Physiological and pathophysiological processes are mediated by
kinins and their receptors. Kinins are liberated by proteolytic
cleavage of the precursor proteins kininogens(1) ; they
decrease blood pressure, induce pain and inflammation, contract smooth
muscles, and regulate ion fluxes(2) . Receptors for kinins are
classified pharmacologically into two major subtypes, B1 and
B2(3) . The B1 receptors are triggered by carboxyl-terminally
truncated kinins such as [des-Arg
]kallidin,
whereas bradykinin is the agonist of B2 receptors. Molecular cloning
has revealed the primary structures of the B1 (4) and the B2
receptors (5) and classified them as members of the
G-protein-coupled receptor family that are thought to contain seven
membrane spanning
-helices.
The signaling pathways of the B2
receptors have been explored in some detail. The bradykinin B2 receptor
is preferentially coupled to G proteins of the G
subtype(6) , which activate the phospholipase C-mediated
cascade. This results in the hydrolysis of inositol-containing lipids,
the generation of inositol phosphates, and the transient rise of the
intracellular free Ca
concentration(7) . The
initial increase of intracellular Ca
is followed by
Ca
extrusion, which counteracts Ca
influx, thereby regulating total cell calcium(8) .
B2-mediated release of diacylglycerol, another hydrolysis product of
phospholipase C, results in the translocation of specific protein
kinase C isoforms(9) . The B2 receptor is also coupled to the
phospholipase A2 pathway, which releases the prostaglandin precursor,
arachidonic acid(10) .
Although the amino acid sequence of
the B2 receptor has been deduced from its cDNA and its transmembrane
topology has been predicted from the corresponding hydropathy plots,
the specific role of the extracellular domains in ligand binding and in
signal transduction is unknown. To address this question, we have
raised antibodies against peptides derived from the ectodomains of the
B2 receptor and used them to probe for the function(s) of the
corresponding structures. Our data show that extracellular domain 3 is
involved in ligand binding and may play an essential role in
communicating the agonist signal through the receptor.
EXPERIMENTAL PROCEDURES
Materials
Na-[
I] (17.4
Ci/mg) and the chemiluminescence detection kit (ECL) were from Amersham
Corp.; [2,3-prolyl-3,4-
H]bradykinin
(specific activity 98 Ci/mmol) was from DuPont NEN; iodogen
(1,3,4,6-tetrachloro-3
-6
-diphenyl-glycoluril) and
1,5-difluoro-2,4-dinitrobenzene were from Pierce; Sephadex-G50 was from
Pharmacia Biotech Inc.; Dowex 1 (1
8), wheat germ agglutinin
(WGA) (
)from Triticum vulgaris, N-acetylglucosamine, and fluorescein isothiocyanate-conjugated
goat anti-rabbit immunoglobulin were from Sigma; rhodamine-conjugated
donkey anti-rabbit immunoglobulin was from Jackson; Centricon 30
filters were from Amicon; keyhole limpet hemocyanin and fura-2/AM were
from Calbiochem; polyvinylidene difluoride sheets were from Millipore;
Affi-Gel 10 and nonfat dry milk was from Bio-Rad; MaxiSorb titer plates
were from Nunc; HOE140 and 3-(4-hydroxyphenyl-propyl)-HOE140
(HPP-HOE140) was from Hoechst; and gelatin was from Merck. All other
chemicals were of analytical grade.
Cell Transfection and Infection with Recombinant
Baculovirus
CHO cells were transfected with the rat B2 receptor
cDNA (rB2CHO12/4) using the Lipofectin transfection method as
described(8, 9) . The pVL1392 vector (kindly provided
by Dr. H. Reiländer, Frankfurt, Germany) contained
the human B2 receptor cDNA such that transcription was directed to the
predicted initiation site(11) . Sf9 cells (2
10
/ml) were infected with the wild type or the recombinant
baculovirus at a multiplicity of infection of 2-5. Cells were
harvested 48-72 h after infection (12) .
Cell Culture
Human foreskin fibroblasts, HF-15 (13) were grown to confluency in Dulbecco's modified
Eagle's medium containing 10% (v/v) fetal calf serum for
2-3 weeks and used at passages 10-15. Chinese hamster ovary
(CHO) cells were grown in Ham's F12 medium and Sf9 cells in TC100
medium, each containing 10% fetal calf serum. The epithelial carcinoma
cell line A431 was maintained in RPMI 1640 medium supplemented with 10%
fetal calf serum, 1 mM sodium pyruvate, 100 units/ml
penicillin and 0.1 mg/ml streptomycin. HF-15, CHO, and A431 cells were
kept in a humidified 5% CO
, 95% air atmosphere at 37
°C, and Sf9 cells were kept in a humidified air atmosphere at 27
°C.
