From The Johns Hopkins University School of Medicine, To determine the role of the cytoplasmic carboxyl
termini of human B1 and B2 kinin receptors (B1KR and B2KR,
respectively) in the internalization of their respective ligands,
des-Arg10-kallidin and bradykinin (BK), both wild
type receptors, as well as truncated B2KRs, a mutated B2KR, and
chimeric receptors were stably expressed in Chinese hamster ovary
cells. Incubation of [3H]BK at 37 °C with cells
expressing wild type B2KR resulted in pronounced and rapid ligand
internalization (~80% after 10 min). By contrast, incubation of
3H-labeled des-Arg10-kallidin with cells
expressing B1KR resulted in a modest, slow internalization of the
ligand (<20% after 10 min). Replacement, from Cys324, of
the cytoplasmic carboxyl terminus of the B2KR with that of the B1KR
from Cys330 (both Cys residues are putative palmitoylation
sites) greatly reduced ligand internalization (~40% after 10 min)
without significantly altering Kd or ligand-induced
signal activation. By marked contrast, the corresponding replacement,
of the sequence from Cys330 of the cytoplasmic carboxyl
terminus of the B1KR with the segment of the B2KR, led to a striking
increase of ligand internalization (~75% within 10 min) without
altering Kd or ligand-induced signal activation.
Truncation of the B2KR to within three amino acids of
Cys324 (truncation at Gly327) led to strongly
reduced ligand internalization (~40% after 10 min). Truncation of
the B2KR up to Lys315 almost completely abolished
internalization of [3H]BK (10% after 10 min). This
additional reduction is apparently not caused by the loss of the
potential palmitoylation site at Cys324, since a B2KR with
a point mutation of Cys324 to Ala internalized
[3H]BK as rapidly as the wild type B2KR.
From these results we conclude that the cytoplasmic carboxyl terminus
of the human B2KR contains sequences that are necessary and sufficient
to permit rapid ligand-induced sequestration of human kinin receptors
and internalization of their agonists.
Kinins are small peptides that are released from high molecular
weight precursors, called kininogens, by limited proteolysis performed
by enzymes known as kallikreins. They exert their large array of
biologic effects at the cellular level via two types of receptors, B1
and B2 kinin receptors (B1KR1
and B2KR, respectively) (1-5). The B2KR is constitutively expressed in
a variety of tissues and cell lines, binding with high affinity to
bradykinin (BK) and to Lys-BK (Kallidin), but not to their respective
carboxypeptidase degradation products des-Arg10-BK and
des-Arg10-kallidin (DAK). The B1KR, in contrast, is
expressed de novo under certain pathological conditions and
exhibits no affinity for BK but a high affinity for DAK and a low
affinity for kallidin and des-Arg9-BK (7). Both human kinin
receptors have been cloned recently (8, 9). Hydrophobicity analysis of
their amino acid sequence revealed that they both have seven
hydrophobic domains that are typical for members of the superfamily of
G-protein-coupled receptors (GPCRs). The amino acid sequences of both
receptors show an overall homology of only 36%, however, with higher
areas of homology in the transmembrane regions (9). Despite their
relatively low homology, the receptors appear to share similar signal
transduction pathways, such as activation of phospholipase C that leads
to the release of inositol phosphates and an increase in intracellular [Ca2+] levels, in response to binding of agonists (9).
While it is already well established that B2KR couples to phospholipase C via the G-protein Gq (10, 11), we demonstrated only
recently that binding of DAK to B1KR leads to the activation of
Gq and Gi1,2 (7). The kinin receptors appear to
differ, however, in mechanisms of signal "termination." Several
studies showed that B2KR responds to agonist binding with
ligand-induced receptor sequestration (12, 13) and ligand
internalization (14, 15). These properties of the B2KR may be
responsible, at least in part, for the rapid decrease in responsiveness
of the involved cells to a second stimulation with BK
(desensitization). The B1KR, in contrast, internalizes its ligand very
slowly and shows almost no short term desensitization (7).
