(Received for publication, May 18, 1995; and in revised form, June 14, 1995)
From the
Chemical cross-linking was used to analyze the binding sites for
the agonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on
the bovine B2 bradykinin receptor. [H]BK and
[
H]NPC17731 bound with high affinity to the same
B2 receptor in bovine myometrial membranes as determined by the total
number of specific binding sites and pharmacological specificity of the
binding of these two radioligands. Cross-linking experiments were done
using a series of bifunctional reagents reactive either primarily to
amines (homobifunctional) or reactive to amines in one end and to
sulfhydryls in the opposite end (heterobifunctional). All the
heterobifunctional reagents plus the homobifunctional arylhalide
1,5-difluoro-2,4-dinitrobenzene were effective in cross-linking the
[
H]BK N terminus specifically to a sulfhydryl in
the receptor, and this cross-linking occurred at 5-100 µM reagent. In contrast, the homobifunctional N-hydroxysuccinimide ester reagents, at
1 mM,
were only able to cross-link [
H]BK to membrane
proteins nonspecifically. The sulfhydryl reagents N-ethylmaleimide, iodoacetamide, and phenylarsine oxide
blocked cross-linking, whereas these reagents did not inhibit
reversible specific [
H]BK binding. Immunoblotting
with anti-BK antiserum revealed that low concentrations of BK
(5-50 nM) were cross-linked to a receptor-specific
species of 65 kDa. All cross-linking of
[
H]NPC17731 was nonspecific with both
homobifunctional and heterobifunctional reagents. The 65-kDa
receptor-specific species was observed on anti-HOE140 immunoblots, but
only when proteins were cross-linked with very high concentrations of
HOE140 (
500 nM). Our results provide direct biochemical
evidence that the binding site for the agonist BK in the bovine B2
receptor is adjacent to a cysteine and is differentiated from the
binding site(s) for the antagonists NPC17731 and HOE140.
The nonapeptide BK ()(Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) belongs to the kinin
family of vasoactive peptides which have dramatic biological activities
on a number of different tissues and organs including smooth muscle
contraction and relaxation, increased vascular permeability, pain, and
cell proliferation(1, 2) . These peptides have also
been implicated as mediators in the development of pathological states
such as asthma, sepsis, viral rhinitis, arthritis, and inflammatory
pain(3, 4, 5, 6, 7, 8, 9) .
Receptors for kinins have been divided into two main subtypes, B1 and
B2, based on the structure-activity of agonists and
antagonists(1) . BK interacts primarily with the B2 receptor
through which this peptide stimulates a number of second messenger
systems including inositol phospholipid
hydrolysis(10, 11, 12, 13) ,
arachidonic acid metabolism(14, 15, 16) ,
tyrosine phosphorylation(17, 18, 19) , and
membrane depolarization and
hyperpolarization(20, 21) .
The recent cloning of the cDNAs for B1 (22) and B2 receptors (23, 24, 25) have revealed that these receptors belong to the superfamily of seven transmembrane-domain, G-protein-coupled receptors(26, 27) . These advances have opened new avenues for identification of specific domains and residues crucial for ligand binding and function of these receptors.
BK antagonists have been synthesized. The first generation of
antagonists were developed around the crucial replacement of L-Pro in BK with a D-aromatic amino acid
residue resulting in moderately high affinities for the B2
receptor(28, 29) . D-Arg
-(Hyp
, D-Phe
)-BK (NPC567) is a typical example of this
generation. In second generation antagonists, a restricted
-turn
was introduced in the C-terminal portion of the peptide, and this
modification substantially increased affinities of these antagonists
for the
receptor(30, 31, 32, 33, 34) . D-Arg
-(Hyp
, Thi
, D-Tic
, Oic
)-BK (HOE140), where Tic is D-1,2,3,4-tetrahydroisoquinoline-3-yl-carbonyl and Oic is L-[(3aS,7aS)-octahydroindol-2-yl-carbonyl],
and D-Arg
-(Hyp
, D-Hype (trans-propyl)
, Oic
)-BK (NPC17731) are
typical examples of this generation.
