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Biochimica et Biophysica Acta 1223 (1994) 202-208

Identification of specific regions of the human endothelin-B receptor required for high affinity binding with endothelin-3 Miki Adachi, Yasuhiro Furuichi, Chikara Miyamoto

*

Department of Molecular Genetics, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa pref. 247, Japan Received 11 March 1994

Abstract

To investigate the endothelin-3 (ET-3) binding region of the endothelin-B (ET B) receptor, we have transiently produced various chimeric endothelin receptors in transfected Chinese hamster ovary cells. Using 125I-ET-1 as the radioactive ligand in the displacement experiment, the replacement of both the second and third extracellular regions including the flanking transmembranes of the ET B receptor with the corresponding domains of the endothelin-A (ETA) receptor, increased the apparent K~ value for ET-3 from 5 • 10-11 M to 10 -s M. The affinity of this chimeric receptor, ETB-BC , for ET-3 was about two orders lower than ET B yet one order higher than ETA. Previously we have reported the involvement of Lys-140 located in the C-terminus of the second transmembrane region of the ETA receptor for ET-1 binding (Eur. J. Biochem., 220, 37-43, 1994). To assess the importance of the corresponding Lys-161 of the ET B receptor in ET-3 binding, we have replaced it with lie in the ET B receptor. The mutant receptor had a 5.6-fold reduction in its affinity for ET-3, but its affinity for ET-1 remained similar. These results demonstrate that Lys-161 of the receptor is important for high affinity binding with ET-3 which, in part, confers the non-selective binding characteristics of the ET B receptor for ET isopeptides.

Key words: Endothelin-3 (ET-3); Receptor; Binding site; Expression 1. Introduction

Endothelin-1 (ET-1), initially described as a 21amino acid vasoactive peptide secreted by vascular endothelial cells [1], is now known to be one of a family of three distinct peptides. ET-2 with two amino acid substitutions closely resembles ET-1, while ET-3 differs from ET-1 in its amino acid sequence at positions 2, 4, 5, 6, 7 and 14, but it shares the C-terminal sequence 16-21 of ET-1 [2]. E T isopeptides are characterized by two disulfide bonds (cysl-cysl5 and cys3cysll), a cluster of polar charged side chains residing within a hairpin loop (residues 6-10) and a hydrophobic C-terminus (residues 16-21) containing the aro-

Abbreviations: ET, endothelin; ETA, endothelin-A receptor subtype; ET B, endothelin-B receptor subtype; ET c, endothelin-C receptor subtype; CHAPS, 3-[(3-eholamidopropyl)dimethylammonio]-lpropanesulfonate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; CHO cell, Chinese hamster ovary cell; G-protein, guanine-nucleotide-binding-regulatory protein. * Corresponding author. Department of Mycology. Fax: + 81 467 465320. 0167-4889/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

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matic indole side chain at Trp-21. Both the hairpin loop and C-terminus seemed to be involved in binding with their distinct receptor [3,4]. After binding, these peptides trigger a common set of short-term responses which include rapid production of inositol 1,4,5-trisphosphate, sustained release of arachidonic acid with synthesis of prostaglandins, mobilization of Ca 2+ from intracellular and extracellular sources, activation of protein kinase C and rapid expression of the c-jun and c-los genes [5,6]. As a result of its potent and long-lasting vasoconstriction, ET-1 is involved in the pathogenesis of delayed cerebral vasosospasm following subarachnoid hemorrhage [7]. Two subtypes of the endothelin receptor, called E T A and E T B, which differ in the order of their binding affinities for three E T peptides have been cloned [8,9]. The E T A receptor has high and equal affinity for ET-1 and ET-2, approximately a 100-1000-fold lower affinity for ET-3, while the E T B receptor subtype displays high and almost equal affinity for all of the E T peptides. Both are included in the superfamily of receptors coupled to a guanine-nucleotide-binding-regulatory protein (G-protein) and span seven transmembrane do-

