The role of helix 8 and of the cytosolic C-termini in
the internalization and signal transduction of B
1
and B
2
bradykinin receptors
Alexander Faussner
1
, Alexandra Bauer
1
, Irina Kalatskaya
1
, Steffen Schu
¨
ssler
1
, Cornelia Seidl
1
,
David Proud
2
and Marianne Jochum
1
1 Ludwig-Maximilians-Universita
¨
t, Abteilung fu
¨
r Klinische Chemie und Klinische Biochemie, Mu
¨
nchen, Germany
2 Department of Physiology & Biophysics, University of Calgary, Alberta, Canada

bradykinin receptor (B
2
wt), stably
expressed in HEK 293 cells, identified a sequence distal to N338
(NSMGTLRTSI, including I347 but not the basally phosphorylated S348)
and in particular the TSI sequence therein, as a major determinant for
rapid agonist-inducible internalization and the prevention of receptor
hypersensitivity. Chimeras of the noninternalizing B
1
bradykinin receptor
(B
1
wt) containing these B
2
wt sequences sequestered poorly, however, sug-
gesting that additional motifs more proximal to N338 are required. In fact,
further substitution of the B
1
wt C-terminus with corresponding B
2
wt
regions either at C330(7.71) following putative helix 8 (B
1
CB
2
) or at the
preceding Y312(7.53) in the NPXXY sequence (B
1
YB
2

B
1
CB
2
, suggesting that putative helix 8 is either directly or indirectly
(e.g. via G protein activation) involved in the interaction between the
receptor and receptor kinases.
Abbreviations
BK, bradykinin; B
x
wt, wild-type B
x
bradykinin receptor; DAK, desArg10kallidin; GPCR, G protein-coupled receptor; GRK, G protein-coupled
receptor kinase; HEK, human embryonic kidney; IP, inositol phosphate.
FEBS Journal 272 (2005) 129–140 ª 2004 FEBS 129
G protein uncoupling are essential. Some of these
desensitization mechanisms involve the translocation
of the stimulated receptor to distinct compartments
and endocytosis after phosphorylation of serine ⁄ threo-
nine residues mostly located in the receptor C-termini
(reviewed in [2]). Little is known so far about the
sequence context in which these residues have to
appear to become phosphorylated by kinases and to
be recognized by the internalization machinery. In par-
ticular, the receptor specificity of these motifs is not
completely understood.
The B
1
bradykinin receptor (B
1

b
-mediated inositol phosphate (IP) release leading to
an elevation of intracellular [Ca
2+
] levels, primarily via
coupling to G protein G
q ⁄ 11
[3,10,11].
They become activated by the kinins, small pro-
inflammatory peptides with great vasoactive potential
implicated as mediators of inflammation, pain and
hyperalgesia [12,13]. The nonapeptide BK and Lys-BK
(kallidin) bind with high affinity to B
2
wt but not B
1
wt.
Removal of the C-terminal arginine through carboxy-
peptidases generates desArg9-bradykinin and desArg10-
kallidin (DAK), two peptides that now bind exclusively
to the B
1
wt [14].
In this study we wanted to exploit the fact that the
B
1
wt does not internalize as part of a gain-of-function
approach to provide insight into the receptor speci-
ficity of the B
2

minimally required for internalization we created two
new B
2
wt truncations, I347* and N338* (Fig. 1). The
former removed the C-terminus including residue
S348, which has been shown to be responsible for the
basal phosphorylation of the B
2
wt, while the latter
truncation deleted all serine and threonine residues
(S339, T342, T345, S346) shown to be phosphorylated
following stimulation of the receptor [17]. In addition,
a triple alanine replacement of
T345-S346-I347(S348)
(mutated residues are underlined) was made, as this
sequence strongly resembles the C-terminal STLS-
motif in the AT
1A
angiotensin II receptor, where a tri-
ple alanine substitution of STL almost completely
abolished receptor sequestration [18].
All of these B
2
receptor constructs were highly
expressed (Table 1). We took care therefore to use
[
3
H]BK concentrations below 1.5 nm, as we have
shown that receptor internalization rates are independ-
ent of agonist concentration in this range [19]. The

