Báo cáo khoa học: Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation pptx

Functional analysis of cell-free-produced human
endothelin B receptor reveals transmembrane segment 1
as an essential area for ET-1 binding and homodimer
formation
Christian Klammt
1
, Ankita Srivastava
2
, Nora Eifler
3
, Friederike Junge
1
, Michael Beyermann
4
,
Daniel Schwarz
1
, Hartmut Michel
2
, Volker Doetsch
1
and Frank Bernhard
1
1 Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt ⁄ Main, Germany
2 Max-Planck-Institute for Biophysics, Department of Molecular Membrane Biology, Frankfurt ⁄ Main, Germany
3 M.E. Mueller Institute for Microscopy, Biocentre, University of Basel, Switzerland
4 Leibniz-Institute of Molecular Pharmacology, Department of Peptide Chemistry & Biochemistry, Berlin, Germany
Keywords
cell-free expression; detergent micelles;
endothelin-1 ligand-binding site; G-protein
coupled receptor; single-particle analysis

Correspondence
F. Bernhard, Centre for Biomolecular
Magnetic Resonance, Institute for
Biophysical Chemistry, University of
Frankfurt ⁄ Main, Max-von-Laue-Str. 9,
D-60438 Frankfurt ⁄ Main, Germany
Fax: +49 69 798 29632
Tel: +49 69 798 29620
E-mail:
(Received 27 March 2007, revised 26 April
2007, accepted 27 April 2007)
doi:10.1111/j.1742-4658.2007.05854.x
The functional and structural characterization of G-protein-coupled recep-
tors (GPCRs) still suffers from tremendous difficulties during sample
preparation. Cell-free expression has recently emerged as a promising alter-
native approach for the synthesis of polytopic integral membrane proteins
and, in particular, for the production of G-protein-coupled receptors. We
have now analyzed the quality and functional folding of cell-free produced
human endothelin type B receptor samples as an example of the rhodop-
sin-type family of G-protein-coupled receptors in correlation with different
cell-free expression modes. Human endothelin B receptor was cell-free pro-
duced as a precipitate and subsequently solubilized in detergent, or was
directly synthesized in micelles of various supplied mild detergents. Purified
cell-free-produced human endothelin B receptor samples were evaluated by
single-particle analysis and by ligand-binding assays. The soluble human
endothelin B receptor produced is predominantly present as dimeric com-
plexes without detectable aggregation, and the quality of the sample is very
similar to that of the related rhodopsin isolated from natural sources. The
binding of human endothelin B receptor to its natural peptide ligand endo-
thelin-1 is demonstrated by coelution, pull-down assays, and surface plas-

mon resonance assays. Systematic functional analysis of truncated human
endothelin B receptor derivatives confined two key receptor functions to
the membrane-localized part of human endothelin B receptor. A 39 amino
acid fragment spanning residues 93–131 and including the proposed trans-
membrane segment 1 was identified as a central area involved in endo-
thelin-1 binding as well as in human endothelin B receptor homo-oligomer
formation. Our approach represents an efficient expression technique for
G-protein-coupled receptors such as human endothelin B receptor, and
might provide a valuable tool for fast structural and functional
characterizations.
Abbreviations
bET-1, biotinylated endothelin-1; C, cytoplasmic; CECF, continuous exchange cell-free; cET-1, Cy3-labeled endothelin-1; CF, cell-free;
CTD, C-terminal domain; E, extracellular; ETB, human endothelin B receptor; ET-1, endothelin-1; GPCR, G-protein-coupled receptor; LMPG,
1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; NTD, N-terminal domain; RM, reaction mixture; SPR, surface plasmon
resonance; TMS, transmembrane segment.
FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3257
G-protein-coupled receptors (GPCRs) form a large
superfamily of membrane proteins, and their genes
comprise an estimated 1–5% of vertebrate genomes.
They modulate the activity of specific targets such as
ion channels or enzymes via G-protein coupling, and
thus initiate intracellular signaling cascades in response
to a broad range of external signals [1,2]. GPCRs have
a similar architecture, composed of seven transmem-
brane segments (TMS1–7) connected by three extracel-
lular (E1–3) and three cytoplasmic (C1–3) loops. In
addition, GPCRs contain a more or less extended
N-terminal domain and C-terminal domain, which are
both often involved either in ligand binding or G-pro-
tein coupling.

Owing to their key role in signal transduction in
eukaryotic cells, GPCRs are estimated to represent the
targets for more than 50% of modern pharmaceutical
drugs [3]. Despite much investigation, the high-resolu-
tion structural evaluation of GPCRs as a prerequisite
for directed drug design is so far still limited to the
naturally abundant phototransducer rhodopsin [4]. As
for many other membrane proteins, the first bottleneck
in structural and functional characterization of GPCRs
is the production of sufficient amounts of protein sam-
ple. Although considerable improvements have been
made, the overproduction of GPCRs in cellular expres-
sion systems based on bacterial, yeast or insect cells is
still complicated and often inefficient [5–11].
Continuous exchange cell-free (CECF) expression
systems based on Escherichia coli cell extracts have
recently been demonstrated to provide a new and
highly promising tool for the preparative-scale produc-
tion of membrane proteins [12–14]. Besides the elimin-
ation of toxic effects upon membrane protein
overproduction, a unique advantage of CECF systems
is the possibility of directly producing soluble mem-
brane proteins in the presence of detergents
[12,13,15,16]. This completely new strategy provides an
artificial hydrophobic environment that is able to inter-
act with membrane proteins during translation. Protein
precipitation is prevented, and functional folding path-
ways can be facilitated [17,18]. The protein–detergent
association is initiated by hydrophobic interactions,
and specific targeting or translocation systems are

