Am J Physiol Heart Circ Physiol 284: H1892–H1898, 2003;
10.1152/ajpheart.01147.2002.
special topic
Regulation of Cardiovascular Signaling by Kinins and Products
of Similar Converting Enzyme System
Expression levels strongly affect ligand-induced sequestration
of B2 bradykinin receptors in transfected cells
Alexander Faussner, Alexandra Bauer, Irina Kalatskaya,
Marianne Jochum, and Hans Fritz
Ludwig-Maximilians-Universitaet Muenchen, Abteilung Klinische Chemie
und Klinische Biochemie, D-80336 Muenchen, Germany
Submitted 7 January 2003; accepted in final form 11 February 2003
tion of the agonist. These processes contribute significantly to the regulation and maintenance of the responsiveness of a cell to external signals because
receptor sequestration represents an important step in
receptor desensitization and/or resensitization. Whereas
the behavior of most wild-type receptors can be investigated in cells expressing them endogenously in significant amounts, only very few naturally expressed
mutants are available for the study of structure-function relationships of receptor domains (11). The artificial expression of receptors and especially of their mutants in cultured cell lines either as transient or as
stable clones has proved to be a very valuable substitute for this purpose.
This approach has also been used quite successfully
with the kinin receptors (1, 13–16) to determine the
role of their COOH-termini in ligand internalization
and signal transduction. However, the various publications reported rather large differences in the internalization rates, even for wild-type B2 receptors ranging from ⬍30% ligand internalization after 10 min in
COS-7 cells (14) to over 80% in Chinese hamster ovary
(CHO) cells (1). This was considered to be at least
partially due to the use of different cell lines and cell
culture techniques, which hindered the comparison of
these data (14). When we could not reproduce our own
data (1) after changing our protocol to generate stable
mutants, we were inspired to look for the molecular
parameters determining the internalization and sequestration rates of the kinin receptors heterologously
or homologously expressed in cultured cell lines and to
define experimental conditions to get reproducible and
comparable data with these cell lines.
receptor-mediated ligand internalization; receptor overexpression
MATERIALS AND METHODS
of a G protein-coupled receptor often triggers the sequestration of the receptor and internaliza-
Materials. CHO cells DUCXB1 were purchased from
American Type Culture Collection, and Flp-In T-REx [human embryonic kidney (HEK)-293] cells were from Invitrogen (Groningen, The Netherlands). [2,3-Prolyl-3,4-3H]brady-
Address for reprint requests and other correspondence: A. Faussner,
Ludwig-Maximilians-Universitaet Muenchen, Abteilung Klinische Chemie und Klinische Biochemie, Nussbaumstrasse 20, D-80336 Muenchen,
Germany (E-mail: [email protected]).
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.
ACTIVATION
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0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society
http://www.ajpheart.org
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Faussner, Alexander, Alexandra Bauer, Irina Kalatskaya, Marianne Jochum, and Hans Fritz. Expression levels strongly affect ligand-induced sequestration of B2 bradykinin receptors in transfected cells Am J Physiol Heart Circ
Physiol 284: H1892–H1898, 2003; 10.1152/ajpheart.01147.
2002.—Transfection of cells with expression vectors is one of
the most important tools used to assess the effects of receptor
mutations on ligand-induced receptor sequestration. Most
transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels
that may also depend on the mutations made. It is, therefore,
important to determine how the regulation of the receptors
depends on their numbers per cell. In Chinese hamster ovary
(CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B2 kinin receptors, we observed poor
sequestration indicated by ⬍20% reduction in cell surface
receptor number after 10 min of stimulation with 1 ␮M
bradykinin (BK) compared with ⬎70% in low-expressing
cells. Whereas the rate of [3H]BK internalization (internalized [3H]BK in percentage of total bound [3H]BK) in lowexpressing cells was independent of the ligand-concentration
used, in high-expressing cells a strong rate decrease was
observed with higher (⬎1 nM) concentrations. Lower ligand
concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently
transfected HEK and COS-7 cells showed results similiar to
those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B2 kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells
with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can
be measured, provided that the ligand concentrations used
are below a threshold at which the internalization rate is still
independent of the ligand concentration.