For GPCRs with very long third intracellular loops, such as the
muscarinic receptor (almost 300 amino acids), important sequences for
ligand-induced receptor sequestration and ligand internalization have
been identified in this third loop (16, 17). In GPCRs with short third
intracellular loops, other domains may contribute to the signal for
ligand internalization. For receptors including the The results of our study demonstrate for the first time (i) that the
information encoded in the cytoplasmic tail of the B2KR is necessary
and sufficient for ligand-induced sequestration of B2 and also B1 kinin
receptors; (ii) that loss of a putative palmitoylation site in the
cytoplasmic tail of B2KR does not interfere with ligand internalization
or receptor sequestration; (iii) that the B1KR, in contrast to B2KR,
does not respond to binding of an agonist with receptor sequestration;
and (iv) that exchange of the cytoplasmic tail does not transfer the
desensitization pattern.
Materials--
Chinese hamster ovary cells DUKXB1 (CHO) and
human fetal lung fibroblasts IMR 90 were obtained from ATCC.
[2,3-prolyl-3,4-3H]Bradykinin (100-108 Ci/mmol),
[3,4-prolyl-3,4-3H]des-Arg10-kallidin (84-89
Ci/mmol), and myo-[2-3H]inositol (22 Ci/mmol)
were purchased from NEN Life Science Products. Unlabeled peptides were
bought from Bachem. All primers were synthesized by Life
Technologies, Inc. and delivered desalted and lyophilized. Restriction enzymes and phosphoramidon were from Boehringer Mannheim or
New England Biolabs. Pfu DNA polymerase was obtained from
Stratagene. Lipofectamine, culture media, most additives, Opti-MEM,
Geneticin (G418 sulfate), and fetal calf serum were bought from Life
Technologies, Inc. Penicillin/streptomycin was obtained from Biofluids
Inc. Captopril was purchased from Sigma. All other reagents were
of analytical grade and commercially available. A pcDNA3 vector, harboring the cDNA encoding the B1 receptor (pcDNA3-B1), was
kindly provided by Dr J. Fred Hess (Merck).
Cell Culture--
Stock cultures of IMR 90 lung fibroblasts were
grown in Dulbecco's modified Eagle's medium with high glucose, 2 mM glutamine, 10% fetal calf serum, and
penicillin/streptomycin. Half of the spent medium was replaced by fresh
medium every 4 days. For subcultures, cells were lifted with trypsin
and seeded in six-well trays in medium composed of two parts of fresh
medium and one part of used (conditioned) medium. Naive CHO cells and
transfected cells were cultivated in Construction of pcDNA3-B2, B2KR Truncations, and the B2KR
Mutation C324A--
mRNA from human synovial fibroblasts was used
as starting material to obtain the cDNA encoding the B2KR gene by
reverse transcriptase polymerase chain reaction (PCR). The PCR product
was then subcloned between the BamHI and XhoI
sites of the pcDNA3 vector (Invitrogen). The sequence of the insert
was determined by sequencing selected clones using the
dideoxynucleotide method (24) and was identical with the published
human B2KR sequence (8). This pcDNA3 vector harboring the B2KR
(pcDNA3-B2) was consequently used as a template to generate the
truncated mutants Gly327 and Lys315 and the point mutation C324A using
standard PCR methodology with appropriate oligonucleotides.
