The Human B1 Bradykinin Receptor Exhibits High
Ligand-independent, Constitutive Activity
ROLES OF RESIDUES IN THE FOURTH INTRACELLULAR AND THIRD
TRANSMEMBRANE DOMAINS*
L. M. Fredrik
Leeb-Lundberg
,
Dong Soo
Kang,
Maria E.
Lamb, and
Dana B.
Fathy
From the Department of Biochemistry, the University of Texas Health
Science Center, San Antonio, Texas 78229-3900
Received for publication, August 14, 2000, and in revised form, December 19, 2000
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ABSTRACT |
The B1 bradykinin (BK) receptor (B1R) is a
seven-transmembrane domain, G protein-coupled receptor that is induced
by injury and important in inflammation and nociception. Here, we show
that the human B1R exhibits a high level of ligand-independent,
constitutive activity. Constitutive activity was identified by the
increase in basal cellular phosphoinositide hydrolysis as a function of the density of the receptors in transiently transfected HEK293 cells.
Several B1R peptide antagonists were neutral antagonists or very weakly
efficacious inverse agonists. Constitutive B1R activity was further
increased by alanine mutation of Asn121 in the third
transmembrane domain of the receptor (B1A121). This mutant
resembled the agonist-preferred receptor state since it also exhibited
increased agonist affinity and decreased agonist responsiveness. A
dramatic loss of constitutive activity occurred when the fourth
intracellular C-terminal domain (IC-IV) of the human B2 BK receptor
subtype (B2R), which exhibits minimal constitutive activity, was
substituted in either B1R or B1A121 to make B1(B2ICIV) and
B1(B2ICIV)A121, respectively. Activity was partially
recovered by subsequent alanine mutation of a cluster of two serines
and two threonines in IC-IV of either B1(B2ICIV) or
B1(B2ICIV)A121, a cluster that is important for B2R
desensitization. The ligand-independent, constitutive activity of B1R
therefore depends on epitopes in both transmembrane and intracellular
domains. We propose that the activity is primarily due to the lack of
critical epitopes in IC-IV that regulate such activity.
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INTRODUCTION |
The B1 and B2 bradykinin
(BK)1 receptors are
seven-transmembrane domain, G protein-coupled receptors (GPCR) (1, 2),
which mediate the actions of kinins (3), pro-inflammatory peptides formed in response to tissue injury from kininogen precursors (4, 5).
Kinin actions include pain, inflammation, and hyperalgesia (4, 6). The
B2 receptor mediates the actions of BK and Lys-BK or kallidin (KD), the
first set of bioactive kinins formed following injury, whereas the B1
receptor is thought to mediate the actions of des-Arg9-BK
and des-Arg10-KD, the carboxypeptidase products of BK and
KD and the second set of bioactive kinins formed (3). Despite binding
very similar ligands, leading to their classification as receptor
subtypes, these receptors exhibit only 36% identity. Furthermore, the
B2 receptor is constitutively expressed, whereas the B1 receptor is
expressed at very low levels, if at all, in healthy tissue but is
induced by inflammatory stimuli such as interleukin-1
(2, 7-9) and
by kinins themselves (8, 10). This pattern of expression is consistent
with the fact that the B2 receptor appears to be the principal kinin
receptor under healthy conditions and in the acute stage of the
inflammatory response (4, 6, 11, 12), whereas the B1 receptor is
important primarily in the chronic stage of the response (6, 13,
14).
The rationale for restricting B1 receptor expression primarily to
conditions of injury is not understood. One explanation is that this
receptor serves to provide a sustained kinin signal (7), which may be
necessary in inflammation but detrimental under healthy conditions.
This explanation is consistent with the fact that the B2 receptor
response is transient and rapidly desensitizes upon agonist
stimulation, whereas the B1 receptor response is sustained and
desensitizes very slowly (15, 16). However, restricting B1 receptor
expression to prohibit sustained agonist stimulation seems unnecessary
since kinin production in the absence of injury is probably low, and
the in vivo production of the B1 receptor agonists
des-Arg9-BK and des-Arg10-KD by
carboxypeptidases from BK and KD, respectively, is relatively inefficient (17). On the other hand, limiting B1 receptor expression would be important if this receptor is constitutively active since constitutive GPCR activity, which occurs both spontaneously and in
response to mutations in these receptors, can lead to disease (18, 19)
including tumorigenesis (20, 21).