EXPRESSION LEVELS AFFECT B2 RECEPTOR SEQUESTRATION
AJP-Heart Circ Physiol • VOL
␮M captopril) for 15 to 45 min. Subsequently, the plates were
incubated on ice with 0.3 ml of ice-cold incubation buffer
containing increasing concentrations of [3H]BK (10 concentrations ranging from 0.01 to 40 nM) for at least 90 min. The
incubation was stopped by rinsing the cells four times with
ice-cold PBS. The cell monolayer was lysed in 0.2 ml 0.3 M
NaOH and transferred with another 0.2 ml buffer to a scintillation vial, and the radioactivity was measured in a betacounter after the addition of 2 ml scintillation fluid. Nonspecific binding was determined in the presence of 5 ␮M of
unlabeled BK and subtracted from total binding being determined with [3H]BK alone to calculate specific binding.
Receptor sequestration assay. Monolayers of cells on 12well plates were rinsed three times with PBS and incubated
on ice with 5 ␮M unlabeled BK in 0.5 ml incubation buffer.
After 60 min, the cells were placed in a water bath at 37°C to
start receptor sequestration. At the indicated times, the trays
were placed on ice, washed two times with ice-cold wash
buffer, and treated with a solution of 0.05 M glycine, pH 3.0
(8) for 10 min on ice to remove all unlabeled extracellular
ligand. The cells were again washed two times with ice-cold
wash buffer, and remaining specific surface binding was
determined with 1–2 nM [3H]BK at 0°C as described above.
Internalization of [3H]BK. Cells on 12-well plates were
rinsed three times with PBS, preincubated with incubation
buffer (15–45 min), and incubated with 0.3 ml of [3H]BK on
ice for 90 min to reach equilibrium binding. To start [3H]BK
internalization, the plates were placed in a water bath at
37°C. The internalization process was stopped after the indicated time by putting the plates on ice and washing the
monolayers four times with ice-cold PBS. Surface-bound
[3H]BK was then separated by treating the cell monolayer
with 0.2 ml of an ice-cold dissociation solution (0.2 M acetic
acid, 0.5 M NaCl, pH 2.7) for 10 min on ice. The supernatant
with formerly surface-bound [3H]BK was quantitatively
transferred to a scintillation vial by rinsing the cells with
another 0.2 ml of PBS (surface-bound [3H]BK). The remaining cell monolayer containing the internalized [3H]BK was
lysed in 0.2 ml 0.3 M NaOH and transferred with another 0.2
ml of water to a scintillation vial. The radioactivity of both
samples was determined in a betacounter after 2 ml scintillation fluid were added. Nonreceptor-mediated [3H]BK internalization and surface binding were determined in the presence of 5 ␮M unlabeled BK and subtracted from total binding
to give specific values.
Protein determination. Total protein was quantified with
the Micro BCA Protein assay reagent kit from Pierce (Rockford, IL) using bovine serum albumin as the standard.
RESULTS
Sequestration of stably expressed B2KRs in HEK-293
and CHO cells. Using the Flp-In system (Invitrogen),
we obtained clones of CHO and HEK-293 cells that
stably expressed the human wild-type B2KRs or eGFP
chimera thereof in high numbers exhibiting a Bmax of
⬃4.5 pmol/mg protein for B2KRs and B2eGFP in CHO
cells and 11 pmol/mg protein in HEK-293 cells and
values of dissociation constant (Kd) of 1.2–2.9 nM for
both cell types and both constructs. Surprisingly, however, the B2KRs and the eGFP chimera of both the
HEK-293 and the CHO cells responded to stimulation
with 1 ␮M BK with only very poor sequestration. These
data obviously deviated from those we had published
earlier with respect to CHO cells (1) (Fig. 1).
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kinin (108 Ci/mmol) was from Perkin Elmer Life Sciences
(Boston, MA). Peptides were bought from Bachem (Heidelberg, Germany). The primers were synthesized by Invitrogen
and delivered desalted and lyophylized. Plaque-forming unit
DNA polymerase was obtained from Stratagene Europe.
