Báo cáo khoa học: Complement factor 5a receptor chimeras reveal the importance of lipid-facing residues in transport competence doc

Complement factor 5a receptor chimeras reveal the
importance of lipid-facing residues in transport
competence
Jeffery M. Klco
1,
*, Saurabh Sen
1,
*
,
, Jakob L. Hansen
2,3,
*, Christina Lyngsø
2,3
,
Gregory V. Nikiforovich
4,
à, Soren P. Sheikh
5
and Thomas J. Baranski
1
1 Departments of Medicine and Molecular Biology & Pharmacology, Washington University School of Medicine, St Louis, MO, USA
2 Laboratory for Molecular Cardiology, Danish National Research Foundation Centre for Cardiac Arrhythmia, The Heart Centre, Copenhagen
University Hospital, Denmark
3 Laboratory for Molecular Cardiology, Danish National Research Foundation Centre for Cardiac Arrhythmia, Department of Neuroscience
and Pharmacology, University of Copenhagen, Denmark
4 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA
5 The Laboratory of Molecular and Cellular Cardiology, Department of Biochemistry, Pharmacology and Genetics, University Hospital of
Odense, Denmark
Keywords
BRET; C5aR; G protein-coupled receptor;
lipid-facing; transmembrane helix

Correspondence
T. J. Baranski, Departments of Medicine
and Molecular Biology & Pharmacology,
Washington University School of Medicine,
Campus Box 8127, 660 South Euclid
Avenue, St Louis, MO 63110, USA
Fax: +1 314 362 7641
Tel: +1 314 747 3997
E-mail:
*These authors contributed equally to this
work
Present addresses
Neurology, CNET, University of Alabama at
Birmingham, AL, USA
àMolLife Design LLC, St Louis, MO, USA
(Received 14 January 2009, revised 3 March
2009, accepted 12 March 2009)
doi:10.1111/j.1742-4658.2009.07002.x
Residues that mediate helix–helix interactions within the seven transmem-
branes (TM) of G protein-coupled receptors are important for receptor
biogenesis and the receptor switch mechanism. By contrast, the residues
directly contacting the lipid bilayer have only recently garnered attention
as potential receptor dimerization interfaces. In the present study, we
aimed to determine the contributions of these lipid-facing residues to recep-
tor function and oligomerization by systemically generating chimeric com-
plement factor 5a receptors in which the entire lipid-exposed surface of a
single TM helix was exchanged with the cognate residues from the angio-
tensin type 1 receptor. Disulfide-trapping and bioluminescence resonance
energy transfer (BRET) studies demonstrated robust homodimerization of
both complement factor 5a receptor and angiotensin type 1 receptor, but

no evidence for heterodimerization. Despite relatively conservative substitu-
tions, the lipid-facing chimeras (TM1, TM2, TM4, TM5, TM6 or TM7)
were retained in the endoplasmic reticulum ⁄ cis-Golgi network. With the
exception of the TM7 chimera that did not bind ligand, the lipid-facing
chimeras bound ligand with low affinity, but similar to wild-type comple-
ment factor 5a receptors trapped in the endoplasmic reticulum with brefel-
din A. These results suggest that the chimeric receptors were properly
folded; moreover, native complement factor 5a receptors are not fully com-
petent to bind ligand when present in the endoplasmic reticulum. BRET
oligomerization studies demonstrated energy transfer between the wild-type
complement factor 5a receptor and the lipid-facing chimeras, suggesting
that the lipid-facing residues within a single TM segment are not essential
for oligomerization. These studies highlight the importance of the
lipid-facing residues in the complement factor 5a receptor for transport
competence.
Abbreviations
AT
1
, angiotensin type 1; BFA, brefeldin A; BRET, bioluminescence resonance energy transfer; C5aR, complement factor 5a receptor; CaR,
calcium-sensing receptor; CCR5, CC chemokine receptor 5; CHO, Chinese hamster ovary; CI, confidence interval; CuP, cupric
orthophenanthroline; EndoH, endo-b-N-acetylglucosaminidase H; GFP
2
, green fluorescent protein 2; GPCR, G protein-coupled receptor; IP
3,
inositol 1,4,5-triphosphate; Rluc, Renilla luciferase; TM, transmembrane helix; YFP, yellow fluorescence protein.
2786 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS
G protein-coupled receptors (GPCRs) are seven
transmembrane (TM) spanning receptors that catalyze
the exchange of GTP for GDP on the a subunit of
heterotrimeric G proteins, ultimately leading to the

activation of multiple intracellular signaling cascades
[1]. The seven a-helical domains of GPCRs are orga-
nized into a tightly packed barrel-like structure, as
demonstrated by the high-resolution structure of
bovine rhodopsin in the inactive state [2,3] and the
structure of b-adrenergic receptor [4,5], and opsin
bound to a transducin peptide [6,7]. Each a-helix has
a surface with a strong packing moment and the vast
majority of the packing moments are oriented toward
the helix bundle [8]. These intramolecular interactions
between the TMs are considered to stabilize the inac-
tive state of the receptor. Disruption of the involved
amino acids often has a functional consequence. For
example, in the complement factor 5a receptor
(C5aR), interfering with a hydrophobic pocket
between TM3, TM6 and TM7 can induce constitu-
tive activity [9,10].
Less is known about the contributions of the lipid-
facing residues in the TM helices with respect to the
structure and function of GPCR. A correlated muta-
tion analysis of rhodopsin-like GPCRs revealed that a
number of correlated mutations map to the lipid-facing
surfaces of the TMs, suggesting a functional signifi-
cance [11]. Our saturation mutagenesis analyses of the
seven TMs of the C5aR functionally mapped essential
residues on lipid facing surfaces in TM2, TM4, TM6
and TM7 [9,12]. These residues may be important in
protein–lipid interactions with respect to aiding mem-
brane insertion and stability. Alternatively, these resi-
dues may mediate the many protein–protein

interactions observed for GPCRs, including those with
endoplasmic reticulum (ER) chaperones such as caln-
exin [13] to regulate membrane expression or the inter-
action of the calcitonin-receptor-like receptor and the
receptor-activity modifying proteins to dictate the
ligand specificity of the receptor [14].
Another possibility is that the lipid-facing residues
may mediate GPCR oligomerization. Although classi-
cally viewed as monomers, biochemical and biophysi-
cal evidence of GPCR oligomerization has become
available at a surprising pace in recent years [15,16].
Most reports, especially for receptors in the rhodopsin
family of GPCRs, suggest that the oligomerization
interface resides in the TMs, although a conserved
oligomerization interface has yet to be identified.
Indeed, different TMs have been frequently implicated:
TM1 in the yeast GPCR Ste2 [17], TM4 in the dopa-
mine D2 receptor [18,19], TM5 in the adenosine A2A
receptor [20] and TM6 in both the b
2
-adrenergic recep-
tor [21,22] and the leukotriene B4 receptor [23]. Other
studies [16,24,25], including our own previous work
[26], have implied that more than one helix is responsi-
ble and that GPCRs likely form larger oligomeric
complexes through multiple oligomeric interfaces.
Taken together, these studies, in which the majority of
the TMs were implicated in oligomerization, suggested
the need for a comprehensive and unbiased analysis of
the TM helices.