H]BK much slower than the B
2
wt, N338* neverthe-
less was able to induce an accumulation of total IPs
identical to that observed for the B
2
wt (Table 1). This
truncated receptor even became hypersensitive, as its
EC
50
for the IP response was 10-fold lower than
that of B
2
wt (0.072 ± 0.038 nm vs. 0.79 ± 0.34 nm;
Table 1). Most interestingly, the effects of a truncation
at N338 could also be achieved in part by the triple
mutation TSIfiAAA as this construct displayed sim-
ilar properties to truncation N338*. It exhibited a
markedly reduced capacity to internalize [
3
H]BK albeit
not as diminished as truncation N338* and was at
least as hypersensitive with an EC
50
¼ 0.058 ±
0.06 nm (Table 1). This sequence obviously contributes
significantly to agonist internalization and signaling of
B
2
wt.

H]BK (Fig. 2B).
As it was obviously not sufficient to simply add the
B
2
wt phosphorylation sites to the B
1
wt to gain full
receptor sequestration as observed in the B
2
wt, we fur-
ther substituted the C-termini of the B
2
wt into the
B
1
wt at two residues conserved in both receptor sub-
types (Fig. 1); specifically at the conserved cysteine
[Cys330(7.71) in B
1
wt, Cys324(7.72) in B
2
wt] that in
the B
2
wt is palmitoylated (chimera B
1
CB
2
) and at
Y7.53 within the NPXXY sequence (chimera B

wt is indicated.
A. Faussner et al. Role of helix 8 and C-termini in bradykinin receptors
FEBS Journal 272 (2005) 129–140 ª 2004 FEBS 131
at the end of the seventh transmembrane domain. We
have shown previously that a B
1
CB
2
chimera stably
expressed in Chinese hamster ovary cells was seques-
tered rapidly upon activation [16]. This was confirmed
in human embryonic kidney (HEK) 293 cells (Fig. 2B).
As the chimera B
1
YB
2
exhibited a slightly attenuated
internalization compared to B
1
CB
2
(Fig. 2B), and the
latter apparently did not gain the full internalization
capability of the B
2
wt, we next tested the possibility
that there is an optimum site for creating rapidly inter-
nalizing chimeras at K7.63 between these two residues
and generated the chimera B
1

wt and B
2
wt. The
different internalization of B
1
KB
2
and B
1
CB
2
was
therefore even more surprising given that these two
chimeras have only minor sequence differences
(Fig. 3A). Therefore we considered three possibilities
to explain the cause of this drop in the internalization
of B
1
KB
2
as compared to B
1
CB
2
. First, that the two
residues (KQ) preceding the cysteine were pivotal; sec-
ond, that the cysteine itself needs to be at a specific
position in the C-terminus; or third, that the B
1
residue

KB
2
⁄ VCfiCV (Fig. 3B).
A major effect, however, was seen with the change
of the polar serine (back) to the nonpolar valine
(B
1
KB
2
⁄ SfiV), the amino acid that is normally found
in this position in the B
1
wt. This replacement led to a
chimera exhibiting rapid internalization (60% after
10 min) that was comparable to that of B
1
CB
2
and
B
1
YB
2
(Fig. 2B).
Phosphorylation patterns of B
2
wt and of B
1
⁄ B
2

max
), receptor affinity (K
d
), basal and stimulated total IP accumulation, and EC
50
of B
2
wt, B
1
wt and B
1
⁄ B
2
receptor chimera. ND, not determined.
Receptor construct
B
max
a
(fmolÆmg protein
)1
)
K
d
(nM)
IP accumulation
EC
50
± SEM
(n
M)Unstimulated (30 minÆbasal

