(Received for publication, September 23, 1996, and in revised form, January 23, 1997)
From the Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224-6801 and § Scios Inc., Mountain View, California 94043
To delineate ligand binding and functional
characteristics of the human B1 kinin receptor, a
stable clone of Chinese hamster ovary cells expressing a single class
of binding sites for
[3H]des-Arg10-lysylbradykinin with a
Kd of 0.3 nM and a
Bmax of 38 fmol/mg protein (~40,000
receptors/cell) was isolated. Studies with peptide analogs showed that
a lysine residue at position 1 (based on the lysylbradykinin sequence)
of ligands was essential for high affinity binding to the human
B1 receptor. In marked contrast to cloned Chinese hamster
ovary cells expressing the human kinin B2 receptor, which
internalized approximately 80% of the ligand within 5 min upon
exposure to 2 nM [3H]bradykinin, exposure of
cells expressing the B1 receptor to 1 nM
[3H]des-Arg10-lysylbradykinin resulted in
minimal ligand internalization. Stimulation of the B1
receptor led to inositol phosphate generation and transient increases
in intracellular calcium, confirming coupling to phospholipase C, while
immunoprecipitation of photoaffinity-labeled G-proteins from membranes
indicated specific coupling of the receptor to Gq/11 and
G
i1,2. The B1, unlike the B2,
receptor does not desensitize (as demonstrated by continuous
phosphoinositide hydrolysis), enhancing the potential role of this
receptor during inflammatory events.
Bradykinin (BK)1 and lysylbradykinin (LBK) are potent vasoactive peptides that have been implicated as mediators of inflammation, pain, and hyperalgesia (1, 2). Two subtypes of kinin receptors, designated B1 and B2 based upon their pharmacological properties, were described in animal tissues (3). BK and LBK are equipotent agonists at the B2 receptor, but BK is ineffective at the B1 receptor. The carboxypeptidase metabolites of BK and LBK, des-Arg9-BK and des-Arg10-LBK, are the prototypical B1 receptor agonists (4). The existence of B1 and B2 receptors was confirmed using selective antagonists (3, 5, 6) and by cloning studies (7-9).
Kinin B2 receptors are constitutively expressed on many cell types and are responsible for the majority of the observed effects of kinins (10), but B1 receptor expression requires induction. Thus, des-Arg9-BK alters smooth muscle tone only after incubating tissues for several hours in vitro (3-5). Exposure in vitro to proinflammatory cytokines also induces B1 receptor-mediated responses via a mechanism that requires protein synthesis (11, 12). Similarly, de novo expression of B1 receptors in animals in vivo occurs upon exposure to noxious stimuli, including bacterial lipopolysaccharide (13) and ultraviolet light (14), or to proinflammatory cytokines (15, 16).
The ability of injurious and proinflammatory stimuli to induce B1 receptors in animal models implies that this receptor may play a role in the actions of kinins in chronic inflammatory conditions (4, 17). Support for this concept comes from studies showing that B1 receptor antagonists have antinociceptive effects in rodent models of persistent hyperalgesia (2, 18, 19).
Despite the potential importance of B1 kinin receptors in chronic inflammation, little is known regarding either the distribution and importance of this receptor system or the signaling systems to which the receptor is coupled in humans. Although two human embryonic lung fibroblast cell lines, IMR90 and WI38, express small numbers of B1 receptors (20, 21), they are poor models to characterize B1 receptor properties because they also express much larger numbers of B2 receptors (9). The recent cloning of the human B1 receptor (9), therefore, provided the first opportunity to characterize this receptor. Binding studies, using transient expression in COS-7 cells, demonstrated species variation in B1 receptor ligand affinities. In contrast to the rabbit receptor, the affinity of des-Arg9-BK for the human B1 receptor was more than 1000-fold lower than that of des-Arg10-LBK (9), indicating that des-Arg9-BK is an ineffective agonist for the human receptor. This species variation emphasized the need to further define the properties of the human receptor to delineate its potential role in inflammatory disorders. To date, however, functional studies of the human B1 receptor have been limited to studies in Xenopus laevis oocytes, showing that stimulation induced aequorin-mediated luminescence (9).
