optimal diastolic tension (2 g) in 5-mL tissue baths containing oxygenated Krebs’ buffer maintained at pH 7.4 and 37°C. Bath solutions were changed every 15 minutes. Isometric contractions were measured with a Grass model 7B polygraph. The rings were allowed to equilibrate 1 hour and were then sequentially exposed to 70 mmol/L KCl and 1 μmol/L acetylcholine and washed. Contractile responses to the synthetic and recombinant peptides in concentrations from 1 to 50 nmol/L were measured, and the tension generated in these responses was expressed as the percentage of the maximal KCl response. To further identify the receptor subtype present on coronary artery rings, the dose-response curves of synthetic ET-1 were repeated in the presence of 1 μmol/L BQ-123, an ETA receptor antagonist.
Receptor Binding Experiments
A-10 cells were seeded in 12-well tissue culture plates at a density of 2×105 cells per well the day before the experiment. The next day, cells, almost 80% confluent, were rinsed twice with cold HBSS supplemented with 0.1% BSA and incubated at room temperature for 2 hours with 25 pmol/L [125I]ET-1 in the presence of unlabeled ET-1, ET-3, and ETB receptor subtype–selective agonists S6c, [Ala1,3,11,15]ET-1, or ETA receptor antagonist BQ-123. To further characterize the receptor subtypes, the total binding of [125I]ET-1 was repeated in the presence of 1 μmol/L BQ-123 or 0.1 μmol/L S6c to block ETA or ETB receptors, respectively. The cells were washed twice with ice-cold HBSS and solubilized in 1N NaOH, and cell-bound radioactivity was measured. After the receptor subtype was identified, similar experiments were performed by using transfection medium containing various concentrations (5 pmol/L to 50 nmol/L) of either wild-type ET-1, [Ala9]ET-1, [Glu9]ET-1, [Ala20]ET-1 or [Ala21]ET-1 recombinant peptides or synthetic ET-1. The receptor binding experiments with Girardi heart cells were performed in glass tubes, because the cells detached from the tissue culture plates during the incubation period. On the day of the experiment, cells were trypsinized and aliquoted into the glass tubes at a density of 5×105 cells per tube. Binding was carried out as described above. Specific binding was calculated as total binding minus nonspecific binding, which was measured in the presence of excess unlabeled ET-1 (0.1 μmol/L). The binding experiments were repeated four times each in duplicate, and the data were analyzed by In-Plot (GraphPad Software).
Expression of Recombinant Peptides
The wild-type and mutant cDNAs were transiently transfected into COS-7 cells, and the media collected 48 hours after transfection were assayed for ir-ET-1 and ir-big ET-1 by RIA. To test the sensitivity of the antibody for the recombinant wild-type and mutant peptides, dilution curves for each peptide were performed and compared with the standard curve for synthetic ET-1. The equivalent slopes of the log-logit plots for synthetic ET-1 and the recombinant peptides indicated that the antibody used in the RIA recognizes the mutant peptides as well as wild-type and synthetic ET-1 (Table 1⇓). On this assay, total ir-ET-1 secreted into the medium collected from mock-transfected cells and cells transfected with wild-type PPET-1, [Ala9-ET-1]PPET-1, [Glu9-ET-1]PPET-1, and [Ala20-ET-1]PPET-1 cDNAs were 50±30, 2704±104, 2870±372, 3206±246, and 3190±340 pg/mL, respectively, suggesting that these mutations had no impact on biosynthesis and processing of the mutant PPET-1s. The corresponding value in the medium obtained from [Ala21-ET-1]PPET-1 cDNA–transfected cells, however, was only 302±44 pg/mL (Fig 2⇓). We recently showed that there is a corresponding increase in the ir-big ET-1 in the cell extracts obtained from cells transfected with this mutant cDNA, suggesting that this residue is involved in the processing and secretion of [Ala21]ET-1 and that the metabolism of this mutant does not differ from that of wild-type ET-1.18
ir-ET-1 in the transfection media was further characterized by HPLC coupled to RIA (Fig 3⇓). In the medium obtained from cells transfected with wild-type PPET-1 cDNA, a major peak coeluted with standard ET-1 at fraction 47, and a minor peak coeluted with standard big ET-1 at fraction 37 (Fig 3A⇓). As shown in Fig. 3B⇓ [Ala9]ET-1 and [Glu9]ET-1 eluted at fraction 46, and the corresponding big ET-1 mutants were detected in fraction 36. For the [Ala20]ET-1 and [Ala21]ET-1 recombinant peptides, the major peak was located at fraction 45, and the minor peak was located at fraction 34 (Fig 3C⇓). The differences in the elution profiles of the mutant peptides are attributable to the replacements made.