To systematically evaluate the contributions of the
individual TMs of the C5aR to receptor function and
oligomerization, we generated chimeras of the C5aR
and the rat angiotensin type 1 (AT
1
) receptor in which
only residues on the proposed lipid-exposed face of the
TM helices were exchanged. The side chains making
intramolecular contacts with the rest of the TM bundle
were not disrupted, aiming to minimize alterations to
the overall receptor 3D structure. Surprisingly, five to
six substitutions on the outer face of TM1, TM2,
TM4, TM5, TM6 or TM7 led to retention of the
chimeric C5aRs in the ER. The chimeric receptors
displayed weaker binding affinity than the wild-type
receptor at the plasma membrane; however, the bind-
ing affinities are similar to the wild-type receptor that
is located in the ER. This suggests that the decrease in
binding affinity is more likely to be a product of recep-
tor localization in the ER and not the result of an
overall structural alteration. Despite all of the chime-
ras being retained in the ER, all of the individual
lipid-facing chimeras demonstrated energy transfer
with the wild-type C5aR. These data are consistent
with the studies mentioned above, suggesting that
GPCRs use more than just a single oligomerization
interface, most likely to generate multimeric GPCR
complexes, at the same time as emphasizing the overall
importance of the lipid-facing residues in the receptor
life cycle.

Results
To monitor the contributions of the TM helices to
receptor function, a chimeric C5aR strategy was
devised to exchange residues only on the lipid-exposed
regions of the TMs. An important aim of these stud-
ies is to determine which lipid-facing residues partici-
pate in C5aR oligomerization. The strategy employs
substituting the lipid-facing residues from one GPCR
into the corresponding TM helix of the C5aR and
monitoring whether oligomerization is affected. TM3
was avoided because, in rhodopsin, it has the highest
helix packing value via its contacts with TM2, TM4,
TM5, TM6 and TM7; TM3 also has the lowest lipid
accessibility surface area [8]. A chimeric strategy was
J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR
FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2787
favored over an alanine or tryptophan scan, which
has successfully been used to study the TM helices of
other membrane proteins, such as the lactose perme-
ase [27], because we suspected that individual amino
acid mutations into the hydrophobic faces of the heli-
ces might not be sufficient to alter the packing or
oligomerization properties of the receptor. The inter-
pretation of these studies relies on selecting a chimeric
partner that does not oligomerize with the C5aR. We
chose the angiotensin AT
1
receptor, which has been
reported previously not to interact with the chemoki-
ne receptor, CC chemokine receptor 5 (CCR5) [28].

The AT
1
R homo-oligomerizes [29] and forms hetero-
oligomers with the AT
2
receptor [30], b
2
-adrenergic
receptor [31], D1 dopamine receptor [32] and the
bradykinin receptor [33], although this interaction has
not consistently been observed [34]. The C5aR has
been shown to form homo-oligomers [35–37] and het-
ero-oligomers with CCR5 [28]. In the present study,
oligomerization between AT
1
R and C5aR was evalu-
ated first by disulfide trapping, which uses cysteine
residues as collisional probes to assess for proximity.
We have previously used this technique to demon-
strate homo-oligomerization of C5aR [26]. As with
C5aR, exposure of membranes from Chinese hamster
ovary (CHO)-K1 cells stably expressing AT
1
R with a
carboxy terminal yellow fluorescence protein (YFP) to
the oxidation catalyst cupric orthophenanthroline
(CuP) produced disulfide-linked oligomers and
decreased the amount of monomeric AT
1
R-YFP

(Fig. 1A). Indeed, a significantly greater fraction of
AT
1
R underwent cross-linking compared to the
C5aR. Furthermore, AT
1
R appears to undergo spon-
taneous cross-linking in the absence of CuP, which is
in good agreement with our previous observations
[29]. Our previous studies on C5aR demonstrated that
fusion of YFP to the carboxy terminus did not alter
the cross-linking kinetics or receptor activity [26], sug-
gesting that the observed cross-linking in AT
1
Ris
dependent on the receptor and not YFP. In cells
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0
100
200
300
400
C5aR-Luc/C5aR-GFP
2
C5aR-Luc/C5aR-GFP
2
/WT-C5aR
GFP
2
/Rluc ratio

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
GFP
2
/Rluc ratio
BRET (x1000)
0
100
200
300
400
C5aR-Luc/C5aR-GFP
2
AT
1
R-Luc/C5aR-GFP
2
CaR-Luc/C5aR-GFP
2
BRET (x1000)
A
B
C
Fig. 1. Oligomerization of C5aR and AT
1
R. (A) Total membranes
were prepared from CHO-K1 cells stably expressing C5aR, AT
1
R-
YFP, or both C5aR and AT
1

