Investigation of the Extracellular Accessibility of the Connecting Loop between Membrane Domains I and II of the Bradykinin B2 Receptor*

Ursula QuittererDagger , Essam Zaki§, and Said AbdAlla§

From the Dagger  Institut für Pharmakologie und Toxikologie der Universität, Versbacher Straße 9, 97078 Würzburg, Germany and the § Genetics Engineering and Biotechnology Research Institute (GEBRI), Alexandria, Egypt

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In analogy to the structure of rhodopsin, the seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven membrane-spanning alpha -helices. To analyze the topology of the bradykinin B2 receptor, we raised site-directed antibodies to peptides corresponding to the loop regions and the amino and car- boxyl terminus of this receptor. We found that a segment with predicted intracellular orientation according to the rhodopsin model, the connecting loop between membrane domains I and II of the bradykinin B2 receptor, was accessible to site-directed antibodies on intact fibroblasts, A431 cells, or COS cells expressing human B2 receptors. Extracellular orientation of this loop was further confirmed by the substituted cysteine accessibility method which showed that exchange of cysteine 94 for serine on this loop by point mutagenesis suppressed the effect of thiol modification by a membrane impermeant maleimide. In addition, this segment seemed to be involved in B2 receptor activation, since (i) thiol modification of cysteine 94 partially suppressed B2 receptor activation, and (ii) site-directed antibodies to the connecting loop between membrane domains I and II were agonists. The agonistic activity of the antibodies was suppressed by the B2 antagonist HOE140 confirming the B2 specificity of the antibody-generated signal. The extracellular orientation of the connecting loop between membrane domains I and II suggests a topology of the B2 receptor different from rhodopsin, consisting of five (instead of seven) transmembrane domains and two hydrophobic segments with both ends facing the extracellular side.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven transmembrane-spanning alpha -helices in analogy to the three- and two-dimensional crystal structures of bacteriorhodopsin (1) and rhodopsin (2). For the beta -adrenergic receptor and the vasopressin V2 receptor the rhodopsin-like topology has been confirmed by site-directed antibodies (3) or by a gene fusion approach (4). Charged amino acids determine the orientation of integral membrane proteins. Both prokaryotic and eukaryotic membrane spanning stretches generally have a net positive charge on the cytoplasmic side and few arginine or lysine residues in extracellular domains (5-7). The connecting loop between membrane domains I and II of the rat and human B2 receptor lacks a net positive charge (8, 9), although the rhodopsin model predicts the intracellular orientation of this loop. Thus, for the bradykinin B2 receptor the seven transmembrane domain topology is in conflict with the "positive inside" rule (7). We therefore determined the orientation of this loop on the intact receptor. We chose an approach which did not alter the receptor's primary sequence since insertion of additional reporter sequences may disturb the correct orientation and/or membrane insertion of the protein (10, 11). To this end antibodies to a peptide corresponding to the connecting loop region between membrane domains I and II were raised. Site-directed antibodies have already proven useful to elucidate the "classical" extracellular regions of the B2 receptor (12) and to determine the agonist-binding site (13). We present here that the connecting loop between membrane domains I and II faces the extracellular side suggesting a membrane topology of the B2 receptor with five membrane spanning and two re-entrant membrane segments which is different from rhodopsin.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Na125I (17.4 Ci/mg), the chemiluminescence detection kit (ECL), and [2,3-prolyl-3,4-3H]bradykinin (specific activity 78 Ci/mmol) were from Amersham; IODO-GEN (1,3,4,6-tetrachloro-3a-diphenyl-glycoluril) and disuccinimidyl tartarate were from Pierce; Dowex AG 1-X8, wheat germ agglutinin, N-acetylglucosamine, and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin were from Sigma; myo-[2-3H]inositol (specific activity 17 Ci/mmol) was from NEN Life Science Products Inc.; stilbenedisulfonate maleimide1 (4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid) was from Molecular Probes; bradykinin, kallidin, and HOE140 were from Bachem.