Previously, we presented a three-state model of agonist binding to the B2 receptor(35) , and this model was subsequently shown to accommodate negative antagonistic/inverse agonistic activity of a number of first and second generation BK antagonists(36) . The classification of at least some BK antagonists as inverse agonists, and, consequently, as having intrinsic functional activity on their own, has increased the importance of delineating the binding sites for B2 receptor agonists and antagonists. Chemical cross-linking has been used extensively to covalently attach ligands to receptors. In this study, we used this technique to investigate the relationship between the binding sites for these two classes of ligands in the B2 receptor.
The effectiveness of the cross-linkers was assessed in
[H]BK binding experiments (Fig. 1).
Membranes were allowed to bind [
H]BK before
addition of cross-linker. [
H]BK cross-linked to
the membranes was determined by dissociating noncovalently bound
[
H]BK. Each panel shows the increase in total
[
H]BK binding, nonspecific
[
H]BK binding, and cross-linked
[
H]BK with increasing concentration of
cross-linker. Nonspecific [
H]BK binding increased
in parallel with total [
H]BK binding indicating
that some [
H]BK was cross-linked to nonreceptor
sites. The effectiveness of the cross-linkers is seen in the difference
between cross-linked [
H]BK and nonspecific
[
H]BK binding. The heterobifunctional reagents
were considerably more effective than the homobifunctional reagents in
cross-linking B2 receptor-bound [
H]BK (Fig. 1). MBS was the most effective reagent, resulting in
nearly maximal cross-linking at concentrations as low as 5
µM. SMPB and SMCC, functionally identical to MBS but with
longer spacer arms, were also significantly effective although to a
lesser extent than MBS. The sulfhydryl alkylating reagent N-ethylmaleimide (NEM) contains the same maleimide functional
group as the above cross-linkers. When membranes were reacted with NEM,
no cross-linking was observed. Thus, the heterobifunctional reagents
were truly cross-linking BK rather than changing BK dissociation
kinetics. SPDP, another heterobifunctional cross-linker which reacts
via disulfide exchange to form a relatively labile disulfide bond, was
also slightly effective in cross-linking. While the homobifunctional N-hydroxysuccinimide ester reagents DSS, DST, and DSP were
able to cross-link [
H]BK to the membranes, the
cross-linking with these reagents at <1 mM was nonspecific,
although minimal specific DSS cross-linking was observed at >1
mM. Interestingly, DFDNB displayed significant specific
cross-linking ability.
Figure 1:
Cross-linking of
[H]BK to bovine myometrial membranes by
homobifunctional and heterobifunctional reagents. Membranes (240 µg
of protein) were incubated with [
H]BK (2-5
nM) for 60 min. Increasing concentrations of cross-linkers
were then added and the incubations continued for 10 min. Total binding
(
), nonspecific binding (
), and cross-linked ligand
(
) were then determined as described under ``Experimental
Procedures.'' Cross-linked ligand was the amount of radioligand
remaining after dissociation. The results are the averages ±
S.E. of at least three experiments with each point assayed in
triplicate.
Figure 2:
Effect of NEM pretreatment on
cross-linking of [H]BK to bovine myometrial
membranes. Membranes (240 µg of protein) were pretreated without (open bars) and with (cross-hatched bars) 10 mM NEM. The membranes were then incubated with a constant
concentration of [
H]BK (2.5 nM) followed
by cross-linking with MBS (50 µM), SMCC (100
µM), SPDP (100 µM), and DFDNB (1 mM)
as described under ``Experimental Procedures.'' Cross-linking
is expressed as percent of total binding which was as shown in Fig. 2. The results are the averages ± S.E. of four
experiments with each point assayed in
triplicate.
Figure 3: Western blot of solubilized preparations of bovine myometrium cross-linked to BK with MBS. Solubilized preparations were incubated in the absence (lane 1) and presence of 5 nM BK (lane 2), 50 nM BK (lane 3), 500 nM BK (lane 4), and 5000 nM BK (lanes 5 and 6) for 60 min and then cross-linked with 50 µM MBS (lanes 1-5) or 1 mM DFDNB (lane 6) as indicated. Samples were electrophoresed under nonreducing conditions and immunoblotted with anti-BK antiserum as described under ``Experimental Procedures.'' The molecular mass standards are shown on the left. The 65-kDa species is indicated by the arrow on the right.