Characterization of ET Receptor Subtypes on A-10 Cells and Girardi Heart Cells
The presence of ETA and ETB receptors on A-10 cells and Girardi heart cells, respectively, was confirmed by competitive binding experiments between [125I]ET-1 and unlabeled ET-1, ET-3, the ETA receptor antagonist BQ-123, and ETB receptor subtype–selective agonists S6c and [Ala1,3,11,15]ET-1. The rank order of the Ki values for ET-1 (0.13 nmol/L), BQ-123 (1 nmol/L), [Ala1,3,11,15]ET-1 (125 nmol/L), ET-3 (500 nmol/L), and S6c (520 nmol/L) demonstrated the presence of a typical ETA receptor subtype (Fig 4A⇓). Although the competition curve with BQ-123 suggested that there might be two classes of binding sites, the analysis of the data from binding experiments with all the unlabeled ligands resulted in a best fit for one class of binding sites. In addition, 1 μmol/L BQ-123 totally blocked the binding of [125I]ET-1, and Northern analysis of mRNA obtained from A-10 cells showed the presence of only one band corresponding to the ETA receptor subtype (data not shown), providing further evidence that only one type of receptor (ETA) is present in this cell line. Similar experiments were performed on Girardi heart cells, and all peptides displaced [125I]ET-1 in a monophasic manner with Ki values of 0.14, 0.2, 0.2, and >900 nmol/L for ET-1, ET-3, [Ala1,3,11,15]ET-1, and BQ-123, respectively (Fig 4B⇓). These results indicated that this cell line possesses mainly ETB receptors, confirming the results of others.17 Moreover, the addition of 1 μmol/L BQ-123 had no effect on total binding, whereas 0.1 μmol/L S6c totally blocked the binding of [125I]ET-1.
Receptor Binding Properties of the Mutant ETs
The concentrations of the recombinant mutant and wild-type ETs in the transfection media ranged between 0.1 and 0.7 nmol/L. To obtain reasonable dose-response curves with the two different cell lines, the transfection media were lyophilized and resuspended in a smaller volume. The concentrated samples were then assayed by RIA. The highest concentration in all the samples was determined to be ≈50 nmol/L. Then, serial dilutions were prepared between 5 pmol/L and 50 nmol/L. The media were used directly in the competitive binding experiments with A-10 and Girardi heart cells, which are enriched in ETA and ETB receptor subtypes, respectively. The medium obtained from mock-transfected cells did not displace any of the [125I]ET-1 bound to both cell types.
As shown in Fig 5A⇓, binding of recombinant ET-1 (Ki, 0.2 nmol/L) to A-10 cells was identical to that of synthetic ET-1 (Ki, 0.2 nmol/L). All of the mutants displayed a different competition curve. When Lys9 was replaced with Ala, the competition curve shifted to the left (Ki, 0.04 nmol/L), suggesting that the [Ala9]ET-1 mutant has a higher affinity for ETA receptors. When the same residue was replaced by Glu, the binding decreased, as evidenced by an increased Ki value of 1.4 nmol/L. [Ala20]ET-1 also exhibited decreased binding with a Ki of 1.6 nmol/L. In contrast, [Ala21]ET-1 exhibited very little binding and displaced only 15% of the radiolabeled ET-1.
The recombinant mutant peptides exhibited different binding curves to Girardi heart cells relative to A-10 cells (Fig 5B⇑). Replacement of Lys9 with Ala or Glu had no measurable effect on binding. These mutant peptides displaced labeled ET-1 in a manner similar to synthetic ET-1 and recombinant ET-1. The competition curve with [Ala20]ET-1 was shifted to the right with a Ki value of 1.4 nmol/L. The [Ala21]ET-1 mutant was again characterized by very weak binding and displaced only 18% of the radiolabel. The binding data obtained from both cell lines are summarized in Table 2⇓.
Effects of Synthetic and Recombinant ETs on Canine Coronary Artery Contraction
When synthetic ET-1 was tested on canine coronary artery rings, which are reported to be rich in ETA receptors, a sharp dose-response curve, starting at 1 nmol/L and reaching 80% of the maximum at 10 nmol/L, was produced (Fig 6⇓). Recombinant ET-1 and synthetic ET-1 produced similar contractions, and [Ala9]ET-1 was found to be even more potent, which is consistent with the binding studies. Interestingly, replacement of Lys9 by Glu caused a dramatic decrease in the contractile response. Only 21% contractility was observed at the highest concentration used (50 nmol/L). The [Ala20]ET-1 mutant peptide gave similar results (ie, only 23% contractility was obtained), whereas [Ala21]ET-1 failed to induce any contraction at all. The EC50 values for each mutant peptide are given in Table 2⇑. The control medium of the mock-transfected cells did not cause any contraction, nor did it interfere with the contractility of synthetic ET-1. Since big ET-1 is 100-fold less potent than ET-1 and since the amount of big ET-1, even in the concentrated media, was always <10 nmol/L, it was concluded that the contractile response was mediated by recombinant ET-1 present in the tranfection media. This result was further supported by the finding that ETA-specific antagonist BQ-123 blocked the contractile response to recombinant wild-type ET-1 (data not shown).