Roche (Mannheim, Germany) delivered Fugene and Invitrogen Hygromycin B and Blasticidin. Polylysine, captopril, 1.10
phenanthroline, and bacitracin were purchased from Aldrich
(Taufkirchen, Germany). Fetal calf serum, culture media,
and penicillin-streptomycin were delivered by PAA Laboratories (Cölbe, Germany). All other reagents were of analytic
grade and are commercially available.
Expression vectors. The sequence of the human B2 kinin
receptor, starting with the third encoded methionine, was
cloned into the BamHI and the XhoI sites of the pcDNA5/
FRT vector (where FRT represent Flp recombinant target)
from Invitrogen. The receptor sequence was preceded at the
NH2 terminus by either a single hemagglutinin tag (MGYPYDVPDYAGSA) or a double-tag sequence (MGRSHHHHHH-GYPYDVPDYAGSA) cloned into the HindIII and
BamHI site of the vector. To obtain lower expression levels of
the B2 kinin receptor (B2KR) in HEK-293, the cytomegalovirus (CMV) promotor sequence was removed from the
pcDNA5/FRT by cleavage with MfeI and HindIII and replaced by the simian virus 40 (SV40) promotor sequence
derived from the pFRT/lacZeo vector (Invitrogen) using standard PCR technology. Alternatively, the tagged B2KR coding
sequences were cloned into a modified pIRESneo vector from
Clontech. For B2eGFP chimera, the sequence for enhanced
green fluorescent protein (eGFP) taken from the pEGFP-C1
vector (Clontech) was added to the COOH terminus of the
B2KR with standard PCR methodology using a chimeric
primer that excluded the stop codon of the B2KR but included
the first methionine of the eGFP-construct. For transient
expression in COS-7 cells, the pCDNA3.1 (Invitrogen) was
used because the other vectors do not contain the SV40 origin
needed for plasmid amplification.
Cell culture. CHO cells were cultivated in MEM-␣ with
ribonucleosides and deoxyribonucleosides supplemented
with 10% fetal calf serum and penicillin-streptomycin. A
CHO host cell line harboring the FRT site was created with
the pFRT/lacZeo-vector (Invitrogen) according to the instructions of the supplier. HEK-293 cells were grown in DMEM
with 10% fetal calf serum and penicillin-streptomycin.
Transfection of these cells was done using Fugene (Roche) by
following the instructions of the producer [2 ␮g plasmid(s)
plus 5 ␮l Fugene per 6-well dish]. Single, stably transfected
clones were obtained after selection with either 300 ␮g/ml
hygromycin B (pcDNA5/FRT-vectors) or after selection with
G418 (800 ␮g/ml) for cells transfected with the pIRESneovector.
Cell transfection using electroporation. Four million cells
(HEK-293 or COS-7) detached with trypsin were mixed together with 10 ␮g DNA in Bio-Rad gene pulser cuvettes (0.4
cm) and electroporated at 260 V, ⬁ ⍀ , 960 ␮FD using a
Bio-Rad gene pulser. The cells from up to six electroporations
were pooled subsequently on a cell culture dish (10 cm),
trypsinized after 24-h incubation in normal culture medium,
and seeded on 12-well plates for binding experiments performed the next day.