1823 ± 664 ND 1.84 ± 0.25 4.1 ± 0.2
b
(n ¼ 5) 0.7 ± 0.3 (n ¼ 3)
B
1
KB
2
⁄ SfiV 1758 ± 150 1.59 ± 0.44 1.31 ± 0.12 7.2 ± 0.8 (n ¼ 3) 1.7 ± 0.2 (n ¼ 3)
B
1
KB
2
⁄ QGVfiKQ 2142 ± 623 ND 1.33 ± 0.12 4.6 ± 0.9
b
(n ¼ 3) 2.0 ± 0.2 (n ¼ 3)
B
1
KB
2
⁄ VCfiCV 1786 ± 320 ND 1.42 ± 0.06 4.3 ± 0.1
b
(n ¼ 3) 0.8 ± 0.1 (n ¼ 3)
B
1
YB
2
2957 ± 1041 1.85 ± 1.4 1.50 ± 0.15 8.7 ± 0.8 (n ¼ 6) 2.2 ± 0.2 (n ¼ 3)
B
1
V323S 846 ± 128 ND 1.44 ± 0.08 4.59 ± 0.84

37 °C compared to the IP content of control cells that
had remained at 4 °C. There was a clear correlation
between the agonist-inducible internalization and the
IP accumulation it could induce when stimulated
(Fig. 5). All chimeric constructs displaying rapid agon-
ist-inducible internalization (B
1
CB
2
,B
1
YB
2
,B
1
KB
2
⁄
SfiV) showed an IP response similar to that seen for
B
1
wt (8.41 ± 0.52 fold for B
1
wt and 7.2–8.7-fold for
the chimera). In contrast, the chimera that internalized
poorly (B
1
KB
2
,B

have to appear, or the receptor specificity of their
function is not very well understood.
0 5 10 15 20 25 30
0
20
40
60
80
100
Internalization [% of total]Internalization [% of total]
N338*
TSI->AAA
B
2
wt
I347*
A
Time [min]
0 5 10 15 20 25 30
0
20
40
60
80
100
B
1
RB
2
B

wt truncations
or mutations were preincubated with the appropriate[
3
H] agonist:
(A) < 1.5 n
M [
3
H]BK; (B) 2 nM[
3
H]DAK) for 90 min on ice. Internal-
ization was started by placing the cells in a 37 °C water bath and
stopped at the indicated times. Surface-bound and internalized
agonist were determined as described in Material and methods.
Agonist internalization was expressed as percentage of total bound
agonist. Results are given as mean ± SEM of at least three inde-
pendent experiments performed in triplicate.
Fig. 3. [
3
H]DAK internalization of B
1
KB
2
derived constructs. (A) Align-
ment of the relevant sequences of the B
1
CB
2
and B
1
KB

1
wt does not become
sequestered [3]. As both receptors couple preferentially
to the same Ga subunit (G
q ⁄ 11
) differential signaling is
less likely to explain differences in internalization than
in two receptors signaling through different G proteins.
Internalization patterns of truncations I347* and
N338*, and the triple point mutant TSIfiAAA more
closely defined the sequence necessary for the internal-
ization of B
2
wt. Because I347* was internalized as rap-
idly as B
2
wt, while the TSIfiAAA mutant showed
reduced internalization and N338* almost none, the
nine residues from S339 to I347 (SMGTLRTSI) must
play a key role in B
2
wt sequestration. The following
results from our gain-of-function approach, however,
led us to conclude that additional motifs in the more
proximal portion of the C-terminus also play a role
in receptor internalization. First, transfer of the B
2
wt
C-terminus starting with the nine residues containing
all known B

