A Single Position in the Third Transmembrane Domains of the Human B1 and B2 Bradykinin Receptors Is Adjacent to and Discriminates between the C-terminal Residues of Subtype-selective Ligands*

Dana B. Fathy, Sandra A. Mathis, Tosso LeebDagger , and L. M. Fredrik Leeb-Lundberg§

From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

In order to identify agonist- and antagonist-binding epitopes in the human B1 and B2 bradykinin (BK) receptors, we exploited the ability of these receptors to discriminate between peptide ligands that differ only by the absence (B1) and presence (B2) of a C-terminal Arg. This was done by constructing chimeric proteins in which specific domains were exchanged between these receptors as recently described by us (Leeb, T., Mathis, S. A., and Leeb-Lundberg, L. M. F. (1997) J. Biol. Chem. 272, 311-317). The constructs were then expressed in HEK293 and A10 cells and assayed by radioligand binding and by agonist-stimulated inositol phospholipid hydrolysis and intracellular Ca2+ mobilization. Substitution of the third transmembrane domain (TM-III) of the B1 receptor in the B2 receptor (B2(B1III)) dramatically reduced the affinities of B2-selective peptide ligands including both the agonist BK and the antagonist NPC17731. High affinity binding of both ligands to B2(B1III) was fully regained when one residue, Lys111, in TM-III of this chimera was replaced with the corresponding wild-type (WT) B2 receptor residue, Ser (B2(B1IIIS111)). Replacement of Ser111 with Lys in the WT B2 receptor decreased the affinities of BK and NPC17731 and increased the affinity of the B1-selective des-Arg10 analog of NPC17731, NPC18565. The results show that the C-terminal residue of peptide agonists and antagonists when bound to the B2 receptor is adjacent to Ser111 in the receptor. A Lys at this position, as is the case in the WT B1 receptor, provides a positive charge that repels the C-terminal Arg in B2-selective peptides and attracts the negative charge of the C terminus of B1-selective peptides, which lack the C-terminal Arg. Therefore, the residues at this one single position are crucial in determining the peptide selectivity of B1 and B2 BK receptors.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Seven-transmembrane domain GPCR1 constitute by far the largest family of plasma membrane receptors. These receptors bind ligands of widely diverse origins, and are unsurpassed as therapeutic targets. Consequently, much effort has been devoted to mapping of the binding sites for agonist and antagonist ligands in these receptors (1, 2). Even though peptides are the most common class of ligands for GPCR, few peptide GPCR have been investigated thus far, and in most of those cases the identity of the peptide-binding epitopes remains elusive.

Receptors for kinins, pro-inflammatory peptides 8-10 amino acids in length, have been classified into two subtypes, termed B1 and B2 (3), and are members of the GPCR superfamily (4, 5). These receptor subtypes, although only 36% identical, discriminate between peptide agonists that differ only in their C-terminal residue; BK binds to the B2 receptor, whereas the C-terminally truncated carboxypeptidase fragments des-Arg9-BK and des-Arg10-Lys-BK, or des-Arg10-KD, bind to the B1 receptor. Several high affinity B2 receptor-selective decapeptide antagonists structurally derived from BK have been developed, including NPC17731 and HOE140 (6-8). Interestingly, the fact that the des-Arg10 analogs of these peptides act as high affinity B1 receptor-selective antagonists emphasizes the significance of the C-terminal Arg in receptor subtype selectivity (9-11).

In the B2 receptor, extensive analysis of most of the TMs and a significant amount of the ECs by alanine-scanning mutagenesis has yielded no information about residues important for antagonist binding and has identified only a few residues important for agonist binding (12-15). We recently developed a novel, potentially more effective strategy for mapping the binding sites in kinin receptors, which is based on the identification of receptor epitopes that enable these receptors to discriminate between ligands (11). This strategy involves the exchange of individual TMs between the B1 and B2 receptor subtypes and the subsequent exchange of non-conserved residues that are possible candidates for discriminatory action. This approach is intrinsically more reliable than alanine-scanning mutagenesis as it yields in sequence both loss-of-function and gain-of-function mutations.

In an initial study, we used this strategy to identify specific residues in TM-VI of the human WT B1 and B2 receptors that are partially responsible for enabling these receptor subtypes to discriminate between peptide agonists (11). In the present study, we analyzed the role of TM-III in peptide ligand discrimination. Our results show that TM-III enables BK receptors to discriminate between both peptide agonists and antagonists, and the discrimination involves specifically the C-terminal amino acid of the peptides, the hallmark for BK receptor subtype selectivity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- [2,3-prolyl-3,4-3H]Bradykinin (114 Ci/mmol), [prolyl-3,4-3H]NPC17731 (53.5 Ci/mmol), des-Arg10-[3,4-prolyl-3,4-3H]kallidin (107 Ci/mmol), and [3H]myo-inositol (10-20 Ci/mmol) were obtained from NEN Life Science Products. NPC17731 and NPC18565 were generous gifts from Donald J. Kyle, Scios, Inc., Sunnyvale, CA. Kallidin was obtained from Peninsula Laboratories (Belmont, CA), and des-Arg10-kallidin was from Bachem (Torrance, CA). LipofectAMINE, DMEM, Leibovitz's L-15 medium, and Hanks' balanced salt solution were from Life Technologies, Inc. Reagents for calcium phosphate transfections were purchased from 5Prime right-arrow 3Prime, Inc. (Boulder, CO). Fura-2/AM was from Molecular Probes (Eugene, OR). Enzymes were obtained from Life Technologies, Inc. and New England Biolabs (Beverly, MA). Sera and all other peptides and chemicals were from Sigma.