The aim of the present studies was to delineate ligand binding and functional characteristics of the human B1 kinin receptor. To achieve this, we isolated a clone of Chinese hamster ovary (CHO) cells expressing the receptor in a stable manner, permitting binding and functional properties to be studied in the same cell type. Given the reported difference in affinities of des-Arg9-BK and des-Arg10-LBK for the human B1 receptor (9), we tested the hypothesis that the presence of a lysine residue at position 1 of ligands is essential for high affinity binding. We also sought to establish whether the human B1 receptor, like the B2 receptor, is coupled to phospholipase C, and, if so, which G-proteins are responsible for this coupling. Finally, we determined if human B1 receptor-mediated actions are regulated by rapid ligand-induced desensitization or internalization.
Materials
The following materials were purchased: minimum essential
medium-, Opti-MEM, trypsin, lipofectamine, and fetal calf serum from
Life Technologies, Inc.; penicillin/streptomycin from Biofluids Inc;
cell culture flasks and dishes from Costar Corp; G418 sulfate, ionomycin, bacitracin, captopril, PIPES, human serum albumin, MES, and
1,4-dioxane from Sigma; polyclonal rabbit peptide antisera specific for
G
s (RM/1, NEI-805), G
q/11 (QL, NEI-809), G
o/i3 (GC/2, NEI-804), and G
i1,2 (AS/7, NEI-801),
[3,4-prolyl-3,4-3H]des-Arg10-LBK (89 Ci/mmol), and [
-32P]GTP (3000 Ci/mmol) from DuPont
NEN; 1-(3-dimethylaminopropyl)-3-ethylenecarbodiimide and
4-azidoaniline hydrochloride from Aldrich;
myo-[2-3H]inositol (20 Ci/mmol) from ICN
Pharmaceuticals Inc.; Fura-2AM from Molecular Probes, Inc.;
des-Arg10-LBK, des-Arg10-Leu9-LBK,
BK, des-Arg9-Leu8-BK, des-Arg9-BK,
des-Arg10-HOE 140, and LBK from Bachem; and AG 1-X8 resin,
formate form, 100-200 mesh from Bio-Rad. All other chemicals were
obtained from Sigma.
The cDNA for the human kinin B1 receptor was generously provided by Dr. J. Fred Hess (Merck). Hoe 140 was a kind gift of Drs. Bernward Schölkens and Klaus Wirth (Hoechst AG).
Peptide Synthesis
The following peptides were synthesized: D-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; Lys-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; D-Lys-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe; Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro; Lys-Arg-Pro-Pro-Gly-Phe-Ser; D-Arg0-Hyp3-D-Hype(trans-3-phenylpropyl)7-Oic8-BK-(0-8); D-Arg0-Hyp3-D-Hype (cis-3-phenylpropyl)7-Oic8-BK-(0-8); Hyp3-D-Hype(trans-3-phenylpropyl)7-Oic8-BK-(1-8); Hyp3-D-Hype (cis-3-phenylpropyl)7-Oic8-BK-(1-8).
All peptides were synthesized on an automated synthesizer using t-butyloxycarbonyl chemistry, according to previously published procedures (22). Boc-Oic-OH and Boc-D-4-hydroxyproline ethers were prepared as described previously (23, 24). The purity of peptides was confirmed using analytical high pressure liquid chromatography and electrospray ionization mass spectroscopy.
Expression and Cloning of Human Kinin Receptors
A cDNA for the human kinin B2 receptor was isolated from human synovial cell RNA using reverse transcriptase and the polymerase chain reaction. Recognition sequences for BamHI and XhoI were inserted to allow for unidirectional cloning of the cDNA. The polymerase chain reaction product was cloned into pBluescript vector (Stratagene), transformed into XL-1 Blue cells (Stratagene), and shown to have a sequence identical to that previously published (8) by dideoxy sequencing (25). The cDNA was then cloned into the pcDNAneoI eukaryotic vector (Invitrogen) and transformed in XL-1 Blue cells, and large scale preparations of the vector containing the cDNA (pcDNAneoI/B2) were purified using "maxi prep" kits (QIAGEN). The cDNA for the human B1 kinin receptor was provided in the eukaryotic vector pcDNA3 (Invitrogen).
