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Volume 270, Number 35, Issue of September 01, pp. 20591-20598, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Agonist Binding Site on the Bovine Bradykinin B2 Receptor Is Adjacent to a Sulfhydryl and Is Differentiated from the Antagonist Binding Site by Chemical Cross-linking (*)

(Received for publication, May 18, 1995; and in revised form, June 14, 1995)

Maryanne C. S. Herzig ,  L. M. Fredrik Leeb-Lundberg (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Chemical cross-linking was used to analyze the binding sites for the agonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on the bovine B2 bradykinin receptor. [^3H]BK and [^3H]NPC17731 bound with high affinity to the same B2 receptor in bovine myometrial membranes as determined by the total number of specific binding sites and pharmacological specificity of the binding of these two radioligands. Cross-linking experiments were done using a series of bifunctional reagents reactive either primarily to amines (homobifunctional) or reactive to amines in one end and to sulfhydryls in the opposite end (heterobifunctional). All the heterobifunctional reagents plus the homobifunctional arylhalide 1,5-difluoro-2,4-dinitrobenzene were effective in cross-linking the [^3H]BK N terminus specifically to a sulfhydryl in the receptor, and this cross-linking occurred at 5-100 µM reagent. In contrast, the homobifunctional N-hydroxysuccinimide ester reagents, at leq1 mM, were only able to cross-link [^3H]BK to membrane proteins nonspecifically. The sulfhydryl reagents N-ethylmaleimide, iodoacetamide, and phenylarsine oxide blocked cross-linking, whereas these reagents did not inhibit reversible specific [^3H]BK binding. Immunoblotting with anti-BK antiserum revealed that low concentrations of BK (5-50 nM) were cross-linked to a receptor-specific species of 65 kDa. All cross-linking of [^3H]NPC17731 was nonspecific with both homobifunctional and heterobifunctional reagents. The 65-kDa receptor-specific species was observed on anti-HOE140 immunoblots, but only when proteins were cross-linked with very high concentrations of HOE140 (geq500 nM). Our results provide direct biochemical evidence that the binding site for the agonist BK in the bovine B2 receptor is adjacent to a cysteine and is differentiated from the binding site(s) for the antagonists NPC17731 and HOE140.

INTRODUCTION

The nonapeptide BK (^1)(Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) belongs to the kinin family of vasoactive peptides which have dramatic biological activities on a number of different tissues and organs including smooth muscle contraction and relaxation, increased vascular permeability, pain, and cell proliferation(1, 2) . These peptides have also been implicated as mediators in the development of pathological states such as asthma, sepsis, viral rhinitis, arthritis, and inflammatory pain(3, 4, 5, 6, 7, 8, 9) . Receptors for kinins have been divided into two main subtypes, B1 and B2, based on the structure-activity of agonists and antagonists(1) . BK interacts primarily with the B2 receptor through which this peptide stimulates a number of second messenger systems including inositol phospholipid hydrolysis(10, 11, 12, 13) , arachidonic acid metabolism(14, 15, 16) , tyrosine phosphorylation(17, 18, 19) , and membrane depolarization and hyperpolarization(20, 21) .

The recent cloning of the cDNAs for B1 (22) and B2 receptors (23, 24, 25) have revealed that these receptors belong to the superfamily of seven transmembrane-domain, G-protein-coupled receptors(26, 27) . These advances have opened new avenues for identification of specific domains and residues crucial for ligand binding and function of these receptors.

BK antagonists have been synthesized. The first generation of antagonists were developed around the crucial replacement of L-Pro^7 in BK with a D-aromatic amino acid residue resulting in moderately high affinities for the B2 receptor(28, 29) . D-Arg^0-(Hyp^3, D-Phe^7)-BK (NPC567) is a typical example of this generation. In second generation antagonists, a restricted beta-turn was introduced in the C-terminal portion of the peptide, and this modification substantially increased affinities of these antagonists for the receptor(30, 31, 32, 33, 34) . D-Arg^0-(Hyp^3, Thi^5, D-Tic^7, Oic^8)-BK (HOE140), where Tic is D-1,2,3,4-tetrahydroisoquinoline-3-yl-carbonyl and Oic is L-[(3aS,7aS)-octahydroindol-2-yl-carbonyl], and D-Arg^0-(Hyp^3, D-Hype (trans-propyl)^7, Oic^8)-BK (NPC17731) are typical examples of this generation.

Previously, we presented a three-state model of agonist binding to the B2 receptor(35) , and this model was subsequently shown to accommodate negative antagonistic/inverse agonistic activity of a number of first and second generation BK antagonists(36) . The classification of at least some BK antagonists as inverse agonists, and, consequently, as having intrinsic functional activity on their own, has increased the importance of delineating the binding sites for B2 receptor agonists and antagonists. Chemical cross-linking has been used extensively to covalently attach ligands to receptors. In this study, we used this technique to investigate the relationship between the binding sites for these two classes of ligands in the B2 receptor.