[3H]BK binding studies. For the determination of dissociation constant Kd and receptor number Bmax, confluent
monolayers on 24-well plates were rinsed three times with
PBS and equilibrated on ice with incubation buffer (40 mM
PIPES, 109 mM NaCl, 5 mM KCl, 0.1% glucose, 0.05% BSA,
2 mM CaCl2, and 1 mM MgCl2, pH 7.4; degradation inhibitors: 2 mM bacitracin, 0.8 mM 1.10 phenanthroline, and 100
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EXPRESSION LEVELS AFFECT B2 RECEPTOR SEQUESTRATION
dence of ligand internalization, we tried to generate
clones with lower receptor expression. For this purpose, the pIRES expression system (Clontech) was employed. Usage of this expression vector, which inserts
itself rather randomly into the cell genome, resulted in
several stable CHO clones with receptor expression
levels below 1 pmol/mg protein. Internalization of
B2KRs in these low-expressing CHO cells no longer
showed a dependence on the concentration of [3H]BK
Internalization of [3H]BK by stably and highly overexpressed B2KRs. Internalization of [3H]BK in these
overexpressing HEK-293 cells (expressed as internalized radioactivity in percentage of specificly total
bound radioactivity) is strongly dependent on the concentration of ligand applied. Fast internalization occurred with low amounts of ligand (⬍1 nM), which
slowed down drastically with higher concentrations of
[3H]BK, from more than 80% internalization at 0.4 nM
[3H]BK after 5 min to as low as 10% at 10 nM [3H]BK
(Fig. 2A).
The apparent decrease of the internalization rate
was due in part to a much less pronounced decrease of
surface binding at higher concentrations. Whereas at a
low concentration of [3H]BK (0.4 nM) almost all surface-bound ligand was internalized within 5 min, at 8
nM of [3H]BK, there was still a considerable amount of
[3H]BK bound to the surface even after 30 min (Fig.
2B). As indicated in Fig. 2C, the uptake rate by which
the cells internalize the ligand is obviously limited,
because almost no difference exists in the amount of
internalized [3H]BK within the first 10 min when either 2 or 10 nM of [3H]BK were applied, despite the
fact that at equilibrium on ice, there was approximately three times more ligand bound to the surface at
the higher concentration (Fig. 2, B and C). Figure 3
shows a similiar concentration dependence of the internalization both for highly expressed B2KRs wild
types and B2eGFP-chimera in HEK-293 cells as well as
in CHO cells.
[3H]BK internalization in low-expressing CHO and
HEK-293 cells. To test the hypothesis that overexpression of the kinin receptors is responsible for the poor
receptor sequestration and the concentration depenAJP-Heart Circ Physiol • VOL
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Fig. 1. B2 kinin receptor (B2KR) sequestration in high-expressing
cells. Human embryonic kidney (HEK)-293 and Chinese hamster
ovary (CHO) cells, stably expressing high numbers of B2KRs, were
preincubated with 1 ␮M bradykinin (BK) for the indicated times at
37°C. Remaining surface binding was then determined at 0°C with
⬃2 nM [3H]BK as described in MATERIALS AND METHODS. The results
are given as means ⫾ SD of triplicate determinations. Curves shown
are representative of two [CHO; B2KR and B2 enhanced green
fluorescent protein (B2eGFP)] and three [HEK-293; B2KR (twice)
and B2eGFP] other experiments with similar results. CHO publlabeled curve obtained with a B2KR construct is taken from Faussner
et al. (1).
Fig. 2. Internalization rate in high-expressing cells strongly dependent on concentration of [3H]BK. HEK-293 cells, expressing high
amount of B2KRs, were preincubated with the indicated concentrations of [3H]BK for 90 min on ice. The internalization was started by
placing the plates in a water bath at 37°C. At the indicated times, the
internalization process was stopped, and surface-bound and internalized ligand were determined as described in MATERIALS AND METHODS. A: internalization of [3H]BK in percentage of total bound
[3H]BK; B: remaining surface binding; C: amount of internalized
[3H]BK in counts per minute (cpm) per well. The results are given as
means ⫾ SD of triplicate determination. A representative experiment of two experiments is shown.
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EXPRESSION LEVELS AFFECT B2 RECEPTOR SEQUESTRATION
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pmol/mg (COS-7) was apparently in the range of our
low-expressing cells. This dependence indicates that
most of the transiently transfected cells highly overexpressed the receptor. Yet, applying lower amounts of
[3H]BK (0.6 nM) in both cell lines gave rise to internalization curves similar to those seen for endogenously
expressing cells and for stable clones expressing lower
amounts of B2KRs.
DISCUSSION
(Fig. 4A) and led to internalization rates similar to
those described for endogenously expressing cells (7).