Cell Culture and Cell Transfection-- Human foreskin fibroblasts, HF-15 (14), A431, and COS cells (ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum and kept in a humidified 5% CO2, 95% air atmosphere at 37 °C. COS cells at 80-90% confluency were transfected using LipofectAMINE according to the manufacturer's instructions (Life Technologies, Inc.) and used 48 h after transfection. The transfection efficiency varied between 30 and 40% as determined by 5-bromo-4-chloro-3-indoyl beta -D-galactoside staining of cells co-transfected by a plasmid coding for beta -galactosidase.

Construction of Expression Vectors-- The cDNAs coding for rat B2 receptor mutants (B2-88Ser; B2-94Ser; B2-88/94Ser) were constructed by overlap extension using the polymerase chain reaction as described (15). Identity of the constructs was confirmed by DNA sequencing.

Determination of Inositol Phosphate Levels-- Inositol phosphate levels of transfected COS cells were determined on adherent cells (15) with minor modifications. Adherent COS cells on gelatin-coated (0.1% in phosphate-buffered saline) 12-well plates were labeled with myo-[2-3H]inositol (2 µCi/ml, specific activity 17 Ci/mmol) for 12 h in inositol-free RPMI medium supplemented with 1% (v/v) fetal calf serum. Prior to the experiment, cells were washed twice with incubation buffer (138 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.6 mM CaCl2, 20 mM Na+-HEPES, pH 7.2) and stored for 5 min in incubation buffer with 10 mM LiCl. Then the cells were placed to 37 °C and the experiment was started by the addition of ligand or buffer as indicated. After 20 min total inositol phosphates were extracted (15). For thiol modification, cells were preincubated with 100 µM of the membrane impermeant thiol-specific reagent stilbenedisulfonate maleimide (Molecular Probes) for 5 min at room temperature.

Determination of Changes in [Ca2+]i-- Bradykinin- or antibody-induced changes in the intracellular free Ca2+ concentration, [Ca2+]i of adherent HF-15 or COS cells seeded on glass coverslips was determined on cells loaded with 2 µM fura-2/AM as described previously (16). Changes in [Ca2+]i are given as the the ratio between 340/380 nm.

Synthesis of Peptides and Production of Antibodies-- Production of domain-specific antisera to the putative intracellular domains of the bradykinin B2 receptor was performed as described previously (12). Briefly, peptides derived from the rat B2 receptor sequence (see Fig. 1A) were synthesized by solid phase peptide synthesis using the Fmoc (N-(9-fluorenyl)methyloxycarbonyl) or the t-butyloxycarbonyl chemistry. Peptides purified by high performance liquid chromatography were routinely analyzed by Edman degradation and electrospray mass spectrometry. Peptides were covalently coupled to the carrier protein keyhole limpet hemocyanin by maleimidocaproyl N-hydroxysuccinimide (peptides I-II and III-IV,) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (peptides V-VI and CTer) (Fig. 1A). Antisera to a peptide derived from the connecting loop between membrane domains III and IV and to the carboxyl terminus (CTer) have been described previously (12). Rabbits were immunized with the conjugates, and the antisera were tested for cross-reactivity with the respective human or rat peptides by the indirect enzyme-linked immunosorbent assay. Immunoselection of the antibodies was routinely performed as described (12).

Western Blotting-- Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride sheets using semidry blotting (17). The blotting membrane was treated with 50 mM Tris, 0.2 M NaCl, pH 7.4 (buffer A), containing 5% (w/v) of nonfat dry milk and 0.1% (w/v) of Tween 20 for 1 h. Antisera were diluted 1:1000 in buffer A containing 2% (w/v) of bovine serum albumin. After 30 min, the blotting membranes were washed five times for 15 min each with buffer A and incubated for another 30 min with peroxidase-labeled F(ab')2 fragments of goat anti-rabbit antibody (Sigma). After extensive washing, bound antibody was visualized with a chemiluminescence detection kit (ECL, Amersham).