The localization of many natural and unnatural constitutively
activating GPCR mutations in transmembrane domains has led to the
belief that interhelical contacts are important in regulating the
ligand-independent isomerization of these receptors between the
inactive and the activated states and, consequently, constitutive activity (22). Residues at a position about two helical turns into
TM-III, occupied by an asparagine in the B2 BK (23) and AT1 angiotensin
II receptors (24) and by a cysteine in the
1-adrenergic receptor (25) and glutamate in rhodopsin (26), are thought to
participate in such a contact. It has been reported that epitopes in
IC-IV also regulate constitutive GPCR activity (27-31), yet little is
known about the underlying mechanism. We recently reported that the B2
receptor exhibits minimal ligand-independent activity. Mutation of a
cluster of serines and threonines in IC-IV, which is important for
agonist-promoted B2 receptor phosphorylation and internalization (32)
and desensitization (31), significantly elevated this activity (31). In
the present study, we show that the human B1 receptor exhibits a high
level of constitutive activity in HEK293 cells. B2R IC-IV strongly
suppresses B1R activity, and this occurs independently of the
activation state of the receptor. The suppression is partially due to
the presence of critical serines and threonines, but other epitopes are
also involved. Thus, we propose that the high constitutive B1R activity
is due to the lack of regulatory epitopes in IC-IV.
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EXPERIMENTAL PROCEDURES |
Materials--
[prolyl-3,4-3H]NPC17731
(53.5 Ci/mmol),
des-Arg10-[3,4-prolyl-3,4-3H]kallidin
(69-105 Ci/mmol),
des-Arg10-[Leu9][3,4-prolyl-3,4-3H]kallidin
(67-105 Ci/mmol), and myo-[3H]inositol
(10-20 Ci/mmol) were obtained from PerkinElmer Life Sciences. M2
monoclonal antibodies against the FLAG epitope were obtained from
Eastman Kodak and Sigma. Des-Arg10-kallidin and
des-Arg10-[Leu9]kallidin were from Bachem
(Torrance, CA), whereas HOE140 and des-Arg10-HOE140 were
from Peninsula (Belmont, CA). NPC17731 and NPC18565 were gifts from
D. J. Kyle, Purdue Pharma, Princeton, NJ. R715 was a gift from D. Regoli, Universite de Sherbrooke, Quebec, Canada. B9858 was a gift from
F. Marceau, L'Hotel-Dieu de Quebec, Quebec, Canada. The original human
and rabbit B1 receptor and human B2 receptor cDNAs were from F. Hess, Merck Research Laboratories, West Point, PA. All other chemicals
were obtained as described previously (33, 34).
Mutation and Transfection--
Mutations were done using a
polymerase chain reaction-ligation-polymerase chain reaction protocol
as described previously (33). The FLAG epitope was inserted at the
receptor N terminus immediately following the initial methionine.
HEK293 human embryonic kidney cells were grown in DMEM supplemented
with 10% heat-inactivated horse serum in 10% CO2 at
37 °C. The cells were transiently transfected with varying amounts
of DNA using the calcium phosphate precipitate method as described
previously (32).
Particulate Preparation--
Transfected HEK293 cells were
washed twice with ice-cold phosphate-buffered saline and then pelleted
by centrifugation at 2000 × g for 10 min. The cells
were then resuspended in a buffer containing 25 mM
2-{[2(hydroxymethyl)ethyl]amino}ethanesulfonic acid, pH 6.8, 0.5 mM EDTA, 0.2 mM MgCl2,
and 1 mM 1,10-phenanthroline and homogenized using an
T25 Ultra-Turrax tissue homogenizer (IKA-Works, Wilmington, NC) at
20,500 rpm for 10 s. Membranes were isolated by centrifugation at
45,000 × g for 30 min at 4 °C and washed 1-3 times
in the above buffer depending on the experiment. The pellets were then
resuspended in the same buffer supplemented with 0.1% bovine serum
albumin and 0.014% bacitracin (binding buffer) and used immediately.
The preparation was washed one time in the experiments determining the
pharmacological profiles of agonist and antagonist binding to the B1
and B2 receptors (Figs. 1 and 4A and Table I). In all other
binding experiments, the preparation was washed three times. Increasing
the number of washes from one to three resulted in an ~10-fold
increase in the value of the binding constants, Kd
and Ki, for des-Arg10-KD on the various
B1 receptor constructs.
Radioligand Binding--
Radioligand binding assays were
performed essentially as described previously (31, 33). Receptor
density was determined on intact HEK293 cells by incubating cells in
Leibovitz's L-15 medium, pH 7.4, 0.1% bovine serum albumin including
the protease inhibitors bacitracin (140 µg/ml) and
1,10-phenanthroline (1 mM) and a saturating concentration
of [3H]des-Arg10-KD or
[3H]NPC17731 (3-5 nM) at 4 °C for 60-90
min. Competition binding was done on particulate preparations by
incubating the preparations in binding buffer including ~0.2-0.5
nM [3H]des-Arg10-KD or
[3H]des-Arg10-[Leu9]KD with and
without various concentrations of competitor at 25 °C for 60-90 min.
Receptor Activity--
Activities of various receptor constructs
were assayed by monitoring PI hydrolysis in HEK293 cells (31). PI
hydrolysis was assayed in cells pre-labeled with 1 µCi/ml
myo-[3H]inositol in DMEM containing 5%
heat-inactivated horse serum. After washing, the cells were incubated
in Leibovitz's L-15 medium containing 5 mM LiCl in the
absence and presence of an agonist or antagonist for 30 min at
37 °C. When agonists and antagonists were coincubated, the
antagonist was added 5 min before the addition of the agonist and then
further incubated for 30 min.