In contrast, we were not able to produce HEK-293
cells with low B2KR expression using the pIRES system. Therefore, we exchanged the strong CMV-promotor regulating the expression of the gene of interest in
the pcDNA5/FRT vector of the Flp-In system (Invitrogen) via PCR for the distinctly weaker SV40 promotor
(see MATERIALS AND METHODS). With this vector applied,
the Flp-In system enabled the production of identical
clones with lower expression (Bmax 4.2 pmol/mg protein, Kd ⫽ 2.26 ⫾ 0.16 nM). Internalization of [3H]BK
was totally independent of the ligand concentration
(Fig. 4B) in these new HEK-293 clones.
B2KR sequestration in low-expressing CHO and
HEK-293 cells. Both low-expressing CHO and HEK293 cells responded to stimulation with 1 ␮M BK with
rapid reduction of the receptor number on the cell
surface, reaching a plateau of remaining surface binding of under 30% within 10 min, similar to the results
published by us previously (Fig. 5).
Internalization of transiently expressed B2 kinin receptors. Transient transfection of cells with receptor
genes is a very common approach to studying receptorassociated mechanisms because results can be obtained rather quickly. However, this method usually
does not allow to discriminate between causes of the
radioactive binding activity: whether this activity derives from a few cells expressing high amounts or from
many cells expressing minor amounts of receptors. In
[3H]BK-internalization studies with transiently B2KRtransfected HEK-293 cells (Fig. 6A) and COS-7 (Fig.
6B), cells we observed a clear dependence of the internalization rate on the ligand concentration despite the
fact that Bmax with 1 pmol/mg protein (HEK-293) or 1.9
AJP-Heart Circ Physiol • VOL
Fig. 4. Internalization of [3H]BK in low-expressing cells. CHO cells
(A) and HEK-293 cells (B) expressing low amounts of B2KRs [⬍1
pmol/mg protein (CHO), 4.2 pmol/mg protein (HEK-293)] were
treated as described in Fig. 2. All time points represent the means ⫾
SD of triplicate determinations. For the representative experiments
shown, five (CHO) and two (HEK-293) other experiments giving
similar results were performed.
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Fig. 3. Internalization of [3H]BK in overexpressing CHO and HEK293 cells after 10 min at 37°C. Cell monolayers on 12-well plates
were incubated with the indicated amount of [3H]BK on ice for 90
min and thereafter placed in a water bath at 37°C for 10 min. The
amount of surface-bound and internalized [3H]BK was determined
as described in MATERIALS AND METHODS. Each experiment was done
in triplicate. Results are given as means ⫾ SD. The exact concentrations of [3H]BK used were the following: 0.4, 1.7, and 9.2 nM (HEK293; B2eGFP); 0.4, 2, and 10 nM (HEK; B2); 0.5, 2.1, and 10.2 nM
(CHO; B2 and B2eGFP).
Transfection of various mammalian cell lines such as
CHO, HEK-293, or COS cells, either transiently or
stably with receptor genes and their variants, is a
powerful and therefore widely applied tool for studying
the function of the different domains of GPCRs. The
same holds true for the (altered) behavior of these
receptors when they are coexpressed with other proteins. The effects that mutations, deletions, and truncations of these receptors exert on their regulation
often provide deeper insights into regulation mechanisms involving these receptors.
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EXPRESSION LEVELS AFFECT B2 RECEPTOR SEQUESTRATION
Stable and transient transfection often results in
receptor numbers per cell that are far above those
observed in endogenously expressing cells. The advantage of such an overexpression is the relatively low
background. However, it has to be made certain that
the overexpressed receptors do not differ too much in
their behavior from naturally expressed ones. For this
reason, experimental conditions have to be defined that
enable the correlation of results obtained from artificially expressed wild-type receptors with those from
naturally expressed ones, so that the probable different
behavior of mutated receptors may be interpreted
properly.