EXPERIMENTAL PROCEDURES

Materials

[2,3-prolyl-3,4-^3H]Bradykinin (110 Ci/mmol) and [prolyl-3,4-^3H]NPC17731 (53.5 Ci/mmol) were purchased from DuPont NEN. Fresh uteri from pregnant cows were from a local slaughterhouse. HOE140 and NPC567 were from Bachem, Inc. (Torrance, CA), while NPC17731 was the kind gift of Dr. S. Farmer of Zeneca Pharmaceuticals Group (Wilmington, DE). BK was purchased from Sigma. Gpp(NH)p was from Boehringer Mannheim. The cross-linkers MBS, SMPB, SMCC, SPDP, DSS, DSP, DST, and DFDNB were from Pierce. Polyclonal anti-BK antiserum was from Peninsula Laboratories, Inc. (Belmont, CA), while monoclonal anti-HOE140 antiserum was kindly provided by Dr. W. Muller-Esterl of the University of Mainz (Mainz, Germany). All other biochemicals were of the highest grade available.

Membrane Preparation

Membranes were prepared essentially by a procedure originally described by Fredrick et al.(37) and subsequently by Leeb-Lundberg and Mathis (35) with a few modifications. After excision, the uteri were immersed in ice. The myometrium was dissected free of the endometrium and other extraneous tissue fragments, chopped in a commercial meat grinder, and then homogenized in ice-cold buffer (10 mM NaH(2)PO(4), pH 6.7, 115 mM NaCl, 4.6 mM KCl, 22 mM NaHCO(3), 0.9 mM MgSO(4)-7H(2)0, 5.5 mM glucose, 13.4 mM Na(2)EDTA; 1:2 w/v) in a Waring Commercial CB-6 blender for 4 min (medium speed) at 4 °C. The homogenate was centrifuged at 3,600 g for 15 min at 4 °C. The pellets were recovered, frozen in liquid N(2), and stored for up to 2 months before further processing. Upon thawing, the pellets were homogenized again in buffer (1:4, v/v) as described above and centrifuged at 1,000 g for 10 min at 4 °C. The pellet was discarded, and the supernatant was decanted through cheesecloth and then centrifuged at 30,000 g for 2 h at 4 °C. The membrane pellet was frozen in liquid N(2), thawed, and washed once by homogenization in 25 mM TES, pH 6.8, 0.5 mM EDTA, 1 mM 1,10 phenanthroline, then centrifuged at 25,000 g for 1 h at 4 °C. The washed membranes were stored in the same buffer in aliquots of 15-25 mg of protein/ml at -80 °C until use.

Membrane Solubilization

Aliquots of frozen membranes were thawed, diluted 2.5-fold with ice-cold solubilization buffer (25 mM TES, pH 7.2, 0.5 mM EDTA, 1 mM 1,10-phenanthroline, 140 µg/ml bacitracin), then solubilized as described previously(38) .

Radioligand Binding

Radioligand binding assays were done essentially as described previously(35) . Membrane preparations were thawed and diluted to 50-500 µg of protein/ml in binding buffer (25 mM TES, pH 7.2, 0.5 mM EDTA, 1 mM MgCl(2), 1 mM 1,10-phenanthroline, 140 µg/ml bacitracin). Binding assays were initiated by addition of radioligand (50 µl) with and without excess nonradiolabeled ligand to the receptor preparation (450 µl). In assays following pretreatment with sulfhydryl reagents, the preparation (225 µl) was incubated with reagent (25 µl) as described in the figure legends prior to addition of ligand (250 µl). After incubation for 60-90 min at 24 °C, assays were terminated by dilution with 4 ml of ice-cold phosphate-buffered saline 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 phosphate-buffered saline. Filters were counted for tritium in a Beckman LS50000TD scintillation counter.

Protein Cross-linking

Aliquots of membrane or solubilized preparations were allowed to bind ligand for 60-90 min at 24 °C as described above before addition of Me(2)SO (2% maximum final concentration) with and without cross-linking reagent; the incubation with cross-linker was continued for an additional 10 min. The cross-linking reaction was then quenched by a 1:1 dilution with 2 M glycine in binding buffer (quench buffer). In the case of solubilized preparations, the quench buffer included also the protease inhibitors phenylmethylsulfonyl fluoride (1 µM), aprotinin (1 µg/ml), and leupeptin (1 µg/ml). In order to dissociate noncovalently bound radioligand, quenched samples were diluted 4-fold with buffer containing 1 µM BK and 10 µM Gpp(NH)p and incubated for 60-120 min at 24 °C followed by filtration on GF/C filters as described above. Cross-linked ligand was the amount of radioligand remaining after dissociation. Specific cross-linked ligand was cross-linked radioligand minus nonspecific binding (as determined in the presence of 1 µM BK in the binding assay). Cross-linking efficiency was the specific cross-linking as percent of total specific binding.