Construction of Receptor cDNA-- The original human WT B1 and B2 receptor clones in vector pcDNA3 (Invitrogen) were kindly provided by J. Fred Hess, Merck Research Laboratories, West Point, PA. Fusions between the B1 and the B2 receptor cDNA clones were made using a modified PCR-ligation-PCR protocol (16). The B2 clone was modified by introducing a XhoI site at the 3' end of the insert. Appropriate B1 and B2 clone fragments were amplified in 100-µl PCR reactions containing 10 ng of template DNA, 200 µM dNTPs, 100 pmol of each primer, 2.5 units of Pfu polymerase, and the reaction buffer supplied by the manufacturer (Stratagene, La Jolla, CA). T7 and SP6 were chosen as flanking primers, and internal primers were designed according to the desired fusion point in the chimeric construct. Appropriate gene fragments were amplified for 20 cycles and purified using 1% agarose gels and the QiaExII kit (Qiagen, Chatsworth, CA). The isolated PCR products were combined, phosphorylated, and ligated using T4 DNA ligase. In a second PCR, the fusion product was amplified for 25 cycles. Amplification products were purified as described above, cut with HindIII and XhoI, and ligated into the pcDNA3 vector. The identity of the chimeric insert was confirmed by cycle sequencing. Pure plasmid DNA for transfections into mammalian cells was isolated with the Qiagen Plasmid Maxi kit (Qiagen).

Cell Culture and Transfection-- Transiently transfected HEK293 cells were used for analysis of radioligand binding and agonist-stimulated inositol phospholipid hydrolysis. These cells were grown in DMEM supplemented with 10% heat-inactivated horse serum at 37 °C in 10% CO2. At 24 h before transient transfections, the cells were seeded into 100-mm dishes or six-well plates at 60-80% confluence. The cells were then transfected using the calcium phosphate precipitate method with overnight incubation in the presence of 15 µg of cDNA/dish. The cells were then further incubated for an additional 72-96 h after transfection.

Transiently transfected A10 cells were used for analysis of intracellular Ca2+ mobilization. These cells were grown in DMEM supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 10% CO2. At 24 h before transfection, the cells were seeded at ~ 80% confluence in six-well plates containing 25-mm glass coverslips, which had been pretreated by incubation in growth medium. Cells were transfected by incubation for 6 h in 1 ml of DMEM containing 6 µl of LipofectAMINE and 2 µg of cDNA per well. The cells were allowed to recover for about 48 h before loading with fura-2.

Membrane Preparation-- Transfected HEK293 cells were washed twice with ice-cold PBS and then pelleted by centrifugation at 2,000 × g for 10 min. The cells were then resuspended in a buffer containing 25 mM TES, pH 6.8, 0.5 mM EDTA, 0.2 mM MgCl2, and 1 mM 1,10-phenanthroline and homogenized using an Ultra-turrax at 20,500 rpm for 10 s. Membranes were isolated by centrifugation at 45,000 × g for 30 min at 4 °C. The pellets were then resuspended in the above buffer supplemented with 0.1% BSA and 0.014% bacitracin (binding buffer).

Radioligand Binding-- Membranes were diluted in binding buffer to give a signal of 1,000-4,000 dpm/assay of specific radioligand binding. Binding assays were performed in a total volume of 0.5 ml with either [3H]BK, [3H]NPC17731, or [3H]des-Arg10-KD with or without varying concentrations of non-radioactive kinin peptides. After incubation for 60-90 min at room temperature, assays were terminated by dilution with 4 ml of ice-cold PBS, 0.3% BSA, and rapid vacuum filtration on Whatman GF/C filters previously soaked in 1% polyethyleneimine. The trapped membranes were then washed with an additional 2 × 4 ml of ice-cold PBS, 0.3% BSA. The filters were then counted for radioactivity in a Beckman LS5000TD scintillation counter. Binding constants were calculated using Radlig (Biosoft). We have shown previously that the above radioligands do not detect any endogenous B1 or B2 receptor in either naive or mock-transfected cells (11).

Intracellular Ca2+ Mobilization-- Using a previously described protocol (17), A10 smooth muscle cells transfected with various cDNAs were incubated in modified Leibovitz's L-15 medium at room temperature with 4 µM fura-2/AM. The fura-2-loaded cells were equilibrated to 37 °C in Hanks' balanced salt solution with 1.3 mM CaCl2, pH 7.4, and stimulated by the addition of BK as noted in Fig. 4. The cytosolic free Ca2+ signal from individual cells was acquired and processed by an integrated digital imaging fluorescence microscopy system. Fura-2 was alternately excited at 340 and 380 nm, and the emissions were collected at 510 nm. The signals are presented in the figure as the ratio of bound/free Ca2+, F340/F380. We have shown previously that kinin agonists fail to elicit an intracellular Ca2+ signal in naive and mock-transfected A10 cells (11).