Immunoprecipitation and Immunoblotting--
HEK293 cells
transfected with FLAG epitope-tagged receptors were subjected to
immunoprecipitation and immunoblotting essentially as previously
described (34). In short, cells were solubilized in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM sodium
phosphate, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 µg/ml
leupeptin, 0.1 mM phenylmethylsulfonyl fluoride) for 30 min
at 4 °C. The lysate was centrifuged at 13,000 × g
for 15 min at 4 °C. The supernatant (1 ml) was then incubated 12-18 h with anti-FLAG M2 antibody (1:200) followed by incubation with protein A-Sepharose beads precoupled to rabbit anti-mouse IgG for an
additional 2 h at 4 °C. The beads were then washed with 2 × 1 ml of lysis buffer and then with 1 ml of 10 mM
Tris-HCl, pH 7.4. The pellet was heated in SDS-polyacrylamide gel
electrophoresis buffer containing 6%
-mercaptoethanol for 5 min at
100 °C and then electrophoresed on 12% polyacrylamide gels. The gel
was then electroblotted onto 0.45-µm nitrocellulose membranes and
stained with anti-FLAG M2 antibody (1:1000). Immunoreactive bands were visualized with an immunodetection kit using peroxidase-labeled sheep
anti-mouse antibody according to the procedure described by the
supplier (PerkinElmer Life Sciences).
Data Analysis--
Where indicated, data are presented as the
mean ± S.E. and were compared using the Student's t
test. The receptor binding constants, Kd and
Ki, for various ligands were calculated by the
Radlig program (Biosoft, Ferguson, MO) using data from radioligand
binding experiments.
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RESULTS |
Agonist Profile of the Human B1 Receptor Expressed in HEK293
Cells--
The human WT B1 receptor transiently expressed in HEK293
cells exhibited a typical agonist profile as determined by both
receptor binding and receptor-mediated PI hydrolysis. Fig.
1, A and B, shows
that des-Arg10-KD, a B1 receptor-selective agonist, bound
to the B1 receptor with an affinity (Kd = 0.098 nM) and stimulated PI hydrolysis with a potency
(EC50 = 1.2 nM) which were ~4 and 3 orders of
magnitude, respectively, higher than those of BK (Kd = 2880 nM, EC50 = 630 nM), a B2
receptor-selective agonist. This profile was clearly different from
that of the human WT B2 receptor subtype transiently expressed in
HEK293 cells to which BK bound with an affinity (Kd = 0.190 nM) and stimulated PI hydrolysis with a potency
(EC50 = 2.0 nM) which were >4 orders of
magnitude higher than those of des-Arg10-KD
(Kd >5000 nM, EC50 >5000
nM).

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Fig. 1.
Pharmacological profiles of the human B1 and
B2 receptors. A, particulate preparations of HEK293
cells transfected with WT B1 (open symbols) or WT B2
(closed symbols) receptors were washed one time, incubated
in the absence and presence of increasing concentrations of
des-Arg10-KD ( , ) and BK ( , ) as indicated, and
assayed for radioligand binding with Kd
concentrations of [3H]des-Arg10-KD (WT B1) or
[3H]BK (WT B2). B, intact cells transfected
with WT B1 or WT B2 receptors were incubated with increasing
concentrations of des-Arg10-KD and BK as indicated in
A and assayed for PI hydrolysis. B, the results
are presented as percentage of maximum, where 100% maximum is the
maximum response of the B1 and B2 receptors to des-Arg10-KD
and BK, respectively. The results are averages ± S.E. of three
independent experiments with each point assayed in duplicate.
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Ligand-independent and Agonist-dependent Activities of
the Human B1 Receptor--
Ligand-independent, constitutive B1
receptor activity was monitored by assaying for basal cellular PI
hydrolysis as a function of the density of the receptors in transiently
transfected HEK293 cells. Fig. 2 shows a
representative experiment in which cells were transfected with
increasing amounts of cDNA (0.5-6 µg) of a FLAG-tagged human B1
receptor (B1R-FLAG) and then assayed in parallel for receptor
expression by specific [3H]des-Arg10-KD
binding and anti-FLAG immunoblotting and for ligand-independent activity by basal PI hydrolysis. PI hydrolysis increased with increasing amounts of des-Arg10-KD binding and the B1
receptor-specific 35-kDa peptide. Fig. 3A shows that the slope factor
of the increase in PI hydrolysis as a function of the human WT B1
receptor, which may be considered as an index of ligand-independent
receptor activity and which we previously termed the index of basal
activity, or IB (31), was 0.58. By comparison, the human B1
receptor activity was considerably higher than that of the human B2
receptor, which had an IB of 0.03 (31). These results show
that the B1 receptor is constitutively active, and this activity is
evident at receptor densities that would be expected to occur in
vivo. Similar results were obtained with the rabbit WT B1 receptor
indicating that the constitutive activity was not a peculiarity of the
human receptor (data not shown).