To confirm the presence of ETA receptors on coronary artery rings, contractility studies were performed with the ETA receptor antagonist BQ-123. As shown in Fig 7B⇓, 1 μmol/L BQ-123, when added in combination with various concentrations of recombinant ET-1 (7.5 to 50 nmol/L), totally blocked the contractile response. Similar results were obtained when the coronary artery rings were pretreated with 1 μmol/L BQ-123 (data not shown). Moreover, 1 μmol/L BQ-123 completely antagonized the contractile response induced by 50 nmol/L ET-1 (Fig 7C⇓). These results are consistent with canine coronary arteries containing mainly ETA receptors.
To determine whether the weak agonists [Glu9]ET-1 and [Ala20]ET-1 have any antagonist properties, the contractile response to ET-1 was tested on coronary artery rings preconstricted with 50 nmol/L of these mutant peptides. As shown in Fig 8⇓, the addition of 10 nmol/L ET-1 to the rings pretreated with [Ala20]ET-1 caused a 100% contractile response similar to that obtained with 10 nmol/L ET-1 on untreated rings. However, when similar experiments were performed on [Glu9]ET-1–pretreated rings, only a 35% increase in the response, reaching a maximum of just 56% relative to 70 mmol/L KCl, was observed. This finding suggests that [Glu9]ET-1 binds to the receptor, causing ≈21% contractility, and the addition of 10 to 50 nmol/L ET-1 cannot displace this mutant peptide completely, resulting in only a 56% contractile response.
In the present study, we have obtained full-length analogues of ET-1, containing single amino acid residue replacements at positions 9, 20, and 21, by using site-directed mutagenesis and a mammalian expression system. This approach rather than the use of synthetic peptides enabled us to study the contribution of certain amino acid residues to the biosynthesis and processing of ET-1 as well as to the receptor binding and bioactivity. With the exception of the [Ala21]ET-1 mutant, the mutations introduced had no apparent effect on the synthesis and posttranslational modifications of the peptides. The results of the binding and contractility experiments with these mutant recombinant peptides have demonstrated that all three residues are involved in the binding and activation of ETA receptors. The substitutions we made at position 9 did not affect the binding to the ETB receptor subtype. To the best of our knowledge, this is the first report on the single amino acid replacements in full-length ET-1 on binding to ETB receptors.
Structure-function studies have revealed that the charged portion of the loop region, Asp8-Lys9-Glu10, is crucial for vasoconstrictor activity mediated by ETA receptors.11 For example, the replacement of Asp8 by Asn and of Glu10 by Gln resulted in a total loss of activity.11 To determine the effect of a charge change at position 9, we replaced the codon for the positively charged Lys by one that encodes a neutral amino acid residue, Ala, and also an oppositely charged residue, Glu. The results of the binding and contractility experiments involving ETA receptors with [Ala9]ET-1 are in good agreement with the report on Ala screening using synthetic peptides of ET-1.19 The replacement of Lys9 by Ala yields a peptide that has an approximately fivefold higher affinity for the ETA receptor. The effect of this substitution was also tested on binding to Girardi heart cells, which have only ETB receptors. This mutant peptide exhibited a binding curve similar to that of wild-type ET-1, suggesting that this change does not alter ETB receptor binding. However, it should be noted that the IC50 of ET-1 for ETB receptors is ≈0.2 nmol/L and that a C-terminal fragment of ET-1-(16-21), ET-1, acts as an agonist for the ETB receptors with an EC50 of 0.3 μmol/L.16 Therefore, it is reasonable to speculate that the loop structure contributes to the receptor affinity.
The replacement of Lys9 by Glu to produce a net −3 charge instead of the normal net −1 charge in the loop region yielded very interesting results. The decrease in the ETA receptor binding and bioactivity with [Glu9]ET-1 could be explained in two ways: (1) Lys9 is directly involved in just receptor binding or in receptor binding and signaling. Replacement of this amino acid with Glu causes a decrease in both. (2) The conformation of the loop region is important for presenting the C-terminal region of the molecule to the receptor in the proper conformation. In this case, the introduction of an extra negative charge could disrupt this structure and indirectly result in a decrease in binding and bioactivity. Kimura et al13 demonstrated that the vasocontractile response to Lys9-nicked ET-1 develops and decays much faster than that of ET-1. In the present study, we did not observe a change in the kinetics of vasocontractility. However, when ET-1 was added to the coronary artery rings preconstricted with this mutant, only a partial response (35%) was detected. On the other hand, when similar experiments were performed on coronary rings pretreated with another mutant peptide, [Ala20]ET-1, ET-1 caused 100% contraction. These findings suggest that once [Glu9]ET-1 is bound to the ETA receptor, ET-1 cannot displace it completely, at least within the time frame of the experiment, and it partially blocks the effects of ET-1.