The vectors containing the cDNA for each of the human kinin receptors were transformed into CHO-DUKXB1 cell monolayers of approximately 50% confluency in 100-mm dishes using lipofectamine reagent (Life Technologies, Inc.). Cells were incubated for 20-24 h with 20 µg of pcDNA3 containing the human B1 cDNA or with 5-10 µg of pcDNAneoI/B2. Clonal selection was performed in G418 (0.8-1 mg/ml). Stable transformants were cloned by limiting dilution and maintained in the same concentration of G418. Clones were selected on the basis of specific binding of the appropriate radiolabeled ligand. The clones selected for further analysis were designated CHO-B1/3 for the B1 receptor and CHO-B2/20 for the B2 receptor. The two clones expressed comparable numbers of their respective receptors (40,000 versus 35,000 sites/cell, respectively).
Radioligand Binding Studies
Binding assays were performed at 4 °C in PIPES buffer, pH
7.4, containing 7.7 g/liter PIPES, 6.4 g/liter NaCl, 0.37 g/liter KCl,
and 1.0 g/liter glucose, supplemented with 0.1% fatty acid-free human
serum albumin, 104 M bacitracin,
10
4 M captopril, and 0.1% sodium azide.
Cells from confluent flasks were washed with this buffer, removed from
flasks by gentle scraping, and centrifuged at 1000 × g
for 10 min. Cells were resuspended in buffer at a density of
approximately 106 cells/ml. Binding assays were performed
in a total volume of 400 µl. For saturation curves,
[3H]des-Arg10-LBK was used in a concentration
range from 0.1 to 10 nM. Displacement binding was performed
using 1 nM [3H]des-Arg10-LBK and
competing ligands in the range of 0.01-10,000 nM.
Nonspecific binding was determined in each case in the presence of 1 µM des-Arg10-LBK. Binding reactions were
terminated, as described previously (26), by filtration and rapid
washing of the cells three times with ice-cold 14% 2-propanol using a
cell harvester (Brandel). Radioactivity associated with the filters was
quantified by scintillation spectrometry. Data were analyzed using
GraphPad computer software (GraphPad Software Inc.).
Ligand Internalization Studies
Confluent monolayers of intact CHO-B2/20 or CHO-B1/3 in 12-well culture dishes were incubated at 37 °C in incubation buffer (40 mM PIPES, 109 mM NaCl, 5 mM KCl, 0.1% glucose, 0.05% human serum albumin, 2 mM CaCl2, 1 mM MgCl2, 2 mM bacitracin, 10 µM phosphoramidon, 100 µM captopril, pH 7.4) containing 2 nM [3H]BK or 1 nM [3H]des-Arg10-LBK, respectively. At the indicated times, cells were washed four times with 0.5 ml of ice-cold buffer and treated for 10 min at 4 °C with 0.3 ml of a solution of 0.5 M NaCl and 0.2 M acetic acid, pH 2.7 (27, 28). The supernatant, containing the dissociated, formerly surface-bound, radioligand was then transferred with another 0.3 ml of buffer to scintillation vials containing 6-ml scintillation fluid, and the radioactivity was quantified. The remaining cells were lysed in 0.3 ml of 0.3 M NaOH, and the radioactivity measured was regarded as internalized ligand. Nonspecific binding and internalization was determined in the presence of 3 µM unlabeled ligand.
Inositol Phosphate Measurement
Total inositol phosphate (IP), defined as IP1,
IP2, and IP3, was measured based on the method
of Berridge (29). Cells were grown to 80% confluency in six-well
dishes and labeled overnight in minimum essential medium- with HEPES
containing 2 µCi/ml myo-[3H]inositol. Cells
were washed and equilibrated for 20 min in medium containing 20 mM LiCl and then stimulated with the appropriate peptide.
Reactions were terminated by removal of medium and the addition of
ice-cold 15% trichloroacetic acid. Cell supernatants were collected
and neutralized by diethyl ether extraction. Total inositol phosphates
were isolated using AG 1-X8 anion exchange columns (formate form).
Total inositol phosphates were eluted with 1.2 M ammonium
formate, 0.1 M formic acid. The effects of pertussis toxin
(PTX) on total IP generation were determined after preincubation for
24 h with 100 ng/ml of PTX.