Lectin Affinity Chromatography of the Human B2 Receptor-- Enrichment of the B2 receptor from HF-15 cells was performed as described previously (12).

Flow Cytometric Analyses-- HF-15, A431, or COS cells (90% confluency) were detached by 0.5 mM EDTA in phosphate-buffered saline and washed twice with ice-cold RPMI 1640 containing 0.1% (w/v) bovine serum albumin, 20 mM Na+-HEPES, pH 7.4 (incubation medium). Cells (1 × 106) were suspended in the incubation medium containing the immuno-selected antibodies (1 × 10-7 M) and incubated for 1 h at 4 °C. After washing three times, fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (diluted 1:80) was added to the cells. The cells were incubated for 1 h on ice, washed, fixed by 2% (v/v) formaldehyde, and analyzed on a FACScan (Becton Dickinson) using the LYSIS program.

Competition Studies with Iodinated Antibodies-- Confluent HF-15 cells, A431 cells, or transfected COS cells on 24-well plates were washed twice with incubation buffer (138 mM NaCl, 5 mM KCl, 1.6 mM CaCl2, 1 mM MgCl2, 20 mM Na+-HEPES, pH 7.2). Then 0.5 ml of incubation buffer was added to each well. Cells were incubated with 1 × 10-8 M immuno-selected anti-I-II antibodies in the presence or absence of 1 × 10-5 M bradykinin, kallidin, or HOE140. Nonspecific binding was determined in the presence of 1 × 10-5 M of the cognate peptide and was usually less than 5% of total binding. After 2 h of incubation at 4 °C, cells were washed three times with ice-cold incubation buffer and 125I-labeled goat anti-rabbit antibodies (specific activity 0.02 Ci/mg) were added. After another incubation step (1 h, 4 °C) and washing, cells were dissolved in 1% (w/v) NaOH and radioactivity was determined.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cross-reactivity of Site-directed Antibodies with B2 Receptors-- Previous results with site-directed antibodies to the B2 receptor showed that the putatative extracellular B2 receptor regions according to the rhodopsin model were indeed accessible to site-directed antibodies on intact cells as determined by fluorescence-activated cell sorting (12). To further analyze the topology of this receptor, we raised site-directed antibodies to the putative intracellular loop regions of the B2 receptor (Fig. 1A). Peptides corresponding to the respective receptor loops (Fig. 1A) were covalently coupled to keyhole limpet hemocyanin and used for immunization in rabbits. The anti-peptide antisera strongly reacted with their respective antigens as determined by indirect enzyme-linked immunosorbent assay (not shown). B2 receptor cross-reactivity was analyzed by immunoblotting using primary human fibroblasts or COS cells expressing native or recombinant B2 receptors, respectively. Similarly to previous results (12), the site-directed antibodies to the connecting loops between membrane domains I-II, III-IV, V-VI, and to the carboxyl terminus (CTer) cross-reacted with B2 receptors of HF-15 cells and stained a protein of 69 ± 3 kDa of HF-15 cells (Fig. 1B, lanes 1-4). The presence of the immunizing peptide (20 µM) extinguished the specific signal of the antibodies in Western blotting as exemplified for antibodies to the connecting loop between membrane domains I-II (Fig. 1B, lane 5). As a positive control, the B2 receptor was visualized by affinity cross-linking of HOE140 to the B2 receptor and detected with anti-HOE140 antibodies (18) (Fig. 1B, lane 6). To further control the B2 receptor specificity of the antisera, B2 receptors were recombinantly expressed in COS cells. The antisera also cross-reacted with B2 receptors recombinantly expressed in COS cells. As exemplified for antibodies to the connecting loop between membrane domains I and II, the antibodies identified a protein of 60 ± 5 kDa (Fig. 1B, lane 7). This finding is in agreement with previous results (13). There was no significant cross-reactivity with proteins of mock-transfected COS cells under the conditions applied, further confirming the B2 receptor specificity of the site-directed antibodies (Fig. 1B, lane 8). The cross-reactivity with a slowly migrating band in B2 receptor-transfected and in mock-transfected cells (Fig. 1B, lanes 7 and 8) may reflect low level expression of endogenous B2 receptors of COS cells as detected previously (15).