YB
2
showed a slightly lower internal-
ization compared to B
1
CB
2
. We therefore tested whe-
ther there was an optimum chimeric exchange point
between these two mutation sites. Intriguingly,
exchange at a conserved lysine (K7.68) between these
two sites resulted in a poorly internalizing chimera
(B
1
KB
2
, Fig. 2B). The crystal structure of inactive
bovine rhodopsin [24] suggested an explanation for this
result by revealing an additional helix 8 close to the
seventh transmembrane domain with a cytosolic local-
ization parallel to the cell membrane. Structure predic-
tion programs [25] indicated that both B
1
wt and B
2
wt
may also contain a helix 8. Our results show that chi-
control
Mr
75

1
⁄ B
2
-chi-
mera. Upper panel: HEK293 cells expressing B
2
wt, B
1
YB
2
,B
1
KB
2
,
or B
1
CB
2
were labeled for 10 h with [
32
P]orthophosphate before sti-
mulation with 1 l
M BK and 1 lM DAK, respectively, for 5 min. Cells
were lysed and proteins were solubilized, immunoprecipitated and
visualized by autoradiography. Molecular size markers are indicated
to the left. Lower panel: protein phosphorylation, given as optical
densities of the bands in the area between 50 and 85 kDa, is pre-
sented as mean ± SD from three independent experiments; un-
stimulated B

1
KB
2
/S
V
B
1
V323S
KQ
2
4
6
8
10
12
Total inositol phosphate
release/control
unstimulated
stimulated
***
***
***
***
Fig. 5. Total IP accumulation of B
1
wt and chimera. HEK293 cells
expressing the indicated receptor constructs were preincubated
with 50 m
M LiCl, and then with (stimulated) or without (unstimula-
ted) 1 l

1
YB
2
– this is probably caused by an
impaired interaction with, or activation of, receptor
kinases resulting in the observed slow internalization.
Further examination of helix 8 revealed that S316 in
the B
2
wt sequence of B
1
KB
2
is responsible for the slow
sequestration of this chimera (Fig. 3B). Helices 8 of
the two receptor subtypes show different charge distri-
butions despite their high sequence identity (Fig. 6).
The B
2
wt exhibits a highly charged N-terminal half
(two arginines and three lysines) but due to S316 does
not display a clear amphipathic structure. The N-ter-
minal half of B
1
wt, by contrast, is less positively
charged (two arginines and one lysine) but B
1
wt has
a strict amphipathic arrangement of the amino acid
residues. This arrangement is probably important in

at position 323 in B
1
wt is not necessary for G
q ⁄ 11
activa-
tion but rather that a polar serine there interferes with
this process. This group also described a strongly
increased basal activity for a B
2
receptor construct
where several C-terminal serine and threonine residues
Fig. 6. Structural comparison of helix 8 in B
1
wt, B
2
wt and chimera. Helix 8 (N-terminus on the left hand side) from both bradykinin receptor
subtypes was modeled along the structure of bovine rhodopsin by means of
DEEPVIEW ⁄ Swiss-PdbViewer v3.7 [34]. The dark green ribbon
presentation belongs to B
1
wt, light green ribbon-parts to B
2
wt. The residues different in B
1
wt and B
2
wt are indicated in larger bold labels.
Basic amino acid residues are in blue, acidic residues in red, polar residues are yellow, and unpolar residues are colored in grey. The black
lines in B
1

d
val-
ues were not significantly different this indicates that
apparently relatively few receptors have to be occupied
to achieve half-maximal stimulation. It is important, of
course, to keep in mind that the K
d
was determined at
4 °C where coupling to G proteins does not play a role,
whereas the EC
50
was obtained by determining the IP
accumulation after 30 min at 37 °C. Nevertheless, it is
likely that this hypersensitivity is related to the fact that
the mutated residues play an important role in the inter-
nalization (Fig. 2A) and in the desensitization of B
2
wt
[17]. Much lower BK concentrations than with B
2
wt
may therefore be sufficient to activate enough receptors
for half-maximal IP accumulation.
In our experiments, we did not observe a strong
constitutive B
1
wt signaling activity as compared to
B
2
wt, nor any significant differences between the B