Construction of B1/B2 Receptor Chimera--
For the synthesis of
the chimera, in which the cytoplasmic tail of the B1KR was replaced
with that of the B2KR (B2CB1), a first PCR was performed with the
pcDNA3-B1 vector as template using a T7 primer and a
chimeric antisense primer
5 Transfection and Selection--
20 µg of plasmid DNA in 300 µl of Opti-MEM and 30 µl of Lipofectamine (Life Technologies, Inc.)
in 270 µl of Opti-MEM were combined and kept at room temperature for
15 min to obtain a precipitate. After the addition of 4.6 ml of
Opti-MEM, the whole mixture was added to subconfluent (40-70%) CHO
cells in a 80-cm2 flask that had been rinsed with 5 ml of
Opti-MEM and incubated with another 5 ml for 15 min at 37 °C. After
16-36 h, the medium was changed with 10 ml of complete medium
containing G 418 (800 µg of geneticin/ml) to start the selection of
stably transfected cells. Medium was changed every 4 days, and after
about 15 days individual clones were isolated by limiting dilution and
screened for binding of the appropriate ligand. All stock cultures were kept under constant selection pressure of G418 (800 µg/ml), whereas cells seeded in dishes/wells were maintained without G418 and used
within 2-5 days.
3H-Ligand Binding Studies--
For the determination
of dissociation constants (Kd) and receptor numbers
(Bmax), cell monolayers in 24- or 12-well (CHO)
or 6-well trays (IMR 90) were washed three times with wash buffer (40 mM PIPES, 109 mM NaCl, 5 mM KCl,
0.1% glucose, 0.05% bovine serum albumin, 2 mM
CaCl2, 1 mM MgCl2, pH 7.4) followed by preincubation at 37 °C with incubation buffer (wash buffer supplemented with protease inhibitors: 2 mM bacitracin, 10 µM phosphoramidon, and 100 µM captopril).
The trays were subsequently placed on ice, and the indicated
concentrations of [3H]BK or [3H]DAK were
added for determination of total B2KR and B1KR binding, respectively.
Nonspecific binding was determined in the presence of 5 µM of the appropriate unlabeled ligand. After an
incubation time of at least 90 min, the cell monolayers were washed
four times with ice-cold incubation buffer, lysed with 0.3 M sodium hydroxide, and transferred quantitatively into
scintillation vials. Radioactivity of the samples was measured in a
3H-Ligand Internalization (14, 25)--
To determine
the internalization of receptor-bound ligand, cell monolayers were
washed three times with prewarmed (37 °C) wash buffer and
preincubated at 37 °C with incubation buffer for at least 30 min.
Internalization was started by replacing the buffer with incubation
buffer containing the indicated concentrations of 3H-ligand
with or without 5 µM unlabeled ligand. After the
indicated time, the incubation was stopped by washing the monolayers
four times with ice-cold wash buffer. The trays were placed on ice, and
the cells were treated with an ice-cold solution of 0.2 M acetic acid and 0.5 M NaCl, pH 2.7 (dissociation solution)
for 10 min in order to dissociate the surface-bound
3H-ligand (0.3 ml for 12-well trays). The supernatant was
transferred to scintillation vials, and the radioactivity was
quantified. The acid-resistant 3H radioactivity, considered
to be internalized 3H-ligand, was quantified after lysis of
the cells with 0.3 M sodium hydroxide.
Receptor Sequestration--
Monolayers of cells in 12-well
dishes were washed twice with prewarmed (37 °C) wash buffer,
preincubated in incubation buffer for at least 30 min at 37 °C, and
incubated with the same buffer containing 1 µM of the
appropriate unlabeled ligand (BK or DAK). After the indicated times,
the cells were washed four times with 0.5 ml of ice-cold wash buffer
and treated on ice for 10 min with dissociation solution to remove all
unlabeled surface-bound ligand. The monolayers were washed again four
times with ice-cold wash buffer, and specific B2KR or B1KR binding was
determined at 0 °C as described above using approximately 2 nM [3H]BK or 1 nM
[3H]DAK.