Membrane Preparation
HF-15 cells, CHO cells, or
Sf9 cells were washed twice with ice-cold phosphate-buffered saline
(PBS). Cells were harvested and homogenized in PBS containing protease
inhibitors, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml N-[N-(L-3-transcarboxyoxiran-2-carbonyl)-L-leucyl]-agmatin
(E64), and 2 µM leupeptin. After centrifugation (3000
g, 15 min) membranes were recovered from the
supernatant, sedimented by another centrifugation step (15,000
g, 30 min), and washed twice with PBS. Crude membranes at a
protein concentration of 1 mg/ml were stored at -80 °C until
use. The binding activities of the membranes for
[
H]bradykinin were 0.5 pmol/mg of protein for
HF-15 cells, 1.5-2 pmol/mg of protein for rB2CHO12/4 cells, and
3-5 pmol/mg of protein for recombinant baculovirus-infected Sf9
cells.
Extraction of Membrane Proteins by Triton X114
CHO
cells were washed twice with ice-cold PBS and harvested in 0.5% (v/v)
of Triton X-114 in PBS containing protease inhibitors; the yield was
approximately 1 mg of protein/0.5 ml of Triton X-114. After
centrifugation (3 min, 4 °C, 13,000 rpm) the pellet debris was
discarded. The supernatant was heated to 30 °C for 4 min and
centrifuged (3 min, 24 °C, 3,000 rpm) to cause phase separation (14) . The supernatant was discarded, and the Triton X-114
phase was dissolved in ice-cold PBS. After another phase separation
step, the Triton X-114 phase was diluted with 2 volumes of SDS sample
buffer (15) and applied to polyacrylamide gel electrophoresis
(PAGE).
Radioiodination of Peptides and
Antibodies
Peptides or antibodies (1 µg each) dissolved in
100 µl of PBS were incubated with 2 mCi of carrier-free
Na-[
I] on a solid phase of Iodogen (100
µg/tube) for 10 min(16) . Unreacted iodine was separated by
gel filtration over Sephadex G50 colums or by anion exchange
chromatography over Dowex-1.
Synthesis of Peptides and Production of Anti-peptide
Antibodies
Peptides derived from the rat or human B2 receptor
sequence (Fig. 1) were synthesized by solid phase peptide
synthesis using the Fmoc (N-(9-fluorenyl)methyloxycarbonyl) or
the t-Boc (t-butyloxycarbonyl) chemistry (Table 1). 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(17) . Rabbits were immunized with
the conjugates(18) . Peptide MLN33 was used for immunization
without prior coupling to a carrier protein. Antisera were tested for
antigen specificity and cross-reactivity with homologous human or rat
peptides by the indirect enzyme-linked immunosorbent assay (ELISA) (19) using microtiter plates (MaxiSorb, Nunc) coated with 2
µg/ml of the peptide or 0.5 µg/ml of the conjugate.
Figure 1:
Positions
of the peptides in the rat B2 receptor. A model of the B2 receptor
topology based on hydrophobicity plots and a transmembrane
-helix
hypothesis is presented. The positions of the peptides from ED and ID
domains used to raise antisera are marked by filled or hatched circles; sequences of overlapping peptides are marked
by mixed filled and hatched circles. For nomenclature and
compositions of the synthetic peptides, see Table 1.
Western Blotting and Immunoprinting
Proteins were
resolved by SDS-PAGE and transferred to polyvinylidene difluoride
sheets using semidry blotting(20) . The sheets were 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 of incubation at 37 °C the
polyvinylidene difluoride sheets were washed five times for 15 min each
with buffer A and incubated for 30 min with peroxidase-labeled
F(ab`)
fragments of goat anti-rabbit antibody (Sigma,
1:5000). After extensive washing, bound antibody was visualized using
the ECL chemiluminescence detection kit (Amersham).
Purification of Anti-peptide Antibodies by Affinity
Chromatography
Peptides were covalently coupled to Affi-Gel 10
(5 mg/ml of gel) according to the manufacturer's instructions
(Bio-Rad). The antiserum (5 ml/ml of gel) was applied and incubated
under gentle agitation for 12 h at 4 °C. The affinity matrix was
washed three times with PBS, and the bound antibodies were eluted with
0.2 M glycine, pH 2.5, and immediately neutralized with 1 M KOH. Antibodies were desalted and concentrated using a
Centricon filtration unit, exclusion limit 30,000 Da. The purity and
specificity of the antibodies were analyzed by SDS-PAGE and
enzyme-linked immunosorbent assay, respectively.
Lectin Affinity Chromatography of B2 Receptor
WGA
was covalently coupled to Affi-Gel 10 (10 mg/ml of gel). B2 receptors
from HF-15 cell membranes were solubilized with 4 mM CHAPS in
20 mM PIPES, pH 6.8. The solution was diluted with an equal
volume of 20 mM PIPES, pH 6.8, adjusted to 1 M NaCl,
100 mM MnCl
, 100 mM CaCl
, and
incubated for 4 h at 4 °C with the WGA affinity matrix. After
extensive washing, bound proteins were eluted by a 30-min incubation
with an equal volume of 20 mM PIPES, 1 M NaCl, 100
mM MnCl
, 100 mM CaCl
, 0.5 MN-acetyl glucosamine, pH 6.8. Proteins were
desalted and precipitated by 80% (v/v) acetone (21) and
recovered by centrifugation. The protein pellet was dissolved in 2%
(w/v) SDS, 5 mM EDTA, 5% (v/v) 2-mercaptoethanol, 20% (v/v)
glycerol, 0.01% (w/v) bromphenol blue, 67.5 mM Tris-HCl, pH
6.7 by boiling for 5 min, and proteins were resolved on 10% (w/v)
polyacrylamide gels containing 0.1% (w/v) of SDS(15) .