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Fig. 1.   Cross-reactivity of site-directed antibodies with the B2 receptor. A, topology model of the human bradykinin B2 receptor (9) with the extended amino terminus as determined by AbdAlla et al. (30). Filled black circles indicate the position of the peptides used to generate domain-specific antisera of this study and dotted circles indicate positions of peptides used to generate antibodies by which the orientation of the extracellular segments was determined previously (12). Arrows indicate the analogous positions of cysteines 88 and 94 of the rat B2 receptor sequence. B, immunoblot of B2 receptors enriched by wheat germ agglutinin affinity chromatography from HF-15 cells. Twenty µg of protein containing 0.1 pmol of B2 receptor were applied per lane. The blots were probed by antisera (dilution 1:1000) to the connecting loop I-II (lanes 1 and 5), to the connecting loop III-IV (lane 2), to the connecting loop V-VI (lane 3) and to the carboxyl terminus (lane 4) in the absence (lanes 1-4) or presence of 20 µM immunizing peptide (lane 5). Bound antibody was visualized by the chemiluminescence detection method. As a control, HOE140 was cross-linked by disuccinimidyl tartarate (1 mM) to the B2 receptor and visualized by anti-HOE140 antibodies (lane 6). Immunoprint of B2 receptors transiently expressed in COS cells (lane 7) probed by anti-I-II antibodies. As a control membranes of mock-transfected COS cells were applied (lane 8). The membrane preparation of transfected COS cells contained about 10-15 fmol of B2 receptor/µg of protein.

Fluorescence-activated Cell Sorting Analysis of Native B2 Receptors on Intact HF-15 Cells-- Since the positive inside rule (7) predicted the extracellular orientation of the connecting loop between membrane domains I and II, we attempted to stain B2 receptors on intact cells with antibodies to this loop. To this end intact HF-15 cells which express high amounts of endogenous B2 receptors, were incubated with immuno-selected site-directed antibodies and fluorescence-activated cell sorting analysis was performed. Antibodies to the connecting loop between transmembrane domains I-II bound to B2 receptors on intact cells as demonstrated by a 100-fold increase in fluorescence intensity (Fig. 2A, panel 1). A similar increase in fluorescence intensity was observed with antibodies to the extracellular domains of the receptor as exemplified for antibodies to the connecting loop between membrane domains II-III (Fig. 2A, panel 2). By contrast, site-directed antibodies to the residual putative intracellular loops of the B2 receptor, i.e. the connecting loop between membrane domains III-IV, V-VI, and to the carboxyl terminus (CTer) failed to stain B2 receptors on intact cells (Fig. 2A, panels 3-5), although all the antisera cross-reacted with similar intensity with B2 receptors in Western blotting (cf. Fig. 1B). The staining of intact cells by anti-I-II antibodies was suppressed by the presence of 20 µM of the immunizing peptide (Fig. 2A, panel 6). Thus, the epitope recognized by the antibodies raised against the connecting loop between membrane domains I-II was accessible from the extracellular side, whereas the epitopes recognized by the antibodies raised against the connecting loops III-IV, V-VI, and the carboxyl terminus were not accessible on intact cells.