Stimulation of Total Inositol Phosphate Release--
80%
confluent cell monolayers of stably transfected clones grown in 12-well
trays were labeled for 24-48 h with 2 µCi of
myo-[3H]inositol in 0.5 ml of
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-adrenergic,
angiotensin II, gastrin-releasing peptide, parathyroid hormone
receptors, and
-opioid receptor, domains have been localized to the
cytoplasmic tail, particularly to sequences enriched in serine and
threonine residues (18-23). Both kinin receptor subtypes have short
third intracellular loops, and their cytoplasmic COOH termini share no
homology after the putatively palmitoylated cysteine. Therefore, we
asked whether their opposite patterns of internalization are the
consequence of either a positive sequence (in the case of the B2KR) or
an inhibitory sequence (B1KR) for sequestration and/or ligand
internalization provided by their cytoplasmic tails.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-minimum essential medium
supplemented with 10% fetal calf serum and penicillin/streptomycin
(complete medium).
-CCTGCAGCCCCCTTTCTGGCATTGTTTATAAAGTTCC-3
composed
of 16 base pairs from the B1 sequence upstream of Cys330
(encoded by GCA) and 18 base pairs of the B2 sequence downstream of Cys324 (GCA). A second PCR was
executed with pcDNA3-B2 as template with the sense primer
5
-CAATGCCAGAAAGGGGGCTGCAGG-3
corresponding to the B2
sequence, starting at Cys324 (encoded by TGC)
and one triplet of the B1 sequence downstream stream of
Cys330, and the antisense primer Sp6. The products of both
reactions, displaying the predicted sizes, were gel-purified and used
with T7 and Sp6 primers in a third PCR. The product of this reaction, the chimeric receptor gene, was subcloned between the BamHI
and XhoI sites of the pcDNA3 vector. The chimeric B2CB1,
in which the cytoplasmic tail of the B2KR was replaced with that of the B1KR was generated in the same way using
5
-GCAAGACTTTTAGGGGTGCACACTCCCTGGTACACC-3
as
antisense primer for the first reaction with pcDNA3-B2 as template and 5
-GTGTGCACCCCTAAAAGTCTTGC-3
as sense
primer in the second PCR with pcDNA3-B1 as template. The
accuracy of the sequence of the chimeras was confirmed by sequencing
selected clones using the dideoxynucleotide method (24). Plasmids were
obtained using JM109 bacteria (Promega) and a plasmid purification midi
kit from Qiagen.
-counter after the addition of a 10-fold volume of scintillation
fluid. Specific binding was calculated as the difference between total
binding and nonspecific binding.
-minimum
essential medium supplemented with 0.05% bovine serum albumin and
penicillin/streptomycin. Monolayers were rinsed with wash buffer,
equilibrated in prewarmed incubation buffer containing 50 mM LiCl for 15-30 min, and stimulated with the appropriate
agonist at 37 °C. The stimulation was terminated by exchanging the
buffer for 0.75 ml of an ice-cold solution of 20 mM formic
acid and by placing the trays on ice for 30 min. After transferring the
supernatants quantitatively with another 0.75 ml of 20 mM
formic acid to tubes, 0.2 ml of a 3% ammonium hydroxide solution was
added, and the mixtures were applied to AG 1-X8 anion exchange columns
(1-ml volume). The columns were washed with 1 ml of 1.8% ammonium
hydroxide, followed by 9 ml of 60 mM sodium formate, 5 mM tetraborate buffer, and total inositol phosphates were
eluted with 1.5 ml of 4 M ammonium formate, 0.2 M formic acid.
Statistics-- All points in the figures represent the mean ± S.E. of at least three experiments done in triplicate. For internalization and sequestration experiments with transfected CHO cells, G418-selected, but not cloned, cell populations and at least one stably transfected clone were evaluated. Data were analyzed using paired or unpaired t tests, as appropriate. Significance was assumed for values of p < 0.05.