Immunoaffinity Chromatography of the B2
Receptor
Affinity-purified domain-specific antibodies were
covalently bound to Affi-Gel 10 (15 mg/ml gel). Membranes of Sf9 cells
infected with baculovirus encoding the human B2 cDNA (100 pmols of B2
receptor/20 mg of total membrane protein) were solubilized with 2%
(w/v) sodium deoxycholate in PBS including 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml E64, and 2 µM leupeptin. The deoxycholate was diluted to 0.1% (w/v) by the
addition of 20 mM HEPES, pH 7.4, containing 150 mM NaCl, 1 mM EDTA (buffer B). Then 10% (v/v) glycerol and
0.1% (w/v) Triton X-100 were added, and the solution was applied to the
immunoaffinity matrix for an overnight incubation. The affinity matrix
was extensively washed with buffer B, and bound proteins were eluted
with 0.2 M glycine, pH 2.5 supplemented with 10% (v/v)
1,4-dioxane. The eluted protein fraction was neutralized with 1 M Tris, pH 8.0, and concentrated by Centricon filtration (exclusion
limit 30,000 Da). The purity of the enriched B2 receptor was assessed
by SDS-PAGE and silver staining. For NH
-terminal
sequencing, proteins from three experiments were pooled, applied to a
ProSpin sample preparation cartridge, and sequenced on a 477 A protein
sequencer equipped with an on-line 120A PTH Analyzer (Applied
Biosystems).
Affinity Cross-linking of the B2 Receptor
B2
agonist or antagonist was cross-linked to the B2 receptor as described
previously (22) with minor modifications. B2 receptors of
HF-15 cells were enriched by WGA affinity chromatography, and the
eluted proteins were desalted by dialysis prior to ligand binding and
cross-linking with 1 mM difluorodinitrobenzene. Cross-linking
to recombinant B2 receptors of CHO (1.5 pmol/mg of protein) and Sf9
cells (4-5 pmol/mg of protein) was performed on intact cells
without prior enrichment of receptor protein.
Competition Studies with Radiolabeled
Ligands
Membranes or confluent HF-15 cells on 24-well plates in
0.5 ml of RPMI 1640 including protease inhibitors and buffered with 20
mM Na
-HEPES, pH 7.4 (binding buffer) were
incubated with [
I]HPP-HOE140 (0.5 nM,
specific activity 1367 Ci/mmol) or with
[
H]bradykinin (2 nM, specific activity
98 Ci/mmol) in the presence of increasing concentrations of
affinity-purified antibodies (5
10
M to 1
10
M). After 2 h of
incubation at 4 °C, the cells were washed three times with ice-cold
medium. The cells were dissolved in 1% (w/v) NaOH, and radioactivity
was determined.
Competition Studies with Iodinated
Antibodies
Confluent HF-15 cells on 24-well plates were washed
twice with binding buffer (see above). Then 0.5 ml of binding buffer
was added to each well. For competition studies cells were incubated at
4 °C with
I-labeled immunoselected antibodies (1
10
M; specific activity 0.02 Ci/mg)
in the presence or absence of 1
10
M bradykinin or HOE 140. After 2 h of incubation at 4 °C, the
cells were washed three times with ice-cold medium and dissolved in 1%
(w/v) NaOH, and radioactivity was determined.
Immunofluorescence Studies of A431 Cells
The human
epithelial cell line, A431 was grown on glass coverslips for 48 h.
Three h before the experiment, the medium was replaced by RPMI 1640
supplemented with 0.5% (v/v) fetal calf serum. Prior to
immunofluorescence, cells were washed three times with 60 mM PIPES, 25 mM HEPES, 10 mM EDTA, 2 mM Mg(CH
COO)
, pH 6.9, and fixed for 30 min
with 3% (w/v) paraformaldehyde in the same buffer adjusted to pH 7.5.
Excess paraformaldehyde was quenched by the addition of 50 mM NH
Cl in PBS, pH 7.4; this was followed by 30 min of
incubation with PBS, pH 7.4, containing 0.3% (w/v) gelatin. The cells
were treated for 1 h at room temperature with anti-peptide antisera,
1:100 in 0.3% gelatin/PBS. The first antibody was detected using a
rhodamine-coupled donkey anti-rabbit immunoglobulin, 1:100 in 0.3%
gelatin/PBS. Controls included antisera preincubated for 2 h with 20
µM of their respective antigens. The coverslips were
embedded in Moviol
and viewed with an Orthoplan
microscope (Leitz).
Flow Cytometric Analysis
Confluent HF-15 cells
(0.5-1 pmol of B2 receptor/mg of protein) were harvested using
PBS, 0.5 mM EDTA, pH 7.4, 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
10
) were suspended in the incubation medium
containing the antisera, 1:100 (v/v), and incubated for 1 h at 4
°C. After washing three times, fluorescein
isothiocyanate-conjugated goat anti-rabbit immunoglobulin (Sigma),
diluted 1:80 (v/v), was added to the cells. The cells were incubated
for 1 h at 4 °C, washed, fixed with 2% (v/v) of formaldehyde, and
analyzed on a FACScan (Becton Dickinson) using the LYSIS program.