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Fig. 2.   Fluorescence-activated cell sorting analysis of B2 receptors on intact cells. A, intact fibroblasts were incubated by immuno-selected domain-specific antibodies to the connecting loop regions between membrane domains I-II (A, panel 1-6), II-III (A, panel 2), III-IV (A, panel 3), V-VI (A, panel 4), and to the carboxyl terminus (CTer), (A, panel 5) for 1 h at 4 °C in the absence (A, panels 1-5) or presence (A, panel 6) of 20 µM immunizing peptide. Bound antibodies were detected by fluorescence-activated cell sorting analysis after application of a fluorescein isothiocyanate-labeled secondary antibody. Cell integrity after incubation with the first antibody was verified by Amido Black. B, staining of B2 receptors on intact HF-15 (B, panel 1), A431 cells (B, panel 2), or on B2 receptor-transfected COS cells (B, panels 3 and 4) by antibodies to the connecting loop between membrane domains I-II. Bound antibody was visualized by fluorescence-activated cell sorting analysis. To control B2 receptor specificity of the staining, B2 receptors were internalized by a 30-min preincubation of the cells by 1 µM bradykinin (BK) at 37 °C (open traces).

Redistribution of B2 Receptors Detected by Antibodies to the Connecting Loop I-II-- We further analyzed the orientation of the connecting loop between membrane domains I-II on two different B2 receptor expressing cells: A431 cells and B2 receptor-transfected COS cells (Fig. 2B). In addition to HF-15 cells, intact A431 cells were stained by antibodies to the connecting loop between membrane domains I-II (Fig. 2B, panel 2) suggesting that the B2 receptor topology was similar on fibroblasts and on epithelial cells. Relative fluorescence intensity of A431 cells was increased only 10-fold above control compared with a 100-fold increase on HF-15 cells (Fig. 2B, panels 1 and 2). This finding may indicate that the amount of B2 receptors/cell of A431 cells is much lower than that of HF-15 cells, a conclusion which is in agreement with the 75-fold increased EC50 value for the bradykinin-induced rise in [Ca2+]i of A431 cells compared with HF-15 cells (6 × 10-9 M for A431 and 0.08 × 10-9 M for HF-15 cells) (not shown). The antibody staining was reduced to control levels on cells where B2 receptors had been internalized by pretreatment with 1 µM bradykinin at 37 °C (Fig. 2B, panels 1 and 2) confirming that the site-directed antibodies stained B2 receptors on the surface of intact cells. On COS cells which were transfected by B2 receptors with a transfection efficiency of 30-40% as determined by beta -galactosidase assay, fluorescence intensity of transfected cells increased 10-fold (Fig. 2B, panel 3). Again, pretreatment with 1 µM bradykinin suppressed the staining of the B2 receptor expressing COS cells by anti-I-II antibodies (Fig. 2B, panel 4). Together these experiments showed that the antibodies to the connecting loop between membrane domains I and II specifically stained B2 receptors on transfected cells, and that the epitope(s) recognized by these antibodies is (are) similarly accessible from the extracellular side of cells expressing recombinant or native B2 receptors.