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RESULTS |
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Ligand Internalization of Endogenously and Heterologously Expressed Kinin Receptors-- Human embryonic lung fibroblasts IMR 90 represent one of the few human cell lines that naturally express both types of kinin receptors, but B1KR expression is low (5,000 sites/cell) compared with that of B2KR (70,000 sites/cell) (9). To date it is not known whether the B1KR becomes sequestered upon ligand stimulation as has been described for the human B2KR (12, 13), and it was only very recently that we reported the almost complete lack of internalization of DAK after binding to a B1KR expressed in CHO cells (7). Since this lack of internalization might have been due to the expression of the B1KR in a noncompatible cell system, we used the acetic acid treatment method (25) that has been used to study the internalization of a variety of hormones to examine the behavior of the B1KR in IMR 90 cells. An added benefit to the study of IMR 90 cells is the ability to compare internalization/sequestration of both kinin receptor types in the same cell line. The B2KR of IMR 90 cells internalized [3H]BK very quickly reaching a plateau of 75% internalization of bound ligand after 10 min (Fig. 1). This is similar to what has been described for the B2KR in other cell lines (14, 15). In contrast, even after 30 min of incubation, less then 20% of bound [3H]DAK was found in an acid-resistant compartment (Fig. 1). We considered that the low level of B1KR expression in IMR 90 cells might be responsible for slow [3H]DAK internalization. We addressed this possibility using two approaches. First, we enhanced expression of B1KR in IMR 90 cells by preincubation with IL-1 (1 ng/ml) for 24 h as previously reported (not shown) (9). Second, we expressed high numbers of each receptor type in CHO cells, resulting in up to 67 fmol of B1KR/106 cells and up to 94 fmol of B2KR/106 cells. In each case, internalization rates for the respective ligands for each receptor were nearly identical to those observed in untreated IMR 90 cells (Fig. 1 and Ref. 7). These studies confirmed that the markedly different internalization rates of the B1KR and B2KR ligands are a property of the receptors themselves rather than simply a reflection of variable receptor density. Nontransfected CHO cells showed no uptake (internalization) of labeled ligand in these conditions (not shown).
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3H-Ligand Internalization of Receptor Chimeras-- In view of the role of the cytoplasmic tail in conveying a signal for internalization in a number of nonkinin receptors (18-23, 26), we next asked (i) if the domains responsible for the rapid internalization of BK by the B2KR and the slow internalization of DAK by the B1KR were contained in their respective cytoplasmic tails and (ii) if so, whether exchange of the tails between the two kinin receptor types would alter their respective ligand internalization rates. To address these questions, we created chimeric receptors in which the cytoplasmic tails were switched at a highly conserved (putative) palmitoylation site (Cys324 in B2KR and Cys330 in B1KR) (Fig. 2)
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Ligand Internalization of Truncated B2KRs-- To examine whether the low internalization rate of the B2CB1 chimera was due to an inhibitory sequence in the cytoplasmic tail of B1KR, we examined the effect of truncation of the B2KR on its rate of ligand internalization. Cys324 was felt to be the ideal site for truncation, since this would maintain consistency with the site of cytoplasmic tail exchange in the previous experiments. Cys324 is presumed to be palmitoylated, however, and several fatty acid transferases require additional amino acids adjacent to the target amino acid (Cys) to function properly. Therefore, we performed a more conservative truncation at Gly327. This truncation resulted in a significant reduction in [3H]BK internalization to a rate comparable with that of the B2CB1 chimera (Fig. 3). Because internalization was still considerably faster in the Gly327 mutant than in the B1KR, which serves as our reference for lack of ligand internalization, we created a second, more extensive truncation at amino acid Lys315. This manipulation of the B2KR resulted in almost complete loss of internalization of [3H]BK (Fig. 3), manifested by a rate very similar to that of B1KR. This additional, marked reduction in the internalization rate, as compared with truncation Gly327, could be caused in part by the loss of the putative palmitoylation site at Cys324. To test this possibility, we mutated Cys324 to Ala (C324A). As Fig. 3 shows, however, this mutation clearly had no influence on the internalization rate. All truncated receptors, and the mutant C324A receptor, had dissociation constants (Kd) similar to WT B2 (Table I).