Inositol Phospholipid Hydrolysis-- Cells were assayed essentially as described (18), with a few modifications. Briefly, transfected HEK293 cells grown in six-well dishes were incubated with 5 µCi/ml myo-[3H]inositol in DMEM, 5% heat-inactivated horse serum at 37 °C for 24 h in 10% CO2. Prior to experimentation, the cells were washed four times with 1 ml of Leibovitz's L-15 medium at room temperature and incubated in Leibovitz's L-15 medium, 50 mM LiCl for 30 min. Following replacement with 2 ml of the same medium, the cells were incubated with or without BK at 37 °C for 20 min. Inositol phosphates were then extracted and isolated using anion exchange chromatography.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Sequence Analysis of B1 and B2 TM-III and Construction of B1/B2 Receptor Chimeras-- As shown in the diagrams in Fig. 1 (A and B), TM-III of the human B1 and B2 BK receptor subtypes exhibit relatively low sequence homology (36%). Following the proposed numbering of residues in GPCR helices (1, 19), an alignment of B1 and B2 receptor residues in a helical wheel diagram reveals that most of the conserved residues between B1 and B2 TM-III reside on the side facing the putative ligand binding pocket in these receptors, whereas the non-conserved residues face the membrane (Fig. 1B). In order to test this orientation and to probe for residues in TM-III that contribute to agonist and antagonist binding in B1 and B2 receptors, we created a chimeric receptor construct in which a region of the B1 receptor encompassing TM-III and IC-II was substituted in the B2 receptor. The nomenclature used for this construct was B2(B1III). This construct was further modified by replacement of the single non-conserved residue in TM-III of B2(B1III) facing the putative ligand binding pocket, Lys111, with the corresponding WT B2 receptor residue, Ser111 (B2(B1IIIS111)). In the proposed helical wheel projection (1, 19), these residues occupy position 9, approximately two turns into the helix of the B1 and B2 receptors (Fig. 1B). Based on results from a previous study (11), we also co-substituted B1 TM-VI, in which Tyr259 and Ala263 were replaced with the corresponding WT B2 receptor residues, Phe259 and Thr263, and B1 TM-III, in which Lys111 was replaced with Ser111, to make B2(B1IIIS111;VIF259,T263). As shown in Fig. 1B, Phe(Tyr) and Thr(Ala) face the binding pocket approximately two turns and one turn into the TM-VI helix, respectively. The pharmacological and functional profiles of the WT and chimeric receptor constructs were determined by radioligand binding and by agonist-stimulated inositol phospholipid hydrolysis and intracellular Ca2+ mobilization in transfected HEK293 and A10 cells using a variety of subtype-selective kinin peptide agonists and antagonists (Table I).


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Fig. 1.   Schematic representations of the human B1 and B2 BK receptors. A, serpentine diagram of the receptors with a magnification of the B1 (filled) and B2 (open) receptor TM-III/IC-II sequences that were exchanged in the chimera. Lys118 in the B1 receptor and Ser111 in the B2 receptors are indicated. B, an outside-inward view of the receptors as a helical wheel diagram. In the magnified diagram, the TM-III residues for the B2 (outside parentheses) and the B1 (in parentheses, where different from B2) receptors are numbered from the start of the helix (B1, Cys110; B2, Cys103) with the helix rotated to optimize the inward orientation of residues believed to be involved in ligand binding in GPCR as previously proposed (1, 19). The region exchanged between the receptors starts at position 3 in the TM-III helix. The residues in the B1/B2 receptors at position 9 (Lys118/Ser111) in TM-III and positions 19 (Tyr266/Phe259) and 23 (Ala270/Thr263) in TM-VI are highlighted (bold).

                              
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Table I
Amino acid sequences of B1 and B2 bradykinin receptor ligands

The Residue at Position 9 in TM-III Discriminates between Subtype-selective Peptide Ligands-- As shown in Fig. 2, Scatchard analysis of saturation binding isotherms reveals that the human WT B2 receptor expressed in HEK293 cells was recognized with high affinity by B2-selective peptide ligands, including both the agonist [3H]BK and the antagonist [3H]NPC17731. The expressed receptor exhibited a pharmacological profile typical of a B2 receptor (Table II). Furthermore, the receptor was functionally active, as revealed by the high potency of BK to stimulate inositol phospholipid hydrolysis in these cells (Fig. 3). The human WT B2 receptor is also capable of mediating BK-stimulated mobilization of intracellular Ca2+ as previously shown by us using transiently transfected A10 cells (11).