HOE140 is a potent B2 receptor antagonist which at
100-fold excess over BK completely displaced specific
[H]BK binding. This antagonist also completely
inhibited BK binding and subsequent MBS cross-linking to the 65-kDa
species in solubilized preparations using 50 nM BK (Fig. 4).
Figure 4: Western blot of solubilized preparations of bovine myometrium cross-linked to BK by MBS: pharmacological specificity. Solubilized preparations were incubated without (lanes 1 and 2) and with 10 µM HOE140 (lane 3) in the absence (lane 1) and presence (lanes 2 and 3) of 50 nM BK for 60 min as indicated and then cross-linked with 50 µM MBS. Samples were electrophoresed under reducing conditions and immunoblotted with anti-BK antiserum as described under ``Experimental Procedures.'' The molecular mass standards are shown on the left. The 65-kDa receptor-specific species is indicated by the arrow on the right.
Figure 5:
Cross-linking of different concentrations
of [H]BK and in the absence and presence of
Gpp(NH)p. A, membranes (100 µg of protein) were incubated
with increasing concentrations of [
H]BK for 60
min and then cross-linked with 50 µM MBS. B,
membranes (240 µg of protein) were pretreated with increasing
concentrations of Gpp(NH)p for 30 min at 24 °C before incubation
with a constant concentration of [
H]BK (0.05
nM) for 60 min and cross-linking with 100 µM MBS.
At 0.05 nM, 5 nM [
H]BK, under
typical binding and cross-linking conditions, the total binding,
nonspecific binding, and cross-linked ligand to 240 µg of membrane
protein were 4,798 ± 197/31,805 ± 951 dpm, 62 ±
14/3,254 ± 160 dpm, and 2,078 ± 35/12,417 ± 318
dpm, respectively. The results are the average ± S.D. of
triplicate assay points from representative
experiments.
Gpp(NH)p, a nonhydrolyzable analog of
GTP, was included to uncouple the G-protein from the high affinity
GTP-sensitive receptors and shift these receptors to a low affinity
state prior to binding of 0.05 nM [H]BK
and cross-linking with 100 µM MBS. Gpp(NH)p
dose-dependently decreased the cross-linking efficiency a maximum of
13.7 ± 2.5% (n = 4) at 100 µM
Gpp(NH)p. A dose-response curve of this effect is shown in Fig. 5B. This result support those above that the
G-protein-coupled receptors exist in a conformation which favors
cross-linking of BK.
Figure 6:
Saturation binding isotherm of
[H]NPC17731 on bovine myometrial membranes.
Membranes (260 µg of protein) were incubated with increasing
concentrations of [
H]NPC17731. Total binding
(
), nonspecific binding (
), determined in the presence of
1 µM BK, and specific binding (
) are shown. The
results are representative of four experiments with each point assayed
in duplicate. The computer-drawn curves represent the best fit to the
experimental data (K
= 51
pM, B
= 462 fmol/mg of
protein).
Figure 7:
Pharmacological specificity of
[H]NPC17731 and [
H]BK
binding to bovine myometrial membranes. Membranes (80-100 µg
of protein) were incubated with a constant concentration of
[
H]NPC17731 (1 nM) (A) and
[
H]BK (2 nM) (B) in the absence
and presence of increasing concentrations of BK (
), HOE140
(
), and NPC17731 (
). The results are presented as % of
Control where control refers to specific
[
H]NPC17731 and [
H]BK
binding to membranes as determined in the presence of 1 µM
BK. 100% control represents 469 ± 48 fmol/mg of protein of
[
H]NPC17731 binding and 534 ± 62 fmol/mg
of protein of [
H]BK binding. The results are the
averages ± S.E. of 2-7 experiments with each point assayed
in duplicate.