To examine receptor desensitization, additional experiments were performed in which CHO-B1/3 or CHO-B2/20 were exposed to buffer or to a maximal concentration (1 µM) of appropriate ligand, and total IP was measured after 5 min. Replicate incubations were then washed four times and exposed to either buffer or appropriate ligand for a second 5-min period, beginning 5 min after removal of the initial stimulus. Total IP was again measured, and the production during each 5-min stimulation period was compared. Data are expressed as mean ± S.D. (n = 3).
Calcium Measurements
Intracellular calcium changes were measured by digital video microscopy as described previously (30, 31).
Immunoprecipitation Studies
Membrane PreparationsMembranes were prepared from
confluent monolayers of CHO-B1/3 cells by a modification of
previously described techniques (32). Cells were incubated for 30 min
at 37 °C in 5 mM HEPES, 0.5 mM EDTA buffer,
pH 8.0, scraped, and centrifuged (27,000 × g for 20 min) at 4 °C. The pellets were resuspended in buffer containing 10 mM Tris-HCl and 1 mM EDTA (pH 8), sonicated for
12 s, and centrifuged as before. The pellet was resuspended in a
solution of 10% sucrose with 20 mM Tris-HCl and 1 mM EDTA (pH 7.5), layered on a 44.5% sucrose cushion, and
centrifuged at 75,000 × g for 30 min. The harvested
membrane layer was resuspended in 20 mM Tris-HCl, 0.25 M sucrose, 1 mM MgCl2 and frozen at
80 °C.
[-32P]GTP-azidoanalide was synthesized
and purified as described (33). Photolabeling was carried out by a
modification of previously described methods (34). Membrane protein
(200 µg) was incubated with 2 µM
des-Arg10-LBK or vehicle and 8 µCi of
[
-32P]GTP-azidoanalide (specific activity 3000 Ci/mmol) in incubation buffer (50 mM HEPES, 5 mM MgCl2, 30 mM KCl, 0.1 mM EDTA, 1 mM benzamidine, pH 7.4) for 3 min at
30 °C. The reaction was stopped by placing the samples on ice.
Samples were then centrifuged at 12,000 × g for 15 min, and pellets were resuspended in 200 µl of photolysis
buffer (50 mM HEPES, 5 mM MgCl2, 30 mM KCl, 0.1 mM EDTA, 1 mM
benzamidine, 2 mM glutathione, pH 7.4). The samples were
irradiated for 15 s at 4 °C with an ultraviolet lamp (254 nm,
150 watts) from a distance of 3 cm. After irradiation, samples were
again centrifuged (12,000 × g for 15 min) and
subjected to immunoprecipitation.
Pellets of photolabeled membranes were
solubilized in SDS (2%) prior to the addition of precipitating buffer
(10 mM Tris-HCl, 1% (w/v) Triton X-100, 1% (w/v) sodium
deoxycholate, 0.5% (w/v) SDS, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.2 mM phenylmethylsulfuronyl fluoride, 10 µg/ml aprotinin,
pH 7.4) and centrifugation at 4 °C for 15 min at 12,000 × g. Aliquots of the supernatant were incubated with an excess
of each G protein antiserum at 4 °C for 4 h. Washed protein
A-Sepharose beads (10% (w/v)) were added, and samples were incubated
overnight. Thereafter, Sepharose beads were precipitated by
centrifugation (12,000 × g for 15 min). Supernatants
were removed, and aliquots were used to compare levels of total
[-32P]GTP-labeled proteins in each sample using two
methods; 1) an aliquot was counted directly via scintillation counting,
and 2) an aliquot of each sample was subjected to SDS-polyacrylamide gel electrophoresis and analyzed using a PhosphorImager. By both methods, there was <10% variation in total [
-32P]GTP
incorporation among samples. Meanwhile, the precipitated protein
A-Sepharose beads were washed with buffer A (50 mM
Tris-HCl, 1% (w/v) Nonidet P-40, 0.5% (w/v) SDS, 600 mM
NaCl, pH 7.4), followed by a wash with buffer B (100 mM
Tris-HCl, 300 mM NaCl, 10 mM EDTA, pH 7.4).
Samples were prepared for SDS-polyacrylamide gel electrophoresis according to standard protocols (35, 36). SDS-polyacrylamide gel
electrophoresis was performed using 12% acrylamide gels (37). Gels
were stained with Coomassie Brilliant Blue, dried, and imaged on
a Molecular Dynamics PhosphorImager plate. Results were analyzed as the
densitometric ratio of bands in stimulated, compared with unstimulated,
membrane preparations.