M. Adachi et al. / Biochimica et Biophysica Acta 1223 (1994) 202-208

mains. The ET A receptor predominantly exists in vascular smooth muscle cells and plays a major role in vasoconstriction [2,10]. The ET B receptor, predominantly present in vascular endothelium cells, mediates endothelium-dependent relaxation by inducing prostacycline and endothelium-derived relaxation factor [11,12], while pharmacological evidence predicts the presence of an ETB-like receptor mediating vasoconstriction [13]. Recently, cloning of the endothelin-C receptor subtype (ET c) cDNA from Xenopus laecis dermal melanophores has been reported. The product of this gene has preferential binding activity with ET-3 rather than with ET-1 or ET-2 [14]. The transmembrane regions and the intracellular loops of these three ET receptors have the greatest degree of sequence identity. The high dissimilarities are found in the Ntermini and extracellular domain 3. Recently we have reported the ligand binding site (Lys-140 located at the end of the second transmembrane) and specific domains (the third and the fourth extracellular regions including the flanking transmembranes) required for ligand selectivity of the human ET A receptor [15]. In this paper, we describe the specific domains of the ET B receptor required for ET-3 binding which confer the non-selective binding of ligand to the receptor.

2. Materials and methods 2.1. Materials

ET-1, ET-2, and ET-3 were from the Peptide Institute (Osaka, Japan). The 125I-labeled ET-1 (86 TBq/mmol) and 125I-labeled ET-3 (82 T B q / mmol) were from New England Nuclear. (CHAPS) 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate was from Dojin Chemical Institute (Kumamoto, Japan). The site-directed mutagenesis system, Mutan-K, was from Takara (Osaka, Japan). 2.2. The chimeric E T receptors

The four extracellular domains of the ET receptors including both flanking transmembrane regions were designated A, B, C, and D. The chimeric ET B receptors made by replacement of particular regions with those of ET A are represented by the name of the replaced region and the numbers of the substituted amino acid sequences, e.g., ETB-A (Met-1 to Ser-120), ETB-B (I1e-138 to Ile-197), ETB-C (Asp-241 to Phe291), and ETB-CD (Trp-206 to C-terminal Ser-442) receptors. For the construction of the expression plasmids for various chimeric ET receptors, DNA fragments encoding the substituted amino acid sequence were amplified by polymerase chain reaction (PCR). The DNA fragments contained suitable restriction sites

203

at the ends, and the sequences that encoded the different domains from the original ET receptor were ligated with the fragments of pCDM8-ET g or pCDM8ET a through several ligation steps for the preparation of chimeric plasmids. For the ETB-D (Asn-351 to Ser362) receptor, we constructed pCDM8-ETA-D using site-directed mutagenesis [16]. 2.3. Cells and transfection

Chinese hamster ovary (CHO) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, New York, USA) supplemented with heat-inactivated 10% fetal bovine serum. The cells were grown and adjusted to 4.106 cells per 90 mm diameter dish 1 day before transfection, and the medium was replaced with 6 ml of fresh DMEM supplemented with heat-inactivated 10% Nu serum. About 8/xg of pCDM8-based chimeric plasmids was dissolved in 100 /xl of distilled water, mixed with 60 /xl of 40 mg/ml of DEAE-dextran in phosphate-buffered saline (PBS) and added to the cell monolayer. After a further addition of 60 ~l of 10 mM chloroquine (Sigma, St. Louis, MO, USA), the cell monolayer was incubated for 3 h. Cells were then treated with 3 ml of 10% dimethyl sulfoxide in PBS for 3 min, fed again with fresh medium, and incubated for 72 h prior to ligand binding. 2.4. Binding assay

CHO cells were removed from the plates and collected by centrifugation. After sonication of CHO cells, the crude membrane was prepared from the cell homogenate by centrifugation at 15 000 rpm for 30 min, and the pellet was dispersed and solubilized in 200/zl of assay buffer (50 mM sodium phosphate buffer (pH 7.4) and 0.1% CHAPS). The inclusion of CHAPS in the assay mixture increased the binding activity and reproducibility. The assay mixture (50/~l) contained 30 /xl of assay buffer, 1-5 /~l of solubilized crude membrane from transfected CHO cells, and 2.5 fmoles of 125I-labeled ET-1 or ET-3. The mixture was incubated for 2 h at room temperature. The receptor-125 I-ligand complex was separated from free 125I-ligand as described before [17], and the radioactivity of the bound ligand was determined. The specific binding was calculated by subtracting non-specific binding in the presence of 1/~.M non-radioactive ET-1.