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Fig. 2.   Saturation binding isotherms of [3H]BK and [3H]NPC17731 on particulate preparations of HEK293 cells expressing human WT B2 BK receptors and B2(B1IIIS111), B2(B1VIF259,T263), and B2(B1IIIS111;VIF259,T263). Particulate preparations of HEK293 cells expressing WTB2, B2(B1IIIS111), B2(B1VIF259,T263), and B2(B1IIIS111;VIF259,T263) were incubated with increasing concentrations of [3H]BK (open circle ) and [3H]NPC17731 (bullet ) and assayed as described under "Experimental Procedures." In each experiment, the two radioligands were assayed in parallel on the same particulate preparation. The saturation binding isotherms and the Bmax ratios shown are representative of three experiments, and the KD values are averages ± S.E. of three experiments with each point assayed in duplicate. KD values are also given in Table II.

                              
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Table II
Agonist and antagonist affinities for WT B2 and B1 BK receptors and chimeric and mutant receptor constructs expressed in HEK293 cells


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Fig. 3.   BK-stimulated inositol phospholipid hydrolysis in HEK293 cells expressing human WT B2 BK receptors, B2(B1III), and B2(B1IIIS111). Cells expressing WTB2 (bullet ), B2(B1III) (black-square), or B2(B1IIIS111) (black-triangle) were incubated with increasing concentrations of BK. The cells were then assayed for [3H]inositol phosphates as described under "Experimental Procedures." The results are presented as percent of maximum, where maximum is the response of WTB2 to BK. 100% Maximum represents 314,231 ± 122,787 dpm (n = 3)

Substitution of B1 TM-III/IC-II in the B2 receptor to make B2(B1III) resulted in a chimera that was not detected in radioligand binding assays by either [3H]BK or [3H]NPC17731 (Table II); nor did this substitution introduce a high affinity binding site for the B1-selective peptide agonist [3H]des-Arg10-KD. To evaluate if B2(B1III) could be expressed and properly folded in mammalian cells, the transfected HEK293 cells were assayed by BK-stimulated inositol phospholipid hydrolysis. As shown in Fig. 3, B2(B1III) was capable of mediating a BK response. However, the potency of BK to stimulate a response through this chimera was significantly less than that through the WT B2 receptor (Fig. 3). B2(B1III) transiently transfected into A10 cells also responded to a high concentration of BK (0.1 µM) with a large increase in the intracellular free Ca2+ concentration (Fig. 4). Mock-transfected cells did not respond to this agonist (Fig. 4). We conclude from these results that B2(B1III) is expressed and properly folded in mammalian cells and that substitution of B1 TM-III/IC-II in the B2 receptor reduces the affinities of both BK and NPC17731 below the level detectable by radioligand binding.


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Fig. 4.   BK-stimulated mobilization of intracellular Ca2+ in A10 cells expressing B2(B1III) and B2(B1IIIR111). At the times indicated by the arrows, the transfected, fura-2-loaded A10 cells were exposed to vehicle or 0.1 µM BK as indicated. The F340/F380 ratio was monitored as described under "Experimental Procedures." Traces are from fields of 14 cells (Mock), 24 cells (B2(B1III)), and 18 cells (B2(B1IIIR111)) and are representative of three experiments.

Substitution of B1 TM-III/IC-II in the B2 receptor results in only one non-conserved change, Ser111 right-arrow Lys, among the residues in TM-III presumed to face the ligand binding pocket (Fig. 1B). These residues are located at position 9, approximately two helical turns into TM-III. To assess if this change was responsible for the decrease in the affinities of the B2-selective ligands, we replaced Lys111 in B2(B1III) with the corresponding WT B2 receptor residue, Ser. As shown in Fig. 2 and Table II, this construct, B2(B1IIIS111), differing in only one amino acid from the B2(B1III) chimera, bound both BK and NPC17731 with affinities virtually identical to those for the WT B2 receptor. The gain of function of the chimera following this replacement was also observed in the increased potency of BK to stimulate inositol phospholipid hydrolysis (Fig. 3). As the binding profile of a chimera in which B1 TM-III without B1 IC-II was substituted in the B2 receptor was identical to that of B2(B1III) containing B1 IC-II (data not shown), we have shown that B1 and B2 TM-III, although structurally only 36% identical, are distinguished pharmacologically by only a single residue.

In addition to the single position in B1 TM-III, B2-selective peptide agonists are also discriminated against by two residues in B1 TM-VI (11). As shown in Fig. 2 and Table II, B1 TM-VI, which is also only 36% identical to its B2 receptor counterpart, becomes pharmacologically compatible with B2 TM-VI following the replacement of two residues in this domain in B2(B1VI), Tyr259 and Ala263, with those in the WT B2 receptor, which are Phe259 and Thr263. To verify the positions of these residues and their contributions to peptide selectivity, a double chimera (B2(B1IIIS111;VIF259,T263)) was made in which appropriately modified B1 TM-III and TM-VI were both substituted in the B2 receptor. This chimera retained the pharmacological profile of a WT B2 receptor (Fig. 2, Table II). Given the low level of sequence homology between human B1 and B2 receptors, these results also illustrate the structural compatibility of GPCR domains.