Immunoblotting

Cross-linked solubilized preparations were concentrated to 30 µl on Centricon 30 filters (Amicon) at 4 °C. Samples of preparations were heated in SDS-polyacrylamide gel elecrophoresis buffer for 3 min at 100 °C. Following electrophoresis on 7.5% polyacrylamide gels, the fractionated proteins were electroblotted onto 0.45-µm nitrocellulose membranes (Schleicher & Schuell) at 500 mA for 45 min at 4 °C using a Genie electroblotter (Idea Scientific) as described by Towbin et al.(39) . The nitrocellulose membranes were blocked by incubation in 10% defatted milk in Tris-buffered saline (TBS) for 1 h. The membranes were then incubated with anti-BK antiserum (1:4) or anti-HOE140 antiserum (1:1000) at 24 °C overnight. The nitrocellulose membranes were washed by a brief rinse in TBS followed by three separate 5-min washes in TBS containing 0.02% Nonidet P-40 followed by a final rinse in TBS. Immunoreactive bands were visualized with ECL (Amersham Corp.) using peroxidase-labeled donkey anti-rabbit antibody according to procedures described by the supplier.

Other Assays

Proteins were assayed using the method of Laemmli(40) . Data is expressed as mean ± S.E. unless otherwise indicated.

RESULTS

Agonist Cross-linking

Bovine myometrial membranes contain a large number of high affinity G-protein-coupled B2 BK receptors(35) . To evaluate the physical relationship between the agonist and antagonist binding sites on this receptor, we used a series of commercially available homo- and heterobifunctional cross-linking reagents of various lengths and reactivities(41, 42) . All of the reagents used, except DFDNB (3Å), contain at least one N-hydroxysuccinimide ester which reacts preferentially with primary amines. The arylhalides of DFDNB exhibit a similar amine reactivity(41, 42) . The homobifunctional reagents DSS (11.4Å), DST (6.8Å), and DSP (12Å) contain an additional N-hydroxysuccinimide ester at the opposite end of the molecule, whereas the heterobifunctional reagents contain either a maleimide (MBS (9.9Å), SMPB (14.5Å), SMCC (11.6Å)) or a pyridyldisulfide (SPDP (6.8Å)), both of which react preferentially with sulfhydryls. Hence, while all of the reagents were capable of reacting with the N terminus of BK, the former reagents were also capable of reacting with membrane primary amines while the latter reagents were capable of reacting with membrane sulfhydryls.

The effectiveness of the cross-linkers was assessed in [^3H]BK binding experiments (Fig. 1). Membranes were allowed to bind [^3H]BK before addition of cross-linker. [^3H]BK cross-linked to the membranes was determined by dissociating noncovalently bound [^3H]BK. Each panel shows the increase in total [^3H]BK binding, nonspecific [^3H]BK binding, and cross-linked [^3H]BK with increasing concentration of cross-linker. Nonspecific [^3H]BK binding increased in parallel with total [^3H]BK binding indicating that some [^3H]BK was cross-linked to nonreceptor sites. The effectiveness of the cross-linkers is seen in the difference between cross-linked [^3H]BK and nonspecific [^3H]BK binding. The heterobifunctional reagents were considerably more effective than the homobifunctional reagents in cross-linking B2 receptor-bound [^3H]BK (Fig. 1). MBS was the most effective reagent, resulting in nearly maximal cross-linking at concentrations as low as 5 µM. SMPB and SMCC, functionally identical to MBS but with longer spacer arms, were also significantly effective although to a lesser extent than MBS. The sulfhydryl alkylating reagent N-ethylmaleimide (NEM) contains the same maleimide functional group as the above cross-linkers. When membranes were reacted with NEM, no cross-linking was observed. Thus, the heterobifunctional reagents were truly cross-linking BK rather than changing BK dissociation kinetics. SPDP, another heterobifunctional cross-linker which reacts via disulfide exchange to form a relatively labile disulfide bond, was also slightly effective in cross-linking. While the homobifunctional N-hydroxysuccinimide ester reagents DSS, DST, and DSP were able to cross-link [^3H]BK to the membranes, the cross-linking with these reagents at <1 mM was nonspecific, although minimal specific DSS cross-linking was observed at >1 mM. Interestingly, DFDNB displayed significant specific cross-linking ability.

Figure 1: Cross-linking of [^3H]BK to bovine myometrial membranes by homobifunctional and heterobifunctional reagents. Membranes (240 µg of protein) were incubated with [^3H]BK (2-5 nM) for 60 min. Increasing concentrations of cross-linkers were then added and the incubations continued for 10 min. Total binding (), nonspecific binding (), and cross-linked ligand (up triangle, filled) were then determined as described under ``Experimental Procedures.'' Cross-linked ligand was the amount of radioligand remaining after dissociation. The results are the averages ± S.E. of at least three experiments with each point assayed in triplicate.