therefore not necessary.
The endothelin (ET) system is involved in many
physiologic processes, such as control of vascular tone,
neurotransmission, embryonic development, renal
function, and regulation of cell proliferation, and it
thus plays an important role in physiopathologic disor-
ders such as congestive heart failure, diabetes, athero-
sclerosis, and primary pulmonary hypertension [19–21].
The human ET receptor type B (ETB) is a prototypic
GPCR distributed among multiple endothelial cell
types as well as in smooth muscle cells, where it trans-
mits vasoactive effects by binding the 21-mer isopep-
tides ET-1, ET-2, and ET-3. ETB has equally potent
affinities for ET-1, ET-2 and ET-3, in contrast to the
homologous ET receptor type A, which has a higher
affinity for ET-1 and ET-2.
We have established protocols for the high-level pro-
duction of ETB and other GPCRs in an individual
CECF system [22]. The GPCRs can be synthesized as
precipitates or in soluble form in micelles of selected
detergents, and apart from small terminal peptide tags
that facilitate detection and purification, no large
fusion proteins are needed for expression and stabiliza-
tion. The functional folding of membrane proteins
overproduced by the new cell-free (CF) approach is of
primary interest, and we therefore further analyzed the
quality of ETB samples obtained after CF production
under different conditions. Ligand-binding and oligo-
mer formation studies demonstrated that CF-produced
ETB is functionally folded when synthesized in the

presence of Brij detergents, and single-particle analysis
revealed nonaggregated proteins that predominantly
form dimeric complexes. On the basis of the functional
in vitro analysis of rationally designed terminal ETB
truncations, we specified a core domain responsible for
ET-1 binding as well as for receptor dimerization in a
relatively small region centered on TMS1.
Results
CF production of ETB
CECF reaction protocols were essentially performed as
previously described [22]. Full-length ETB
cHx
and trun-
cated derivatives were produced as translational
fusions with the small 12 amino acid T7-tag at the
N-terminus and a poly(His)10-tag at the C-terminus
(Fig. 1). To obtain the highest yields of the individual
constructs, optimization of the Mg
2+
and K
+
concen-
trations in the ranges 12–16 mm and 250–340 mm was
critical. Under optimized conditions, ETB
cHx
could by
synthesized in yields of up to 3 mg per mL of reaction
mixture (RM), and it was separated as a prominent
band of c. 46 kDa by SDS ⁄ PAGE (Fig. 2). The full-
length synthesis of ETB

cHx
was verified by immunode-
tection with antibodies directed against the N-terminal
T7-tag and the C-terminal poly(His)10-tag, respectively
(data not shown).
ETB
cHx
was CF produced either as a soluble protein
in the presence of detergents or as a precipitate in the
absence of detergents. We analyzed the yield and
sample quality of ETB synthesized in the presence of
Ligand binding of cell-free endothelin B receptor C. Klammt et al.
3258 FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS
digitonin and long-chain Brij derivatives, as these
detergents have been most effective for the soluble
expression of GPCRs [15,22]. In the presence of 1%
Brij78 and 1.5% Brij58, completely soluble ETB
cHx
with final yields of c. 3 mg proteinÆ(mL RM)
)1
was
produced. With Brij35 (0.1%) and digitonin (0.4%),
only 500 lg and 100 lg of soluble ETB
cHx
per mL of
RM were obtained, respectively, in addition to ETB
cHx
precipitate. The soluble ETB
cHx
produced was purified

in one step by Ni-chelate chromatography, and, on
average, c. 60% of the synthesized ETB
cHx
in the RM
was recovered. ETB
cHx
precipitate CF produced in the
absence of detergents was completely solubilized in
1% 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-
(1-glycerol)] (LMPG) and purified by Ni-chelate chro-
matography.
Coelution of purified ETB with ET-1
The mode of CF expression, i.e. expression as precipi-
tate or as soluble protein, as well as the type of
Fig. 1. Proposed secondary structure of ETB. The proposed seven TMSs are illustrated. Various tags used for the modification of CF-pro-
duced ETBs are indicated. Predicted sites for post-translational modifications are a glycosylation site at N59, a disulfide bridge connecting E1
and E2, and several palmitoylation sites at cysteine residues in the CTD. The first (black) and last (gray) amino acid positions used for the
construction of truncated ETB fragments are indicated.
Fig. 2. PAGE analysis of CF-expressed full-length ETB on a 12%
SDS gel. M, marker. Lane 1: RM control. Lane 2: supernatant of
ETB
cHx
expression in the presence of Brij58. Lane 3: supernatant
of ETB
cHx
expression in the presence of Brij78. Lane 4: ETB
cHx
after Ni–chelate acid chromatography. Lane 5: supernatant of
ETB
Strep

expression in the presence of Brij78. Lane 6: ETB
Strep
after
Strep-Tactin purification. Lane 7: ETB
cHx
precipitate after expression
without detergent. Lane 8: ETB
cHx
precipitate solubilized in 1%
LMPG. Lane 9: ETB
cHx
precipitate solubilized in 1% LMPG after
Ni–chelate acid chromatography. ETB is marked by arrows.
C. Klammt et al. Ligand binding of cell-free endothelin B receptor
FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3259
detergent, could have a significant impact on the fold-
ing of ETB
cHx
into a functional conformation. The
affinity for its natural peptide ligand ET-1 was there-
fore analyzed with purified ETB
cHx
samples produced
under different conditions. Mixtures of the Cy3-dye-
labeled derivative Cy3-labeled endothelin-1 (cET-1)
with purified ETB
cHx
, either CF produced as precipi-
tate and solubilized in 1% LMPG or directly produced
as soluble protein in the presence of 1.5% Brij58, 1%