Thiol Modification of Cysteines 88 and 94 of the B2 Receptor-- Besides site-directed antibodies, cysteine modification by membrane impermeable thiol-modifying reagents is another means to determine the topology of polytopic membrane proteins (19-21) without modifying the receptor's primary sequence. The connecting loop between membrane domains I-II of the B2 receptor contains two cysteines at position 88 and 94 (Fig. 1A). Cysteine modification often alters the function of membrane proteins (20). Therefore we asked whether the B2 receptor contains a free thiol group on its surface which is important for receptor function. We treated intact COS cells expressing the wild-type rat B2 receptor with the membrane-impermeant thiol-specific probe, stilbenedisulfonate maleimide (19) and measured B2 receptor activation. In the presence of 100 µM stilbenedisulfonate maleimide, the bradykinin-induced increase of inositol phosphate levels was reduced by 30 ± 6% (Fig. 3A). The concentration of bradykinin necessary to produce half-maximal activation decreased from 1.4 ± 0.2 × 10-9 M to 9 ± 3 × 10-9 M in the presence of stilbenedisulfonate maleimide (Fig. 3A). These data suggest the importance of an extracellularly accessible cysteine(s) for B2 receptor activation. Most cysteines within the extracellular side of the B2 receptor are supposed to be linked in disulfide bridges (8). Therefore we next asked whether a cysteine(s) in the connecting loop between membrane domains I-II had been modified by stilbenedisulfonate maleimide. To this end three different B2 receptor mutants were created by point mutation of cysteine(s) to serine(s) at positions 88, 94, and 88/94. All three B2 receptor mutants were not different from the wild-type B2 receptor in their affinity for [3H]bradykinin (KD = 0.6 ± 0.2 × 10-9 M) and their EC50 values for the bradykinin-induced rise in inositol phosphate levels (1.5 ± 0.3 × 10-9 M) determined after transient expression in COS cells. Next, the bradykinin-induced rise in inositol phosphate levels was determined in the absence or presence of stilbenedisulfonate maleimide. Similarly as on the wild-type B2 receptor expressing cells, stilbenedisulfonate maleimide decreased the bradykinin-induced rise in inositol phosphate levels on cells expressing the B2 receptor mutant where cysteine 88 was replaced by serine (Fig. 3B). By contrast, on cells expressing mutants B2-94Ser and B2-88/94Ser, stilbenedisulfonate maleimide did not significantly decrease inositol phosphate levels after bradykinin stimulation (Fig. 3B). This finding suggests that cysteine 94 within the connecting loop between membrane domains I-II was accessible to thiol modification on intact cells, and modification of this cysteine suppressed B2 receptor activation (Fig. 3B). Thus, cysteine 94 of the connecting loop between membrane domains I-II is accessible to the membrane impermeant thiol modifying agent stilbenedisulfonate maleimide on intact cells. Similar results were obtained with 10 µM biotin maleimide (3-(N-maleimidopropionyl)biocytin), another membrane impermeant thiol modifying agent (not shown). These findings extend the data obtained with the site-directed antibodies: (i) the connecting loop between membrane domains I and II is accessible from the extracellular side and (ii) the extracellular orientation of this receptor loop seems to be involved in B2 receptor activation.


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Fig. 3.   Thiol modification of cysteine 94 within the loop region between membrane domains I-II. COS cells were transiently transfected by cDNAs coding for wild-type rat B2 receptor (B2), or for B2 receptor mutants B2-88/94Ser (88/94Ser), B2-88Ser (88Ser), or B2-94Ser (94Ser). Cells were labeled with myo-[2-3H]inositol and inositol phosphate levels were determined after stimulation with bradykinin. A, concentration-response relationship for the bradykinin-induced rise in inositol phosphate levels on the wild-type B2 receptor determined in the presence or absence of 100 µM stilbenedisulfonate maleimide. The values are given as % of maximum determined with 10-7 M bradykinin. A representative experiment is given which has been reproduced three times with similar results. B, bradykinin-stimulated (100 nM) increase in inositol phosphate levels of COS cells expressing wild-type B2 receptor, B2-88/94Ser, B2-88Ser, and B2-94Ser determined in the presence of 100 µM stilbenedisulfonate maleimide. The values are given as percent of control determined on cells without thiol modification. The control values were identical on COS cells expressing the four different B2 receptors indicating that equal amounts of B2 receptors were expressed. Data are the means of three different experiments (±S.E.) performed in triplicate.

Agonist-like Activity of Antibodies to the Connecting Loop between Membrane Domains I and II on B2 Receptors-- To further analyze the potential involvement of the connecting loop I-II in B2 receptor activation, the effect of the anti-I-II antibodies on B2 receptor activation was determined. Immuno-selected anti-I-II antibodies (100 nM) activated B2 receptors of transfected COS cells and of HF-15 cells (Fig. 4, panels 1 and 3) as determined by the transient rise in [Ca2+]i of fura-2 labeled cells. No significant signal was obtained with mock-transfected COS cells (Fig. 4, panel 2) under the conditions applied, or after application of unrelated antibodies (Fig. 4, panel 5). The signal was suppressed when the cells had been pretreated for 5 min with a 100-fold molar excess of the B2 antagonist HOE140 thereby confirming the B2 specificity of the signal (Fig. 4, panels 4 and 6). Thus, anti-I-II antibodies are capable to (partially) activate the B2 receptor and therefore are agonists.