Agonist Cross-linking Specifically to a Sulfhydryl

To demonstrate the importance of a sulfhydryl in cross-linking BK, membranes were treated with 10 mM NEM prior to cross-linking (Fig. 2). In control samples without NEM, the level of nondissociated radioligand was equal to the level of nonspecific binding (data not shown). With NEM, radioligand dissociation is decreased, resulting in slightly increased binding (see Table 1). NEM blocked cross-linking by all the heterobifunctional reagents down to control levels. Cross-linking was also blocked by phenylarsine oxide (PAO), which reacts with vicinal sulfhydryls, and iodoacetic acid (IAA), which alkylates free sulfhydryls (data not shown). DFDNB cross-linking was also blocked by the above sulfhydryl reagents, indicating that this cross-linker, which is usually considered to be homobifunctional in activity and structure, is reacting via a sulfhydryl in our system (Fig. 2; data not shown). The cross-linking observed with high concentrations of DSS was not blocked by NEM and therefore probably involves an amino group (data not shown).

Figure 2: Effect of NEM pretreatment on cross-linking of [^3H]BK to bovine myometrial membranes. Membranes (240 µg of protein) were pretreated without (open bars) and with (cross-hatched bars) 10 mM NEM. The membranes were then incubated with a constant concentration of [^3H]BK (2.5 nM) followed by cross-linking with MBS (50 µM), SMCC (100 µM), SPDP (100 µM), and DFDNB (1 mM) as described under ``Experimental Procedures.'' Cross-linking is expressed as percent of total binding which was as shown in Fig. 2. The results are the averages ± S.E. of four experiments with each point assayed in triplicate.


A Sulfhydryl Is Not Required for Agonist B2 Receptor Binding

Since a sulfhydryl is adjacent to the BK binding site, we examined the role of such a group in BK receptor binding. Membranes were pretreated with NEM, IAA, PAO, and the disulfide reducing agent DTT. As shown in Table 1, none of the sulfhydryl reagents inhibited [^3H]BK binding at any of the concentrations tested (0.01-10 mM). Indeed, a small but significant increase in binding was consistently observed with these reagents. Therefore, while a sulfhydryl appears to be located in close proximity to the BK binding site, such a group is not directly involved in BK binding. Disulfide bonds also do not appear to be critical for BK binding to the expressed receptor as DTT only minimally inhibited [^3H]BK binding.

Identification of Cross-linked B2 Receptor-specific Species

The identity of the cross-linked BK species was evaluated by Western blotting using a polyclonal anti-BK antiserum. We performed titration curves of BK (0-5000 nM) cross-linking at 50 µM MBS in solubilized preparations of bovine myometrium (Fig. 3). At both 5 nM and 50 nM BK, the major cross-linked species detected was at 65 kDa. Increasing the BK concentration to 500 nM did not increase the intensity of the 65-kDa species indicating saturation of BK cross-linking. An intense 145-kDa doublet band appeared at 500 nM BK; species of 60-90 kDa appeared at higher BK concentrations. The cross-linking pattern at 5000 nM BK using 1 mM DFDNB was similar to that using 50 µM MBS including the presence of the 65-kDa species. Similar cross-linking patterns were observed in both soluble and membrane preparations (data not shown).

Figure 3: Western blot of solubilized preparations of bovine myometrium cross-linked to BK with MBS. Solubilized preparations were incubated in the absence (lane 1) and presence of 5 nM BK (lane 2), 50 nM BK (lane 3), 500 nM BK (lane 4), and 5000 nM BK (lanes 5 and 6) for 60 min and then cross-linked with 50 µM MBS (lanes 1-5) or 1 mM DFDNB (lane 6) as indicated. Samples were electrophoresed under nonreducing conditions and immunoblotted with anti-BK antiserum as described under ``Experimental Procedures.'' The molecular mass standards are shown on the left. The 65-kDa species is indicated by the arrow on the right.


HOE140 is a potent B2 receptor antagonist which at 100-fold excess over BK completely displaced specific [^3H]BK binding. This antagonist also completely inhibited BK binding and subsequent MBS cross-linking to the 65-kDa species in solubilized preparations using 50 nM BK (Fig. 4).

Figure 4: Western blot of solubilized preparations of bovine myometrium cross-linked to BK by MBS: pharmacological specificity. Solubilized preparations were incubated without (lanes 1 and 2) and with 10 µM HOE140 (lane 3) in the absence (lane 1) and presence (lanes 2 and 3) of 50 nM BK for 60 min as indicated and then cross-linked with 50 µM MBS. Samples were electrophoresed under reducing conditions and immunoblotted with anti-BK antiserum as described under ``Experimental Procedures.'' The molecular mass standards are shown on the left. The 65-kDa receptor-specific species is indicated by the arrow on the right.