The Residue at Position 9 in TM-III Discriminates between Receptor Subtype-selective Peptides through Ionic Interactions-- The low affinities of B2-selective ligands for B2(B1III) may be caused either by the absence of the Ser or by the presence of the Lys at position 9. The importance of the hydroxyl group of Ser111 for high affinity BK and NPC17731 binding was evaluated by substituting Ala at this position (B2(B1IIIA111)). Table II shows that B2(B1IIIA111) retained high affinity binding of both BK and NPC17731. We conclude from the results using B2(B1IIIS111) and B2(B1IIIA111) that Ser111 in the WT B2 receptor probably does not directly interact with either class of B2-selective ligand and that a Lys at this position interferes with the binding of these ligands.

Several other residues were substituted in position 9 in the B2(B1III) chimera to determine if Lys interfered with the binding of B2-selective ligands because of its size or because of its positive charge. The importance of the size of Lys was evaluated by substituting with two other residues of no or less charge but of comparable size, Met and His. B2(B1IIIM111) and B2(B1IIIH111) retained high affinity binding of both BK and NPC17731 (Table II). Thus, Lys is probably not interfering with BK and NPC17731 binding by size-exclusion. An Arg was substituted in this position (B2(B1IIIR111) to partially mimic the positive charge of Lys. Interestingly, the affinities of BK and NPC17731 for B2(B1IIIR111) were 6- and 12-fold lower, respectively, than those for B2(B1IIIS111) (Table II). Fig. 4 shows that B2(B1IIIR111) retained functional activity as determined by single cell Ca2+ imaging. These results indicate that the charge of Lys is responsible for the reduced affinities of BK and NPC17731 for B2(B1III). The relatively smaller inhibitory effect of Arg compared with Lys is most likely due to the partial aromatic character of the guanidinium group of the Arg side chain, which reduces the effect of the positive charge of this residue. Indeed, several studies have also shown that, in other receptors in which ligand binding is affected by the charge of Lys, Arg only partially mimics the effect of Lys (20, 21).

The above results indicate that position 9 in TM-III is oriented toward the binding pocket of these chimeras. In order to show that this orientation is not an artifact of the chimera, a Lys was also substituted in this position in the WT B2 receptor (B2K111). The affinity of NPC17731 for B2K111 was 10-fold lower than that for the WT B2 receptor (Table II). In contrast, the affinity of this ligand for B2A111, in which an Ala was substituted at this position, was not significantly different from that of the WT B2 receptor (Table II). Lys substitution also interfered with BK binding, as observed by the 37-fold decrease in the affinity of this ligand (Table II). These results confirm the inward orientation of position 9 in the B2 receptor and the inhibitory effect on the binding of B2-selective ligands when this position is occupied by a positively charged residue such as Lys, as it is in the WT B1 receptor.

The Residue at Position 9 in TM-III Discriminates between the Charges of the C-terminal Residues of Subtype-selective Peptide Ligands-- B2-selective peptides contain two candidate residues that may be repelled by the charge of Lys: an Arg at the C terminus and an Arg at the N terminus (Table I). We have previously shown by chemical cross-linking that the N terminus of BK when bound to the receptor is oriented extracellularly and adjacent to Cys277 in EC-IV and Cys20 in EC-I (15). Consequently, in B2-selective peptide agonists, the C-terminal Arg is the only residue available to sense the charge of a Lys when present in position 9 in TM-III. The N terminus of NPC17731 is not positioned in the same manner as peptide agonists. To directly investigate the orientation of the peptide antagonist, we made complementary mutations in the ligand and in the receptor. This was done by evaluating the effect of Lys (B2K111) on the binding of NPC17731, which contains a C-terminal Arg, and NPC18565, the des-Arg10 analog of NPC17731. As shown in Fig. 5A and Table II, the presence of a Lys at position 9 of TM-III reduced the affinity of NPC17731 by about 10-fold. In contrast, this substitution increased the affinity of NPC18565 by about 5-fold (Fig. 5B, Table II). An Ala at this position (B2A111) did not perturb significantly the binding of either ligand. These results provide conclusive evidence that the C-terminal Arg, rather than the N-terminal Arg, of the receptor-bound B2-selective peptides is adjacent to position 9 in TM-III. Indeed, this is one of only a few examples in a GPCR where the interaction of a ligand with a specific residue in the receptor has been assessed by complementary mutations in the ligand and in the receptor.


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Fig. 5.   NPC17731 and NPC18565 binding isotherms on particulate preparations of HEK293 cells expressing human WT B2 BK receptors and B2K111, B2A111, and B2E111mutants. Particulate preparations of HEK293 cells expressing WTB2 (bullet ), B2K111 (black-triangle), B2A111 (black-square), and B2E111 (black-down-triangle ) were incubated with KD concentrations of [3H]NPC17731 in the absence and presence of increasing concentrations of NPC17731 (A) and NPC18565 (B) as indicated and assayed as described under "Experimental Procedures." The results are averages ± S.E. of three experiments with each point assayed in duplicate. Some points have error bars that are smaller than the symbols. KD values are given in Table II.