Initial association kinetic experiments showed that specific
binding of [3H]des-Arg10-LBK to
CHO-B1/3 cells reached equilibrium by 60 min at 4 °C
(Fig. 1). All subsequent binding studies were,
therefore, performed using incubation periods of at least 60 min.
Analysis of data from saturation binding indicated the presence of a
single population of binding sites with a Kd of 0.3 nM and a Bmax of 38 fmol/mg protein,
equivalent to approximately 40,000 sites/cell (Fig. 2).
Mock-transfected CHO cells failed to show specific binding of
[3H]des-Arg10-LBK (not shown).
An evaluation of the ability of analogs of bradykinin to displace
[3H]des-Arg10-LBK from the cloned human kinin
B1 receptor demonstrated that the presence of a lysine
residue at position 1 of the LBK sequence was essential for high
affinity binding (Fig. 3, Table I). This is exemplified not only by confirmation of the previously published finding (9) of the much higher affinity (Ki = 0.15 nM) of des-Arg10-LBK for the receptor compared
with des-Arg9-BK (Ki = 180 nM) but also by the similar differences in potency between
the antagonists des-Arg10-Leu9-LBK and
des-Arg9-Leu8-BK. In addition, LBK showed a
moderate affinity (Ki = 88 nM) for the
human B1 receptor, while BK was totally ineffective in
displacing [3H]des-Arg10-LBK. In contrast to
the striking increases in binding affinity observed upon extension of
the amino terminus of BK, or of des-Arg9-BK, with a lysine
residue, extension with a D-lysine residue had minimal
effects (Table I). Extension of the bradykinin sequence with an
amino-terminal arginine did improve binding affinity, but not to the
same degree as lysine. Although the lysine at position 1 is important,
further extension of the LBK sequence with an additional lysine, or of
des-Arg10-LBK with a D-lysine, had little
additional effect on binding affinity. Finally, as expected, truncation
of the carboxyl terminus of des-Arg10-LBK resulted in a
striking loss of binding affinity.
|
Previous studies using conformationally constrained analogs of BK containing an alkyl ether of D-4-hydroxyproline in either the cis or trans geometric state at position 7 of the BK sequence demonstrated that analogs containing trans-propyl ethers had a much higher affinity for the B2 receptor than corresponding cis analogs (38). We examined, therefore, the ability of similar analogs of des-Arg9-BK to displace [3H]des-Arg10-LBK from the cloned B1 receptor (Table II). In contrast to the B2 receptor, the human B1 receptor showed no marked specificity for cis- or trans-propyl ether derivatives.
|
Marked differences between cloned human B1 and
B2 receptors were also observed with regard to
receptor-mediated ligand internalization. Exposure of cloned
CHO-B2/20 cells to 2 nM
[3H]bradykinin resulted in internalization of
approximately 80% of the ligand within 5 min, but exposure of
CHO-B1/3 cells to 1 nM
[3H]des-Arg10-lysylbradykinin resulted in
minimal ligand internalization (Fig. 4). These data were
confirmed in additional clones (data not shown). Exposure of
nontransfected CHO cells to either radiolabeled ligand resulted in no
internalization.
Stimulation of CHO-B1/3 cells with
des-Arg10-LBK resulted in a dose-dependent
generation of total IP (Fig. 5), confirming coupling of
the human B1 receptor to phospholipase C activation.
Preincubation of cells with 100 ng/ml of PTX had no effects on the
dose-dependent generation of total IP in response to
des-Arg10-LBK. Consistent with the coupling of the human
B1 receptor to phospholipase C, stimulation of
CHO-B1/3 cells with 10 nM
des-Arg10-LBK induced a robust transient increase in
intracellular Ca2+ levels (Table III). The
effect of des-Arg10-LBK was receptor-mediated, since it was
inhibited by a brief preincubation of the cells with the B1
receptor antagonist, des-Arg10-Leu9-LBK. BK did
not significantly elevate Ca2+ above basal levels, even at
a concentration of 1 µM.