3. Results 3.1. Displacement o f 125I-labeled ET-1 bound to various chimeric E T A receptors by unlabeled ET-3

We have transiently produced various chimeric ET receptors in CHO cells, in which the extracellular

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M. Adachi et al. / Biochimica et Biophysica Acta 1223 (1994) 202-208

domains A, B, C and D together with flanking transmembranes of the ET a receptor were replaced with the corresponding domain of the ETA receptor. The parental and chimeric ET receptors bound between

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Fig. 1. Displacement of the 125I-labeled ET-1 bound to the parental and chimeric ET receptors by unlabeled ET-1 and ET-3. Transfection of CHO cells with the parental and mutated receptor cDNA-pCDM8 followed by isolation of crude membranes was as described in Materials and methods. Binding of z25I-labeled ET-I (50 pM) to membranes was performed in the absence or presence of increasing concentrations of unlabeled ET-1 ( o ) and ET-3 (e). The ligand binding activities of the parental and chimeric receptors are indicated by relative percentages of those determined in the absence of unlabeled ET-1 and ET-3. The structures of the parental and chimeric ET receptors are also shown with ET A and ET a receptors being represented by closed and open bars, respectively. 1, ETa; 2, ETa-A; 3, ETa-B; 4, ETa-C; 5, ETa-D; 6, ETa-CD; 7, ETB-BC; 8, ETA.

M. Adachi et al. / Biochimica et Biophysica Acta 1223 (1994) 202-208

volved in ET-3 binding with high affinity, we determined the K i values of ET-1 and ET-3 by displacement of 125I-ET-1 bound to various chimeric receptors by unlabeled ET-1 and ET-3. Substitution of the A, B and D domains including the flanking transmembranes of the ET a receptor with the same domain of the ETA receptor did not change the characteristics of nonselective ligand binding. However, replacement of the third extracellular (C) region increased the K i value of ET-3 from 5 • 10 -11 M to 1.3" 10 -9 M (Fig. 1 (panel 4) and Table 1). To understand the specific region of the ET a receptor required for high affinity binding with ET-3, we produced chimera ET B receptors by replacing two regions. When both C and D regions of the ET a receptor were replaced with the corresponding domain of the ETA receptor, the K i value for ET-3 increased from 5" 10 -11 M to 1.6.10 -9 M (Fig. 1 (panel 6), Table 1). In contrast, the chimeric receptor, ETa-BC, revealed a low affinity (K i = 1.3-10 -s M) for ET-3 which was 260-fold lower than that of the wild-type ET a. These results indicate that both the B and C regions of the ET a receptor including the flanking transmembranes are required for high affinity ET-3 binding (Fig. 1 (panel 7), Table 1).

3.2. Binding of 125I-ET-3 to various chimeric ET receptors To confirm the regions of the ET a receptor required for binding with ET-3, we have monitored the binding of 125I-labeled ET-1 and ET-3 to these receptors. The binding activities of ET-3 to ETa, ETa-A , ETB-B and ETa-D receptors were almost equipotent to those of ET-1, which represents the high affinity of ET-3 to these receptors (Fig. 2, lanes 1, 2, 3 and 5).

Table 1 Inhibition constants (K i) of ET-1 and ET-3 for the binding of 125I-labeled ET-1 to the parental and chimeric ET a receptors Chimera receptors

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The parental and chimeric ET B receptors were transiently produced in CHO cells. After preparation of the crude membranes, the binding of 125I-labeled ET-1 to each receptor was determined in the presence and absence of increasing concentrations of unlabeled ET-1 and ET-3. To evaluate iigand selectivity of various chimeric ET receptors, the ratios ( Y / X ) are calculated from the K i values of ET-3 (Y) and ET-1 (X).