Measurement of Changes in Intracellular Free
Ca
Concentration
Intracellullar free
Ca
concentration,
[Ca
]
, of HF-15 cells was
determined by fura-2/AM as described previously (8) with minor
modifications. Confluent HF-15 cells grown on 10-mm-diameter glass
coverslips were washed twice with minimum essential medium buffered
with 20 mM Na
-HEPES, pH 7.4 (HMEM), and
incubated with 2 µM fura-2/AM in HMEM containing 0.04%
(w/v) pluronic F-127. After a 45-min incubation at 30 °C, the cells
were washed twice and incubated in HMEM for another 30 min to allow for
complete deesterification of fura-2/AM. For determination of changes in
[Ca
]
, the coverslips were
mounted in a holder at an angle of 45° and put into a thermostatted
quartz cuvette, and fluorescence at 510 nm was determined. The
excitation wavelength alternated between 340 and 380 nm in intervals of
600 ms. Changes in [Ca
]
are
given as the ratio of 340 and 380 nm.
RESULTS
Selection of Peptides for Immunizations
To raise
antisera that cross-react with four predicted extracellular domains
(EDs) (
)of the B2 receptor, we selected six segments from
ED1 through ED4 of the B2 receptor so that the peptides included a
cysteine residue if available (Table 1). The peptides covered the
entire sequence of the putative ectodomains of the B2 receptor; there
were two overlaps of 1 and 3 residues between the peptides selected
from ED3 and ED4, respectively (Fig. 1). Two additional peptides
chosen from the putative intracellular domains (IDs), ID2 and ID4,
served as controls. The peptides were covalently coupled to a carrier
protein, keyhole limpet hemocyanin, and used for immunization except
for MLN33, which was directly used without prior conjugation. The
resultant antisera recognized their cognate antigens as verified by the
indirect enzyme-linked immunosorbent assay, and cross-reacted with the
sequences from the human or the rat B2 receptor, respectively (not
shown). A single peptide, designated CWN12, which is derived from ED4
and covers the center portion of peptide SGC18, failed to produce a
significant titer of specific antibodies (not shown).
Cross-reactivity with the Denatured B2 Receptor
To
analyze the interaction of the anti-peptide antisera with B2 receptors
we used HF-15 fibroblast-derived B2 receptor, partially purified by WGA
affinity chromatography for immunoprint analysis (Fig. 2A).
Antisera directed against EDs 1, 2, 3, and 4 (lanes 2-7) and
to ID2 or ID4 (lanes 8 and 9) efficiently recognized
a protein of 69 ± 3 kDa. A protein of similar molecular mass, 69
± 5 kDa, was stained when HOE140-labeled B2 receptor was
identified by antiserum to HOE140 (Fig. 2A, lane
1)(22) . Likewise, when
[
I-Tyr
]bradykinin was cross-linked
to the B2 receptor in HF-15 membranes and the sample was resolved by
SDS-PAGE followed by autoradiography, a single radiolabeled band of 69
± 3 kDa was seen (Fig. 2A, lane 10). In
some cases bands of higher molecular mass were seen, which might
represent aggregated B2 receptors (Fig. 2A, lanes
7-9).
Figure 2:
Immunoblotting of B2 receptors with
domain-specific antisera. A, B2 receptor of HF-15 cells was
enriched by WGA chromatography. Twenty µg of protein containing 0.1
pmol of B2 receptor were applied/lane. Immunoblots were probed by
antisera to ED1 (lane 2), ED2 (lane 3), ED3
(lane 4), ED3
(lane 5), ED4
(lane 6), ED4
(lane 7), ID2 (lane 8), and ID4 (lane 9), and bound antibody was
visualized by the chemiluminescence detection method. As controls the
B2 receptor was cross-linked to HOE140 and detected by anti-HOE140 (lane 1), or cross-linked to
[
I-Tyr
]bradykinin and visualized by
autoradiography (lane 10). B, immunoprint of Triton
X-114-enriched B2 receptor from CHO membranes (10 µg of protein
containing 0.1 pmol of B2 receptor/lane) was probed by anti-ED1 (lane 1). Nontransfected CHO cells were used for control (lane 2). For comparison, the B2 antagonist HOE140 was
cross-linked to B2 receptor in the absence (lane 3) or
presence of a 1000-fold molar excess of bradykinin (lane 4)
and detected using anti-HOE140. C, immunoblot of
CHAPS-extracted (20 mM) B2 receptors of Sf9 cells. Ten µg
of protein (0.3 pmol of B2 receptor) were loaded on each lane. The
immunoprint was probed by anti-ED1 (lane 1). As controls we
used Sf9 cells expressing an unrelated human protein,

HS glycoprotein (lane 2) or B2 receptor
cross-linked to the B2 antagonist HOE140 in the absence (lane
3) or presence (lane 4) of a 1000-fold molar excess of
bradykinin; HOE140 was detected by anti-HOE140. All antisera were
applied in a dilution of 1:1000.