|
In light of the lack of receptor-induced ligand internalization
observed for the B1 receptor, we used IP generation as a
tool to examine functional desensitization. Exposure of
CHO-B1/3 cells to 1 nM
des-Arg10-LBK led to a continuous, linear accumulation of
total IP over a 60-min period, indicating a lack of desensitization
(Fig. 6). In contrast, BK-induced generation of total IP
in CHO-B2/20 cells reached a plateau within minutes (not
shown). These data were further supported by studies of repeat
stimulation. Exposure of CHO-B2/20 cells to 1 µM BK resulted in an increase in total IP (1371 ± 136 cpm) over a 5-min incubation, compared with exposure to buffer
alone (389 ± 20 cpm). After washing, a second 5-min exposure to
buffer led to neither a significant increase nor decrease in total IP
levels (1261 ± 235 cpm). Repeated stimulation with BK caused only
a modest further increase in total IP (to 1607 ± 131 cpm) during
the second incubation, consistent with significant receptor
desensitization. By contrast, a different pattern of response was seen
with CHO-B1/3 cells. Exposure to 1 µM
des-Arg10-LBK resulted in a generation of 1313 ± 101 cpm of IP in 5 min, compared with 577 ± 56 cpm in cells exposed
to buffer alone. After repeated washing, however, there was no
significant difference in IP levels after a second 5-min exposure
period to either buffer (2171 ± 110 cpm) or 1 µM
des-Arg10-LBK (2206 ± 321 cpm), but both were
markedly elevated compared with the first 5-min period. Binding studies
using radiolabeled ligand under similar conditions revealed that these
data can be explained by a low off-rate of des-Arg10-LBK
from the human B1 receptor, since over 95% of the ligand remained on the cell surface after 5 min, despite repeated washing. Thus, continued production of IP occurring during the washing and
second incubation periods is due to continued receptor occupancy and
confirms the lack of B1 receptor desensitization.
To determine which G-proteins may be involved in coupling the
B1 receptor to functional responses, we performed
immunoprecipitation studies using membranes incubated in the presence
or absence of des-Arg10-LBK. A representative blot (Fig.
7) shows enhanced GTP binding to Gq/11 and
G
i1,2 but not to G
s or G
o/i3,
indicating receptor-mediated activation of the former two G
subunits. In seven such experiments, des-Arg10-LBK
stimulation led to a 2.6 ± 0.7-fold increase in the intensity of
the immunoprecipitated G
q/11 band compared with that in unstimulated membranes and a 1.5 ± 0.2-fold increase in G
i1,2
but no changes in G
s or G
o/i3.
The cloning of the human B1 kinin receptor provided the first opportunity to examine the properties of this receptor. Initial studies, however, relied on the use of transient transfection of the receptor into COS-7 cells for binding studies, while limited functional data were obtained in X. laevis oocytes (9). The development of a stable clone of CHO cells expressing the human B1 receptor has permitted us to analyze, for the first time, both ligand binding characteristics and functional responses of the receptor in the same cell.
Our studies clearly demonstrate that agonist binding to the human B1 receptor leads to the generation of IP and transient increases in intracellular calcium, demonstrating that the receptor is coupled to phospholipase C activation. This is consistent with studies also demonstrating B1 receptor-mediated activation of phospholipase C in cells from other species (39-42) and confirms that both B1 and B2 receptors couple to this signaling pathway (43-45).
Immunoprecipitation studies indicate that the human B1
receptor is coupled to Gq/11 and G
i1/2 but not to
G
s or G
o/i3. The fold increase in GTP uptake upon
receptor stimulation was most marked for G
q/11. This pattern of
G-protein coupling is very similar to that observed upon stimulation of
human B2 receptors.2 Although
the observation that BK-induced increases in IP can be modified by PTX
exposure in some cell types demonstrates that proteins of the Gi family
can contribute to B2 receptor-mediated activation of
phospholipase C, the failure of PTX to modify IP generation in response
to des-Arg10-LBK stimulation in our studies indicates that
Gi1/2 does not contribute to B1
receptor-mediated activation of phospholipase C. Rather, members of the
Gq/11 subfamily of G-proteins have been shown to link cell surface
receptors to the known
-isoforms of phospholipase C (48-51), and
our data show that these G-proteins serve a similar role for the human
B1 receptor.