Brij78 or 0.4% digitonin, respectively, were separated
by gel filtration, and the elution fractions were analyzed
by taking advantage of the different absorbancies of the
two compounds (Fig. 3). The 52 kDa ETB
cHx
elutes at a
retention volume of 1.6 mL, whereas the 21-mer cET-1
starts to elute at a volume of 2.1 mL. Coelution of
cET-1 with ETB
cHx
therefore indicates complex forma-
tion of the receptor with its ligand, giving evidence of a
native protein conformation. In contrast, CF-produced
ETB
cHx
present in an unfolded or inactive conformation
should result in the separation of the two compounds.
cET-1 was completely separated from ETB
cHx
sam-
ples that were CF produced as precipitate and solubi-
lized in LMPG, indicating that, despite solubilization,
the receptor might not have adopted its native confor-
mation. In contrast, significant amounts of cET-1 co-
eluted with ETB
cHx
synthesized in the soluble mode of
CF expression in the presence of digitonin, Brij58 and
Brij78. The highest apparent binding of cET-1 was
obtained with protein CF expressed in the presence of

Brij78. This expression condition was therefore chosen
for further sample preparations of ETB
cHx
and its
derivatives.
Fig. 3. Functional conformation of full-length ETB
cHx
. ET-1 binding of ETB
cHx
analyzed by coelution. Purified ETB samples produced CF at dif-
ferent conditions were incubated with cET-1 for 3 h at 21 °C, and subsequently analyzed on a Superose 6 PC 3.2 ⁄ 30 column. The elution
chromatograms show total protein absorption at 280 nm (solid line) and specific absorption of cET-1 at 550 nm (dashed line). The retention
volume of ETB
cHx
is indicated by arrows. (A) ETB
cHx
CF expressed as precipitate and resolubilized in 1% LMPG; (B) soluble ETB
cHx
expressed in the presence of 0.4% digitonin; (C) soluble ETB
cHx
expressed in the presence of 1.5% Brij58; (D) soluble ETB
cHx
expressed in
the presence of 1% Brij78.
Ligand binding of cell-free endothelin B receptor C. Klammt et al.
3260 FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS
The percentage of ligand-binding receptor present in
purified ETB
cHx
samples obtained after soluble CF

expression in the presence of 1% Brij78 was deter-
mined by correlation of the molar ratio of complexed
cET-1 with the amount of supplied ETB
cHx
. After
background subtraction, we estimated the amount of
ligand-binding ETB
cHx
present in samples obtained
under the described conditions as c. 50%. This value is
similar to that for ETB samples obtained after conven-
tional expression in insect cells.
Single-particle analysis of CF-produced ETB
cHx
The quality of CF-expressed and purified ETB
cHx
was
analyzed by negative stain electron microscopy.
ETB
cHx
protein that was CF synthesized in the pres-
ence of Brij78 revealed evenly distributed particles with
no detectable signs of aggregation (Fig. 4A). ETB
cHx
synthesized under these conditions appears to be
predominantly dimeric, and the good quality of the
sample allowed further structural assessment using
single-particle analysis. Five hundred side views were
reference-free aligned, classified, and averaged within
the classes (Fig. 4A). ETB

cHx
side view averages dis-
play a pair of rods with a length of 63–68 A
˚
. The dis-
tance between the centers of the rods corresponds to
35–38 A
˚
, and the rods are closely associated at one
end. These values are in excellent agreement with the
dimensions observed for the rhodopsin dimer [23,24].
Single rods, which presumably represent side views of
dimers but could also be ETB
cHx
monomers, represent
less than 10% of all particles. In contrast, and in
agreement with the observed inability to bind cET-1,
ETB
cHx
produced as a precipitate and solubilized in
LMPG was found to be aggregated, and is therefore
most likely unfolded (Fig. 4B).
Localization of the ET-1-binding site
In order to determine the ETB region that is essential
for binding of ET-1, a series of eight plasmids coding
for terminally truncated ETB fragments and contain-
ing different secondary structural elements were con-
structed (Table 1, Fig. 1). All fragments could be
overproduced in amounts of at least 1 mgÆ(mL RM)
)1

in our CF system as soluble proteins in the presence of
1% Brij78 (Fig. 5A). The fragments were purified after
expression in one step by Ni-chelate chromatography,
and the purity was evaluated by SDS ⁄ PAGE analysis
(Fig. 5B).
The ligand binding of ETB
cHx
and truncated deriva-
tives was characterized by pull-down assays of purified
proteins with immobilized biotinylated ET-1 (bET-1),
as described in Experimental procedures. Fractions
containing complexes of bET-1 with ETB derivatives
were separated by SDS ⁄ PAGE and blotted, and the
proteins were identified by immunodetection with
antibody to T7-tag (Fig. 6). Only fragments containing
TMS1-like full-length ETB
cHx
, ETB
131
[N-terminal
domain (NTD)-TMS1], ETB
168
(NTD-TMS2), ETB
203
(NTD-TMS3) and also the NTD-deleted fragment
ETB
93
(TMS1-TMS3) were detected in the eluted
fractions, and formed complexes with bET-1. Accord-
A