Specificity of the Anti-B2 Antisera
To assess the
specificity of the antisera, we used membranes from CHO cells and Sf9
cells that overexpress the rat or the human B2 receptor for Western
blotting and immunoprinting. The results are exemplified for the
antiserum to the first ectodomain, ED1. In Triton X-114-extracted
membranes of recombinant CHO cells expressing the rat B2 cDNA, anti-ED1
detected a single 69 ± 3-kDa protein (Fig. 2B, lane 1). No specific staining was found in nontransfected
cells, Fig. 2B, lane 2. As control B2 receptor
from CHO cells that was labeled by HOE140 was detected using
anti-HOE140 antiserum (Fig. 2B, lane 3).
Binding of the antibodies to the B2 receptor was suppressed when
cross-linking was performed in the presence of a 1,000-fold molar
excess of bradykinin (Fig. 2B, lane 4). We
further tested the specificity of the anti-ED1 antiserum using
CHAPS-extracted membranes of Sf9 cells infected with recombinant
baculovirus encoding the human B2 cDNA, and detected three protein
bands of 38, 41, and 45 ± 5 kDa (Fig. 2C, lane 1). Proteins with a molecular mass of approximately
75-80 kDa are likely to be dimerized B2 receptors that aggregated
probably during sample preparation of Sf9 cell membranes expressing
high amounts of B2 receptor(22) . Membranes from mock-infected
Sf9 cells did not show any specific bands (Fig. 2C, lane 2). As a control HOE140 was cross-linked to the B2
receptor followed by detection with anti-HOE140 antiserum. Proteins of
similar molecular weight were stained, Fig. 2C, lane 3. This staining was suppressed by a 1,000-fold molar
excess of bradykinin (Fig. 2C, lane 4). The
differences in the apparent molecular masses of recombinant B2
receptors from Sf9 cells and of B2 receptors of HF-15 fibroblasts and
the occurrence of multiple immunoreactive bands in Sf9 cells are likely
caused by incomplete glycosylation, characteristic for glycoproteins
expressed in Sf9 cells(23) .
Affinity Purification and Amino-terminal Sequence
Analysis of the B2 Receptor
Is the protein identified by
immunostaining with anti-peptide antisera the authentic B2 receptor? We
enriched the receptor protein from Sf9 membranes by immunoaffinity
chromatography using a mixture of immunoselected anti-peptide
antibodies to the extracellular domains. Edman degradation of the
protein revealed an amino-terminal sequence of
Met-Leu-Asn-Val-Thr-Xaa-Gln-Gly-Xaa-Thr-Leu-Asn-Gly-Thr-Phe-Ala-Xaa-Ser,
where Xaa stands for an unidentified residue. This sequence is
identical with the human B2 receptor sequence starting at the third
in-frame initiator codon(11) ; note that the construct in
baculovirus had been engineered such that only the most 3`-located
initiator codon was available. These data demonstrate that the
affinity-purified anti-peptide antibodies selectively enrich B2
receptor from solubilized Sf9 membranes.
Fluorescence-activated Cell Sorting (FACS) Analysis of
Native B2 Receptors on HF-15 Fibroblasts
To test the reactivity
of the antibodies with native B2 receptor we performed FACS analysis of
HF-15 cells that were stained by antisera to the various extracellular
domains ED1 to ED4 (Fig. 3A). Antisera to extracellular
domains ED1 to ED4 bound to B2 receptors of intact HF-15 cells (Fig. 3A, I-VI) as demonstrated by increased
fluorescence intensity in comparison with preimmune serum (Fig. 3A, VII), suggesting that the various
anti-peptide antibodies cross-react to similar extents with the B2
receptor. No specific staining was observed with antisera to
intracellular domain ID2 or ID4, exemplified for anti-ID2 (Fig. 3A, VIII). This finding is in agreement
with the hypothetical model of the B2 receptor (cf. Fig. 1).
Figure 3:
Analysis of extracellular domains of the
B2 receptor by flow cytometric analysis. A, intact HF-15 cells
containing 1 pmol of B2 receptor/mg of protein were labeled with six
individual antisera to the extracellular domains ED1 to ED4 of the B2
receptor (I to VI). Preimmune serum (VII) or
antiserum to intracellular domain 2, anti-ID2 (VIII) were
applied as controls. The ordinate specifies the relative cell
number, and the abscissa gives the log of fluorescence
intensity. B, effect of agonist and antagonist on cell surface
localization of B2 receptors. Intact HF-15 cells containing
approximately 0.3 pmol of B2 receptor/mg of protein were pretreated
with 1 µM of the agonist bradykinin (III) or of
the antagonist HOE140 (IV) for 60 min at 37 °C. Cells were
cooled to 4 °C, and an equal mixture of the various antisera to the
extracellular domains was applied (I). Preimmune serum served
as the control (II). The antiserum dilution was 1:100
throughout.