Figure 8:
Cross-linking of
[H]NPC17731 and [
H]BK to
bovine myometrial membranes. Membranes were incubated with constant
concentrations of [
H]NPC17731 (2.5 nM)
and [
H]BK (2.5 nM) for 90 min and then
cross-linked with various reagents at 1 mM as described. Total
binding (open bars), nonspecific binding (right-hatched
bars), and cross-linked ligand (left hatched bars) was
then determined as described under ``Experimental
Procedures.'' The results are the averages ± S.D. of
triplicate assay points from a representative
experiment.
Western blot analysis of antagonist cross-linking was done by
probing soluble proteins cross-linked either with MBS to BK or with
DFDNB to HOE140 with the respective antisera (Fig. 9). DFDNB has
been reported to cross-link HOE140 to B2 receptors on human foreskin
fibroblast and, consequently, was chosen in this experiment to maximize
the possibility for antagonist cross-linking(43) . While the
65-kDa band is detected on both anti-BK and anti-HOE140 immunoblots,
HOE140, despite its 10-fold higher affinity for the B2 receptor, is
detected only at concentrations 10-100-fold higher than the
effective BK concentrations. In all, these results support those
obtained using [H]NPC17731 in that even though
these antagonists bind specifically to the B2 receptor with affinities
equal to or higher than that of BK, the binding site(s) for the
antagonists on the B2 receptor is inaccessible to the cross-linker and,
consequently, not identical to the binding site for BK.
Figure 9: Western blots of solubilized preparations of bovine myometrium cross-linked to BK by MBS or to HOE140 by DFDNB. Solubilized preparations were incubated in the absence (lane 1) or presence of 5 nM (lane 2), 50 nM (lane 3), 500 nM (lane 4), and 5000 nM (lane 5) of BK (upper panel) or HOE140 (lower panel) for 60 min and then with 50 µM MBS (upper panel) or 1 mM DFDNB (lower panel). Samples were electrophoresed under reducing conditions and immunoblotted with anti-BK or anti-HOE140 antisera as described under ``Experimental Procedures.'' The molecular mass standards are shown on the left. The 65-kDa receptor-specific species is indicated by the arrows on the right.
The results described in this study show that the binding site on the B2 receptor occupied by the agonist BK is adjacent to a cysteine residue(s) in the receptor, and this binding site is different from the site(s) on the receptor occupied by the antagonists NPC17731 and HOE140.
As shown in Fig. 10, the B2 BK receptor belongs
to the superfamily of seven transmembrane-domain G-protein-coupled
receptors. As such, this receptor contains in addition to seven
transmembrane helices an extracellular N-terminal (N) domain,
three extracellular loops (EL), three intracellular loops, and
a C-terminal domain which by palmitoylation can be attached to the
membrane to form a fourth intracellular loop. The rat B2 receptor
contains four extracellular cysteine residues (Cys,
Cys
, Cys
, Cys
) (23) (Fig. 10), and these residues are conserved in the
mouse (24) and human (24, 25) receptors. Two
of these residues, Cys
and Cys
, are
conserved in most members of this receptor
superfamily(26, 27) , and studies with two members,
rhodopsin and the
-adrenergic receptor, have revealed that these
two residues are probably linked in a disulfide bond and may stabilize
the correctly folded conformation of these
receptors(44, 45, 46, 47) .
Figure 10: Model structure of the rat B2 BK receptor. N, N-terminal domain; EL-1, extracellular loop 1; EL-2, extracellular loop 2; EL-3, extracellular loop 3. Cysteines, lysines, and aspartates 268 and 286 are filled. Specific amino acid residues discussed in the text are filled and numbered. The amino acid sequence was taken from Novotny et al.(47) .