R-YFP. Membranes were treated with
1.5 m
M CuP and the reaction was terminated with NEM and EDTA
after 10 min. The samples were resolved by nonreducing
SDS ⁄ PAGE and immunoblotted (IB) with anti-C5aR serum (left) or
anti-GFP serum (right). In this experimental set-up, the GFP anti-
body cross reacts with the YFP for the detection phenomenon. (B)
BRET
2
saturation curves. COS-7 cells were transiently transfected
with a fixed amount of C5aR-Rluc, AT
1
R-Luc or CaR-Luc and
increasing amounts of C5aR-GFP
2
. BRET
2
-ratios, total lumines-
cence and total fluorescence were measured, as described in the
Experimental procedures. For titration experiments, all of the trans-
fections contained 0.3 lg of Rluc-tagged receptor and 0.1–4 lgof
the GFP
2
-tagged receptor. (C) The specificity of the C5aR homo-oli-
gomer BRET signal was analysed by transiently transfecting a fixed
amount of C5aR-Rluc and increasing amounts of C5aR-GFP
2
alone
or in combination with untagged C5aR. In this experiment, the
specificity of the C5aR homodimer BRET signal was tested by

cotransfecting the titrations with 3 lg of untagged C5a receptor. In
(B) and (C), data represent at least 10 transfections performed on
three experimental days. To illustrate the specificity of the BRET
signal more clearly, we have chosen to report the GFP
2
⁄ Rluc ratios
that are less than 1. The full spectra and quantification are included
in the Supporting information (Fig. S1 and Table S1).
Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al.
2788 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS
stably expressing both C5aR ( 40 kDa) and
AT
1
R-YFP ( 90 kDa), no hetero-oligomeric com-
plexes ( 130 kDa) were present after CuP addition,
despite normal patterns of homo-oligomeric cross-
linking for C5aR and AT
1
R-YFP. Because the rate of
cross-linking depends on the proximity of the side
groups, the flexibility of the structures containing the
cysteine probes, and the oxidizing environment, it is
possible that hetero-oligomers might be less suitable
partners for cross-linking. Therefore, a negative result
in this assay does not preclude that the C5aR and
AT
1
R form hetero-oligomers.
We next used bioluminescence resonance energy
transfer (BRET

2
) technology to evaluate further the
hetero-oligomerization potential of AT
1
R and C5aR.
As shown in Fig. 1B, robust energy transfer is
observed between C5aR molecules tagged with Renilla
luciferase (Rluc) and green fluorescent protein 2
(GFP
2
). The BRET
50
for this interaction is 0.17 [95%
confidence interval (CI) = 0.12–0.22], where BRET
50
is defined as the GFP
2
⁄ Rluc ratio at which 50% of
the maximum BRET value is reached. By contrast,
AT
1
R-Rluc coexpressed with C5aR-GFP
2
demon-
strated a right-shifted BRET
2
signal and a reduction
in the maximum BRET response compared to the
C5aR–C5aR pair (BRET
50

= 3.3, 95% CI = 1.7–4.8;
Fig. 1B).
To control for specificity of the BRET signal, we
performed two sets of experiments. First we controlled
for ‘bystander’ energy transfer. Accordingly, we coex-
pressed C5aR-GFP
2
with the calcium-sensing receptor
(CaR-Luc). The CaR is a member of the metabotropic
glutamate receptor family, which has been shown to
form strong homodimers, and therefore would not be
expected to form oligomers with a rhodopsin family
member [38]. When coexpressed with the C5aR-GFP
2
,
the CaR-Luc demonstrated a low maximum BRET
signal, which is similar to that observed for the
C5aR-GFP
2
⁄ AT
1
R-Rluc coupled with a BRET
50
value
of 0.59 (95% CI = 0.32–0.85). Second, we performed
the BRET titration curve in the presence of untagged
wild-type C5aR and observed a significant right shift
in the signal between C5aR-Rluc and C5aR-GFP
2
(BRET

50
= 0.87, 95% CI = 0.40–1.35 versus BRET
50
= 0.17, 95% CI = 0.12–0.22; Fig. 1C). The maximal
BRET signal was not decreased by coexpressing
untagged C5aR (see Fig. S1), which might reflect the
fact that the C5aRs do not form simple dimers but
rather assemble in larger oligomeric structures.
The BRET results combined with the absence of
cross-linking suggest that little if any oligomerization
occurs between C5aR and AT
1
R and that the AT
1
Ris
a suitable chimeric partner for C5aR.
Selection of lipid-exposed residues in C5aR
Similar to all members of the rhodopsin family of
GPCRs, C5aR, rhodopsin and AT
1
R share many con-
served amino acids within the TM bundle [39], allow-
ing for an alignment of the TM sequences with high
confidence (Fig. 2). Furthermore, it is expected that
the orientation of the TM bundle witnessed in the
high-resolution structure of rhodopsin is similar in
other rhodopsin family GPCRs. This prediction was
validated by the recently determined structures of the
b
2

-adrenergic receptors [4,5]. For our studies, we used
the X-ray structure of the dark-adapted conformation
of bovine rhodopsin as a template for modeling the
orientation of the lipid-facing residues in the C5aR
and AT
1
R. Five to seven lipid-exposed residues in each
helix were selected (Fig. 2).
A 3D model of the TM bundle of C5aR was then
generated to validate the selections of the lipid-facing
residues. The model was constructed as described in
the Experimental procedures. Briefly, the low-energy
conformations of each individual TM were assembled
into a TM bundle using the X-ray structure of dark-
adapted rhodopsin as a template. The resulting 3D
model of the TM region of the C5aR differed from
rhodopsin by a rms value of 2.40 A
˚
, mostly as a result
of less dense packing of TM1 with the other helices,
causing a slight shift of TM1 relative to the rest of the
bundle (Fig. 3A). At the same time, the main kinks in
the TM helices of rhodopsin (e.g. the functionally
important kink in TM6) were preserved in the 3D
model of C5aR. Also, the 3D model of the TM bundle
of C5aR differed from the recently published X-ray
structure of the b
2
-adrenergic receptor [4,5] by a rms
value of only 2.69 A

˚
, although the 3D model was not
built by aligning to this particular X-ray structure.
The identified residues in C5aR were then changed
to the residue found at the cognate position in the
AT
1
receptor, one TM at a time. Of the 39 total posi-
tions targeted in the helices, C5aR and AT
1
R have an
identical side chain at eight positions. Ultimately, six
changes were made in the TM1 lipid-facing chimeric
receptor, whereas five substitutions were introduced in
the TM2, TM4, TM5, TM6 and TM7 chimeras
(Table 1). The 3D models generated for the resulting
chimeras built by the same methodology to generate
the wild-type C5aR TM model verified that the
selected residues point into the lipid bilayer (Fig. 3B).
Furthermore, no significant changes in residue–residue
interactions within the TM bundle were observed for
the six lipid-facing chimeras. Therefore, we assume
that critical intramolecular interactions important for
receptor activity and helix packing are preserved. We
J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR
FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2789
also suggest that the interaction of the chimeras with
the membrane should be unperturbed, in large part
because AT
1