Redistribution of B2 Receptor Detected by Antibodies to
Extracellular Domains
The successful binding of anti-peptide
antisera to cellular B2 receptors allowed us to examine the fate of the
B2 receptor after its activation by an agonist. In these studies we
applied a mixture of the various anti-ED antibodies. HF-15 cells were
preincubated at 37 °C for 60 min in the absence (Fig. 3B, I) or presence of 1 µM bradykinin (Fig. 3B, III). Pretreatment
of the cells by the B2 agonist drastically reduced the antibody binding
to B2 receptors (Fig. 3B, III). Thus, after
agonist treatment the antigenic epitopes are no longer available for
antibody binding. Preincubation with 1 µM of the
antagonist, HOE140, did not change the overall fluorescence intensity (Fig. 3B, IV). Together these data suggest
that our anti-peptide antibodies readily recognize the extracellular
domain(s) of the B2 receptor and that, like other G-protein-coupled
receptors, sequestration and/or internalization modifies the
accessibility of extracellular domains for antibody detection.
Immunofluorescence of A431 Cells
To
immunovisualize the B2 receptor on cells other than fibroblasts, the
epidermoid carcinoma cell line A431 was chosen. A strong immunostaining
of the plasma membrane of fixed, nonpermeabilized cells was observed
with a mixture of antibodies against ED1 to ED4 (Fig. 4a). A
specific staining of the outer rims of the cells was also observed when
the individual antisera against extracellular domains, ED1, ED2,
ED3
, and ED4
, were used (Fig. 4e to 4h). The cells exhibit a punctuated labeling, which
may be due to receptor clustering and/or high receptor density in
pseudopodia and microvilli of A431 cells; this latter notion was
confirmed by electron microscopy (not shown). The presence of the
cognate antigen for each antibody abrogated the specific immunostaining (b). No staining was seen with preimmune serum (c),
with antiserum to an unrelated peptide (d), or with an
antiserum to intracellular domain, ID2 (i). Hence the
anti-peptide antibodies are useful to probe for the B2 receptor on the
surface of various cell types ( Fig. 3and Fig. 4).
Figure 4:
Immunofluorescence of A431 cells. Intact
A431 cells containing 0.3 pmol of B2 receptor/mg of protein were
stained with a mixture of antisera to extracellular domains ED1, ED2,
ED3, and ED4 in the absence (a) or presence (b) of 20
µM each of the cognate peptides. Individual antisera to
ectodomains ED1 (e), ED2 (f), ED3
(g), ED4
(h) or intracellular
domain ID2 (i) were used. For control preimmune serum (c) or antiserum to an unrelated peptide was applied (d). Bar, 10
µM.
Blockade of Bradykinin Binding by Anti-ED
Antibodies
We asked whether our antibodies interfered with the
binding of bradykinin to the B2 receptor. HF-15 cell membranes were
preincubated for 2 h at 4 °C with 250 nM affinity-purified
antibodies to the various extracellular domains. This was followed by
the addition of 2 nM [
H]bradykinin and
further incubation for 60 min at 4 °C. The unbound radioligand was
separated by filtration through GF/C glass filters, and the
filter-bound radioactivity was determined. Controls were done in the
absence (total binding) or presence of 2 µM of
the unlabeled ligand, bradykinin (Fig. 5A, columns 1 and 2). Out of six antisera tested only antibodies
against the amino-terminal portion of extracellular domain 3,
ED3
, and to the carboxyl-terminal segment of extracellular
domain 4, ED4
, interfered with bradykinin binding to the B2
receptor (Fig. 5A, columns 5 and 8).
Antibodies to other segments of the extracellular domains (Fig. 5A, columns 3, 4, 6,
and 7) and to the intracellular domains (not shown) had no
effect on [
H]bradykinin binding. These data
suggest that domains ED3 and ED4 of the B2 receptor might be critically
involved in ligand binding.
Figure 5:
Displacement of radioligands by
domain-directed antibodies. A, membranes of HF-15 cells (100
µl containing 30-50 fmol of B2 receptor) were incubated for 2
h at 4 °C with 250 nM affinity-purified antibodies to ED1,
ED2, ED3
, ED3
, ED4
, or
ED4
, followed by the incubation with 2 nM [
H]bradykinin. Nonspecific binding and total
binding were determined in the presence or absence of 2 µM unlabeled bradykinin. Following filtration and washing,
filter-bound radioactivity was determined. B and C,
competition of increasing concentrations of antibodies with
radioligands. Intact HF-15 cells containing 15-20 fmol of B2
receptor/well were incubated at 4 °C with 2 nM of
[
H]bradykinin (B) or with 0.5 nM of [
I]HPP-HOE140 (C) in the
presence of increasing concentrations of anti-ED3
(
),
anti-ED3
(
), or anti-ED4
(
). The
results (mean ± S.E.) from three independent experiments are
given as percentage of maximum specific binding in the absence of
competitor (100%).
Concentration-dependent Displacement of Radioligand by
Anti-ED Antibodies
To further analyze the involvement of
extracellular domains 3 and 4 in agonist and antagonist binding, we
tested whether the effect of the antibodies is dose-dependent. Intact
HF-15 cells were incubated for 2 h at 4 °C with the radioligand in
the presence of increasing concentrations, 100 pM to 10
µM, of the affinity-purified antibodies (Fig. 5, B or C). Cell-bound radioactivity was determined.