Preference of Agonist Cross-linking for the High Affinity G-protein-coupled B2 Receptor State

Next, we determined if the cross-linking of BK to a sulfhydryl in the receptor is sensitive to association of the receptor with the G-protein. Bovine myometrial membranes contain two populations of B2 receptors(35) . One receptor population (50%) is of high affinity and sensitive to GTP, and another population (50%) is of low affinity and insensitive to Gpp(NH)p. Different concentrations of [^3H]BK were used in experiments to determine if cross-linking of BK at 50 µM MBS occurred preferentially to the high or low affinity receptor subpopulations. Raising the [^3H]BK concentration from 0.05 nM, which occupies 84% and 12% of the receptors, respectively(35) , to 2 nM, which occupies 100 and 84% of the receptors, respectively, decreased the MBS cross-linking efficiency 9.3 ± 2.2% (n = 3). Fig. 5A shows a dose-response curve of this effect. This decrease in efficiency corresponds to the increased BK occupancy of the low affinity receptor (12 to 84%). If both receptor subpopulations were equally available for cross-linking, no change in cross-linking efficiency would be observed with a change in the level of occupancy of the high and low affinity receptor populations. BK is cross-linked to the low affinity receptor population. However, the cross-linking efficiency is less than the efficiency to the high affinity receptor population.

Figure 5: Cross-linking of different concentrations of [^3H]BK and in the absence and presence of Gpp(NH)p. A, membranes (100 µg of protein) were incubated with increasing concentrations of [^3H]BK for 60 min and then cross-linked with 50 µM MBS. B, membranes (240 µg of protein) were pretreated with increasing concentrations of Gpp(NH)p for 30 min at 24 °C before incubation with a constant concentration of [^3H]BK (0.05 nM) for 60 min and cross-linking with 100 µM MBS. At 0.05 nM, 5 nM [^3H]BK, under typical binding and cross-linking conditions, the total binding, nonspecific binding, and cross-linked ligand to 240 µg of membrane protein were 4,798 ± 197/31,805 ± 951 dpm, 62 ± 14/3,254 ± 160 dpm, and 2,078 ± 35/12,417 ± 318 dpm, respectively. The results are the average ± S.D. of triplicate assay points from representative experiments.


Gpp(NH)p, a nonhydrolyzable analog of GTP, was included to uncouple the G-protein from the high affinity GTP-sensitive receptors and shift these receptors to a low affinity state prior to binding of 0.05 nM [^3H]BK and cross-linking with 100 µM MBS. Gpp(NH)p dose-dependently decreased the cross-linking efficiency a maximum of 13.7 ± 2.5% (n = 4) at 100 µM Gpp(NH)p. A dose-response curve of this effect is shown in Fig. 5B. This result support those above that the G-protein-coupled receptors exist in a conformation which favors cross-linking of BK.

Antagonist Binding to B2 Receptors

Fig. 6shows that the BK antagonist [^3H]NPC17731 interacted with specific binding sites in bovine myometrial membranes. Analysis of equilibrium binding revealed that this ligand bound with high affinity (K = 35 ± 6 pM, n = 4) to a finite number of binding sites (B(max) = 416 ± 18 fmol/mg of protein). The B(max) for specific [^3H]NPC17731 binding is virtually identical to that for specific [^3H]BK binding (476 ± 48 fmol/mg of protein)(35) . Various kinin agonists and antagonists competed for [^3H]NPC17731 binding with a specificity which is also virtually identical to that of [^3H]BK binding (Fig. 7, A and B), providing further evidence for recognition of a common B2 receptor by these two ligands. Unlike [^3H]BK binding(35) , [^3H]NPC17731 binding dissociated with a single rate, and, as expected for an antagonist, this rate was insensitive to Gpp(NH)p(36) .

Figure 6: Saturation binding isotherm of [^3H]NPC17731 on bovine myometrial membranes. Membranes (260 µg of protein) were incubated with increasing concentrations of [^3H]NPC17731. Total binding (), nonspecific binding (), determined in the presence of 1 µM BK, and specific binding (up triangle, filled) are shown. The results are representative of four experiments with each point assayed in duplicate. The computer-drawn curves represent the best fit to the experimental data (K = 51 pM, B(max) = 462 fmol/mg of protein).