R has a similar complement of lipid-facing
residues. Furthermore, 18 of the 31 changes occurred
between the hydrophobic residues isoleucine, leucine,
phenylalanine and valine. A statistical analysis of the
lipid-exposed surface of membrane proteins with high-
resolution structures found that these four residues
demonstrate the strongest preference for interacting
with lipids in the hydrocarbon core of the lipid bilayer
[40]. In TM7, for example, all five of the substitutions
involved isoleucine, leucine or phenylalanine.
Analysis of chimeric receptors
Stable transfection of the six chimeric receptors with
a carboxy-terminal YFP fusion in CHO-K1 cells
revealed that all of the chimeric receptors were
expressed, although their migration on SDS ⁄ PAGE
was different from that of the wild-type receptor
(Fig. 4A). C5aR is glycosylated on an asparagine resi-
due in the amino terminus [41] and the carbohydrate
character is dependent on the receptor location in the
secretory pathway. Endo-b-N-acetylglucosaminidase H
(EndoH) removes high-mannose oligosaccharides, and
glycoproteins sensitive to EndoH treatment are found
in the ER or the cis-Golgi [42,43]. Further processing
in the Golgi generates EndoH-resistant complex oligo-
saccharides; therefore, the susceptibility to EndoH can
be used as a marker for transport through the secre-
tory pathway. The majority of the chimeric receptors
were sensitive to EndoH, suggesting that these recep-
tors were found predominantly in the ER. By contrast,
the wild-type receptor had a significant proportion of

Fig. 2. Alignment of rat AT
1
R, human C5aR
and bovine rhodopsin. The outward facing
locations selected for substitution are
shown in bold. Positions with greater than
60% conservation in the rhodopsin family
[39] are shown at the top in italics. TM heli-
ces in rhodopsin are underlined; residue
numbering corresponds to rhodopsin
sequence. The alignment was performed
by
CLUSTALW analysis.
Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al.
2790 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS
EndoH-resistant species (Fig. 4A). Moreover, the lipid-
facing chimeras were frequently expressed at higher
levels and migrated as high-molecular weight aggre-
gates. These slower-migrating species were sensitive to
EndoH, as demonstrated by separation on low
percentage SDS ⁄ PAGE (S. Sen & T. J. Baranski,
unpublished results). It is unclear whether these larger
species represent specific higher-ordered structures,
such as dimers, or whether they are nonspecific aggre-
gates of the chimeric C5aRs. These findings were
supported by strong perinuclear and intracellular
staining by fluorescence microscopy for all of the chi-
meras in a pattern consistent with the endoplasmic
reticulum, which is not seen for the wild-type C5aR
(Fig. 4B). Together with the high levels of EndoH-sen-

sitive receptors in cell lysates, the substitutions in the
chimeric receptors appear to disrupt the ability of
C5aR to reach the cell surface. Surprisingly, this effect
was observed for all of the TMs that were evaluated.
Although the TM4 chimera was expressed in
CHO-K1 cells at lower levels, as assessed by western
blotting, fluorescence microscopy revealed similar
levels of expression compared to the other TM chi-
meras. This difference most likely reflects the hetero-
geneous expression of receptors in cells stably
expressing the TM4 chimera. Furthermore, transient
expression in COS-7 cells revealed comparable levels
of expression for all the TM chimeras, as well as simi-
lar EndoH sensitivities (Fig. 5). Transient expression in
COS-7 cells also resulted in a significant fraction of the
chimeric receptors, as well as the wild-type receptor,
migrating as higher-molecular weight, EndoH-sensitive
complexes. As in CHO-K1 cells, the significance of
these ER complexes remains unclear.
A
B
Fig. 3. Molecular model of C5aR. (A) Sche-
matic representation of spatial arrangement
of the TM helices in C5aR (shaded ribbons)
and rhodopsin (tubes). View from the side
of the membrane; the extracellular surface
is on the top. TM helices are color-coded
as: TM1, white; TM2, blue; TM3, cyan;
TM4, green; TM5, red; TM6, yellow; TM7,
magenta. (B) Sketch of the TM bundle of

C5aR (helices shown as shaded ribbons)
extracted from the 3D models of the lipid-
facing chimeras. The lipid-facing residues
after substitution are shown and color-coded
according to their helix: TM1, white; TM2,
blue; TM4, green; TM5, red; TM6, yellow;
TM7, magenta. The view is from both the
extracellular surface (left) and intracellular
surface (right) perpendicular to membrane
plane. For clarity, the residues are not
labeled. For mutations, see Table 1.
Table 1. Substitutions introduced at lipid-exposed locations in
C5aR.
TM1 TM2 TM4 TM5 TM6 TM7
I38V I73V G151L A201G K242R L284I
V42T V80L I155V V205T A249L F288I
V46I L84C A158I V208I I253F I291F
L49V I91L G162L I220T V260I I295L
L53F S95Y I169L F224L M264L I298L
W60I
J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR
FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2791
Competitive binding studies on the wild-type and
chimeric receptors showed that the receptors were
capable of binding ligand, although with a weaker
affinity than the wild-type C5aR (Fig. 6 and Table 2).
The only exception was the TM7 chimera, which dem-
onstrated no detectable binding. To determine whether
the decrease in binding affinity in the other chimeras
was the result of protein instability and misfolding or

was merely a byproduct of the localization of chimeras
in the ER, cells expressing the wild-type C5aR were
treated with brefeldin A (BFA) to disrupt receptor
maturation and ER to Golgi transport. The apparent
K
d
values for the BFA-treated wild-type receptor are
in the same range as that of the chimeric receptors
(Table 2). Furthermore, the wild-type receptor demon-
strates two distinct binding populations and a two-site
competition binding analysis verified two receptor
populations with distinct binding characteristics. The
high-affinity population (K
d1
and B
max1
) is consistent
with the known binding characteristics of the C5aR
and most likely reflects the receptor population at the
plasma membrane. The lower affinity population (K
d2
and B
max2
) is consistent with the binding kinetics of
the BFA-treated wild-type receptor, as well as the ER
retained chimeras, which suggests that this lower affin-
ity population may comprise the proportion of the
wild-type C5aR in the ER. Thus, all the mutant chime-
ric receptors display a lower binding affinity similar to
the BFA-treated wild-type receptor and, based on our