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The binding activities of ET-3 to ETB-C, ETB-CD and ETB-BC receptors, however, decreased to 0.34, 0.23 and 0.11 pmol/mg protein, as compared with 0.83 pmol/mg protein in the parental ET B receptor (Fig. 2, lanes 4, 6 and 7). Based on these results, the high affinity binding of ET-3 to the ET B receptor seems to be ascribed to both the B and C regions of the ET s receptor.

3.3. Substitution of Lys-161 of the ET a receptor with Ile reduced the binding activity with ET-3 Previously we have reported the importance of Lys140 of the ETA receptor for ligand binding, based on the evidence that the substitution of Lys-140 with lie resulted in a 13-fold reduction in its affinity for ET-1 and a Bmax 3.6-fold lower than that of the original ETA receptor [15]. Lys-140 located at the C-terminal end of the second transmembrane region is conserved among human ETA and ET B [8,18], rat ETA and ET B [19,20], bovine ETA and ET B [21,22] and porcine ET B [23] receptors (Fig. 3). Since the B-domain of the ET B receptor is insensitive to BQ-123 for ligand binding [24], we have examined the binding activity of the ETB(K 161-o I) receptor for ET-1 and ET-3, in which Lys-161 (corresponding to Lys-140 of the human ETA receptor) was replaced with lie. Both the ET B and ETB(K 161 ~ I) receptors were found to have similar K d (1.1 and 1.4 pM) and Bmax values (8.6 and 10 pmol/mg protein) for binding with ET-1; however, the latter mutant showed a 5.6-fold lower affinity for ET-3 than the parental one (Fig. 4). The K d and Bma~ values of the parental receptor for binding with ET-3 were 1.1 pM and 8.4 pmol/mg protein, while these values of the mutant receptor were 6.2 pM and 6.6 pmol/mg protein, respectively. The decreased affinity of ET-3 to the ETB(K161 ~ I) receptor resulted in a decrease in the

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M. Adachi et al. / Biochimica et Biophysica Acta 1223 (1994) 202-208 140

4. Discussion

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K i value from 5" 10 -11 M to 4.5.10 -1° M. Therefore, at least Lys-161 in the B region of the ET a receptor is required for the high affinity binding of ET-3, not of ET-1.

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In this study we have identified the region of the human ET B receptor involved in non-selective ligand binding, namely the regions responsible for binding with ET-3. Substitution of the third extracellular region (C) of the ET B receptor, including the flanking transmembrane domains, with an equivalent portion of the ETA receptor reduced its affinity for ET-3 26-fold (Fig. 1 (panel 4) and Table 1). In addition, when both the second and third extracellular regions, including flanking transmembrane domains, of the ET B receptor were substituted with the corresponding domain of the ETA receptor, the resultant chimeric receptor (ETB-BC) revealed an affinity for ET-3 260-fold lower than that for the ETB receptor (Fig. 1 (panel 7) and Table 1). Sakamoto et al. [3] reported involvement of the region spanning the second intracellular loop through the amino-terminal half of the fourth extracellular region of the human ET B receptor in the high affinity binding with ET-3. We newly added the second extracellular domain including the flanking transmembrane regions to the above mentioned region of the human ET B receptor to render high affinity binding with ET-3. The structure of ET-3 was solved using high-resolution NMR spectroscopy [25]. ET-3 reveals a highly ordered, compact conformation, in which a helical region extending from Lys-9 to Cys-t5 lies in close apposition with the C-terminal hexapeptide largely driven by hydrophobic interaction. In addition, the C-terminal Trp residue was reported to be involved in binding to the ET receptor [26]. Since the ETA-selective antagonist BQ-123 mimicks a structure within the C-terminal linear portion of ET-1, the C-terminal portion of the ET peptide seems to be involved in binding to ET receptors. We have previously reported involvement of the second extracellular (B) region, including the flanking transmembrane domains, especially the Lys-140 residue of the ETA receptor for ET-1 binding with high affinity [15]. The Lys-140 residue of the human ETA receptor was conserved among ETA and ET B receptors (Fig. 3), but not in other G-protein-coupled receptors with seven transmembrane domains. Based on this evidence, we substituted the Lys-161 of the ET 8 receptor corresponding to the Lys-140 of the ETA receptor with lie. This mutant receptor, ETB(K 161 ~ I), revealed an equipotent activity for ET-1 binding, but it had Kj values for ET-3 binding 5.6-fold lower than that of the parental receptor (Fig. 4). Thus, Lys-161 of the ET B receptor is, in part, important for the binding with ET-3 and not for ET-1. Therefore, the ligand binding site located in the B region of the ET B receptor seems to be partly modified from the corresponding site of the ETA receptor to access ET-3. This modification may be the result of the lack of affinity of the ET B receptor to ET A specific antagonists such as BQ-123