Figure 7: Pharmacological specificity of [^3H]NPC17731 and [^3H]BK binding to bovine myometrial membranes. Membranes (80-100 µg of protein) were incubated with a constant concentration of [^3H]NPC17731 (1 nM) (A) and [^3H]BK (2 nM) (B) in the absence and presence of increasing concentrations of BK (), HOE140 (), and NPC17731 (). The results are presented as % of Control where control refers to specific [^3H]NPC17731 and [^3H]BK binding to membranes as determined in the presence of 1 µM BK. 100% control represents 469 ± 48 fmol/mg of protein of [^3H]NPC17731 binding and 534 ± 62 fmol/mg of protein of [^3H]BK binding. The results are the averages ± S.E. of 2-7 experiments with each point assayed in duplicate.


Lack of Antagonist Cross-linking to B2 Receptors

Next, attempts were made to cross-link [^3H]NPC17731 to the receptor. Fig. 8shows that unlike the cross-linking of [^3H]BK, all cross-linking of the antagonist with either homobifunctional or heterobifunctional cross-linkers was nonspecific. As nonspecific cross-linking of NPC17731 clearly occurred with several of the reagents, the N terminus in NPC17731 was accessible for cross-linking.

Figure 8: Cross-linking of [^3H]NPC17731 and [^3H]BK to bovine myometrial membranes. Membranes were incubated with constant concentrations of [^3H]NPC17731 (2.5 nM) and [^3H]BK (2.5 nM) for 90 min and then cross-linked with various reagents at 1 mM as described. Total binding (open bars), nonspecific binding (right-hatched bars), and cross-linked ligand (left hatched bars) was then determined as described under ``Experimental Procedures.'' The results are the averages ± S.D. of triplicate assay points from a representative experiment.


Western blot analysis of antagonist cross-linking was done by probing soluble proteins cross-linked either with MBS to BK or with DFDNB to HOE140 with the respective antisera (Fig. 9). DFDNB has been reported to cross-link HOE140 to B2 receptors on human foreskin fibroblast and, consequently, was chosen in this experiment to maximize the possibility for antagonist cross-linking(43) . While the 65-kDa band is detected on both anti-BK and anti-HOE140 immunoblots, HOE140, despite its 10-fold higher affinity for the B2 receptor, is detected only at concentrations 10-100-fold higher than the effective BK concentrations. In all, these results support those obtained using [^3H]NPC17731 in that even though these antagonists bind specifically to the B2 receptor with affinities equal to or higher than that of BK, the binding site(s) for the antagonists on the B2 receptor is inaccessible to the cross-linker and, consequently, not identical to the binding site for BK.

Figure 9: Western blots of solubilized preparations of bovine myometrium cross-linked to BK by MBS or to HOE140 by DFDNB. Solubilized preparations were incubated in the absence (lane 1) or presence of 5 nM (lane 2), 50 nM (lane 3), 500 nM (lane 4), and 5000 nM (lane 5) of BK (upper panel) or HOE140 (lower panel) for 60 min and then with 50 µM MBS (upper panel) or 1 mM DFDNB (lower panel). Samples were electrophoresed under reducing conditions and immunoblotted with anti-BK or anti-HOE140 antisera as described under ``Experimental Procedures.'' The molecular mass standards are shown on the left. The 65-kDa receptor-specific species is indicated by the arrows on the right.


DISCUSSION

The results described in this study show that the binding site on the B2 receptor occupied by the agonist BK is adjacent to a cysteine residue(s) in the receptor, and this binding site is different from the site(s) on the receptor occupied by the antagonists NPC17731 and HOE140.

As shown in Fig. 10, the B2 BK receptor belongs to the superfamily of seven transmembrane-domain G-protein-coupled receptors. As such, this receptor contains in addition to seven transmembrane helices an extracellular N-terminal (N) domain, three extracellular loops (EL), three intracellular loops, and a C-terminal domain which by palmitoylation can be attached to the membrane to form a fourth intracellular loop. The rat B2 receptor contains four extracellular cysteine residues (Cys, Cys, Cys, Cys) (23) (Fig. 10), and these residues are conserved in the mouse (24) and human (24, 25) receptors. Two of these residues, Cys and Cys, are conserved in most members of this receptor superfamily(26, 27) , and studies with two members, rhodopsin and the beta-adrenergic receptor, have revealed that these two residues are probably linked in a disulfide bond and may stabilize the correctly folded conformation of these receptors(44, 45, 46, 47) .

Figure 10: Model structure of the rat B2 BK receptor. N, N-terminal domain; EL-1, extracellular loop 1; EL-2, extracellular loop 2; EL-3, extracellular loop 3. Cysteines, lysines, and aspartates 268 and 286 are filled. Specific amino acid residues discussed in the text are filled and numbered. The amino acid sequence was taken from Novotny et al.(47) .