Antibodies to ED3
effectively blocked binding of
[
H]bradykinin (Fig. 5B) and of
[
I]HPP-HOE140 (Fig. 5C) with
IC
values of 31.2 ± 1.4 and 79 ± 3.6
nM, respectively. Antibodies against ED4
inhibited
[
H]bradykinin binding to the B2 receptor
(IC
= 226 ± 40.1 nM) (Fig. 5B); however, the antibodies did not block the
binding of [
I]HPP-HOE140 (Fig. 5C). Other antibodies such as anti-ED3
did not interfere with the binding of the ligands (Fig. 5, B and C). We conclude that at least part of the
contact site for bradykinin and/or for HOE140 may be near or within the
amino-terminal portion of ED3. The carboxyl-terminal portion of ED4 may
contribute to the receptor binding of the agonist but not of the
antagonist.
Displacement of
I-Labeled Anti-ED
Antibodies by B2 Ligands
Anti-ED antibodies interfere with
radioligand binding to the B2 receptor. This may either indicate that
they are competitive inhibitors for the binding site or that the
antibodies act allosterically by inducing and/or stabilizing a receptor
conformation unable to bind the ligand. To discriminate between these
possibilities competition binding between the unlabeled ligands,
bradykinin and HOE140, and the
I-labeled antibodies
anti-ED3
and anti-ED4
was done with HF-15
fibroblasts. The binding of 10 nM
I-labeled
anti-ED3
to the receptor was reduced by 80% in the presence
of 10 µM bradykinin, and it was abolished by 10 µM HOE140 (Fig. 6A). At the same concentration the cognate
peptide, KDY13, completely displaced radiolabeled anti-ED3
;
the B1 receptor agonist, [des-Arg
]bradykinin, had
no effect (Fig. 6A). In the case of
I-labeled anti-ED4
antibodies, no inhibition
of binding was seen in the presence of 10 µM bradykinin or
HOE140, whereas displacement was observed by the same concentration of
the cognate peptide, SGC18, Fig. 6B. We conclude that
antibodies to ED3
, but not antibodies to ED4
,
are competitive with B2 receptor ligands. Anti-ED4
may
cause an allosteric alteration and stabilize a conformation of the
receptor unable to bind agonists.
Figure 6:
Competition of radiolabeled anti-ED
antibodies and of anti-idiotypic antibodies with bradykinin or HOE140.
Confluent HF-15 cells were incubated for 2 h at 4 °C with 10 nM
I-labeled anti-ED3
(panel A) or
[
I]anti-ED4
(panel B) in
the absence or presence of 10 µM bradykinin, HOE140,
[des-Arg
]bradykinin, or cognate peptides KDY13 of
anti-ED3
and SGC18 of anti-ED4
. In panel
C, HF-15 cells suspended in medium were incubated with 10 nM [
I]-labeled anti-idiotypic antibodies in
the absence or presence of 10 µM bradykinin or HOE140 or
of a 100-fold molar excess (1 µM) of anti-ED3
or anti-ED4
. Following incubation, the cells were
washed and lysed, and the cell-associated radioactivity was determined.
The results (mean ± S.E.) from three independent experiments are
presented.
Displacement of Anti-idiotypic Antibodies by Antibodies
to ED3
To further address the interaction between
anti-ED3
and the kinin receptor we applied anti-idiotypic
antibodies that had been raised against the idiotype, monoclonal
antibody MBK3 to bradykinin(24) . These antibodies have
previously been shown to bind to and stimulate in an agonist-like
manner the human or mouse B2 receptor(24) . Competition
experiments demonstrate that bradykinin and HOE140 interfere with
I-labeled anti-idiotypic antibodies for receptor binding (Fig. 6C). Antibodies to ED3
displaced the
radiolabeled anti-idiotypes, although not completely, whereas
antibodies to ED1, ED2, and ED4
had no effect
(anti-ED4
is shown in Fig. 6C). These
findings indicate that the majority of the anti-idiotypic antibodies
are likely to bind to ED3
and that interaction sites for
bradykinin, HOE140, anti-idiotypic antibodies, and anti-ED3
antibodies with the external portion of the receptor are mutually
overlapping.
Agonist-like Effects of Antibodies to
ED3
Our finding that antibodies to ED3
interfere with the receptor binding of bradykinin and
anti-idiotypic antibodies prompted us to ask whether anti-ED3
itself is an agonist. Therefore, we measured intracellular free
Ca
in HF-15 fibroblasts treated with
anti-ED3
. At a concentration of 250 nM anti-ED3
transiently increased
[Ca
]
in an agonist-like manner, Fig. 7A. This effect is mediated by the B2 receptor because
a 10-fold molar excess of the B2 antagonist, HOE140, prevented the
Ca
transient (Fig. 7B). Antibodies to
the distal portion of the same domain, ED3
, or to other
ectodomains such as ED4
were without effect (Fig. 7, C and D). Hence polyclonal antibodies to the
amino-terminal portion of ectodomain 3 are agonists.