localization data, fail to reach the plasma membrane.
This internal receptor population is properly folded,
but probably not assembled correctly for transport to
the plasma membrane. These findings suggest that the
lipid-facing residues examined in the present study are
not essential for monomeric receptor folding (with the
exception of the TM7 residues), but rather are vital for
receptor maturation and subcellular transport.
Of note, the TM1-mutated C5aRs display higher
affinity versus the low affinity sites of the wild-type
C5aR and the other TM-mutated receptors (compare
13.5, 47.9 and 15.7 nm versus  150–600 nm; Fig. 6
EndoH
Mock WT TM1 TM2 TM4 TM5 TM6 TM7
100
75
100
*
+
75
50
37
50
37
–+ – +– +–+–+–+–+–+
Fig. 5. Expression and activity in COS-7
cells. (A) Cell lysates were treated with (+)
and without ()) EndoH. C5aR-YFP with
EndoH-resistant complex oligosaccharides
(*) and EndoH-sensitive high-mannose oligo-

saccharides (+) are shown. Western blots
were performed with anti-C5aR serum.
A
B
Fig. 4. Stable expression of lipid-facing chimeras in CHO-K1 cells.
(A) Cell lysates were treated with (+) and without ()) EndoH. C5aR-
YFP with EndoH-resistant complex oligosaccharides (*) and EndoH-
sensitive high-mannose oligosaccharides (+) are shown. Western
blots were performed with anti-C5aR serum. (B) Fluorescence
microscopy of wild-type C5aR or lipid-facing chimeras.
Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al.
2792 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS
and Table 2) and the total number of binding sites is
lower than for the other TM-mutated receptors or the
low affinity site of the wild-type C5aR. The molecular
basis for this interesting difference is not known. It is
also possible that some of the mutated residues in the
TM contribute directly to ligand binding. We do not
favor this interpretation because our modeling of the
C5aR places the TM1 residues (I38, V42, V46, L49,
L53 and W60) on the lipid-facing portion of the TM.
Furthermore, mapping of the essential residues in
TM1 of the C5aR did not identify lipid-facing resi-
dues; however, the scan identified D37 and A40,
which, in our model of the C5aR, are positioned on
one face of the a-helical surface of TM1, pointing
favorably for a potential interaction with ligand
toward the center of the helical crevice [12]. Nonethe-
less, if TM1 is either disordered or highly flexible, it is
possible that some of the lipid-facing residues might

contribute directly to the binding affinity of the recep-
tor for C5a or that these residues might affect the con-
formation of D37 and A40 or other residues involved
in ligand binding.
To investigate whether the mutant C5aRs that did
reach the cell surface were able to activate G proteins,
we performed inositol 1,4,5-triphosphate (IP
3
) accumu-
lation assays in COS-7 cells using a small molecule
C5aR agonist (W5Cha) that is incapable of traversing
the plasma membrane. These studies demonstrated
ligand stimulated activation for all of the chimeras,
with the exception of the TM7-mutated C5aR (Fig. 7).
However, the levels of IP
3
accumulation were consis-
tently lower than the wild-type receptor, which likely
reflects the smaller amount of mutated C5aRs at the
plasma membrane. Based on the EndoH treatment
(Fig. 5), it is difficult to determine the percentage of
the mature receptors that were targeted appropriately
to the plasma membrane. Nonetheless, the ability of
the chimeras to induce IP
3
accumulation after ligand
Fig. 6. Binding analysis of the wild-type and lipid-facing chimeras. Competition binding analysis of the wild-type and lipid-facing chimeras
was performed with isolated membranes as described in the Experimental procedures. The graphs are representative of a typical experi-
ment performed at least in duplicate and repeated three times independently. Calculations were performed using
GRAPHPAD PRISM software.

In the BFA experiment, the compound was added 8 h post-transfection at a concentration of 10 lgÆmL
)1
. (A) Wild-type C5aR; (B) wild-type
C5aR treated with BFA (10 lgÆ mL
)1
); (C) TM1 chimera; (D) TM2 chimera; (E) TM4 chimera; (F) TM5 chimera; (G) TM6 chimera.
J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR
FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2793
treatment argues that, for at least the fraction of the
receptors reaching the plasma membrane, the intro-
duced substitutions do not drastically alter the overall
molecular architecture of the receptors. Interestingly,
the TM7 chimera was neither able to activate G pro-
teins, nor bind ligand, despite the fact that the amino
acid substitutions in TM7 were the most conservative
versus the other TMs (all L, I or F; Table 1). The
inability of TM7 to tolerate even conserved hydro-
phobic substitutions demonstrates a unique folding
requirement for TM7 relative to the other TMs
(excluding TM3, which was not included in the present
study). Similarly, saturation mutagenesis studies dem-
onstrated that TM7 tolerated the fewest substitutions
in the functional mapping of the seven TMs in the
C5aR [9,12].
Oligomerization of the lipid-facing chimeras
Receptor biogenesis has frequently been linked to
GPCR oligomerization, suggesting that defects in olig-
omerization as a result of the introduced amino acid
substitutions may be responsible for the observed
transport incompetence. Unfortunately, an evaluation

of the oligomerization potential of receptors in the ER
by disulfide trapping is complicated by the presence of
high-molecular aggregates subsequent to resolution on
Table 2. Binding parameters for the wild-type and mutant constructs. Apparent K
d
values and the approximate relative B
max
were directly
derived from the raw data of the homologous competition-binding assay by fitting into competition binding models (one site ⁄ two site) and
taking the best-fit R
2
values. Values are represented from each experiment, where the experiments were repeated two or three times at
least in duplicate and the standard errors are representative of data from within the experiment. NA, only a single population of low-affinity
sites were demonstrated for these receptors; ND, not detectable.
Construct
High affinity sites Low affinity sites
K
d1
(nM) B
max1
(pmolÆmg
)1
) K
d2
(nM) B
max2
(nmolÆmg
)1
)
C5aR wild-type 1.05 ± 0.07 13.15 440 ± 45 5.48