Considering the hydrophilic nature of BK, intracellular domains are probably not involved in the binding of this ligand. Indeed, site-directed mutagenesis indicates that the binding of BK (48) as well as several other peptide agonists such as neurokinins(49, 50) , thyrotropin(50, 51) , lutenizing hormone(52) , formyl peptides(53) , and interleukin-8 (54) to their receptors involves interactions with extracellular residues. This is in contrast to the nonpeptidic ligands which seem to interact exclusively with residues located in transmembrane domains(27) . Based on structural homology modeling, molecular dynamics, and systematic conformational searching methods, Kyle has proposed a model of BK bound to the rat B2 receptor(55) . The model suggests that the acidic side chains of Asp and Asp in EL-3 of this receptor (Fig. 10), which are conserved in all B2 receptors for which the cDNAs have been cloned (23, 24, 25) , interact electrostatically with the basic guanidinyl side chain of the N-terminal Arg^1 in BK, which is absolutely crucial for receptor binding(28) . Support for the proposed model was recently provided by the fact that an Ala, Ala double mutation in the receptor reduced BK binding affinity by about 500-fold(48) .

Chemical cross-linking provides a means of identifying the domain of a ligand binding site directly in the native protein without having to rely on factors such as protein expression which may vary in mutagenesis studies. All the heterobifunctional cross-linking reagents and DFDNB were effective in cross-linking BK to the B2 receptor; the link was specifically to a sulfhydryl residue as cross-linking was completely blocked by sulfhydryl alkylation with NEM, IAA, and PAO. In spite of the short lengths of the cross-linking reagents, we examined the possibility that BK was cross-linked to an associated G-protein rather than to the receptor. Indeed, some G-protein alpha subunits contain sulfhydryls and NEM is known to alkylate sulfhydryls in G(56) , G(57) , and G(58, 59) . Therefore, the G-protein was uncoupled from the B2 receptor by addition of Gpp(NH)p, leading to isomerization of the receptor to a low affinity agonist binding state(35) . This treatment decreased but did not abolish the specific cross-linking of all the effective reagents. Essentially the same amount of decrease in the cross-linking efficiency was observed upon increasing the BK concentration to increase BK occupancy of the Gpp(NH)p-insensitive, G-protein-uncoupled receptor subpopulation. Thus, a sulfhydryl in a receptor-associated G-protein does not seem to serve as an anchor for cross-linked BK. Rather, G-protein receptor coupling seems to improve the access of the sulfhydryl through which BK is cross-linked to the receptor. Immunoblotting of the cross-linked BK with anti-BK antiserum revealed a major receptor-specific band at 65 kDa. This band was specifically and dramatically reduced when BK was bound and subsequently cross-linked in the presence of the B2 receptor antagonist HOE140. This molecular mass corresponds to that observed following cross-linking of iodo[I]Tyr analogs of BK to B2 receptors from various species(43, 60, 61) . A band corresponding to that at 145 kDa observed at 500 nM has not been previously reported. This band may correspond to two cross-linked receptor monomers or a receptor monomer cross-linked to a protein closely associated with the agonist-occupied receptor such as a G-protein subunit.

The B2 receptor contains multiple extracellular sulfhydryls that are conserved among species, and any of these sulfhydryls may serve to anchor cross-linked BK. DTT had little effect on the binding of BK to the bovine receptor or to the receptor on intact rat myometrial cells. Thus, even though a putative disulfide bond between the conserved cysteines in EL-1 and EL-2 may be crucial for proper receptor expression (Fig. 10), reducing such a bond in the expressed receptor does not affect BK binding. Furthermore, a disulfide bond between these residues would make them unavailable for cross-linking. Sulfhydryl alkylation by NEM, IAA, or PAO did not inhibit BK binding, indicating that a cysteine is not directly involved in the BK binding reaction. Considering the effectiveness of short cross-linking reagents such as DFDNB (3Å), the anchor residue must be relatively close to the BK binding site in the B2 receptor. As the N terminus of BK is believed to be adjacent to the conserved aspartates in EL-3(55) , the cysteine in the same loop is one likely candidate for covalently anchoring BK to the receptor through cross-linking. On the other hand, as secondary structure models of G-protein-coupled receptors, in part based on electron diffraction data for bacteriorhodopsin(62, 63) , propose close proximity of the N domain and transmembrane helix 7, the cysteine in the N-terminal domain may also be available for cross-linking BK.

Of the three tested homobifunctional cross-linking reagents which rely on N-hydroxysuccinimide ester chemistry, only DSS cross-linked BK to the receptor and did so only at concentrations about 1000-fold higher than those of heterobifunctional reagents. N-Hydroxysuccinimide esters are capable of reacting with sulfhydryls, but these groups are about 1/1000 as reactive as primary amines. The involvement of a sulfhydryl in DSS cross-linking was ruled out by the lack of inhibition by NEM pretreatment. Apart from the N terminus, only one lysine residue (Lys) is exposed extracellularly in the rat B2 receptor that could serve to cross-link BK to the receptor with homobifunctional reagents(23) . This residue is conserved in the human B2 receptor (24, 25) but replaced with an arginine in the mouse B2 receptor(24) ; both of these receptors contain a lysine in the N domain. Our results suggests that a lysine residue is present in the bovine receptor but is not readily accessible for cross-linking BK.