M. Adachi et al. / Biochimica et Biophysica Acta 1223 (1994) 202-208

[3,24]. Although the role of Lys-161 of the ET 8 receptor in binding with ET-1 is probably different from that of the Lys-140 of the ETA receptor, the ET B receptor maintains the high affinity binding activity with ET-1. Further information on the ligand binding site of the ET B receptor must await elucidation of the three-dimensional structure of this receptor followed by clarification of its interaction with the ligands. The Asp-ll3 residue in the third transmembrane region of the/3-adrenergic receptor has been reported to participate in receptor-ligand interactions [27]. The corresponding amino acid residue in the human ET B receptor is Lys-182. Substitution of the corresponding Lys in the third transmembrane region of the rat ET B receptor with Asp reduced its affinity for ET-3 without affecting G-protein coupling [28]. The Lys-182 residue located in the C region of the human ET B receptor is probably important for high affinity binding with ET-3. A comparison of the primary sequence of the human ETA and ET~ receptors reveals 59% identity. Alignment of both ET receptors reveals a high degree of homology in the transmembrane domains and three cytoplasmic loop regions. Low homologies are found in the N-termini and the second and third extracellular domains, suggesting that these regions may be involved in determining the specificity of the receptors for the different ET peptides. The second putative extracelluar region of the ET B receptor is a five amino acid residue shorter than the equivalent region of the human ET A receptor. Homologies of the second and the third extracellular regions are 50% and 41% between ETA and ET B receptors, respectively. Both extracellular regions, including the flanking transmembrane domains of the human ET B receptor, are involved in non-selective ligand binding, especially the binding with ET-3 (Figs. 1 and 2). Both regions are probably involved in the interaction with the charged moieties of the N-terminal loop of ET-3, whose amino acid sequence is different at positions 2, 4, 5, 6 and 7 from that of ET-1. The greatest dissimilarity is found in the N-terminal extracellular region in which the amino acid sequence identity between the human ETA and ET B receptors is only 4%. Indeed, in the human ET B receptor it was found to form the stable complex with ET-1 which can survive in 2% SDS and migrate in SDS-PAGE under low temperature, but not in the human ETA receptor. The N-terminal extracellular region from Lys-73 to Thr-99, especially Asp-75 and Pro-93 of the human ET B receptor, is responsible for the formation of the stable complex [9,29]. This evidence will, in part, explain the functional dissimilarity of the N-terminal extracellular domain between the human ETA and ET B receptors. This region, located in the human ETA and ET B receptors, is required for binding of ET-1 to the human ETA and ET B receptors ([30], and unpublished results). Meanwhile, the identity

207

of the C-terminal intracellular region of the human ETA and ET B receptors is 42%. This region is required for ligand binding, probably by maintenance of the 3-D structure of the receptor [30], and for signal transduction for coupling with the G-protein [31]. The knowledge gained by molecular biology research using the mutated and chimeric ET receptors together with information clarifying the tertially structure of the ET B receptor will lead to a design of an ET B antagonist, which may enable us to demonstrate the functional differences between ET receptor subtypes.

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