Considering that position 9 in the WT B1 receptor is occupied by a Lys, which is Lys118, it is significant to note the substantially higher affinity of NPC18565 for B2K111 compared with the WT B2 receptor. It is feasible that a positively charged residue at this position could provide a counter-ion for the negatively charged C terminus of B1-selective des-Arg peptides. If that were the case, the presence of a negatively charged residue at this position should repel NPC18565. Indeed, and as shown in Fig. 5B and Table II, the affinity of NPC18565 for B2E111 was significantly less than that for the WT B2 receptor (4-fold). The importance of a Lys at this position for the binding of B1-selective ligands was further supported by the complete loss of high affinity des-Arg10-KD binding following substitution of a Ser at this position in the WT B1 receptor (B1S118) (Table II). Thus, position 9 in TM-III in B2K111, and presumably also in the WT B1 receptor, provides a counter-ion for the C terminus of B1-selective des-Arg peptides. The affinities of BK and NPC17731 for B2E111 were not significantly different from those for the WT B2 receptor and B2A111, again indicating that, even though position 9 in the WT B2 receptor is adjacent to the C-terminal Arg of B2-selective peptides, it does not provide a point of interaction for the Arg in these peptides (Fig. 5A and Table II).

B2(B1III) Discriminates between Peptide Agonists and Antagonists-- Analysis of the relative number of high affinity binding sites for BK and NPC17731 and the competition between these two ligands in the WT and chimeric receptors indicates that when B1 TM-III is substituted in the B2 receptor, the construct discriminates between agonists and antagonists. As shown in Figs. 2 and 6 (A and B) and Table II, WT B2 and B2(B1IIIS111) receptors expressed high affinity binding sites for both agonists and antagonists. As would be expected, the Bmax values for high affinity binding of BK and NPC17731 in the WT B2 receptor were approximately equal (Fig. 2). Furthermore, BK and NPC17731 readily competed with each other for binding to this receptor (Fig. 6, C and D, and Table III). In contrast, the ratio of Bmax values for high affinity BK to NPC17731 binding in B2(B1IIIS111) was only 0.34 compared with 1.28 for WT B2 receptors (Fig. 2). Furthermore, the potency of BK to displace NPC17731 binding to this construct and the slope of the BK displacement curve (nH = 0.50 ± 0.14) were dramatically reduced (Fig. 6D, Table III). The ability of the agonist KD to compete with NPC17731 binding to B2(B1IIIS111) was also reduced (nH = 0.54 ± 0.04) (Table III). This phenomenon is agonist-specific as the ability of the antagonist NPC17731 to displace BK binding to this chimera was not significantly different from that on WT B2 receptors (Fig. 6C, Table III). Even though the potencies of the agonists to compete with antagonist binding were dependent to some extent on the type of residue occupying position 9 in TM-III in the B2(B1III) constructs, in all of these constructs, as well as in a B2(B1III) construct that lacked B1 IC-II, were the agonist binding parameters significantly reduced (Table III). This phenomenon was not an artifact of a chimera as substitution of B1 TM-VI in the B2 receptor to make B2(B1VIF259,T263) did not significantly interfere with either agonist or antagonist binding and competition (Figs. 2 and 6 (A-D), Table III). However, substitution of B1 TM-III together with TM-VI to make B2(B1IIIS111;VIF259,T263) again reduced both the relative number of agonist binding sites (Fig. 2) and the ability of agonists to compete with antagonist binding (Fig. 6D, Table III). Indeed, in the double chimera, the effect on the agonist binding was exaggerated.


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Fig. 6.   BK and NPC17731 competition binding isotherms of [3H]BK and [3H]NPC17731 on particulate preparations of HEK293 cells expressing human WT B2 BK receptors and B2(B1IIIS111), B2(B1VIF259,T263), and B2(B1IIIS111;VIF259,T263). Particulate preparations of HEK293 cells expressing WTB2 (bullet ), B2(B1IIIS111) (black-triangle), B2(B1VIF259,T263) (open circle ), and B2(B1IIIS111;VIF259,T263) (triangle ) were incubated with KD concentrations of [3H]BK and [3H]NPC17731 in the absence and presence of increasing concentrations of NPC17731 and BK as indicated and assayed as described under "Experimental Procedures." The results are averages ± S.E. of three experiments with each point assayed in duplicate. Some points have error bars that are smaller than the symbols. KD and KI values are given in Table II and Table III, respectively.

                              
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Table III
Agonist and antagonist competition for WT B2 BK receptors and chimeric and mutant receptor constructs expressed in HEK293 cells

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In this study, we have used a novel mutagenesis approach to identify crucial epitopes in the ligand binding pockets of the B1 and B2 BK receptor subtypes. Specifically, we show that the residues at position 9 in TM-III of these receptors, which is located approximately two turns into the helix, are adjacent to and responsible for discriminating between the C-terminal residues of B1- and B2-selective peptide ligands when bound to these receptors. When this position is occupied by a Lys, which naturally occurs in the WT B1 receptor (Lys118), the Lys repels the positive charge of the C-terminal Arg of B2-selective peptides. On the other hand, the Lys attracts the negative charge of the C terminus of B1-selective peptides that lack the C-terminal Arg. When this position is occupied by a Ser, which it is in the WT B2 receptor (Ser111), B2-selective peptides are not repelled and readily bind. On the other hand, no counter-ion is available to attract B1-selective peptides.