0.67 ± 0.01 8.73 305 ± 26 4.17
1.05 ± 0.08 12.92 163 ± 54 2.01
Wild-type (BFA) NA NA 395 ± 17 3.25
< 0.0008 0.005 598 ± 18 4.10
0.31 ± 0.21 0.93 749 ± 29 8.79
TM1 NA NA 13.5 ± 2.0 0.62
47.9 ± 1.2 0.78
15.7 ± 4.8 0.37
TM2 NA NA 222 ± 10 1.99
233 ± 14 2.40
504 ± 27 3.34
TM4 NA NA 282 ± 22 5.26
294 ± 25 2.07
150 ± 33 1.13
TM5 NA NA 310 ± 34 3.29
289 ± 16 2.92
596 ± 26 7.56
TM6 NA NA 956 ± 37 4.70
332 ± 21 2.49
TM7 ND ND
Fig. 7. IP
3
signaling assay. IP
3
signaling activity of the wild-type
C5aR and lipid-facing chimeras. COS-7 cells were transiently
transfected with Ga
16
plus the wild-type C5aR or and lipid-facing
chimera and treated with 1 l

M W5Cha. Each bar represents the
mean ± SE of at least two independent trials.
Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al.
2794 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS
SDS ⁄ PAGE (Fig. 4A). BRET, however, has been used
to characterize GPCR oligomerization in the ER in
previous studies [22,44]. BRET
2
saturation curves were
generated in which the wild-type C5aR containing a
carboxy terminal Rluc was coexpressed with increasing
amounts of the lipid-facing chimeras fused to GFP
2
in
COS-7 cells. Two different energy transfer patterns
were observed: (a) a slope and maximal BRET signal
similar to the wild-type receptor, such as TM2, TM4
and TM7 (Fig. 8A) or (b) a right-shifted energy trans-
fer relative to the wild-type receptor, such as the TM1,
TM5 and TM6 chimeras (Fig. 8B). However, quantifi-
cation of the BRET
50
values for this group did not
demonstrate significant differences compared to the
wild-type–wild-type interaction (see Table S1). As pre-
viously shown (Fig. 5), a significant fraction of both
the wild-type C5aR and the chimeras are in the ER
when expressed in COS-7 cells. To address the possibil-
ity that lipid facing-mutated receptors are aggregating
and therefore interacting nonspecifically with wild-type

C5aR, we performed additional BRET experiments with
the TM7-mutated C5aR-GFP
2
and AT1R-Luc, or
CaR-Luc. The rationale for these experiments is that if
the TM7-mutated C5aR, which appeared to be the least
well folded considering its failure to bind ligand, forms
large aggregates in the ER, then we might expect that
these aggregates would also trap AT1R and CaR that
are folding and being processed in the ER. We found no
evidence for any interactions between AT1R or CaR
and the TM7-mutated C5aR (see Fig. S2). Although we
cannot rule out that TM7-mutated C5aR is misfolded,
the BRET signal is specific for this mutant and the
wild-type C5aR.
Discussion
Much is known about how the seven TM helix bundle
of GPCRs packs and reorganizes during receptor acti-
vation. This helix packing is mediated by intramolecu-
lar interactions among amino acids whose side chains
are oriented away from the lipid bilayer into the helix
core. Less is known, however, about the roles of the
amino acids that point away from the bundle. These
residues would be expected to be important in mem-
brane insertion, thus regulating protein stability and
folding, and in protein–protein interactions, such as
receptor oligomerization. To systematically evaluate
each TM with a notable lipid exposed surface in
C5aR, we employed a ‘lipid-facing chimera’ approach
in which only five to six residues predicted to orient

away from the TM helix bundle were altered. A poten-
tial advantage to this novel approach was to minimize
the likelihood of altering the overall 3D structure of
the receptors, which is a common side effect of swap-
ping entire TM helices. For example, chimeras of the
a
1b
-adrenergic receptor in which each TM was
replaced in entirety by the corresponding helix from
the b
2
-adrenergic receptor were primarily retained in
the ER [45], likely secondary to global receptor
misfolding. To our knowledge, this is the first study
in which chimeric GPCRs were generated that
exchanged only the lipid-exposed residues.
The binding data reported in the present study on
both the wild-type receptor and the chimeric receptors
illustrate that GPCRs can assume a ligand-binding con-
formation in the endoplasmic reticulum; however, the
overall binding is not as avid as that observed for recep-
tors at the plasma membrane. The low affinity of C5aR
in the ER might illustrate a more general concept;
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0
100
200
300
400
Wild-type

TM1
TM5
TM6
Wild-type
TM2
TM4
TM7
GFP
2
/Rluc ratio
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
GFP
2
/Rluc ratio
BRET (x1000)
0
100
200
300
400 A
B
BRET (x1000)
Fig. 8. Oligomerization of lipid-facing chimeras. BRET
2
saturation
curves of COS-7 cells transiently transfected with a fixed amount
of C5aR-Rluc and increasing amounts the GFP
2
tagged lipid-facing
chimeras. Data represent at least ten transfections performed on

three experimental days. (A) BRET
2
saturation curves of lipid-facing
chimeras (TM2, TM4 and TM7) demonstrating BRET
2
values similar
to the wild-type C5aR (solid black). (B) BRET
2
saturation curves of
lipid-facing chimeras (TM1, TM5 and TM6) with decreased BRET
2
values compared to the wild-type C5aR (solid black).
J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR
FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2795
namely, that GPCRs in the ER are not fully competent
to bind ligand and this might be the basis for a quality-
control or fail-safe mechanism to prevent unwanted
receptor activation. The lower binding affinity of the
receptors in the ER may be the result of several
mechanisms: (a) the cholesterol content of the ER
membrane is different from that of the plasma mem-
brane and the high-affinity binding state of the receptor
might only be achieved in presence of membrane bound
cholesterol, as observed for the oxytocin receptors [46];
(b) receptors in the ER may not be associated with G
proteins and thus may be incapable of high-affinity
ligand binding; and (c) the oligomerization state of
receptors in the ER may be different from those of the
matured population at the surface of the membrane.
Importantly, the subcellular localization and binding