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Fig. 2.
Ligand-independent PI hydrolysis by the human
B1 receptor. HEK293 cells were transfected with increasing amounts
of B1R-FLAG DNA as indicated. Parallel sets of cells were then assayed
for basal cellular PI hydrolysis (open bars), specific
[3H]des-Arg10-KD binding using a saturating
concentration of radioligand (filled bars), and
anti-B1R-FLAG immunoreactivity (top). The position of the
receptor in the Western blot is indicated (right side
arrow). The molecular weights of the IgG heavy and light chains
are indicated (left side arrows). The results are from a
representative experiment performed three times.
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Fig. 3.
Ligand-independent and
agonist-dependent PI hydrolysis by the human B1 and B2
receptors. HEK293 cells transfected with varying amounts of the WT
B1 ( , ) or WT B2 ( , ) receptors were incubated in the
absence (A and B, closed symbols) and
presence (B, open symbols) of 1 µM
des-Arg10-KD and 1 µM BK, respectively, and
then assayed for PI hydrolysis and radioligand binding using a
saturating concentration of radioligand. B, the effects of
des-Arg10-KD and BK on the B1 and B2 receptor,
respectively, are shown (dashed arrows). Note the difference
in the y axis scale in A and B. The
results are from 4 to 8 independent experiments with each point
performed in duplicate.
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Addition of the agonist des-Arg10-KD at 1 µM,
a concentration sufficient to activate maximally the human B1 receptor,
to B1 receptor-expressing cells yielded a slope factor of PI hydrolysis of 16.0. This factor, which we previously termed the index of agonist-promoted receptor activity, or IA (31), represented a 27-fold increase over the IB for the B1 receptor (Fig.
3B). By comparison, a similar increase (23-fold) was
observed following addition of 1 µM BK to the B2
receptor-expressing cells (IA = 0.86) (31).
The Effect of Antagonists on Ligand-independent and
Agonist-dependent Activities of the Human B1
Receptor--
GPCR antagonists range in their actions from partial
agonism to neutral antagonism to inverse agonism. Inverse agonism
requires ligand-independent activity and is thought to involve the
stabilization of the inactive receptor state. Several B1 receptor
peptide antagonists have been developed (Table
I). These peptides bound to the human B1
receptor expressed in HEK293 cells with an order of potency that was
NPC18565 > B9858 ~ des-Arg10[Leu9]KD ~ R715 ~ des-Arg10HOE140 > des-Arg9[Leu8]BK (Fig.
4A and Table I). As expected,
all the peptides, at 1 µM (except
des-Arg9[Leu8]BK which was at 10 µM), antagonized B1 receptor-mediated stimulation of PI
hydrolysis in response to 0.1 µM des-Arg9-BK
in these cells (Fig. 4B, left panel). On the
other hand, none of the peptides significantly inhibited the
constitutive B1 receptor activity, even though
des-Arg10-[Leu9]KD,
des-Arg9[Leu8]BK, and NPC18565 appeared to
have some effect (Fig. 4B, right panel). Thus,
these peptides are neutral antagonists or, at most, very weakly
efficacious inverse agonists on the human B1 receptor in HEK293
cells.

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Fig. 4.
Effects of antagonists on ligand-independent
and agonist-dependent PI hydrolysis of the human B1
receptor. A, particulate preparations of HEK293 cells
transfected with WT B1 receptors were washed one time, incubated in the
absence and presence of increasing concentrations of various peptide
antagonists as indicated, and assayed for competition radioligand
binding using a Kd concentration of
[3H]des-Arg10-KD. The results are
averages ± S.E. of three independent experiments with each point
assayed in duplicate. Binding constants (Ki)
calculated from these experiments are summarized in Table I.
B, HEK293 cells were transfected with WT B1 receptors and
incubated with and without 1 µM
des-Arg10-[Leu9]KD (DALKD), 10 µM des-Arg9-[Leu8]BK (DALBK), 1 µM NPC18565, 1 µM
des-Arg10-HOE140 (DAHOE140), 1 µM R715, and 1 µM B9858 in the presence (left panel) and
absence (right panel) of 0.1 µM
des-Arg9-BK (DBK) as indicated and described
under "Experimental Procedures." The cells were then assayed for PI
hydrolysis. The results are presented as percentage of control where
100% control is the PI hydrolysis in mock-transfected cells and was
5264 ± 390 dpm (n = 11). The density of the
receptors was ~15 fmol/106 cells. The results are
averages ± S.E. of at least six independent determinations with
each point assayed in duplicate. **, p < 0.01 compared
with des-Arg9-BK (left panel).