by rhodopsin [29]. In the angiotensin II receptor
AT
1A
point mutations in the region of putative helix 8
abolished release of inositol trisphosphates and the
GTP-inducible shift in receptor affinity. In addition,
peptides based on its helix 8 sequence stimulated bind-
ing of GTPcStoG
q ⁄ 11
[30]. All of these data point to
an involvement of putative helix 8 in the interaction
with cognate G proteins. As both bradykinin receptors
coupled to the same G
a
subunit G
q ⁄ 11
the different IP
responses obtained with the wild-type receptors and
the chimera let us speculate that each wild-type helix 8
may be specific either for selected bc subunits or for
either G
q
or G
11
. Additional experimental work will be
necessary to test this hypothesis, particularly as the
two receptors, while both coupling to G
q ⁄ 11
(and G
i

cal structure. Mutation of specific residues in their
model led to a strongly reduced propensity for helical
formation with the N-terminus of helix 8 being more
influential than the C-terminal portion. Based on this
model, we could speculate that the two bradykinin
receptor subtypes, and those chimeras with an
intact ⁄ homogenous helix 8, are able to appropriately
switch conformation, whereas the receptors with a chi-
meric helix 8 have lost this capacity.
Taken together, our results demonstrate that almost
full capability for receptor internalization can be con-
ferred to the normally noninternalizing B
1
wt, via trans-
fer of the C-terminus of B
2
wt, provided that the new
chimeric receptors have an intact ⁄ homogeneous helix 8
either from B
2
wt or B
1
wt or a chimeric B
1
⁄ B
2
helix
with a conserved V323. Chimeric receptors with a het-
erogeneous helix 8 exhibited an identical effect on sign-
aling as well as on internalization, i.e. poor signaling

¨
ller-Esterl (University of Frankfurt,
Germany). Unlabeled peptides were bought from Bachem
(Heidelberg, Germany). The primers were synthesized by
Invitrogen and delivered desalted and lyophilized. Pfu DNA
polymerase was obtained from Stratagene Europe (Heidel-
berg, Germany). Fetal bovine serum, culture media, and
penicillin ⁄ streptomycin were purchased from PAA Labor-
atories (Co
¨
lbe, Germany). Fugene 6 was from Roche
(Mannheim, Germany) and Invitrogen supplied hygromycin
B and blasticidin. Poly(lysine), captopril, 1.10-phenanthro-
line and bacitracin were purchased from Aldrich (Taufkir-
chen, Germany). Ion exchange columns AG 1 · 8 (formiate
form) were bought from Bio-Rad (Munich, Germany). All
other reagents were of analytical grade and are commer-
cially available.
Cell culture
HEK 293 cells, host cells harboring an Flp recombinant
target (FRT) site in their genome, were cultivated in Dul-
becco’s modified Eagle’s medium (DMEM) with high glu-
cose, 10% (v ⁄ v) fetal bovine serum and 100 UÆmL
)1
⁄
100 lgÆmL
)1
penicillin ⁄ streptomycin. For binding studies or
the measurement of total inositol phosphate accumulation
cells were seeded on cell culture dishes pretreated with

wt and
of the B
1
⁄ B
2
receptor chimera
Standard PCR techniques using either receptor-specific or
chimeric primers with the B
1
wt and B
2
wt genes as templates
were applied to generate truncated or point-mutated ver-
sions of the B
1
wt, B
2
wt and several B
1
⁄ B
2
chimeras. All
PCR products were ligated between the BamHI and XhoI
sites of the pcDNA5 ⁄ FRT vector. Cells were transfected
using Fugene 6 following the manufacturer’s instructions,
i.e. 2 l g plasmids (0.4 lg gene of interest in pcDNA5 ⁄ FRT
plus 1.6 lg pOG44-vector) and 5 lL Fugene 6 per six-well
dish. Stably transfected clones were obtained after selection
with 250 lgÆmL
)1