Using the Flp-In system, we obtained several clones
expressing high amounts of B2KR: 10–11 pmol/mg
protein in HEK-293 cells and 4.5 pmol/mg protein in
CHO cells. Similar high-expression levels in CHO cells
have been reported by others (15). Our results clearly
demonstrate that such an overexpression prevents the
internalization machinery from rapidly translocating
all occupied receptors. This can be deduced either from
the poor sequestration of B2KRs after stimulation with
saturating amounts of unlabeled agonist or from the
slow ligand internalization when concentrations are
used that lead to occupation of a major part of the
receptor compartment. This happens mostly in internalization assays in which (almost) saturating concentrations of tritiated ligands are used as opposed to
assays in which iodinated ligands are used. In the
latter, the ligand concentrations are usually far below
the Kd due to the high specific labeling.
The data presented indicate that the term “overexpression” has to be interpreted in the context of the cell
type used. Whereas a B2KR expression level of 4.2
pmol/mg protein still enabled fast internalization rates
in HEK-293 cells {even with almost saturating concentrations of [3H]BK (Fig. 4)}, a similar expression level
of 4.5 pmol/mg protein in CHO cells led to a clear
dependence of the internalization rate on the ligand
AJP-Heart Circ Physiol • VOL
Fig. 6. Internalization in transiently transfected cells. HEK-293
cells (A) and COS-7 cells (B) were transfected transiently by electroporation as described in MATERIALS AND METHODS. After 2 days, the
cells expressing ⬃1 pmol/mg protein (HEK) and 1.9 pmol/mg protein
(COS-7) were treated, and the internalization was determined as
described in Fig. 2. Each time point represents the means ⫾ SD of
triplicate determinations. One other experiment with HEK cells was
performed giving similar results.
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Fig. 5. Sequestration in low-expressing cells. CHO cells and HEK293 cells expressing low amounts of B2KR [⬍1 pmol/mg protein
(CHO) and 4.2 pmol/mg protein (HEK-293)] were treated as described in Fig. 1. All points represent the means ⫾ SD of triplicate
determinations. Six other experiments in CHO cells and one in
HEK-293 gave similar results. CHO publ-labeled curve is taken from
Faussner et al. (1). SV40, simian virus 40.
concentration applied (Fig. 3). This difference may be
caused by cell-type specific expression levels of proteins
involved in receptor sequestration, as indicated by the
different expression levels observed for ␤-arrestin in
CHO, COS-7, and HEK-293 cells (9). Although ␤-arrestins are involved in the sequestration of many
GPCRs via clathrin-coated pits (12), it is possible that
they do not play a role in the sequestration of the
human B2KR, because coexpression of the B2KR with a
dominant-negative ␤-arrestin mutant had no influence
on its sequestration (8). Although phosphorylation of
the B2KR is required for its sequestration (14), it is
rather unlikely that the phosphorylation by a specific
receptor kinase is the rate-limiting step in the sequestration of overexpressed B2KRs because this is a very
rapid reaction in which one kinase molecule can phosphorylate many receptors in a very short time. Because
there are several reports indicating that the B2KR is
sequestering via caveolae (3, 10), it is more probable
that the longer-lasting interactions with adaptor pro-
EXPRESSION LEVELS AFFECT B2 RECEPTOR SEQUESTRATION
AJP-Heart Circ Physiol • VOL
nalization assay could be used with a ligand concentration that enables the internalization rate independent of the ligand concentration; meaning that only as
many receptors are occupied and activated as the cell
can normally deal with. Because receptor wild types
and mutants often have quite different expression levels, the highest ligand concentration that still gives
rise to specific internalization rates might be considerably different for each, and therefore each has to be
checked separately, particularly when a reduction in
the internalization rate becomes obvious compared
with the wild type.
We highly appreciate the excellent technical work of Steffen
Schüssler and Cornelia Seidl.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to A. Faussner (FA 288/3-1).
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10. Ostrom RS. New determinants of receptor-effector coupling:
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11. Parnot C, Miserey-Lenkei S, Bardin S, Corvol P, and
Clauser E. Lessons from constitutively active mutants of G
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343, 2002.