B
Fig. 4. Single-particle analysis of CF-produced ETB. Representative
views of electronmicrographs of the negatively stained ETB full-
length construct. (A) ETB produced as soluble protein in the pres-
ence of Brij78. The ETB sample appears to be nonaggregated, and
particles are predominantly dimeric (black arrow); ETB monomers
can be seen occasionally (white arrow). Side view class averages
of reference-free aligned ETB dimers are displayed in the gallery on
the right. (B) ETB produced as precipitate and solubilized in LMPG.
The sample is no longer monodisperse, but rather forms aggre-
gates.
C. Klammt et al. Ligand binding of cell-free endothelin B receptor
FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3261
ingly, proteins devoid of TMS1-like ETB
132
(TMS2-
CTD) and ETB
204
(TMS4-CTD) did not interact with
bET-1.
Analysis of ETB
cHx
–ligand interaction by surface
plasmon resonance (SPR)
Although the coelution approach gives good evidence
for a ligand-binding activity of CF-produced ETB
samples, it is primarily not a quantitative assay. SPR
allows the sensitive detection and quantification of
molecular interactions in real time. We immobilized
bET-1 on the streptavidin surface of the biosensor chip

and analyzed the direct binding of functionally active
ETB
cHx
. ETB
cHx
solutions with increasing concentra-
tions from 10 nm to 250 nm were loaded on the bET-1
chip, and binding kinetics were evaluated using
biaevaluation 3.1 software. In general, signals
obtained from the Biacore assay were lower than
expected for loading of ETB
cHx
as a relatively large
analyte, and this effect is probably due to ligand occlu-
sion by the detergent micelles. Binding constants were
therefore not determined by steady-state kinetics, but
Table 1. Structural characteristics of CF-produced ETB derivatives. With the exception of ETB
DT7
, all proteins contain additionally an N-ter-
minal T7-tag. x, included; –, deleted; ⁄ , partially truncated.
Fragment Region kDa
Included domains
C-terminal
tagNTD T1 C1 T2 E1 T3 C2 T4 E2 T5 C3 T6 E3 T7 CTD
ETB
DT7
M1–S443 50.7 x x x x x x x x x x x x x x x cH6
ETB
cHx
Q2–S443 52.5 x x x x x x x x x x x x x x x cHx

ETB
Strep
E27–S443 49.5 x x x x x x x x x x x x x x x Strep
ETB
131
E27–C131 14.4 x x ⁄ –––––––––––– cHx
ETB
168
E27–P168 18.5 x x x x ⁄ –– –––––––– cHx
ETB
203
E27–V203 22.2 x x x x x x ⁄ –––––––– cHx
ETB
306
E27–G306 34.1 x x x x x x x x x x ⁄ – – – – cHx
ETB
132
M132–S443 38.6 – – ⁄ xxxxxxxx xxxx cHx
ETB
204
A204–S443 30.8 – – – – – – ⁄ xxxxxxxx cHx
ETB
307
M307–S443 18.8 – – – – – – – – – – ⁄ x x x x cHx
ETB
93
P93–V203 15.3 ⁄ xxxxx ⁄ –––– –––– cHx
A
B
Fig. 5. PAGE analysis of CF-expressed ETB fragments on SDS

gels. (A) CF expression of ETB fragments in the presence of Brij78;
0.7 lL of supernatant (S) and 2 lL of eluate (E) after Ni–chelate
chromatography were analyzed. The overproduced ETB truncations
are indicated by arrows. (B) Soluble CF-expressed, purified and
reconstituted ETB fragments. Nine microliters of each sample was
analyzed on a 16.5% SDS gel. 1, ETB
93
; 2, ETB
131
; 3, ETB
168
;4,
ETB
203
, 5, ETB
306
; 6, ETB
132
; 7, ETB
204
; 8, ETB
307
; 9, ETB
cHx
.
Arrows indicate the ETB derivative monomers. Putative oligomeric
forms are also visible.
Fig. 6. Ligand binding of ETB derivatives. Binding of ETB deriva-
tives to bET-1 was analyzed by pull-down assays. Bound proteins
eluted from avidin matrix were separated on 16.5% SDS gels and

detected by immunoblotting with antibody to T7-tag. Arrows indi-
cate detected bET-1-interacting ETB fragments, and asterisks indi-
cate expected positions of noninteracting ETB derivatives. M,
marker.
Ligand binding of cell-free endothelin B receptor C. Klammt et al.
3262 FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS
rather by association and dissociation rates, which
were fitted by using 1 : 1 Langmuir models. The deter-
mined k
d
was used for calculation of k
a
, and the bind-
ing constants K
D
were determined from k
d
⁄ k
a
.We
determined the binding constant K
D
for binding of
ETB
cHx
to bET-1 as 6.2 ± 1.7 · 10
)9
(Fig. 7). Similar
assays with the C-terminal-truncated derivatives
ETB

131
and ETB
93
revealed K
D
values of
(2.7 ± 1.9) · 10
)8
and (1.7 ± 0.5) · 10
)8
, respectively.
Identification of TMS1 as an essential element
for ETB dimerization
Several GPCRs are known to form dimers that remain
stable even after SDS ⁄ PAGE analysis. Protein bands
corresponding to dimers or even higher oligomers of
full-length ETB
cHx
and of most of the truncated deriv-
atives are visible after separation of purified protein
samples by SDS ⁄ PAGE (Fig. 5A,B). In addition, our
single-particle studies provided strong evidence of
ETB
cHx
dimer formation. We therefore attempted to
identify the structural elements responsible for ETB
oligomerization by analyzing heterodimer formation
between full-length ETB
Strep
and the various truncated