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Fig. 4.   Changes in the intracellular free calcium concentration [Ca2+]i of cells stimulated by antibodies to the connecting loop between membrane domains I and II (anti-I-II). Adherent COS cells (panels 1, 2, 4, and 5) or HF-15 cells (panels 3 and 6) on glass coverslips were labeled by fura-2/AM, and changes in [Ca2+]i were monitored. COS cells were transfected by the human B2 receptor cDNA (panels 1, 4, and 5) 48 h before the experiment. As a control mock-transfected COS cells were used (panel 2). At the time indicated by an arrow, 100 nM immuno-selected anti-I-II antibodies (panels 1-4 and 6) or the same concentration of unrelated antibodies (panel 5) were added to the cells. Where indicated, COS or HF-15 cells had been pretreated by the B2 antagonist HOE 140 (10 µM) for 5 min to control the B2 specificity of the signal (panels 4 and 6). Changes in [Ca2+]i are given as the ratio between 340 and 380 nm. A representative experiment is shown which has been reproduced at least four times with similar results.

Displacement of Antibodies to the Connecting Loop between Membrane Domains I and II by B2 Ligands-- We previously demonstrated that antibodies to the connecting loop between membrane domains IV-V (Fig. 1A) also activated the B2 receptor (12). These antibodies were directed to the bradykinin-binding site (13). Therefore we asked whether antibodies to the connecting loop between membrane domains I and II (anti-I-II) also affected agonist or antagonist binding. The binding of 1 nM 125I-labeled HPP-HOE140 or of [125I-Tyr0]bradykinin to HF-15 cells was not altered by the presence of 250 nM immuno-selected anti-I-II antibodies (not shown). Furthermore, the presence of the B2 agonists bradykinin or kallidin did not reduce the binding of anti-I-II antibodies to adherent HF-15 cells, A431 cells, or B2 receptor-transfected COS cells (Fig. 5) indicating that the connecting loop between membrane domains I-II is not involved in the binding of agonists to the B2 receptor. By contrast, the presence of a 1000-fold molar excess of B2 antagonists as demonstrated for HOE140, suppressed the binding of anti-I-II antibodies to HF-15, A431, or B2 receptor-transfected COS cells (Fig. 5). Similar results were obtained with NPC 17773, another B2-specific antagonist (not shown).


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Fig. 5.   Competition binding studies of antibodies to the connecting loop between membrane domains I and II (anti-I-II) with B2 ligands. Confluent HF-15, A431, or B2 receptor-transfected COS cells seeded on 24-well plates were incubated at 4 °C with 10 nM immuno-selected anti-I-II antibodies in the absence (control) or presence of 10 µM bradykinin, kallidin, HOE140, or the cognate peptide. After washing, bound antibodies were detected by iodine-labeled secondary antibodies. Values are given as % of control (= 100%) and are the means of three different experiments (± S.E.). Control values were 46,600 ± 5,800 cpm/well for HF-15 cells, 39,570 ± 2,120 cpm/well for A431 cells, and 320,300 ± 12,030 cpm/well for B2 receptor-transfected COS cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Topological modeling of G-protein-coupled receptors relies on the assumption that the seven hydrophobic segments form seven membrane-spanning alpha -helices according to the structures of rhodopsin (2) or bacteriorhodopsin (1). Since this assumption was in conflict with the positive inside rule (7) for the connecting loop between membrane domains I-II of the bradykinin B2 receptor (8, 9), we attempted to determine the orientation of this loop. Two different approaches were applied: (i) accessibility to site-directed antibodies and (ii) the substituted cysteine accessibility method which combines thiol modification by a membrane impermeant thiol-specific reagent with point mutagenesis. With both methods we found that the connecting loop between membrane domains I and II of the B2 receptor faces the extracellular side. Since the B2 receptor's amino terminus, the connecting loop regions between membrane domains II-III, IV-V, and VI-VII were accessible to site-directed antibodies on intact cells (12), and the loop regions between membrane domains III-IV, V-VI, and the carboxyl terminus were not accessible on intact cells (cf. Fig. 2A), we suggest a topological model for the bradykinin receptor which is different from rhodopsin consisting of five membrane spanning and two re-entrant membrane segments (Fig. 1A). A receptor model with two re-entrant membrane segments is reminiscent of the glutamate GluR3 receptor model (22). The initially proposed topology of this receptor consisting of four membrane-spanning segments had to be abandoned for a model consisting of three transmembrane domains and one re-entrant membrane segment (22). The crystal structure of prostaglandin H2 synthase-1 (23) revealed how a re-entrant membrane segment can anchor a protein to the membrane by monotopic insertion (24) into the membrane.