The results presented in this study provide new and definitive information about the orientation of the peptide agonist when bound to the human B2 receptor. We have previously shown that the N terminus of the bound BK is located within 3 Å of extracellular Cys20 in EC-I and Cys277 in EC-IV (15). BK binding is dependent on two Asp residues, Asp268 and Asp286, located in EC-IV (12) and with which either the N terminus or the guanidinium side chain of Arg1 is believed to interact (22). The binding of BK also requires Phe259 and Thr263 located two and one helical turns, respectively, below EC-IV on the interior face in TM-VI (11, 13, 14). We conclude from these results that BK, when bound to the B2 receptor, reaches from the extracellular surface of the receptor adjacent to EC-IV, down two helical turns along the interior face of TM-VI, and across into a pocket bordered by the interior face of TM-III, and presumably TM-V and -VI, and adjacent to Ser111. These results are in remarkably good agreement with a model of BK bound to the B2 receptor proposed by Kyle based on structural homology modeling, molecular modeling, and systematic conformational searching methods of BK and the receptor (22).

Two-dimensional NMR and molecular modeling of BK in various solvents and micelles have revealed that Ser6 through Arg9 adopt a beta -turn-like structure when in a hydrophobic environment (23, 24). The importance of this beta -turn for receptor binding was exploited by substituting constraining amino acids that mimic a beta -turn at position 8 and 9 in BK antagonists (Table I). This modification yielded a second-generation of BK antagonists, such as NPC17731 and HOE140, with substantially increased affinities for the B2 receptor (6-8). Two-dimensional NMR of the tetrapeptide Ser-D-Hype(trans-propyl)-Oic-Arg, which corresponds to the four C-terminal residues of NPC17731, revealed that this tetrapeptide maintains a beta -turn even in an aqueous environment at neutral pH (25). Our results show that the binding cavity for the C-terminal residue of BK is shared with that of the peptide antagonist NPC17731. This information would suggest that the essential C-terminal beta -turn of both peptide agonists and antagonists exists in a similar location when bound to the B2 receptor. Whether the C-terminal residues of the agonist and antagonist peptides are adjacent or overlap is not yet known. The possibility exists that their C-terminal backbones overlap, but that the side chains, which may confer the distinct activities of the peptides, do not overlap. On the other hand, it is clear that the N-terminal residues of these two classes of ligands do not overlap (26). Our results readily accommodate those obtained in mapping studies using antibodies against extracellular domains of the human B2 receptor (27). In these studies, antibodies against the C-terminal half of EC-IV, which approximates the positioning of the agonist N terminus, inhibited specifically agonist binding. On the other hand, antibodies against the N-terminal half of EC-III, which would be expected to overlay the cavity in which the C-terminal residues of both agonists and antagonists appear to reside, inhibited both agonist and antagonist binding.

The identity of the epitope(s) in the B2 receptor with which the crucial C-terminal Arg interacts is not known. Ser111 is not conserved in the B2 receptor across different species, and the lack of an effect of mutating this residue on both agonist and antagonist binding in the human B2 receptor agrees with the results obtained in a recent rigorous mutational study of the rat B2 receptor (14). In this study, mutation of the corresponding Tyr to Phe or Thr had no effect on the affinity of either BK and HOE140, a high affinity antagonist structurally and functionally related to NPC17731. Indeed, BK and HOE140 binding were not affected by any number of additional mutations made in this helix including those at positions 2 (Arg right-arrow Ala or Gln), 5 (Asn right-arrow Ala), 6 (Thr right-arrow Ala), 7 (Met right-arrow Trp), 11 (Asn right-arrow Ala), 12 (Leu right-arrow Trp), 13 (Tyr right-arrow Ala or Phe), 14 (Ser right-arrow Ala), 15 (Ser right-arrow Ala), and 17 (Cys right-arrow Ala). Of these positions, 2, 5, 9, 11, 12, and 15 would be predicted by the helical wheel projection to face the ligand binding pocket (Fig. 1B). These results suggest that B2 TM-III does not provide any residue with which peptide ligands interact directly. Alternatively, B2 TM-III together with other B2 TMs may provide residues that contribute to a hydrophobic cavity in this receptor that accommodates, e.g., the aromatic properties of the C-terminal Arg. B2-selective antagonists retain significant activity following the replacement of their C-terminal Arg with an aromatic amino acid (28). A single mutation of any one such receptor residue may only have a minimal effect on the binding of a ligand that requires this cavity. It is under such conditions that our approach is particularly effective.

Our results strongly suggest that Lys118 at position 9 in TM-III of the WT B1 receptor serves a dual role in the interaction of subtype-selective peptide ligands with this receptor. That this residue discriminates against B2-selective peptide ligands by repelling their C-terminal Arg was supported by two observations. 1) The loss in affinity of both BK and NPC17731 following substitution of B1 TM-III in the B2 receptor, and the complete recovery of their affinity following the replacement of one residue, Lys111, in TM-III of this chimera with the corresponding WT B2 receptor residue, Ser111; and 2) the loss in affinity of BK and NPC17731, but not the des-Arg10 analog of NPC17731, NPC18565, following substitution of Lys at Ser111 in the WT B2 receptor.