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The Role of Asn121 in TM-III in Ligand-independent and
Agonist-dependent Human B1 Receptor Activity--
Fig.
5A shows the position of
Asn121 in TM-III of the human B1 receptor. This residue is
conserved in some GPCR, and mutation of residues at this position in
several GPCR results in the constitutive activation of the receptors
(22-26). These observations have led to the belief that these residues
participate in an interhelical contact with residues in other
transmembrane domains that restrains the receptor in an inactive
conformational state. One possibility is that the alignment of this
residue is different in the B1 receptor resulting in a weaker contact,
i.e. mutation of Asn121 does not lead to an
increase in constitutive activity. Fig. 5B shows that
B1A121 had a slope factor of PI hydrolysis that was 9-fold
higher than that of the WT B1 receptor. Thus, despite the constitutive
activity of the WT B1 receptor, Asn121 remains involved in
conformational selection in this receptor.

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Fig. 5.
Ligand-independent and
agonist-dependent PI hydrolysis and agonist binding by the
human B1 receptor and B1A121. A, the amino
acid sequence of TM-III of the human B1 receptor is shown. Indicated is
Asn121 (filled circle). B, HEK293
cells transfected with varying amounts of the WT B1 receptors ( ) or
B1A121 ( , ) were incubated in the absence
(closed symbols) and presence (open symbols) of 1 µM des-Arg10-KD as indicated and then assayed
for PI hydrolysis and radioligand binding using a saturating
concentration of radioligand. The effect of des-Arg10-KD is
indicated (dashed arrow). The results are from 4 to 8 experiments with each point performed in duplicate. C,
particulate preparations of cells transfected with WT B1 receptors
( , ) or B1A121 ( , ) were washed three times,
incubated in the absence and presence of increasing concentrations of
des-Arg10-KD ( , ) or
des-Arg10-[Leu9]KD ( , ) as indicated,
and assayed for saturation binding using a Kd
concentration of [3H]des-Arg10-KD and
[3H]des-Arg10-[Leu9]KD,
respectively. The effect of the Asn121 Ala mutation on
des-Arg10-KD binding is indicated (dashed
arrow). The results are averages ± S.E. of three independent
experiments with each point assayed in duplicate. Binding constants
(Kd) calculated from these experiments are
summarized in Table II. D, particulate preparations of cells
transfected with WT B1 receptors ( , , , ) or
B1A121 ( , ) were washed three times, incubated in the
absence and presence of increasing concentrations of
des-Arg10-KD ( , , , ) and
des-Arg10[Leu9]KD ( , ), with and
without 100 µM Gpp(NH)p as indicated, and assayed for
competition binding using Kd concentrations of
[3H]des-Arg10-[Leu9]KD and
[3H]des-Arg10-KD, respectively. The effects
of Gpp(NH)p on des-Arg10-KD binding to WT B1 receptors and
B1A121 are indicated (dashed arrow). The results
are averages ± S.E. of three independent experiments with each
point assayed in duplicate. Binding constants (Ki)
calculated from these experiments are summarized in Table II.
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To determine whether the preferred conformation of B1A121
is related to that normally favored by the agonist
des-Arg10-KD, we studied various parameters of
des-Arg10-KD interaction with the receptor.
Des-Arg10-KD stimulation elevated the slope factor of PI
hydrolysis for this mutant only 3-fold compared with 27-fold for the WT
receptor (Fig. 5B). Furthermore, the Kd
value of des-Arg10-KD binding and the Ki
value of des-Arg10-KD to compete with the binding of the
antagonist des-Arg10-[Leu9]KD to this
construct were 3.3- and 5.5-fold, respectively, lower than for the WT
receptor (Fig. 5, C and D and Table
II). In contrast, the
Kd of the antagonist
des-Arg10-[Leu9]KD was not significantly
perturbed by this mutation (Fig. 5C).
GTP sensitivity of agonist binding signifies the ability of GPCR to
isomerize between conformational states of different degrees of
activity and G protein coupling. Inclusion of 100 µM
Gpp(NH)p caused an increase (2.2-fold) in the Ki
value of des-Arg10-KD to compete with the binding of
des-Arg10-[Leu9]KD binding to the WT B1
receptor (Fig. 5D and Table II). This classical result
indicates that the agonist prefers to bind to and favor the formation
of the G protein-coupled state of the WT B1 receptor and that it is
capable of isomerizing between G protein-coupled and -uncoupled states.
Gpp(NH)p had no effect on the Ki value of
des-Arg10-[Leu9]KD to compete with the
binding of the des-Arg10-KD to the WT receptor (Fig. 5 and
Table II), an observation compatible with the behavior of
des-Arg10-[Leu9]KD as a neutral antagonist,
i.e. it is unable to distinguish between G protein-coupled
and -uncoupled receptor states. In terms of B1A121,
inclusion of 100 µM Gpp(NH)p caused an increase
(2.1-fold) in the Ki value of
des-Arg10-KD to compete with the binding of
des-Arg10-[Leu9]KD binding (Fig.