This study gives strong evidence that the B2 receptor is the first G-protein-coupled receptor with a seven hydrophobic segment structure different from the well established rhodopsin topology. The extracellular localization of the connecting loop between transmembrane domains I and II is in accordance with the positive inside rule governing membrane protein topology (7). The sequences of several other G-protein-coupled receptors also lack a net positive charge in the connecting loop between transmembrane domains I-II (25, 26), suggesting the existence of a distinct class of G-protein-coupled receptors with B2 receptor-like topology.

The extracellular orientation of the connecting loop between membrane domains I and II of the B2 receptor seems to have functional consequences on B2 receptor activation. Thiol modification by a membrane-impermeant maleimide of cysteine 94 within the I-II region partially suppressed B2 receptor activation. In addition, antibodies to the connecting loop between membrane domains I and II were capable of activating the receptor. Thus, extracellular localization of the "intact" connecting loop I-II seems to be a prerequisite for B2 receptor activation. However, this region is not involved in forming the agonist-binding site, since we did not detect any effect of the anti-I-II antibodies on binding of the classical B2 agonists, bradykinin or kallidin to the B2 receptor. Chemical cross-linking studies (27) and site-directed mutagenesis (28, 29) suggest that the agonist- and antagonist-binding sites to the B2 receptor are not identical and may be only partially overlapping. When we tested the effect of a 1000-fold molar excess of the B2 antagonist HOE140 on the binding of anti-I-II antibodies to the B2 receptor, we found that in contrast to B2 agonists, HOE140 almost completely suppressed antibody binding. However, the binding of iodine-labeled HOE140 was not affected by the presence of the antibodies. There are two possible explanations for these findings: either (i) the connecting loop I-II forms a contact site of HOE140 to the B2 receptor which is different from the agonist-binding site, or (ii) the B2 antagonist induces or stabilizes a receptor conformation which is not accessible for the anti-I-II antibodies. Future studies applying site-directed mutagenesis and/or ligand cross-linking will have to determine which of these two possibilities is true thereby shedding more light on the question of how the atypical topology of the B2 receptor may affect receptor functioning.

    ACKNOWLEDGEMENTS

We thank Dr. W. Müller-Esterl (University of Mainz, Germany) for support in the field of bradykinin receptors, Dr. A. A. Roscher (Munich, Germany) for HF-15 cells and Dr. M. AlAwady, (University of Kairo, Egypt) for initial help in raising anti-peptide antisera.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: stilbenedisulfonate maleimide, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; HOE140, D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-Tic-Oic-Arg; kallidin, [Lys0]bradykinin; bradykinin, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; fura-2/AM, 1-[2-(carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid, pentaacetoxymethylester; [Ca2+]i, intracellular [Ca2+].

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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