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Inositol Phosphate Production-- Although the exchange of cytoplasmic carboxyl termini dramatically altered the sequestration pattern of wild type kinin receptors, there was no marked effect on EC50 values for total IP production. Stimulation of both chimeras resulted in values that were not significantly different from the corresponding wild types (Table I). Truncation of the B2KR to Gly327 also did not affect the EC50 for total IP production, but truncation to Lys315 resulted in a significantly higher EC50 value.
A markedly different profile of responses was observed, however, when the time course of IP production by wild type and mutant receptors was examined. Stimulation of the B2KR for a period of 30 min led to a rapid initial rate of IP production, with 35% of total release occurring within the first minute, with a much slower subsequent rate for the remainder of the period (Fig. 5). By contrast, as we have reported previously (7), stimulation of the B1KR produced a more sustained, even rate of IP production over the 30-min period, with less than 10% of total production occurring in the first minute. Stimulation of both chimeric receptors resulted in rates of total IP production that fell between those observed for each of the wild type, although they more closely resembled that of the B1KR, since less than 15% of overall production occurred within the first minute. Truncation of the B2KR to Gly327 led to a time course of IP production that closely resembled that of the B2CB1 chimera, but stimulation of the Lys315 mutant led to a time course of IP production that was almost superimposible with that observed for the B1KR. ![]() |
DISCUSSION |
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The GPCRs are a superfamily of receptors that transmit extracellular signals to cytosolic and membrane-associated effector molecules via coupling to one or more heterotrimeric G-proteins (27, 28). In the case of kinins, ligand activation of the receptor-G-protein-effector complex leads to a variety of profound responses, including vasodilatation, increased vascular permeability, inflammation, and pain in the affected organs or tissues (1-5). Termination of the intracellular signal pathways is, therefore, critical to limit the biological consequences of an individual agonist such as bradykinin. Termination can be accomplished at the cellular level by several processes that, in principle, may apply to all components of the receptor-G-protein-effector complex, although those for the receptor are best studied. These include desensitization (reduced cellular response to a second stimulation by agonist), resensitization (recovery of a response), and down-regulation (loss of the involved protein due to degradation).
In this study, we focused on the identification of receptor domains that are involved in the ligand-induced sequestration of the kinin receptors and the structural requirements for this function. We used chimeric kinin receptors and mutant B2KR receptors in a dual approach to determine what structural domains of the B2KR are critical for B2KR sequestration (to cause loss of function) and to determine if transfer of these domains to B1KR can confer the ability of the receptor to sequester (gain of function).
Our results show that the cytoplasmic tail of the B2KR contains a sequence(s) that is necessary and sufficient for the sequestration of kinin receptors. There have been several studies demonstrating sequestration of a variety of GPCRs and/or ligand internalization as a response to ligand binding (16-23, 26). For non-GPCRs, such as the low density lipoprotein receptor or the transferrin receptor, distinct small peptide sequences in their cytoplasmic domains, all containing a tyrosine residue, have been identified as carriers of information for receptor internalization (29). In addition, for at least one GPCR, the neurokinin 1 receptor, tyrosine, but not serine or threonine, residues, in the cytoplasmic tail, have been demonstrated to be important for ligand internalization (30). Additional internalization motifs or mechanisms for GPCRs must exist, however. Tyrosine residues obviously do not play a critical role in kinin receptor sequestration, since the cytoplasmic tail of the B2KR that sequesters very rapidly, contains only one tyrosine residue (Tyr320, Fig. 3), and this tyrosine is conserved in the B1KR (Tyr327, Fig. 3), which does not internalize/sequester. An inhibitory sequence in the cytoplasmic tail of B1KR, as reported for other receptors (22, 31), that might counteract a possible positive effect of this tyrosine is not likely, because the chimera B2CB1 did not exhibit further reduction of the internalization rate compared with the corresponding Gly327 truncation.
The chimera B1CB2 did not internalize as rapidly as WT B2 (Fig. 2), and the Lys315 truncation showed distinctly less internalization than the Gly327 truncation (Fig. 3). Therefore, some additional requirement for receptor internalization appears to be located in the sequence between Lys315 and Gly327. The putative palmitoylation site at Cys324 is not involved, because the mutant C324A internalizes and sequesters very similarly to the WT B2 (Fig. 3 and Fig. 4, top).