ETB fragments in two different pull-down assays.
First, purified ETB fragments and full-length ETB
Strep
were incubated at equimolar concentrations and then
loaded on Strep-Tactin columns. In a second assay,
the full-length ETB
Strep
receptor was coexpressed
with the various truncated fragments in CF reactions,
and the RMs were then loaded on Strep-Tactin col-
umns. In both assays, the interacting protein fragments
were identified after washing, elution and SDS ⁄ PAGE
separation by immunoblotting with antibody to T7-tag
(Fig. 8).
In the coexpression assays, the synthesis of full-
length ETB
Strep
and that of the corresponding ETB
fragment was always visible by immunoblotting
(Fig. 8A). After loading of the RMs on Strep-Tactin
columns, the fragments ETB
93
(TMS1-TMS3), ETB
131
(NTD-TMS1), ETB
168
(NTD-TMS2), ETB
203
(NTD-
TMS3) and ETB

306
(NTD-TMS5) were coeluted
together with ETB
Strep
, indicating an interaction of the
proteins. However, fragment ETB
132
(TMS2-CTD)
lacking the TMS1 region was not detectable in the
eluted fraction, and therefore seems not to interact
with ETB
Strep
. After mixing of purified proteins, again
the fragments ETB
131
(NTD-TMS1), ETB
168
(NTD-TMS2), ETB
203
(NTD-TMS3) and ETB
306
(NTD-TMS5) were found to interact with ETB
Strep
,
whereas fragments lacking TMS1, such as ETB
132
(TMS2-CTD) and ETB
204
(TMS4-CTD), could not be
coeluted with full-length ETB

Strep
and were localized
only in the flow-through of the Strep-Tactin column
(Fig. 8B).
A
B
C
Fig. 7. SPR response curves for the interaction of immobilized
bET-1 with full-length ETB, ETB
131
and ETB
93
. (A) Interaction of
ETB between 10 and 250 n
M. (B) Interaction of ETB
131
between
200 and 1600 n
M. (C) Interaction of ETB
93
between 10 and 200 nM.
C. Klammt et al. Ligand binding of cell-free endothelin B receptor
FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3263
Discussion
The high-level production of GPCRs in conventional
in vivo systems such as E. coli or Pichia pastoris cells
can be very difficult and inefficient. Successful approa-
ches require the construction of large fusion proteins or
intensive optimization [11,25]. In addition, a variety of
steps in conventional expression and purification proto-

cols, such as kinetics of membrane insertion, saturation
of biosynthetic translocation machinery, control of pro-
teolysis, growth conditions, and extraction of recom-
binant membrane proteins from cellular membranes,
are highly critical and need intensive optimization.
We have established a fast and efficient protocol for
the high-level production of functionally folded human
ETB and other GPCRs that eliminates most of the
critical steps of conventional expression systems, as
membranes and living cells are no longer involved.
Furthermore, the proteolysis of synthesized membrane
proteins can easily be prevented by protease inhibitors.
N-terminal digestion of ETB, which is frequently
observed upon in vivo expression, was not detectable
by CF expression [26]. Also, terminal truncated deriva-
tives of ETB, which are often very difficult to express
in vivo, due to proteolysis or translocation problems,
can be produced at high levels in the CF system [27].
A unique advantage of CF expression systems is the
possibility of inserting membrane proteins directly into
detergent micelles upon translation. The efficiency of
this solubilization mode was nearly 100% in the case
of ETB, as no residual precipitate was detectable and
expression levels were similar to those obtained in the
absence of detergent. The CF approach is very
straightforward, and purified ETB protein in sufficient
amounts for structural analysis can now be obtained
in less than 2 days. It should also be mentioned that
the production of labeled membrane proteins, even
with complicated label combinations, is easily feasible

by CF expression without the need for extensive opti-
mization screens and without any loss of productivity
[28–30]. The CF expression technique might therefore
become applicable also for the production of other
GPCRs. In this regard, we have already produced the
porcine vasopressin type 2 receptor and the rat corti-
cotropin-releasing factor precursor receptor at high
levels of several milligrams per milliliter of RM by
using protocols very similar to that for ETB [15,17,22].
In addition, larger thioredoxin fusions of the human
M2 muscarinic acetylcholine receptor, of the human
b
2
-adrenergic receptor and of the rat neurotensin
receptor have been produced in CF systems in yields
approaching 1 mg of protein [16].
As observed for production of the porcine vasopres-
sin type 2 receptor [15,22], only the steroid detergent
digitonin and several long-chain polyoxyethylene deriv-
atives such as Bri35, Brij58 and Brij78 were suitable for
the CF synthesis of soluble ETB in milligram amounts.
Detergents of the Brij family are extremely mild deter-
gents, being unable to disintegrate membranes, and
they are tolerated by the CF transcription ⁄ translation
machinery in amounts far exceeding 100 · critical
micellar concentration [15]. The quality of synthesized
receptor can vary with the type of detergent, and the
highest apparent ET-1 binding was obtained with
Brij78, with some lower activity being seen in digitonin
and other Brij derivatives. In cellular systems, it is also

known that the binding activity and structural integrity
of GPCRs can be sensitive to the supplied detergents
during solubilization [31]. The extraction of active lig-
and-free ETB from cell membranes was only possible
with digitonin [27]. Accordingly, specific detergents
A
B
Fig. 8. Analysis of dimerization of truncated ETB fragments with
full-length ETB
Strep
containing a StrepII-tag. Interacting proteins
eluted from Strep-Tactin spin columns were separated on 16.5%
SDS gels and immunoblotted with an antibody against the T7-tag.
(A) Interaction of ETB
Strep
and truncated ETB fragments after
coexpression in the CF system in the presence of 1% Brij78. S,
supernatant of the RM; E, corresponding eluted fractions from the
Strep-Tactin columns. (B) In vitro interaction of purified ETB frag-
ments with purified ETB
Strep
. Bound fractions or flow-throughs (F)
were analyzed. 1, ETB
131
–ETB
Strep
; 2, ETB
168
–ETB
Strep