3
H]BK (10 concentrations ranging from 0.01 to  40 nm)
or [
3
H]DAK (0.01–10 nm) for at least 90 min. The incuba-
tion was stopped by rinsing the monolayers three times
with ice-cold PBS and lysing the monolayers by addition
of 0.2 mL of 0.3 m NaOH. The bound radioactivity was
transferred quantitatively into scintillation vials with
another 0.2 mL of water and measured in a b-counter
after addition of scintillation fluid. Nonspecific binding
was determined in the presence of 5 lm unlabeled agonist
and subtracted from the total binding to calculate the spe-
cific binding.
Internalization of [
3
H]BK and [
3
H]DAK
To determine the internalization of receptor-bound agonist,
cell monolayers on 12-well plates were rinsed three times
with ice-cold PBS (pH 7.2) and incubated with the indi-
A. Faussner et al. Role of helix 8 and C-termini in bradykinin receptors
FEBS Journal 272 (2005) 129–140 ª 2004 FEBS 137
cated concentration of [
3
H]agonist in 0.3 mL incubation
buffer on ice to reach equilibrium binding. To start the
internalization of [
3

3
H]inositol in 0.5 mL
DMEM with fetal bovine serum and 100 UÆmL
)1
⁄ 100 lgÆ
mL
)1
penicillin ⁄ streptomycin. The monolayers were then
placed on ice, rinsed three times with ice-cold PBS (pH 7.2)
and incubated with or without the appropriate agonist in
incubation buffer containing 50 mm LiCl. Basal and stimu-
lated IP accumulation was started by placing the tray in a
water bath at 37 °C for 30 min. It was stopped by exchan-
ging the buffer with 0.75 mL of ice-cold 20 mm formic acid
and by transferring the tray onto ice for additional 30 min.
As a baseline control one tray was left on ice with LiCl incu-
bation buffer without agonist. The EC
50
was determined by
adding escalating concentrations of agonist (10
)12
to 10
)6
m)
for 30 min at 37 °C. The supernatant was then applied
together with another 0.75 mL of 20 mm formic acid and
0.2 mL of a 3% (w ⁄ v) ammonium hydroxide solution to AG
1-X8 anion exchange columns, followed by 1 mL 1.8% (w ⁄ v)
ammonium hydroxide solution, 9 mL of 60 mm sodium
formiate, 5 mm sodium tetraborate buffer and 0.5 mL of 4 m

Tris-buffered saline with 0.1% (v ⁄ v) Tween 20 (TBST) fol-
lowed by addition of the corresponding secondary peroxi-
dase-labeled rabbit anti-rat Ig (1 : 1000) for 1 h. After
washing in TBST three times each for 15 min antibody
binding was detected using the Western Blot Chemolumi-
nescence Reagent Plus.
Receptor phosphorylation
Confluent cells on 6-well plates were washed twice with
phosphate-free DMEM, incubated for 3 h at 37 °C in the
same medium, and labeled with 0.2 mCiÆmL
)1
[
32
P]ortho-
phosphate for 10–12 h. After exposure to 1 lm BK or
DAK for 5 min at 37 °C, monolayers were scraped into
0.5 mL of RIPA buffer containing protease inhibitors (see
above) and phosphatase inhibitors (25 mm NaF, 1 mm
sodium orthovanadate, 0.3 lm okadaic acid). Immunopre-
cipitation and separation on a 10% (w ⁄ v) SDS polyacryl-
amide gel were carried out as described previously. The
proteins of interest were electroblotted onto nitrocellulose
membranes and identified by autoradiography.
Protein determination
Total protein per well was quantified by lysing the cells
with 0.3 mL of 0.3 m NaOH. The protein content of this
solution was determined with the Micro BCA Protein assay
reagent from Pierce (Rockford, IL, USA) using bovine
serum albumin as standard.
Data analysis

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