5D and Table II). The increase was not significant indicating that it is still unclear whether or not this mutant is
readily capable of isomerizing between conformational states.
Taken together, the above results imply that mutation of
Asn121 in the B1 receptor favors an activated conformation
of the receptor. The decrease in des-Arg10-KD
responsiveness of and increase in des-Arg10-KD affinity for
B1A121 strongly suggest that the favored conformation is
the one preferred by des-Arg10-KD. Given that the B1
receptor was sensitive to mutation of Asn121, this residue
is most likely aligned properly in this receptor. Consequently, other
reasons may be responsible for the elevated ligand-independent activity
of this receptor.
The Role of IC-IV in Ligand-independent and
Agonist-dependent Human B1 Receptor Activity--
The
human B2 receptor contains a cluster of serines and threonines (Ser/Thr
cluster), including Thr342, Thr345,
Ser346, and Ser348 in IC-IV, that is conserved
among B2 receptors from four different species (Fig.
6), and this cluster is important for
regulating agonist-promoted receptor activity by receptor
phosphorylation and internalization (32) and desensitization (31).
Alanine mutation of this cluster significantly increased
ligand-independent B2 receptor activity leading to the thought that it
is also important for regulating constitutive receptor activity (31).
The human B1 receptor contains several serines and threonines in IC-IV
including Thr321, Thr331, Ser339,
Ser340, and Ser341 that are conserved in the
rabbit receptor (Fig. 6). On the other hand, the rat and mouse B1
receptors, being severely truncated in their IC-IV, only contain a
threonine corresponding to Thr321. Furthermore, the human
B1 receptor does not appear to serve as a substrate for phosphorylation
(35). Thus, another reason for the high constitutive activity of the B1
receptor could be that this receptor lacks critical serines and
threonines in IC-IV that regulate such activity. To test this
possibility, we first substituted IC-IV of the B2 receptor, which
contains the Ser/Thr cluster, in the B1 receptor to make B1(B2ICIV). As
shown in Fig. 7A, this
substitution dramatically suppressed the constitutive B1 receptor
activity. The agonist-dependent activity was also suppressed (Fig. 7B). This substitution also inhibited the
enhanced constitutive activity and agonist-dependent
activity of B1A121 (Fig. 7, C and D).
Subsequent alanine mutation of the Ser/Thr clusters in both B1(B2ICIV),
to make B1(B2ICIVASer/Thr), and B1(B2ICIV)A121,
to make B1(B2ICIVASer/Thr)A121, significantly
restored the receptor activities, including both the ligand-independent
(Fig. 7, A and C) and
agonist-dependent activities (Fig. 7, B and
D). Thus, the Ser/Thr cluster in B2 IC-IV represents one
epitope that negatively regulates constitutive receptor activity.
Furthermore, this regulation appears to occur independently of the
activation state of the receptor since it was observed both with and
without the Asn121
Ala activating mutation.

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Fig. 6.
Amino acid sequence alignment of IC-IV of B2
and B1 receptors from different species. Indicated is the
predicted junction of the seventh transmembrane domain and IC-IV
(vertical line), species conservation of residues in each
receptor subtype (bold), species conservation of serines and
threonines in each receptor receptor subtype (*), and the cluster of
serines and threonines conserved among B2 receptors from different
species that were mutated into alanines to create the receptor
constructs B1(B2ICIVASer/Thr) and
B1(B2ICIVASer/Thr)A121 (Ser/Thr
cluster). The numbering of residues in the B2 and B1 receptors is
according to Hess et al. (1) and Menke et al.
(2).
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Fig. 7.
Ligand-independent PI hydrolysis by the human
B1/B2 receptor IC-IV chimeras. A and B,
HEK293 cells transfected with varying amounts of the WT B1 ( , ),
B1(B2ICIV) ( , ), or B1(B2ICIVASer/Thr) ( , )
were incubated in the absence (A and B,
closed symbols) and presence (B, open
symbols) of 1 µM des-Arg10-KD as
indicated and then assayed for PI hydrolysis and radioligand binding
using a saturating concentration of radioligand. C and
D, cells transfected with varying amounts of
B1A121 ( , ), B1(B2ICIV)A121 ( ,
), or B1(B2ICIVASer/Thr)A121 ( , ) were
incubated in the absence (C and D, closed
symbols) and presence (D, open symbols) of 1 µM des-Arg10-KD as indicated and then assayed
for PI hydrolysis and radioligand binding using a saturating
concentration of radioligand. B and D, the
effects of des-Arg10-KD are shown (dashed
arrows). Note the difference in the y axis scales. The
results are from 3 to 8 independent experiments with each point
performed in duplicate.
|
|
 |
DISCUSSION |
In this report, we show that the human B1 BK receptor subtype
exhibits high ligand-independent, constitutive activity. Despite the
constitutive activity of the WT receptor, alanine mutation of
Asn121 in TM-III results in a further increase in receptor
activity. On the other hand, substitution with IC-IV of the B2
receptor, which exhibits minimal ligand-independent activity, strongly
suppresses constitutive activity of the B1 receptor as well as the
mutation-enhanced activity of B1A121. The suppressive
effect of B2 IC-IV is partially due to the presence of serines and
threonines that are important for B2 receptor desensitization, but
other epitopes also seem to be involved. These observations show that
constitutive activity depends on epitopes in both transmembrane and
intracellular domains. We propose that the high constitutive B1
receptor activity is due primarily to the lack of epitopes in IC-IV
that regulate such activity.