The homobifunctional N-hydroxysuccinimide reagents used in this study have been used by other investigators to cross-link radioiodinated BK analogs to B2 receptor-like proteins in various systems. Lee et al.(60) detected a 69-kDa species when cross-linking [I-Tyr^8]BK to neuroblastoma-glioma cell membranes with 2 mM DSP, while Yaqoob and Snell (61) detected a 81-kDa species when cross-linking the same radioligand to rat uterine membranes with 0.3 mM DSS. Also, Abd Alla et al.(43) observed a 69-kDa reduced and a 59-kDa nonreduced species when cross-linking I-Tyr^0-BK to human foreskin fibroblast membranes with 1 mM DST. These investigators did not report efficiencies of cross-linking by these reagents. We have found that as much as 40% of the specific receptor binding of [I-Tyr^8]BK in bovine membranes was cross-linked using only 50 µM of the homobifunctional reagent DST, (^2)strongly indicating that the lack of cross-linking of BK to the bovine B2 receptor with these reagents is not due to the lack of an available primary amine in the receptor. Instead, either the addition of or replacement with a tyrosine residue in BK or iodination of the tyrosine must alter the positioning of the ligand in the receptor to make it more accessible for cross-linking with a primary amine(s). The functional properties of iodinated BK analogs have not been investigated. In cultured rat myometrial cells [^3H]BK is an excellent probe for detecting the B2 receptor, but we have found that the B2 receptor affinity of [I-Tyr^8]BK on these cells is too low for detection of any specific binding.^2 This is direct evidence of a significant change in the binding parameters of the ligand following incorporation of a tyrosine and/or iodination. These results stress the importance of using BK rather than a synthetic analog such as [I-Tyr^8]BK when probing the native agonist binding site on the B2 receptor.

Abd Alla et al.(43) reported that, in human foreskin fibroblasts, HOE140 was cross-linked to the B2 receptor with DFDNB and DST. However, in analogy with our results, no cross-linking of HOE140 with heterobifunctional reagents such as MBS to this receptor was observed. (^3)The human receptor contains an additional Lys in the N-terminal domain that may serve as an anchor for HOE140 in this receptor with DFDNB and DST. Even though the antagonists NPC17731 and HOE140 specifically occupy B2 receptors in bovine membranes, these ligands are not capable of being cross-linked to the bovine receptor by either homo- or heterobifunctional reagents as determined by both the lack of specific cross-linked [^3H]NPC17731 binding and the data from immunoblots of proteins cross-linked to HOE140. One could invoke purely conformational constraints to explain differences in cross-linking of agonists and antagonists. However, this explanation does not correlate with receptor mutagenesis studies. In contrast to BK binding, antagonist binding is not reduced following mutation of the conserved Asp and Asp in the rat B2 receptor(48) . Considering the competitive nature of agonists and antagonists at B2 receptors, it is likely that these two classes of ligands occupy at least in part the same space in the receptor. However, our cross-linking results provide further direct biochemical evidence that the binding of these ligands in the B2 receptor involve in part different determinants.

FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM41659 and Robert A. Welch Foundation Grant AQ-1087. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760. Tel.: 210-567-3766; Fax: 210-567-6595.

(^1)
The abbreviations used are: BK, bradykinin; G-protein, guanine nucleotide regulatory protein; Gpp(NH)p, guanylyl-imidodiphosphate; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; SMPB, succinimidyl 4-(p-maleimidophenyl)butyrate; SMCC, succinimidyl 4(N-maleimidomethyl)cyclohexane-1-carboxylate; SPDP, N-succinimidyl 3(2-pyridyldithio)propionate; DSS, disuccinimidylsuberate; DSP, dithio-bis(succinimidylpropionate); DST, disuccinimidyl tartarate; DFDNB, 1,5-difluoro-2,4-dinitrobenzene; NEM, N-ethylmaleimide; IAA, iodoacetic acid; PAO, phenylarsine oxide; DTT, dithiothreitol; TES, 2-{[2-hydroxymethyl)ethyl]amino}ethanesulfonic acid; TBS, Tris-buffered saline.

(^2)
M. C. S. Herzig and L. M. F. Leeb-Lundberg, unpublished observations.

(^3)
S. Abd Alla, personal communication.

ACKNOWLEDGEMENTS

We thank Dr. W. Muller-Esterl for donating the anti-HOE140 antiserum, Dr. S. Farmer for donating NPC17731, W. DeWeerd, C. Sewel, and Dr. M. Flynn for technical assistance, and S. A. Mathis for carefully reading the manuscript.

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