; 3, ETB
203
–
ETB
Strep
; 4, ETB
306
–ETB
Strep
; 5, ETB
132
–ETB
Strep
, flow-through; 6,
ETB
132
–ETB
Strep
; 7, ETB
204
–ETB
Strep
, flow-through; 8, ETB
204
–ETB-
Strep
. M, marker; dotted arrow, full-length ETB
strep
; solid arrow,
truncated ETB fragments; gray arrow, putative ETB

Strep
–ETB
xx
heterodimers.
Ligand binding of cell-free endothelin B receptor C. Klammt et al.
3264 FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS
were required for the functional folding of other mem-
brane proteins, such as the nucleoside transporter Tsx,
during CF expression [15]. CF-produced precipitates of
Tsx as well as of ETB did not adopt functional confor-
mations upon solubilization. An initial screen for suit-
able detergents is therefore most important for the
production of functionally folded membrane proteins
during CF expression in the soluble mode. ETB is
known to become post-translationally modified by
palmitoylation, phosphorylation, and glycosylation.
However, these modifications do not play a role in the
ligand-binding capacities of ETB [32], and they are
most likely absent in CF-produced ETB, resulting in
more homogeneous sample preparations that might be
even more suitable for crystallization studies. On the
other hand, disulfide bridge formation is very likely to
occur in CF systems as long as no specific chaperones
are required [33].
SPR studies of GPCRs are generally difficult to per-
form, due to the intrinsic properties of these proteins.
Hydrophobic environments are necessary, and the SPR
sensitivity level requires high receptor concentrations
on the biosensor surface in order to detect the binding
of low molecular weight ligands. Therefore, only a few

SPR measurements with GPCRs have been successful
so far [31,34], but these reports have shown that lig-
ands can bind solubilized GPCRs even in lipid-free
environments and without the need for membrane
reconstitution. Most recently, a modified assay that
employs the detergent-solubilized neurotensin receptor
as the analyte has been described [35], and we success-
fully applied this approach to the characterization of
ETB. Interestingly, for both GPCRs, the amplitude of
the observed response was lower than might be expec-
ted if the relatively high mass of the receptor used as
analyte is considered. Ligand occlusion by immobiliza-
tion on the sensor chip surface, as well as limited
access to the ligand-binding site of the receptor due to
the presence of detergent molecules, might account for
this effect. Although there is still some potential for
the optimization of this technique, e.g. by systematic
evaluation of sensor chip surfaces or of linker struc-
tures, the good correlation of the findings presented
here with the published results obtained with neuroten-
sin receptor indicate that the SPR technique could
become a promising tool for the optimization of
GPCR expression conditions, for the localization of
ligand-binding sites, and for the identification of com-
pounds with new properties that could be important
for the pharmaceutical industry.
Human ETB forms a very tight complex with ET-1
that remains stable even in 2% SDS [36]. ET-1 binds
with high affinity to purified ETB in Brij78 micelles, as
indicated by the determined K

D
of 6 nm, which is even
lower than the value of 29 nm previously determined
by total internal reflection fluorescence spectrometry
with linear fluorescent labeled ET-1 [22]. ETB ⁄ ET-1
dissociation constants determined in vivo in various
cellular environments range between 40 pm and
300 pm [37–41]. It is known that the ligand-binding
kinetics of ETB in intact cells are different from those
in corresponding membrane preparations [42]. In addi-
tion, the interaction of ETB in vivo with other pro-
teins, such as G-proteins or receptor activity-modifying
proteins, might dramatically increase the affinity for
distinct ligands [43]. In this work, we determined the
dissociation constant of pure ETB in the environment
of detergent micelles, and this is also the first analysis
of ETB by SPR measurement. The different assay con-
ditions, in addition to the use of a modified biotinyl-
ated ET-1 derivative as a ligand, have therefore most
likely resulted in modified binding kinetics.
The localization of ligand-binding sites in ETB is
still a subject of controversy. Labeling of ETB with
radioactive ET-1, followed by chemical crosslinking
and trypsin-digest analysis, located the ET-1-binding
domain between residues I85 in the NTD and Y200 in
the second cytoplasmic loop C2 [44]. In addition, dele-
tions, mutations and the lack of glycosylation in the
NTD were found to have no effect on ET-1 binding to
ETB [27]. Our direct in vitro analysis of purified N-ter-
minally and C-terminally truncated ETB derivatives

confined the ET-1-binding site to a 39 amino acid area
between P93 in the NTD and C131 in the first cyto-
plasmic loop C1. These data are in agreement with the
above-mentioned findings, and they further define
TMS1 as a central determinant for ET-1 binding. On
the basis of chimeric ETB derivatives and binding of
antagonists, Wada et al. proposed a 60 amino acid
area spanning I138-I197, and thus covering TMS2 and
TMS3, as the ET-1-binding site [44]. In addition, other
regions, such as TMS5, have been proposed to be
involved in ligand binding as evaluated by photoaffini-
ty labeling with ETB-specific agonists [45]. This result
might have been caused by side-effects of the crosslink
approaches, different binding sites of the supplied
antagonists, or conformational changes of the analyzed
chimeric ETB derivatives. We showed that ETB
truncations devoid of TMS1 but still retaining TMS2
and TMS3 are not able to bind ET-1 in detectable
amounts. Nevertheless, the affinity of ETB
93
and
ETB
131
for ET-1 was reduced by approximately one
order of magnitude, indicating that other regions of
ETB still might contribute to the ligand binding. Evi-
dence for several and partially overlapping binding sites
of ETB for different ligands has been documented [46].
C. Klammt et al. Ligand binding of cell-free endothelin B receptor
FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3265