The low but distinct internalization rate of [3H]DAK in WT B1 or IMR 90 fibroblasts may best be explained by a slow continuous turnover of B1KRs that, in a ligand-independent manner, transports bound ligand inside the cell, since there is no significant reduction of surface binding even after incubation with 1 µM DAK for 30 min (Fig. 4, bottom). In other words, DAK has no direct influence on the number of B1 surface receptors. In contrast, after the exchange of the cytoplasmic tail of the B1KR with that of the B2KR, rapid internalization of [3H]DAK was observed (Fig. 3), and DAK exposure resulted in a reduction of the number of B1KRs on the cell surface (Fig. 4, bottom). This clearly indicates that the cytoplasmic tail of the B2 receptor, on the one hand, provides the B1KR with the ability to undergo internalization but, on the other hand, now is under the control of the remaining domains of B1KR (i.e. induction of sequestration is dependent on DAK, and the receptor has not simply achieved a higher rate of constitutive recycling). Thus, despite the low homology of only 36% between both receptors (9), nearly complete transfer of the internalization pattern of B2KR is possible via transfer of its cytoplasmic tail.
Obviously, it is important to understand what sequence(s) in the
cytoplasmic tail of B2KR provides signals for sequestration and how it
becomes accessible for the cellular machinery mediating receptor
sequestration. Several groups have shown the importance of serine and
threonine residues for receptor internalization in the cytoplasmic
tails of GPCRs (18-23). A good possibility for explaining the marked
reduction in ligand internalization exhibited by the Gly327 truncation,
therefore, is the removal of the sequence Thr-Ser-Ile-Ser in the
cytoplasmic tail, a sequence that is similar to the sequence
Ser-Thr-Leu in the angiotensin AT1 receptor that was determined to play
an essential role in its internalization (19). Phosphorylation of
serine and threonine residues of other GPCRs by receptor kinases has
been shown to lead to coupling of -arrestins, which are involved in
receptor internalization (32). A similar mechanism might apply for the
internalization of the B2KR, since transient phosphorylation of serine
and threonine residues of the B2KR within minutes after stimulation
with BK has been reported (33). This phosphorylation took place at
least in part, if not exclusively, in the cytoplasmic tail of the B2KR. Partial inhibition of B2KR internalization by concanvalin A led to
reduced dephosphorylation of the B2KR, suggesting that a role for
receptor internalization is to facilitate receptor dephosphorylation, as similarly proposed for the
-adrenergic receptor (18, 33). A study
with
-adrenergic receptors demonstrated that receptors that were
resistant to phosphorylation, either because of a truncation proximal
to the palmitoylation site or because of point mutations of all
critical phosphorylation sites (i.e. serines and
threonines), displayed markedly reduced receptor sequestration (32). A
phosphorylated cytoplasmic tail, however, may facilitate receptor
internalization through increased interaction with
-arrestins but
cannot be the only contact site in GPCRs for
-arrestins, since
overexpression of
-arrestins has been shown to lead to
internalization of receptors that previously were prevented from
internalization due to resistance to phosphorylation or COOH-terminal
truncation (32). One might speculate, assuming a similar mechanism for
kinin receptor internalization, that overexpression of
-arrestin
should also lead to internalization of WT B1 receptors.
Truncation of the cytoplasmic tail to Lys315 leads to a significant increase of the EC50 for total IP release determined after 30 min of stimulation. It is not yet clear whether this is because the cytoplasmic tail is directly involved in G-protein coupling or because this noninternalizing truncated receptor remains in the membrane in an active, but low affinity, state.