Our data also provide insight into the regulation of B1 receptor-mediated responses. Exposure of cloned human B2 receptors to ligand results in a rapid receptor-mediated ligand internalization (Fig. 4), a process that is accompanied by a loss of surface receptors.3 B2 receptor-mediated ligand internalization, associated with receptor sequestration, has also been noted to occur in a hamster vascular smooth muscle cell line (52, 53) and in human dermal fibroblasts (28). This rapid internalization and sequestration process leads to a reduction in the ability of cells to respond to further ligand exposure. In marked contrast, B1 receptor-mediated ligand internalization is very slow (Fig. 4) and is not associated with any loss of surface receptor binding.3 It is tempting to suggest, therefore, that this slow internalization represents a constitutive, ligand-independent turnover of a fraction of the expressed receptors. Not only does the B1 receptor fail to show rapid ligand internalization or receptor sequestration, but the continuous generation of total IP observed with time clearly demonstrates that the receptor does not desensitize. Thus, once B1 receptors are induced during an inflammatory event, the receptor cannot be rapidly down-regulated and can continue to activate second messenger pathways as long as ligand is available. This provides further support for the idea that B1 receptors could be important in inflammatory diseases and suggests that primary regulation of B1 receptor activation occurs at the level of ligand availability.
In terms of ligand specificity, our data demonstrate that the human B1 receptor requires the presence of a lysine residue at position 1 of the LBK sequence to bind a ligand with high affinity. This is not simply a requirement for a charged amino acid, since a bulky arginine residue is much less effective than lysine. Moreover, there is a stereochemical requirement for the lysine to be in the L-configuration, since D-lysine does not confer the same high affinity. This requirement for a lysine residue at position 1 probably explains why des-Arg9-BK, the "classical" B1 receptor agonist, has a 10-fold lower affinity for the human receptor than has been reported for the rabbit B1 receptor (41). The 1000-fold difference in affinity for the human B1 receptor between des-Arg10-LBK and des-Arg9-BK is in agreement with previously published data (9) and indicates that des-Arg10-LBK must be considered the natural ligand for the human B1 receptor in vivo. Although we find that LBK is a much better ligand than BK, the affinity of LBK for the human B1 receptor observed by us was 10-fold lower than that reported by Menke and colleagues (9). The reasons for this are unclear, but one potential explanation could be that these authors performed binding studies at 23 °C. If carboxypeptidase activity was not fully inhibited at this temperature, conversion of a small percentage of LBK to des-Arg10-LBK could lead to a higher apparent affinity.
The knowledge that des-Arg10-LBK is the likely natural ligand for the human B1 receptor has significant implications. First, those studies that have used des-Arg9-BK to try to delineate the role of B1 receptors in human tissue responses in vivo (54, 55) must now be considered flawed and should be repeated using the appropriate ligand. As mentioned above, however, even if future studies demonstrate expression of B1 receptors in human tissues during inflammatory diseases, this will not, by itself, constitute proof of biological importance but must be considered in light of the fact that receptor-mediated activity is also dependent upon ligand availability. To generate des-Arg10-LBK, an extremely specific environment must exist. First, tissue kallikrein must be expressed in the tissue in question in an active form, since this is the only enzyme that is known to produce LBK in humans (1). Then, LBK must be converted to des-Arg10-LBK by the actions of an appropriate carboxypeptidase that must also be present. Moreover, this latter step must occur before the LBK is acted upon by other peptidases, such as aminopeptidase M (which would remove the lysine that is critical for high affinity binding to the human B1 receptor, converting LBK to BK), or neutral endopeptidase or angiotensin-converting enzyme, both of which would further truncate the carboxyl terminus of the ligand to a form that would be inactive on the human B1 receptor. These extremely specific requirements provide exquisite control mechanisms for activation of the receptor.
In summary, we have used a stable clone of CHO cells expressing the human B1 receptor to characterize the binding properties and functional responses of this receptor. Des-Arg10-LBK appears to be the natural ligand for the human receptor. Activation of the receptor by this ligand results in inositol phosphate generation and increases in intracellular calcium levels via mechanisms that are coupled to phospholipase C via Gq/11. In contrast to the B2 receptor, activation of the human B1 receptor is not associated with ligand-induced receptor internalization, nor does the receptor show desensitization. The availability of a stable clone expressing the human B1 receptor should greatly facilitate future studies of the regulation of this receptor, its interaction with G-proteins, and its potential role in inflammation.