Constitutive receptor activity provides a rationale for restricting the
expression of the B1 receptor primarily to conditions of inflammation.
Indeed, the IB for the B1 receptor is almost as high as the
IA for the B2 receptor. It is unlikely that this activity
completely substitutes for agonist stimulation of the receptor. In
fact, the ability to promote the formation of a receptor state with
features of a des-Arg10-KD-preferred state by the
Asn121
Ala mutation argues that this agonist has a role
in B1 receptor function. However, since the formation of B1 agonists is
relatively inefficient (17), constitutive receptor activity may serve a role prior to the formation of a sufficient amount of agonist or,
similarly, in anatomical areas where receptors are required but
insufficient amounts of agonists are formed. In addition to sustaining
the receptor signal, constitutive receptor activity may synergize with
interleukin-1
to induce rapidly the receptor, as described
previously for receptor agonists (8).
The models explaining ligand-independent, constitutive GPCR activity
assert that these receptors spontaneously isomerize between inactive
and activated conformational states termed R and R*, respectively, and
that R* associates with a G protein to form R*G that triggers the
intracellular signal (36, 37). In these models, agonists act by
preferentially binding to and stabilizing R*. Even though the basic
features of these models prevail, there is emerging evidence that the
above equilibrium is not limited to two receptor states but harbors
multiple microscopic equilibria with a number of partially/fully
activated states. The isomerization constant J that governs
the spontaneous formation of the activated state in these models is an
important determinant of the level of constitutive activity of a GPCR.
The transmembrane location of several natural and unnatural
constitutively activating mutations suggests that this process involves
the breaking of interhelical contacts necessary to constrain the
receptor in an inactive state (22). Residues at a position in TM-III
occupied by an asparagine in the AT1-angiotensin II
receptor (Asn111) and the B2 BK receptor
(Asn113) have been proposed to participate in such a
contact since their mutation yields an increase in ligand-independent
activity (23, 24). This residue is conserved in the B1 receptor
(Asn121). Alanine mutation of this residue led to a 9-fold
increase in the ligand-independent receptor activity on PI hydrolysis.
This mutation also caused the fold stimulation of receptor activity by
the agonist des-Arg10-KD to decrease from 27- to 3-fold,
the Kd value of des-Arg10-KD to decrease
3.3-fold, and the Ki value of
des-Arg10-KD for competing with the binding of the
antagonist des-Arg10-[Leu9]KD to decrease
5.5-fold. The lack of an effect of the mutation on the
Kd value of the antagonist
des-Arg10-[Leu9]KD indicates that this is an
agonist-specific effect. The resemblance of this mutant to a
des-Arg10-KD-preferred conformational state argues that
des-Arg10-KD is a bona fide B1 receptor agonist
and that the receptor does not solely rely on constitutive activity for
function. The sensitivity of the B1 receptor to the mutation of this
residue argues that the normal elevated activity of the receptor is not
due to the misalignment of Asn121.
IC-IV represents another domain that may be responsible for regulating
constitutive receptor activity. Epitopes that interact with effectors
involved in both signaling (e.g. G protein) and desensitization (e.g. GPCR kinase and arrestin) are
generally present in this domain, and both have been proposed to
influence ligand-independent GPCR activity. Thus, receptor activity
should be sensitive to the presence and absence of receptor epitopes through which such effectors interact. Indeed, we recently used the B2
receptor to provide evidence for a role of IC-IV in regulation of
ligand-independent receptor activity (31). This receptor exhibits
minimal ligand-independent activity. However, a significant rise in
activity was observed following alanine mutation of a cluster of
serines and threonines in IC-IV of this receptor. This cluster is a
substrate for agonist-promoted receptor phosphorylation (32) and
important for B2 receptor desensitization (31). Thus, the Ser/Thr
cluster exerts a suppressive effect on the ligand-independent activity
of this receptor. The presence of other suppressive epitopes in IC-IV
of this receptor was not readily obvious since the maximal level of
ligand-independent B2 receptor activity is unknown.