The second proposed role of Lys118 in the WT B1 receptor is that of a counter-ion for the C terminus of B1-selective peptides, which lack the C-terminal Arg. Support for this role was provided by the fact that substitution of Lys at the corresponding position in the B2 receptor increased the affinity of NPC18565, whereas substitution of Glu at this position reduced the affinity of this peptide. On the other hand, substitution of Ala at this position had no effect on the affinity of this ligand. The complete loss in high affinity des-Arg10-KD binding following substitution of Ser at Lys118 in the B1 receptor and the conservation of this residue in this receptor across different species provide additional evidence for such a role of this residue. Furthermore, methylation of the C-terminal carboxyl group, which would disrupt an ionic interaction with the primary amine on Lys118, is detrimental for peptide action through the B1 receptor (29).

In light of the proposed discriminatory role of Lys118, it is appropriate to briefly discuss the binding of KD. This peptide, which carries a C-terminal Arg (Table I), binds to the B1 receptor with an affinity that is 2 orders of magnitude lower than that of des-Arg10-KD but 3 orders of magnitude higher than that of BK. Hence, this peptide is considerably less selective for the two receptor subtypes than both BK and des-Arg10-KD. We believe that the accommodation of the C-terminal Arg of KD in the B1 receptor is possible due to additional binding energy provided by the interaction of an epitope in this receptor with the N-terminal Lys in KD. Indeed, the dramatically lower affinity of the B1-selective peptide des-Arg9-BK compared with that of des-Arg10-KD for the B1 receptor clearly demonstrates this point.

The role of TM-III in ligand binding to GPCR is well described and has been used to orient this TM in helical wheel projections (1, 19). Position 8 is occupied by an Asp in the beta 2-adrenergic receptor (30), M1 muscarinic acetylcholine receptor (31), and D1 and D2 dopamine receptors (32, 33), which serves as a counter-ion for the cationic amine when bound to these receptors. An Asp at this position is also important for agonist binding to the SST1 somatostatin receptor (34). A Lys at position 9 has been shown to be important for both agonist and antagonist binding to endothelin A and B receptors (21, 35), and a Tyr at this position is absolutely crucial for agonist binding to the thyrotropin-releasing hormone receptor (36). These results emphasize the central positioning and role of TM-III in ligand binding pockets of GPCR.

Substitution of B1 TM-III in the B2 receptor interfered with several agonist binding parameters in a way that was independent of agonist affinity. Replacement of Lys111 in B2(B1III) with several residues of both polar and hydrophobic nature was sufficient to recover high affinity agonist binding to this chimera. On the other hand, the ability of agonists to compete with antagonist binding in this chimera was dramatically reduced. In addition, the number of high affinity agonist binding sites in the chimera was only approximately 1/3 of the number of high affinity antagonist binding sites. This effect was not an artifact of a chimera since substitution of B1 TM-VI in the B2 receptor did not interfere with these parameters. Even though the significance of these observations is not yet known, they provide some clues to the relationship between the agonist and antagonist binding sites in the B2 receptor. First, these results confirm that the binding sites for these two classes of ligands are not identical. Second, TM-III must contain an epitope(s) upon which only agonists are dependent but that is not directly required for agonist binding. This epitope may be involved in the isomerization between inactive, antagonist-preferred and activated, agonist-preferred conformations (37). In addition to the central role of TM-III in ligand binding, this helix also appears to play a central role in receptor activation. Of the helices in rhodopsin that have been investigated for photoactivation-induced motion by site-directed nitroxide spin-labeling, which include TM-III, -IV, -V, and -VI, motion was apparently localized specifically in TM-III and TM-VI (38, 39). The proposed orientation of BK in the binding pocket places this agonist at a potentially pivotal point between TM-III and TM-VI. Furthermore, in the model proposed by Kyle, Pro7 in BK is located in a hydrophobic cavity made up of residues from TM-III and TM-VI (22). This observation is also interesting considering that the replacement of Pro7 with a D-aromatic residue or an ether of D-4-hydroxyproline is the fundamental requirement for converting the BK peptide to an antagonist. One possibility is that the introduction of a heterologous TM-III in the B2 receptor causes the misalignment of interhelical contacts, which are required for the formation of a conformational state of the receptor to which agonists preferentially bind. A similar conclusion was drawn following the observation that mutations along TM-II in the NK-1 receptor selectively interfered with the same agonist binding parameters (40).

In summary, we have identified a single position in TM-III of the human B1 and B2 receptors that is adjacent to and discriminates between the C-terminal residues of subtype-selective peptide ligands. Our results also provide evidence that the ligand binding pockets in these structurally divergent GPCR subtypes may indeed be very similar.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM41659.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Institute of Veterinary Medicine, University of Göttingen, 37073 Göttingen, Germany.

§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7760. Tel.: 210-567-3766; Fax: 210-567-6595; E-mail: lundberg{at}bioc02.uthscsa.edu.

1 The abbreviations used are: GPCR, G-protein-coupled receptor; BK, bradykinin; KD, kallidin; TM, transmembrane domain; EC, extracellular domain; IC, intracellular domain; WT, wild-type; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TES, 2-{[2-(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PCR, polymerase chain reaction.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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