studies also demonstrated that the lipid facing residues
are important, although not absolutely essential, for
trafficking the C5aR to the plasma membrane.
Retention of GPCRs in the ER has been linked to
defects in receptor oligomerization [47]. A number of
studies have now observed GPCR oligomerization in
the ER, such as with CCR5 [48], Ste2 [49], the vaso-
pressin and oxytocin receptors [44], C5aR [35] and
5-HT
2C
[50], demonstrating that GPCR oligomeriza-
tion is a constitutive process occurring early in receptor
biogenesis. Furthermore, for the GABA
B
receptor, olig-
omerization in the ER is a prerequisite for transport to
the plasma membrane [51]. The results obtained in the
present study also suggest that oligomerization occurs
early in the biosynthetic process. However, our results
demonstrate that oligomerization is not sufficient for
membrane localization. In addition, we anticipated that
some of the lipid-facing chimeras would disrupt oligo-
merization, thereby identifying potential oligomeriza-
tion surfaces. Surprisingly, substituting the lipid-facing
residues of any of the TMs does not significantly alter
oligomerization of the C5aR, as assessed by BRET.
This is an important conclusion and fits with the idea
that oligomerization surfaces occur between multiple
TMs and that alterations within any individual TM are
not sufficient to disrupt oligomerization. These results

may have the greatest impact on the interpretation of
other studies that correlate oligomerization and cell
surface expression.
Experimental procedures
Generation of chimeric C5aR
Restriction sites engineered into the gene for C5aR at the
approximate boundaries of the TM helices were described
previously [9,12]. The following sites were present at the 5¢
and 3¢ ends of each TM: TM1, Mlu1 and BstEII; TM2,
Nde1 and BsrGI; TM4, Sfi1 and BsrGI; TM5, BssHII and
BspE1; TM6, BglII and Xho1; and TM7, HindIII and FseI.
Single-stranded oligonucleotides for both the sense and
antisense strands of the TM helices of the C5aR incorpo-
rating the mutations from the AT
1
R receptor were synthe-
sized. Each TM was split into five different segments and
the oligonucleotides were annealed in annealing buffer (final
concentration, 10 mm Tris, pH 7.5, 50 mm NaCl, 1 mm
EDTA) following incubation at 95 °C for 3 min and cool-
ing at room temperature for 1 h. Annealing of the oligonu-
cleotides generated the 5¢ and 3¢ overhangs for the
subsequent ligations. The annealed oligonucleotides were
ligated into the gene for C5aR in the pBS-SK Bluescript
vector. W60I, I73V, G151L and A201G mutations were
later incorporated by site-directed mutagenesis because
these locations overlapped with the restriction sites required
for subcloning. Each TM chimera was then subcloned into
the pcDNA3.1 vector for expression in mammalian cells.
All chimeric receptors contain a carboxy-terminal YFP and

an A66C substitution in the first intracellular loop and a
C144S mutation in the second intracellular loop. These
mutations have no impact on the function of C5aR [26].
For BRET
2
studies, the appropriate receptors were subcl-
oned into the pGFP
2
or pRluc vectors (Biosignal Packard,
Montreal, Canada).
Stable cell line generation and maintenance
All cells were maintained under standard conditions in
Ham’s F-12 media (CHO-K1) or DMEM media (COS-7)
containing 10% fetal bovine serum supplemented with
50 unitsÆmL
)1
of penicillin and streptomycin and 2 mm
l-glutamine. Transfections were performed with Lipofecta-
mine 2000 (Invitrogen, Carlsbad, CA, USA) in accordance
with the manufacturer’s instructions. Cells were selected in
1mgÆmL
)1
of G418 and ⁄ or 500 lgÆmL
)1
of hygromycin.
Membrane preparation and cross-linking
Membranes were prepared exactly as described previously
[26]. For cross-linking, Cu-(O-phenanthroline)
3
(Sigma, St

Louis, MO, USA) was added to membrane preparations at
a final concentration of 1.5 mm and the reaction was termi-
nated after 10 min with SDS sample buffer (final concentra-
tion, 50 mm Tris, pH 6.8, 2% SDS and 10% glycerol)
supplemented with a final concentration of 10 mm
EDTA, 10 mm N-ethylmaleimide, 5 lgÆmL
)1
of aprotinin,
5 lgÆmL
)1
of leupeptin and 0.50 mm phenylmethanesulfo-
nyl fluoride. The samples were heated at 50 °C for 10 min
and then resolved by SDS ⁄ PAGE in the absence of reduc-
ing agents. The effects of cross-linking were examined by
immunoblotting with anti-C5aR [26] or anti-GFP sera,
which cross-react with YFP (Santa Cruz Biotechnology,
Santa Cruz, CA, USA).
Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al.
2796 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS
EndoH treatment of C5aR
Equal number of CHO-K1 cells were lysed in 1 · SDS sam-
ple buffer supplemented with 2% b-mercaptoethanol,
1 lgÆmL
)1
of leupeptin, 1 lgÆmL
)1
of aprotinin and 5 m m
phenylmethanesulfonyl fluoride by aspirating ten times
through a 27-gauge needle. The lysate was then heated at
50 °C for 10 min. If samples were treated with endo-b-

N-acetylglucosaminidase (EndoH; New England Biolabs,
Beverly, MA, USA), 250 units were added and the mixture
was incubated at 37 °C for 3 h.
Binding assay
Binding assays on isolated membranes were performed
using [
125
I]C5a (40 lCiÆmL
)1
; Perkin Elmer, Waltham, MA,
USA) as a radioligand. Using the membranes prepared as
described above, the binding assays included 5 lg of total
protein for each data point. After the membranes were
thawed on ice, the protein concentration was determined
using BSA as standard. The binding reaction was set up
using the Binding Buffer (Hanks buffer, 25 mm Hepes, pH
7.5, BSA 0.1%) containing the membrane fraction and cold
C5a (concentration varying between 10
)11
and 10
)5
m). The
reaction was initiated upon addition of 100 pm of [
125
I]C5a.
After incubation for 45 min, the reaction was terminated
through filtration with a Millipore harvestor using a GF ⁄ C
filter pre-soaked in Binding Buffer containing 0.1% poly-
ethylenimine (Sigma). After three rounds of washing, the
filters were dried, folded into quarters and added to scintil-

lation tubes with 3 mL of the scintillation liquid. For the
BFA experiment, cells were treated were BFA (10 lgÆmL
)1
;
Sigma) 8 h post transfection and membranes were prepared
after 48 h in accordance with the protocol described above.
BRET
2
assay
Except for minor differences, the BRET
2
assay was per-
formed as described previously [38,52]. Briefly, 2.5 million
COS-7 cells were seeded onto a p10 plate, grown overnight
and then transfected. For titration experiments, all of the
transfections contained 0.3 lg of Rluc-tagged receptor and
0.1–4.0 lg of the GFP
2
-tagged receptor. In Fig. 1C, the
specificity of the C5aR homodimer BRET signal was tested
by cotransfecting the titrations with 3 lg of untagged C5a
receptor. The expression levels of the tagged or nontagged
C5aRs correlate well with the amount of cDNA used for
the transient transfections [26; unpublished results]. After
48 h, the cells were washed twice with NaCl ⁄ P
i
before
detachment in NaCl ⁄ P
i
supplemented with 1 mm EDTA.