12. Perry SJ and Lefkowitz RJ. Arresting developments in heptahelical receptor signaling and regulation. Trends Cell Biol 12:
130–138, 2002.
13. Pizard A, Blaukat A, Michineau S, Dikic I, Müller-Esterl
W, Alhenc-Gelas F, and Rajerison RM. Palmitoylation of the
human Bradykinin B2 receptor influences ligand efficacy. Biochem 40: 15743–15751, 2001.
14. Pizard A, Blaukat A, Müller-Esterl W, Alhenc-Gelas F, and
Rajerison RM. Bradykinin-induced Internalization of the hu-
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teins, either those involved in the translocation of the
B2KR into the caveolae or those in the following sequestration into intracellular compartments is the
rate-limiting step. So far, however, little is known
about the exact mechanism of GPCR sequestration via
caveolae and therefore little about the kinds of proteinprotein interactions that may be responsible for this
rate limitation.
Our results indicate that published data on reduced
internalization rates of receptor mutants have to be
carefully interpreted in particular when these data
were obtained from transiently transfected cells,
COS-7 cells in particular, or from stable clones with
very high expression. For example, it was reported that
at an expression level of 8.59 pmol/mg protein in COS-7
the B2 receptor double mutant C324A/C329A reveals a
30% reduction in internalization compared with the
wild type (13). Yet, we found a perfectly normal internalization behavior for this mutant in HEK-293 cells
with lower expression (not shown).
Cautious interpretation is also necessary when one
wants to draw conclusions concerning the recycling of
the B2KRs from differences between the receptor sequestration and ligand internalization rates (8). Because determination of receptor sequestration, i.e., the
change of the number of receptors on the cell surface
after exposure to an agonist, always involves the full
receptor complement, it has to be compared with ligand
internalization rates under identical conditions, i.e.,
with (almost) saturating ligand concentrations. Our
results show that under these conditions the relative
reduction of receptors on the cell surface of high- and
low-expressing cells (Figs. 1 and 5) corresponds well
with the relative amount of ligand internalized (Figs.
2A and 4, respectively), indicating that there is no
significant receptor recycling in the first 30 min after
receptor activation.
Whereas the present report deals only with the loss
of receptor sequestration in cells that overexpress
B2KRs, this loss might be a phenomenon that occurs in
other GPCRs systems, too. For example, the vasopressin receptor V1a responds to 20-min exposure to an
agonist with an 80% reduction of the number of receptors on the cell surface, when stably expressed in HEK293 cells (5). However, only a 30% reduction under
otherwise identical experimental conditions was observed when V1a is transiently expressed (4).
Another interesting aspect of the phenomenon described here concerns the possibility that these stably
overexpressing cells may represent partially sequestration-deficient cell lines. Therefore, one could use
these cell lines to identify proteins involved in receptor
internalization by transiently overexpressing these
proteins in these cells and trying to find out whether
they are capable of rescuing receptor-sequestration.
In summary, our results demonstrate that highly
expressed B2KRs in HEK and CHO cells no longer
show receptor-specific sequestration and internalization rates. To overcome this problem, usage of weaker
or inducible expression promotors to obtain lower expression levels might be useful. Alternatively, a inter-
H1897
H1898
EXPRESSION LEVELS AFFECT B2 RECEPTOR SEQUESTRATION
man B2 receptor requires phosphorylation of three serine and
two threonine residues at its carboxyl tail. J Biol Chem 274:
12738–12747, 1999.
15. Pizard A, Marchetti J, Allegrini J, Alhenc-Gelas F, and
Rajerison RM. Negative cooperativity in the human bradykinin
B2 receptor. J Biol Chem 273: 1309–1315, 1998.
16. Yu J, Prado GN, Taylor L, Piserchio A, Gupta A, Mierke
DF, and Polgar P. Global chimeric exchanges within the intracellular face of the bradykinin B2 receptor with corresponding
angiotensin II type Ia receptor regions: generation of fully functional hybrids showing characteristic signaling of the AT1a receptor. J Cell Biochem 85: 809–819, 2002.
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