Considering the hydrophilic nature of BK, intracellular domains are
probably not involved in the binding of this ligand. Indeed,
site-directed mutagenesis indicates that the binding of BK (48) as well as several other peptide agonists such as
neurokinins(49, 50) ,
thyrotropin(50, 51) , lutenizing hormone(52) ,
formyl peptides(53) , and interleukin-8 (54) to their
receptors involves interactions with extracellular residues. This is in
contrast to the nonpeptidic ligands which seem to interact exclusively
with residues located in transmembrane domains(27) . Based on
structural homology modeling, molecular dynamics, and systematic
conformational searching methods, Kyle has proposed a model of BK bound
to the rat B2 receptor(55) . The model suggests that the acidic
side chains of Asp and Asp
in EL-3 of this
receptor (Fig. 10), which are conserved in all B2 receptors for
which the cDNAs have been cloned (23, 24, 25) , interact electrostatically
with the basic guanidinyl side chain of the N-terminal Arg
in BK, which is absolutely crucial for receptor
binding(28) . Support for the proposed model was recently
provided by the fact that an Ala
, Ala
double mutation in the receptor reduced BK binding affinity by
about 500-fold(48) .
Chemical cross-linking provides a means
of identifying the domain of a ligand binding site directly in the
native protein without having to rely on factors such as protein
expression which may vary in mutagenesis studies. All the
heterobifunctional cross-linking reagents and DFDNB were effective in
cross-linking BK to the B2 receptor; the link was specifically to a
sulfhydryl residue as cross-linking was completely blocked by
sulfhydryl alkylation with NEM, IAA, and PAO. In spite of the short
lengths of the cross-linking reagents, we examined the possibility that
BK was cross-linked to an associated G-protein rather than to the
receptor. Indeed, some G-protein subunits contain sulfhydryls and
NEM is known to alkylate sulfhydryls in
G
(56) , G
(57) , and
G
(58, 59) . Therefore, the G-protein
was uncoupled from the B2 receptor by addition of Gpp(NH)p, leading to
isomerization of the receptor to a low affinity agonist binding
state(35) . This treatment decreased but did not abolish the
specific cross-linking of all the effective reagents. Essentially the
same amount of decrease in the cross-linking efficiency was observed
upon increasing the BK concentration to increase BK occupancy of the
Gpp(NH)p-insensitive, G-protein-uncoupled receptor subpopulation. Thus,
a sulfhydryl in a receptor-associated G-protein does not seem to serve
as an anchor for cross-linked BK. Rather, G-protein receptor coupling
seems to improve the access of the sulfhydryl through which BK is
cross-linked to the receptor. Immunoblotting of the cross-linked BK
with anti-BK antiserum revealed a major receptor-specific band at 65
kDa. This band was specifically and dramatically reduced when BK was
bound and subsequently cross-linked in the presence of the B2 receptor
antagonist HOE140. This molecular mass corresponds to that observed
following cross-linking of iodo[
I]Tyr analogs
of BK to B2 receptors from various
species(43, 60, 61) . A band corresponding to
that at 145 kDa observed at 500 nM has not been previously
reported. This band may correspond to two cross-linked receptor
monomers or a receptor monomer cross-linked to a protein closely
associated with the agonist-occupied receptor such as a G-protein
subunit.
The B2 receptor contains multiple extracellular sulfhydryls that are conserved among species, and any of these sulfhydryls may serve to anchor cross-linked BK. DTT had little effect on the binding of BK to the bovine receptor or to the receptor on intact rat myometrial cells. Thus, even though a putative disulfide bond between the conserved cysteines in EL-1 and EL-2 may be crucial for proper receptor expression (Fig. 10), reducing such a bond in the expressed receptor does not affect BK binding. Furthermore, a disulfide bond between these residues would make them unavailable for cross-linking. Sulfhydryl alkylation by NEM, IAA, or PAO did not inhibit BK binding, indicating that a cysteine is not directly involved in the BK binding reaction. Considering the effectiveness of short cross-linking reagents such as DFDNB (3Å), the anchor residue must be relatively close to the BK binding site in the B2 receptor. As the N terminus of BK is believed to be adjacent to the conserved aspartates in EL-3(55) , the cysteine in the same loop is one likely candidate for covalently anchoring BK to the receptor through cross-linking. On the other hand, as secondary structure models of G-protein-coupled receptors, in part based on electron diffraction data for bacteriorhodopsin(62, 63) , propose close proximity of the N domain and transmembrane helix 7, the cysteine in the N-terminal domain may also be available for cross-linking BK.