Desensitization of a receptor is generally demonstrated by a decrease in the amplitude of a response upon sequential stimulation with agonist. This approach is not meaningful for evaluating the B1KR, however, because, as we have previously reported, the B1KR agonist DAK has a very slow dissociation rate (7). Consequently, cells exposed to an initial stimulation with DAK that are then washed extensively and allowed to sit for an additional 10 min in buffer continue to exhibit a steady rise in IP upon a second incubation in buffer alone. This renders a repeat stimulation with DAK uninterpretable because of the limited number of unoccupied receptors available for binding. We chose, therefore, to compare the relative rates of accumulation of IP over time in the continued presence of agonist and were able by this method to demonstrate a clear difference in the rates of IP accumulation between B1KR and B2KR.
We expressed the data in these experiments, as in the EC50 calculations, as a percentage of "maximal" response, defined arbitrarily as the 30-min time point, rather than as Emax values. This was done, in part, because absolute increases in total IP varied significantly even within the same clones from experiment to experiment, presumably as a result of variability in cell densities, receptor numbers, and length of incubation with [3H]inositol. In addition, defining Emax (using a cumulative assay such as total IP) in a way that will allow meaningful comparison of a receptor that desensitizes and one that does not is difficult, if not impossible. Because the IP responses in B1KR and Lys315 do not plateau over the time points examined, defining maximal responses by an arbitrary time point is potentially misleading. Even in electing to use an arbitrary time (30 min) as "maximal" response, we are left with the additional problem of widely varying receptor densities among the clones examined. Although it has been reported that BK-stimulated IP production was not receptor number-dependent in cells possessing between 25,000 and 140,000 receptors/cell (34), we cannot confirm these data as yet for our own cell system.
The fact that both chimera, the two truncations of B2KR, and the point mutation C324A have dissociation constants at 0 °C that are similar to those of the wild type receptors indicates that the cytoplasmic tails play only a minor role in regulating ligand binding characteristics. As shown above, however, they play a significant role in signal termination. This is exemplified by the observation that truncation of the cytoplasmic tail of the B2KR to Lys315 results in a receptor that, like the B1KR, gets neither sequestered nor desensitized. It remains unclear, however, if this latter property of Lys315 and B1KR is due, in part, to the lack of a rapid, pronounced ligand-induced reduction in the number of available cell surface receptors or results from the absence of sequences that are important for desensitization by mechanisms other than removal of receptors from the cell surface. If such sequences do exist in the tail of the B2KR downstream of Cys324, their information apparently cannot be transferred, since the chimera B1CB2 does not show a desensitization pattern that resembles that of the B2KR (Fig. 5).
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This study shows the importance of the COOH terminus as an independent domain for ligand-induced internalization of kinin receptors. The availability of chimeras of receptor subtypes that show completely different sequestration behavior but otherwise similar G-protein coupling and of an internalization-deficient mutant (Lys315) of B2KR should help us to gain greater understanding of the interaction of the different processes of receptor sequestration, signaling, and desensitization/resensitization of kinin receptors in particular and, hopefully, of peptide receptors in general.
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ACKNOWLEDGEMENT |
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We thank Dr. Santa J. Ono for help with the design of the PCR primers.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL32272 and AR01856 and by grants from the Arthritis Foundation, Maryland Chapter, and the American Heart Association, Maryland Affiliate.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.
Recipient of a fellowship from the Deutsche
Forschungsgemeinschaft. To whom correspondence should be addressed:
Johns Hopkins Asthma & Allergy Center, 5501 Hopkins Bayview Circle,
Baltimore, MD 21224. Tel.: 410-550-2061; Fax: 410-550-2090.
1
The abbreviations used are: B1KR, B1 kinin
receptor; B2KR, B2 kinin receptor; BK, bradykinin; DAK,
des-Arg10-kallidin (des-Arg10-Lys-BK); CHO,
Chinese hamster ovary; GPCR, G-protein-coupled receptor; PCR,
polymerase chain reaction; PIPES,
piperazine-N,N-bis(2-ethanesulfonic acid); IP,
inositol phosphate; WT, wild type.
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REFERENCES |
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