Homo-oligomerization of rhodopsin-like GPCRs is
an increasingly recognized mechanism, and might rep-
resent an important platform for the modulation of
GPCR activities such as ligand binding, signaling or
trafficking [24,47–49]. Even SDS-resistant dimerization
of b
2
-adrenergic receptor and vasopressin type 2 recep-
tor has been reported [47], and SDS-resistant dimers of
CF-produced porcine vasopressin type 2 receptor have
also been detected [15]. The ETB dimer bands
observed during our SDS ⁄ PAGE analysis indicate a
similar stable association. The first evidence of the for-
mation of ETB homodimer and also of its homolog
human endothelin A in vivo was recently obtained by
fluorescence resonance energy transfer analysis in
HEK293 cells [50]. Interestingly, ETB dimer formation
in vivo did not depend on the presence of ET-1. This is
in accordance with our observed oligomerization of
CF-produced ETB in the absence of any ligand. Fur-
thermore, ETB dimer formation is strongly supported
by single-particle analysis, and the bilobed structures
described are almost identical to that of rhodopsin [24]
and to those of the vasopressin type 2 receptor and
corticotropin-releasing factor receptor type 1 [22]. By
analyzing truncated ETB derivatives, we confined the
site that was essential for dimer formation to the
TMS1 fragment, which was also identified as covering
the ET-1-binding site. The two fragments ETB
131

and
ETB
93
, which overlapped in that region, did still form
homodimers as well as heterodimers with full-length
ETB. Our results therefore indicate that TMS1 is a
key area for two main functions of ETB: the binding
of ET-1 as one of the main natural peptide ligands,
and ETB dimerization. This close colocalization raises
the question of whether dimer formation could modu-
late the ligand-binding activity of ETB.
In summary, the presented work provides an
interesting alternative approach for the generation of
high-quality samples for the functional and structural
characterization of ETB and similar GPCRs. Further
analysis of the identified ETB
131
and ETB
93
fragments
will help to identify residues involved in ligand binding
and dimerization, and they might even represent suit-
able targets for structural studies by high-resolution
NMR analysis.
Experimental procedures
CF expression
Proteins were produced in CECF systems essentially as pre-
viously described [14,22]. Analytical-scale reactions for the
optimization of reaction conditions were performed in mic-
rodialyzers (Spectrum Laboratories, Rancho Dominguez,

CA, USA) with a molecular mass cut-off of 25 kDa in an
RM volume of 70 lL with an RM ⁄ feeding mixture ratio of
1 : 14. Preparative-scale reactions were carried out in dispo-
dialyzers (Spectrum Laboratories) in an RM volume of
1 mL with an RM ⁄ feeding mixture ratio of 1 : 17. The
reaction was optimized for the concentrations of the ions
Mg
2+
(15 mm) and K
+
(290 mm). For soluble expression,
detergent was supplied during the reaction at the following
final concentrations: Brij35, 0.1%; Brij58, 1.5%; Brij78,
1%; and digitonin, 0.4%.
Cloning procedures and protein analysis
Coding regions of full-length ETB and its derivatives were
amplified from cDNA by standard PCR techniques, and
the fragments were inserted into the expression vector
pET21a(+) (Merck Biosciences, Darmstadt, Germany).
Additional codons for extended poly(His)10-tags or for
StrepII-tags were inserted by the Quickchange procedure
(Stratagene, La Jolla, CA, USA).
Protein separated on 12% or 16.5% (w⁄ v) Tris ⁄ gly-
cine ⁄ SDS gels were transferred to 0.45 lm Immobilon-P
poly(vinylidene difluoride) membranes (Millipore, Eschborn,
Germany) blocked for 1 h in blocking buffer containing
1 · Tris-buffered saline, 7% skim milk powder (Fluka,
Buchs, Switzerland), and 0.1% (w ⁄ v) Triton X-100. Horse-
radish peroxidase-conjugated T7-tag antibody (Merck Bio-
sciences) was diluted 1 : 5000 and incubated for 1 h with the

membrane. Washed blots were analyzed by chemilumines-
cence in a Lumi-Imager F1 (Roche Diagnostics, Penzberg,
Germany). Protein concentrations were determined by the
bicinchoninic acid assay (Sigma, Taufkirchen, Germany).
Soluble fractions diluted 1 : 10 in column buffer (20 mm
Tris, pH 8.0, 500 mm NaCl) were applied to 1 mL His-
trapHP columns (GE Healthcare, Freiburg, Germany) equil-
ibrated in column buffer with 0.1% Brij78. Chromatography
was performed at a flow rate of 1 mLÆmin
)1
with washing
steps of six column volumes of column buffer supplemented
with 10 mm,20mm and 50 mm imidazole, respectively, and
bound protein was eluted with 375 mm imidazole. ETB
Strep
was purified on Strep-Tactin Spin columns (IBA, Go
¨
ttingen,
Germany) according to the manufacturer’s recommenda-
tions. Precipitates produced CF in the absence of detergent
were suspended in 1% LMPG in 20 mm phosphate buffer
(pH 7.0), in volumes equal to the RM volume. Suspensions
were incubated for 1 h at room temperature with gentle sha-
king, and this was followed by centrifugation for 10 min at
20 000 g (using an Eppendorf table top centrifuge 5810) in
order to remove residual precipitate.
Ligand-binding analysis
The Cy3 dye was attached at Lys9 of cET-1. Biotin was
covalently attached to Cys1 of ET-1 and Lys-9, result-
ing in bET-1. For coelution studies of ET-1 with ETB,

