Investigation of the Extracellular Accessibility of the
Connecting Loop between Membrane Domains I and II of the Bradykinin
B2 Receptor*
Ursula
Quitterer
,
Essam
Zaki§, and
Said
AbdAlla§
From the
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 |
In analogy to the structure of rhodopsin, the
seven hydrophobic segments of G-protein-coupled receptors are supposed
to form seven membrane-spanning
-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.
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INTRODUCTION |
The seven hydrophobic segments of G-protein-coupled receptors are
supposed to form seven transmembrane-spanning
-helices in analogy to
the three- and two-dimensional crystal structures of bacteriorhodopsin
(1) and rhodopsin (2). For the
-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 |
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
-D-galactoside
staining of cells co-transfected by a plasmid coding for
-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 |
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.
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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).
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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
-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.
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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.
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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.
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DISCUSSION |
Topological modeling of G-protein-coupled receptors relies on the
assumption that the seven hydrophobic segments form seven membrane-spanning
-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+].
 |
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