Takai et al20 reported that substitution of Glu at position 9 in a modified ET-1 fragment yields a potent and specific ETB agonist, IRL 1620, Suc-[Glu9,Ala11,15]ET-1-(8-21), with an IC50 of 16 pmol/L. Interestingly, another ET-1 analogue, IRL 1038, [Cys11,15]ET-1-(11-21), acts as an ETB receptor antagonist.21 Since IRL 1620, but not IRL 1038, has the charged residues Asp8, Glu9, and Glu10, it was suggested that these residues are involved in the vasoconstrictor activity and that replacement of Lys9 by Glu increases the affinity for the ETB receptor. As discussed above, this region of ET-1 might contribute to the selectivity and affinity of the ETB receptor. However, under the experimental conditions of the present study, Glu9 replacement did not increase the affinity to ETB receptors. Although S6c, in which Lys9 is naturally replaced by Glu, is known to be an ETB-selective agonist,22 its IC50 value (0.2 to 1 nmol/L) is very similar to that of [Glu9]ET-1 reported in the present study. This difference in the IC50 values between IRL 1620 and S6c is most likely due to the linear structure of IRL 1620 or the absence of the first eight amino acid residues.
Reports from several research groups have demonstrated that the C-terminal region is crucial for vasoconstrictor activity and that removal of Trp21 causes a 103-fold decrease in the contractile response mediated by the ETA receptor.13 15 Also, replacement of Trp21 by other aromatic residues, Phe or Tyr, caused a fivefold decrease in the vasoconstrictor activity,11 indicating that the indole ring of Trp is important for recognition and/or signaling by the ETA receptor. In the present study, we demonstrated that Ile20 and Trp21 are important for binding to ETB receptors as well. In addition, our findings that [Ala20]ET-1 exhibited decreased binding and bioactivity and that [Ala21]ET-1 showed only 15% binding and no bioactivity suggest that these residues are involved in both receptor binding and activation.
On the basis of studies with chimeric receptors between ETA and ETB receptors, it was proposed that the ETs have two distinct domains that determine the selectivity for ET receptors.23 According to this model, the so-called address domain containing the N-terminal loop structure is involved in the recognition and binding to ETA receptors by interacting with transmembrane domains IV to VI. The C-terminal region was postulated to act as a message domain for the ET receptors and to be responsible for signaling and subsequent bioactivity via interaction with transmembrane domains I, II, III, and VII. However, our data showing that residues 20 and 21 are involved in receptor binding and vasoconstriction do not support this classification of the domain structure of ET-1. Although the mechanism(s) by which the C-terminal region of ET-1 interacts with the receptors is not known, one possibility, as shown with some other G protein–coupled receptors,24 25 is that the hydrophobic C-terminal region of ET-1 may fold back, starting at about His16, and intercalate into the membrane. If so, the side chains of Lys140 in the second transmembrane domain or of Lys159 in the third transmembrane domain of the ETA receptor might be potential sites for an ion-ion interaction involving the ET-1 α-carboxyl group. Although the [Ala21]ET-1 mutant has this carboxyl group, because of the lack of the indole ring of Trp21, the peptide may not localize correctly on the membrane and thus cannot bind to the receptor.
In conclusion, using recombinant point mutants of ET-1, we have shown that Ile20 and Trp21 are involved in binding to both receptor subtypes. In addition, Lys9 is an important residue for binding and activation of the ETA subtype, whereas replacement of this residue either by Ala or Glu does not affect its binding to the ETB receptor subtype.
Selected Abbreviations and Acronyms
|HPLC||=||high-performance liquid chromatography|
This study was supported by the American Heart Association, Georgia Affiliate, Inc (Grant-in-Aid awards to Drs Tackett and Puett). Dr Ergul is the recipient of a Graduate Student Fellowship (92GSF/4) from the American Heart Association, Florida Affiliate, Inc. We would like to thank Dr Prema Narayan for critically reading the manuscript and Sondra Ricci and Kanili Shoemaker for expert technical assistance.
- Received April 21, 1995.
- Accepted August 21, 1995.
- © 1995 American Heart Association, Inc.
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