Ligand binding of cell-free endothelin B receptor C. Klammt et al.
3266 FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS
receptor samples produced CF under different conditions
were bound on HistrapHP columns equilibrated in 20 mm
Hepes (pH 7.4), 150 mm NaCl and 0.02% dodecyl malto-
side, and eluted as described above. Ten to thirty micro-
grams of the purified receptor was mixed with cET-1
dissolved in 20% dimethylsulfoxide. The mixtures were
incubated for 2 h at 21 °C, filtered, and then separated on
a pre-equilibrated Superose 6 PC column (3.2 mm ⁄ 30 cm)
(GE Healthcare) at a flow rate of 0.05 mLÆ min
)1
on a
SMART chromatography station (GE Healthcare). The
cET-1 ligand was detected by specific absorption at
550 nm. Peak area values were calculated by smart
manager software and plotted with kaleidagraph 3.52
software. Nonspecific binding of cET-1 was monitored by
saturation of the ETB sample with unlabeled ET-1 for 2 h
at 21 °C, followed by incubation with cET-1 for an addi-
tional 2 h and subsequent gel filtration. The nonspecific
binding of ET-1 was determined to be below 10% of the
total binding.
For pull-down assays, bET-1 was mixed with ETB deriv-
atives in 20 mm Tris (pH 8.0), 500 mm NaCl and 0.1%
Brij78 at a molar ratio of 5 : 1, and incubated for 1 h at
25 °C. Mixtures were added to 100 lL of presaturated
monomeric avidin matrix (Pierce, Rockford, IL, USA), and
incubated for 1 h at 4 °C with gentle mixing. The matrix
was subsequently packed in 2 mL gravity flow columns,

washed with five column volumes of 20 mm Hepes
(pH 7.4), 150 mm NaCl and 0.02% dodecyl maltoside, and
eluted with 2 mm biotin in a total volume of 1 mL. The
eluate was mixed with 25 lL of 1% sodium deoxycholate,
and incubated for 15 min at 25 °C. Then, 1 mL of 12% ice-
cold trichloroacetic acid was added, and the mixture was
centrifuged at 10 000 g for 20 min at 4 °C (Eppendorf table
top centrifuge 5810). The resulting pellet was dried, suspen-
ded in 50 lL of 0.1% SDS, and analyzed by SDS ⁄ PAGE.
Biacore measurements
Kinetic measurements were done with a Biacore T100
(Uppsala, Sweden) in 20 mm Hepes ⁄ NaOH (pH 7.4),
500 mm NaCl and 0.1% Brij78 at 25 °C. The ligand bET-
1 was loaded at 400–450 resonance units on Biacore Sen-
sor Chips streptavidin, with 60 s contact time, a flow rate
of 10 lLÆmin
)1
, and a stabilization time of 1.500 s.
Responses obtained for the reference flow cell were
directly subtracted from the curves, and revealed negligible
nonspecific binding to the control surface. ETB binding
was analyzed at a flow rate of 30 lLÆmin
)1
, with 480 s
contact time and 1500 s dissociation time. Data were proc-
essed with biaevaluation 3.1 software.
Single-particle analysis
Different concentrations of purified ETB particles were
adsorbed to glow-discharged 400 mesh carbon-coated
Parlodion grids (SPI-Supplies, West Chester, PA, USA)

and negatively stained with 2% (w ⁄ v) uranyl acetate. For
this, a dilution series of the protein at constant detergent
concentration was generated to obtain an optimal protein
concentration on the grid. Images were recorded at a
magnification of · 50 000 on Kodak SO163 film (Roche-
ster, NY, USA), using a Hitachi H-8.000 microscope
(Tokyo, Japan) operating at an acceleration voltage of
200 kV. For image processing, negatives were digitized on
a Heidelberg PrimescanD 7100 (Heidelberg, Germany)
drum scanner at a resolution of 2 A
˚
⁄ pixel at the speci-
men level. The eman boxer program [51] was used to
select a total of approximately 500 particles from electron
micrographs. Particle projections were subjected to refer-
ence-free alignment [52] and classification by multivariate
statistical analysis [53], employing the spider package
[54].
Protein interaction studies
Soluble fractions containing ETB
Strep
and the various His-
tagged ETB fragments were obtained from analytical CF
reactions in the presence of Brij78 after centrifugation of
the RM at 20 000 g for 10 min (Eppendorf table top centri-
fuge 5810). After dilution 1 : 20 with 100 mm Tris ⁄ HCl
(pH 8.0) and 150 mm NaCl to a final volume of 1.4 mL,
the solution was split into 2 · 0.7 mL volumes and loaded
on pre-equilibrated Strep-Tactin Spin columns (IBA).
Washing and elution was performed essentially according

to the manufacturer’s recommendations with the exception
that all buffers were adjusted to 0.05% Brij78. For interac-
tion of purified proteins, approximately equimolar concen-
trations of ETB
Strep
and ETB fragments were combined in
20 mm Tris ⁄ HCl (pH 8.0), 150 mm NaCl, and 0.05%
Brij78, incubated for 1 h at 25 °C, and purified on Strep-
Tactin columns as described above.
Acknowledgements
We are grateful to Clemens Glaubitz and Andreas
Engel for valuable discussions, and we thank Walter
Rosenthal for the cDNA of human ETB. We further
thank Robert Tampe
´
and Katrin Schulze for their help
with SPR analysis. The work was financially supported
by SFB 628 ‘Functional Membrane Proteomics’.
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