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-LundbergDagger, 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1beta (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 alpha 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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% beta -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (black-triangle, triangle ) and BK (, open circle ) 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.

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 (black-triangle, triangle ) or WT B2 (, open circle ) 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.

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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Table I
Amino acid sequences of B1 receptor peptide antagonists and their binding constants on WT B1 receptors


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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).

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 (black-triangle) or B1A121 (black-down-triangle , down-triangle) 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 (triangle , black-triangle) or B1A121 (down-triangle, black-down-triangle ) were washed three times, incubated in the absence and presence of increasing concentrations of des-Arg10-KD (triangle , down-triangle) or des-Arg10-[Leu9]KD (black-triangle, black-down-triangle ) 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 right-arrow 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 (triangle , black-triangle, open circle , ) or B1A121 (down-triangle, black-down-triangle ) were washed three times, incubated in the absence and presence of increasing concentrations of des-Arg10-KD (triangle , black-triangle, down-triangle, black-down-triangle ) and des-Arg10[Leu9]KD (open circle , ), 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.

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).

                              
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Table II
Binding constants of B1 receptor peptide agonists and antagonists on WT B1 receptors and B1A121a

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 right-arrow 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 (black-triangle, triangle ), B1(B2ICIV) (, open circle ), or B1(B2ICIVASer/Thr) (black-square, ) 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 (black-down-triangle , down-triangle), B1(B2ICIV)A121 (black-diamond , diamond ), or B1(B2ICIVASer/Thr)A121 (star , ) 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 right-arrow 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-1beta 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 right-arrow 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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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