The human B1 receptor contains several serines and threonines in
IC-IV, but none of them are conserved in B1 receptors from different
species. In fact, the B1 receptor is apparently not phosphorylated
(35), and it is subject to very limited desensitization (16) and
internalization (38). The dramatic inhibition by B2 receptor IC-IV on
the constitutive activity of the WT B1 receptor and B1A121
reiterates the suppressive effect of this domain. Furthermore, this
effect provides us with a crude sense of the upper and lower limits of
activity of this receptor. Subsequent mutation of the Ser/Thr cluster
in this chimera partially restored the constitutive activity. This
gain-of-function mutation corroborates that serines and threonines are
important epitopes for regulating constitutive activity. However,
equally important is that these sequential loss- and gain-of-function
mutations reveal the existence of an additional, currently unknown,
epitope(s) in B2 receptor IC-IV that regulate ligand-independent
receptor activity. Furthermore, this regulation occurs independently of
the state of receptor activation since it was observed with the
constitutive activity of both the WT B1 receptor and
B1A121. Taken together, we conclude that the high
constitutive activity of the B1 receptor is at least in part a
consequence of the lack of epitopes in IC-IV that regulate such activity.
Although our understanding of the precise physiological and
pathophysiological role of the B1 receptor is still relatively sparse,
past studies have indicated that this receptor is generally silent or
absent in healthy tissues but is induced in response to pathological
insults during which it mediates cardiovascular and nociceptive
responses to the insult (7). The recent study of mice lacking the B1
receptor gene has confirmed past conclusions as well as shed new light
on the significance of this receptor (14). These mice are healthy,
fertile, and normotensive. On the other hand, endotoxin-induced
hypotension and accumulation of polymorphonuclear leukocytes in
inflamed tissue is dramatically attenuated. B1 receptors apparently are
not involved in the noxious heat sensitivity of isolated nociceptors
but instead facilitate a more complex nociceptive reflex. Further
evidence for the pathophysiological role of the B1 receptor has come
from the identification of two allelic polymorphisms of this receptor
(39, 40). One of these polymorphisms involves a G
C
substitution in a positive control region of the promoter, and the
C allele yields an increase in the activity of the B1 receptor
promoter. Interestingly, the C allele is significantly less
prevalent in individuals with end-stage renal failure and inflammatory
bowel disease.
The interest in using the B1 receptor as a target for novel
anti-inflammatory agents has grown with the understanding of the function of this receptor in inflammation and nociception (41). A
number of B1 antagonists are available, even though all of them are
peptides and structurally highly related. Several of these antagonists
exhibit remarkably high affinities and selectivities for the B1
receptor. The identification of constitutive B1 receptor activity
prompted the necessity to investigate inverse agonism in the
interaction of these antagonists with this receptor. In receptor
models, this pharmacological parameter is defined as the ability of a
ligand to bind preferentially to and stabilize the inactive state of
the receptor and, consequently, inhibit not only
agonist-dependent activity but also ligand-independent activity of the receptor. This action is in contrast to neutral antagonists, which are nonselective for various receptor states and,
consequently, only inhibit agonist-dependent activity. None of the antagonists significantly inhibited the constitutive B1 receptor
activity even though des-Arg9-[Leu8]BK,
des-Arg10-[Leu9]KD, and NPC18565 seem to
elicit a small effect. Thus, available B1 receptor antagonists are
either neutral or very weakly efficacious inverse agonists. It is
appropriate to briefly discuss the effect of NPC18565 and
des-Arg10-HOE140 since these antagonists were derived from
the high affinity and selective B2 receptor antagonists NPC17731 and
HOE140, respectively, by simply truncating the C-terminal arginine. The
parent B2 antagonists act as highly efficacious inverse agonists on the
B2 receptor (23, 31, 42). In addition, Gpp(NH)p increases the potency of these ligands to compete for [3H]BK binding indicating
that they stabilize the G protein-uncoupled state of the B2 receptor.
The lack of significant inverse agonism of
des-Arg10-[Leu9]KD was reinforced by the fact
that the binding of this antagonist was not perturbed by Gpp(NH)p.
Considering the constitutive activity of the B1 receptor described in
this report, it is clear that inverse agonism has to be considered
seriously in the search for effective anti-inflammatory agents acting
through the B1 receptor. Furthermore, inverse agonists will be key to
the identification of constitutive activity of native B1 receptors
in vitro and in vivo.
 |
ACKNOWLEDGEMENTS |
We thank D. J. Kyle for donating
NPC17731 and NPC18565, D. Regoli for donating R715, and F. Marceau for
donating B9858.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM41659.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
the University of Texas Health Science Center, Mail Code 7760, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-3766; Fax:
210-567-6595; E-mail: lundberg@biochem.uthscsa.edu.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M007396200
 |
ABBREVIATIONS |
The abbreviations used are:
BK, bradykinin;
GPCR, G protein-coupled receptor;
KD, kallidin;
PI, phosphoinositide;
WT, wild-type;
IC-IV, fourth intracellular domain;
TM-III, third
transmembrane domain;
DMEM, Dulbecco's modified Eagle's medium;
IA, index of agonist-promoted receptor activity;
IB, index of basal receptor activity.
 |
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