Figure 7:
Effect of anti-ED antibodies on the
[Ca
]
of human
fibroblasts. HF-15 cells were loaded with fura-2/AM, and the change in
the ratio of fluorescence at 340/380 nm was followed. At the time
points indicated 250 nM affinity-purified antibodies to
ED3
(panels A, B), ED3
(C), or ED4
(D) were applied. In panel B, 2.5 µM HOE140 was added 50 s prior to
application of the antibody. The results were similarly obtained with
three different antibody preparations, each derived from two different
rabbits.
DISCUSSION
In these studies antibodies directed to putative
extracellular domains of the bradykinin B2 receptor were prepared.
These antibodies were used to map extracellular domains involved in
ligand binding. This approach of ligand binding site mapping is
complementary to the site-directed mutagenesis
studies(25, 26, 27) . The antibody approach
reduces the commonly voiced concern about site-directed mutagenesis,
that the mutation changes the receptor structure and binding ability
without being located at the ligand binding site.
Our experiments
show that the amino-terminal portion of ectodomain 3, ED3
,
is involved in agonist binding and sensing because (i) antibodies to
this segment competed with bradykinin for binding to the B2 receptor,
(ii) bradykinin almost completely abolished the binding of radiolabeled
anti-ED3
to B2 receptors, and (iii) anti-ED3
antibodies were agonists. A direct contact between the ED3
segment and bradykinin is uncertain; however, the mutual
competition of ED3
antibodies and bradykinin suggests such
a possibility. The ED3
region is also involved in binding
of the antagonist, HOE140, because (i) anti-ED3
blocked
HOE140 binding to B2 receptors, (ii) HOE140 completely abolished the
binding of radiolabeled anti-ED3
to B2 receptors, and (iii)
HOE140 nearly completely blocked anti-ED3
-induced cytosolic
Ca
increase, i.e. anti-ED3
agonism.
The agonistic effect of anti-ED3
antibodies demonstrates that this receptor region can assume or
can be induced to assume conformation(s) that transmit the signal to
the G-protein. If one considers a two-domain model of G-protein-coupled
receptors as suggested by the observation that transmembrane regions
(TMs) 1-5 and TMs 6-7 need not be covalently connected for
G-protein-coupled receptors to bind and
signal(28, 29) , then anti-ED3
might push
apart the two domains, allowing access of the G-protein to the
intracellular loops. Alternatively, anti-ED3
might
stabilize the R* activated form of the receptor which is at equilibrium
with the R inactive form under basal conditions(30) .
Autoantibodies to extracellular domains of the adrenergic and
muscarinic receptors have been detected in the serum of patients with
myocardial diseases or malignant
hypertension(31, 32, 33) . These antibodies
are directed to the same extracellular loop as is anti-ED3
,
interfere with ligand binding, and are agonists. These similarities
between antibodies to extracellular domains of cationic amine receptors
and of a peptide receptor emphasize the common molecular mechanisms
governing the action of G-protein-coupled receptors.
A few attempts
have been made to elucidate the binding site of the B2 receptor using
site-directed mutagenesis(26, 27) . (
)Alanine substitutions of negatively charged residues were
made at the TM7/ED4 boundary, D286A contained in the ED4
epitope, and at the TM6/ED4 boundary, D268A contained in the
ED4
epitope. The D268A change reduced slightly the affinity
of bradykinin and did not change the affinity with the related
antagonists HOE140 or NPC17761(26) .
However,
anti-ED4
antibodies had no effect on bradykinin or HOE140
binding. The D286A mutation had larger effects on the bradykinin
affinity and small effects on antagonist binding affinity. In
accordance with that observation, anti-ED4
antibodies
reduced bradykinin binding but had no effect on antagonist binding.
Finally an alanine substitution, E179A, contained in the ED3
epitope, also caused a small reduction in bradykinin
affinity(26) . Thus the mutagenesis and the antibody methods
for probing ligand binding sites concur in their indication that the
charged residues Asp
and Glu
and associated
peptide regions may be involved in agonist binding.
Our
anti-ED4
antibody does not confirm the involvement of
Asp
in bradykinin binding; however, we note that D268 is
the amino-terminal residue of the ED4
peptide, DTL12; and
thus, the anti-ED4
antibodies may not bind the Asp
residue as part of an extended protein chain in the same way as
when it is the first residue of a peptide. The finding that
anti-ED4
inhibits bradykinin binding but is unable to
inhibit HOE140 binding suggests that the binding sites for peptidic
agonists and antagonists on B2 receptors do not perfectly overlap. This
conclusion agrees with the suggestions, derived from site-directed
mutagenesis studies, that agonists and antagonists do not bind to
identical sites on the receptor(26, 34) .
These studies, which used anti-extracellular domain antibodies
covering all the extracellular domains of the bradykinin receptor,
demonstrate that the binding of agonists by the receptor involves
extracellular regions at the top of TM4, ED3
, and to a
lesser extent at the top of TM7, ED4
. In contrast, the
binding of antagonists is only affected by antibodies directed to the
top of TM4, ED3
. Furthermore, the anti-ED3
antibodies are agonists, suggesting that the TM4 to TM5 loop,
ED3, is important for signal transduction. These studies also point to
the importance of extracellular domains for binding and signal
transduction in this member of the G protein-coupled receptor family
and demonstrate the utility of epitope-specific antibodies in defining
functionally important regions of receptors.