Of the three
tested homobifunctional cross-linking reagents which rely on N-hydroxysuccinimide ester chemistry, only DSS cross-linked BK
to the receptor and did so only at concentrations about 1000-fold
higher than those of heterobifunctional reagents. N-Hydroxysuccinimide esters are capable of reacting with
sulfhydryls, but these groups are about 1/1000 as reactive as primary
amines. The involvement of a sulfhydryl in DSS cross-linking was ruled
out by the lack of inhibition by NEM pretreatment. Apart from the N
terminus, only one lysine residue (Lys) is exposed
extracellularly in the rat B2 receptor that could serve to cross-link
BK to the receptor with homobifunctional reagents(23) . This
residue is conserved in the human B2 receptor (24, 25) but replaced with an arginine in the mouse B2
receptor(24) ; both of these receptors contain a lysine in the
N domain. Our results suggests that a lysine residue is present in the
bovine receptor but is not readily accessible for cross-linking BK.
The homobifunctional N-hydroxysuccinimide reagents used in
this study have been used by other investigators to cross-link
radioiodinated BK analogs to B2 receptor-like proteins in various
systems. Lee et al.(60) detected a 69-kDa species
when cross-linking [I-Tyr
]BK to
neuroblastoma-glioma cell membranes with 2 mM DSP, while
Yaqoob and Snell (61) detected a 81-kDa species when
cross-linking the same radioligand to rat uterine membranes with 0.3
mM DSS. Also, Abd Alla et al.(43) observed a
69-kDa reduced and a 59-kDa nonreduced species when cross-linking
I-Tyr
-BK to human foreskin fibroblast
membranes with 1 mM DST. These investigators did not report
efficiencies of cross-linking by these reagents. We have found that as
much as 40% of the specific receptor binding of
[
I-Tyr
]BK in bovine membranes was
cross-linked using only 50 µM of the homobifunctional
reagent DST, (
)strongly indicating that the lack of
cross-linking of BK to the bovine B2 receptor with these reagents is
not due to the lack of an available primary amine in the receptor.
Instead, either the addition of or replacement with a tyrosine residue
in BK or iodination of the tyrosine must alter the positioning of the
ligand in the receptor to make it more accessible for cross-linking
with a primary amine(s). The functional properties of iodinated BK
analogs have not been investigated. In cultured rat myometrial cells
[
H]BK is an excellent probe for detecting the B2
receptor, but we have found that the B2 receptor affinity of
[
I-Tyr
]BK on these cells is too low
for detection of any specific binding.
This is direct
evidence of a significant change in the binding parameters of the
ligand following incorporation of a tyrosine and/or iodination. These
results stress the importance of using BK rather than a synthetic
analog such as [I-Tyr
]BK when probing the native
agonist binding site on the B2 receptor.
Abd Alla et al.(43) reported that, in human foreskin fibroblasts, HOE140
was cross-linked to the B2 receptor with DFDNB and DST. However, in
analogy with our results, no cross-linking of HOE140 with
heterobifunctional reagents such as MBS to this receptor was observed. ()The human receptor contains an additional Lys in the
N-terminal domain that may serve as an anchor for HOE140 in this
receptor with DFDNB and DST. Even though the antagonists NPC17731 and
HOE140 specifically occupy B2 receptors in bovine membranes, these
ligands are not capable of being cross-linked to the bovine receptor by
either homo- or heterobifunctional reagents as determined by both the
lack of specific cross-linked [
H]NPC17731 binding
and the data from immunoblots of proteins cross-linked to HOE140. One
could invoke purely conformational constraints to explain differences
in cross-linking of agonists and antagonists. However, this explanation
does not correlate with receptor mutagenesis studies. In contrast to BK
binding, antagonist binding is not reduced following mutation of the
conserved Asp
and Asp
in the rat B2
receptor(48) . Considering the competitive nature of agonists
and antagonists at B2 receptors, it is likely that these two classes of
ligands occupy at least in part the same space in the receptor.
However, our cross-linking results provide further direct biochemical
evidence that the binding of these ligands in the B2 receptor involve
in part different determinants.