The cells were spun down at 50 g, resuspended in NaCl ⁄ P
i
and then split into two portions of approximately
0.5 · 10
6
cells. The first portion was used to examine the
GFP
2
levels, by fluorescent measurements, and the Rluc
expression by measuring the Coelenterazine h induced lumi-
nescence. The second portion of the cells was submitted to
DeepBlueC excitation, and the luminescence at the dual
bands (515 ⁄ 30 nm and 410 ⁄ 80 nm) was measured on a
Fusion Reader (Hewlett-Packard, Palo Alto, CA, USA).
The BRET
2
ratio was determined according to a previously
described principle [53]: ([emission (515 ⁄ 30))emis-
sion (410 ⁄ 80)] · Cf) ⁄ [emission (410 ⁄ 80)]; where Cf denotes
the Rluc luminescence cross-talk ratio defined as emis-
sion (515 ⁄ 30) ⁄ emission (410 ⁄ 80) when Rluc expressed alone
is excited. Data were analysed by ‘one site binding ⁄ hyper-
bola’, nonlinear regression curve fitting in prism (GraphPad
Software Inc., San Diego, CA, USA). The BRET
50
values
are defined as the GFP
2
⁄ Rluc ratio where 50% of the
maximum BRET value is reached.

Fluorescence microscopy
Subconfluent CHO-K1 cells grown overnight on coverglass
were fixed in 4% formaldehyde for 20 min. Cells were
washed three times in 1 · NaCl ⁄ P
i
. Coverglass was briefly
inverted to remove excess liquid and mounted on a micro-
scope slide with Prolong Antifade (Molecular Probes,
Carlsbad, CA, USA). All images were recorded on a Zeiss
color AxioCam HRc mounted on a Zeiss Axioscop micro-
scope equipped with a Zeiss CP-achromat · 100 objective
(Carl Zeiss, Oberkochen, Germany) using a standard
fluorescein isothiocyanate filter set.
Molecular modeling
Modeling was performed essentially as described previously
[54,55]. All energy calculations were performed using the
standard ECEPP ⁄ 2 force field with rigid valence geometry
and the value of dielectric macroscopic constant of 2.0.
Only trans-conformations of Pro residues were considered,
and residues of Arg, Lys, Glu and Asp were present as
charged species. Packing of the 7TM bundle for the 3D
model(s) of C5aR was performed according to a previously
described procedure [56]. Packing consisted of minimization
of the sum of all intra- and interhelical interatomic energies
in the multidimensional space of parameters that included
the ‘global’ parameters (i.e. those related to movements of
individual helices as rigid bodies; namely, translations along
the coordinate axes X, Y, Z and rotations around these
axes T
X

,T
Y
and T
Z
) and the ‘local’ parameters (i.e. the
dihedral angles of the side chains for all helices; the starting
values of those angles were optimized prior to energy mini-
mization by an algorithm described previously [57]). The
coordinate system for the global parameters was selected:
the long axial X coordinate axis for each TM helix (TM1
to TM7) was directed from the first to the last C
a
-atom;
the Y-axis was perpendicular to X and went through the
C
a
-atom of the ‘middle’ residue of each helix; and the
Z-axis was built perpendicular to X and Y to maintain
J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR
FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2797
the right-handed coordinate system. The above ‘reference
points’ in TM helices were defined by sequence alignment
with rhodopsin TM helices using the clustalw procedure:
TM1, I38-V50-A63 (the first, middle and last residue,
respectively); TM2, N71-L84-Q98; TM3, A107-A122-V138;
TM4, A150-W161-F172; TM5, E199-F211-F224; TM6,
R236-F251-F267; and TM7, L281-Y290-Y300.
Generating the 3D model for the resting state of C5aR
started from finding the low-energy conformations for each
individual TM helical segment. The 3D structures for indi-

vidual helices were found by energy minimization starting
from the all-helical backbone conformations (i.e. the values
of all dihedral angles F and W were initially )60°). All
dihedral angles were involved in the minimization process;
however, some limitations on the F, W and x values
()20°‡F, W ‡ )100°; x = 150° ±30°) were placed dur-
ing energy minimization to mimic, to some extent, limita-
tions on the intrahelical mobility of TM segments
immobilized in the membrane. The rms differences between
the found helical conformations and those of rhodopsin
varied from 0.89 A
˚
(TM4) to 2.04 A
˚
(TM5); here (and
throughout the text), the rms values were calculated for C
a
atoms only. The ‘global’ starting point for assembling the
TM bundle for C5aR was the X-ray structure of dark-
adapted rhodopsin (Protein Databank entry: 1F88, chain
A). At the stage of helical packing, the dihedral angles F
and W were ‘frozen’ at the values previously obtained by
energy minimization of the individual helices. The resulting
3D model of the TM region of the C5aR differed from the
corresponding region of rhodopsin by a rms value of
2.40 A
˚
.
Acknowledgements
This work was supported by an award from the Amer-

ican Heart Association (J.M.K.), as well as by grants
from National Institutes of Health, GM63720-01
(T.J.B.) and GM068460 (G.V.N.); the John Meyer
Foundation (S.P.S.); the Danish Medical Research
Council, The Novo Nordisk Foundation and The
Købmand i Odense Johan og Hanne Weimann f. Seed-
orffs legat (J.L.H.); and the Danish National Research
Foundation (J.L.H. and S.P.S.).
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Supporting information
The following supplementary material is available:
Fig. S1. Full-scale BRET data for oligomerization of
C5aR and AT
1
R.
Fig. S2. BRET data for oligomerization of C5aR,
AT
1
R, CaR and TM7-mutated C5aR.
Table S1. BRET best-fit values of one-site binding
(hyperbola).
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary

materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al.
2800 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS