From the Institute of Medical Microbiology, Hannover
Medical School, D-30623 Hannover, Germany and the Departments of
¶ Molecular Biology and
Pulmonary Pharmacology, SmithKline
Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406
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ABSTRACT |
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Chimeras were generated between the human
anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to
define the structural requirements for ligand binding and
discrimination. Chimeric receptors were generated by systematically
exchanging between the two receptors four receptor modules (the N
terminus, transmembrane regions 1 to 4, the second extracellular loop,
and transmembrane region 5 to the C terminus). The mutants were
transiently expressed in HEK-293 cells (with or without G Activation of the complement cascade leads to the generation of
the 74-77-amino acid anaphylatoxins C3a and C5a. Acting as potent
signaling molecules, C3a and C5a exhibit a broad spectrum of
proinflammatory effects on different organs and cell types, which
include smooth muscle contraction, increase in vascular permeability,
chemotaxis, cell activation, and granule secretion. Despite a high
overall similarity in their structure and functional effects, C3a and
C5a exhibit qualitative and quantitative differences in their ability
to elicit the above-mentioned reactions, suggesting distinct roles of
each anaphylatoxin in the initiation and maintenance of inflammatory
reactions. Increased serum levels of C3a and C5a have been reported in
a number of disease states, like the adult respiratory distress
syndrome, chronic polyarthritis, and psoriasis, suggesting an important
role of the anaphylatoxins in the pathophysiology of these and other
inflammatory diseases (for review see Refs. 1 and 2). A more detailed
knowledge about the mode of action of the anaphylatoxins on their
target cells might suggest novel and valuable therapeutic approaches
for the treatment of many of these disease states.
C3a and C5a have similar solution structures consisting of a globular
core composed of four tightly folded Several mutagenesis experiments, including chimeras with the fMLP
receptor, N-terminal deletion mutants, and site-directed mutagenesis of
individual residues have revealed a two-point binding model for the
C5aR (10-14). According to this model, a first binding site is located
in the C5aR N terminus that binds core residues of the C5a ligand. This
interaction provides about half of the overall binding energy and
facilitates the interaction of the C terminus of the ligand with the
second binding site, which encompasses several residues scattered among
the extracellular loops and TM helices. Two of these,
Glu199 in EL2 and Arg206 in TM5, have already
been identified (15, 16). This model is confirmed by studies using
synthetic peptides mimicking the C5a C terminus, which are able to bind
to the C5aR and to induce signal transduction, although with much lower
affinity and potency than the wild type ligand. As might be expected,
the activity of such peptides is not affected by mutations in the C5aR
N terminus, in contrast to what is observed with native C5a (12,
13).
The sites of interaction between C3a and the C3aR are not known. Like
C5a, synthetic C3a analogue peptides of the C terminus are full
receptor agonists, although most of these investigations have been
performed in the heterologous guinea pig system. As the peptide length
is extended toward the C3a N terminus, the activity of the peptides
gradually increases. A sequence-optimized 21-mer "superagonist"
peptide (17) has been identified that exhibits ~10% of the activity
of wild type C3a versus the cloned human C3aR (18). These
data would also indicate a two-point binding site in the C3aR, where
the C terminus of the ligand is bound by the effector site, whereas the
overall affinity is increased by additional interactions. However, no
mutagenesis experiments of the C3aR have been reported yet.
Receptor chimeras between the C5aR and the fMLP receptor have been
generated and found to be expressed on the cell surface allowing for
the delineation of sites involved in fMLP binding (19). Because the
human C3aR also has high homology to the C5aR, we used an analogous
approach to identify sequence modules in the anaphylatoxin receptors
that are involved in ligand binding. Using a panel of 19 receptor
mutants, we show that the binding behavior of C3a to the C3aR differs
from the C5a-C5aR interaction, culminating in the generation of a true
hybrid C3a/C5a receptor.
Materials--
Human C3a was obtained from Advanced Research
Technologies (San Diego, CA), human C5a was from Sigma,
125I-labeled C3a and C5a were from NEN Life Science
Products, and the synthetic peptides H-1264 and H-8135 were from Bachem
(King of Prussia, PA). The monoclonal mouse IgG1 anti-C3aR antibody 8H1
was generated in our
laboratory,2 the monoclonal
mouse IgG1 anti-C5aR antibody W17/1 (20) was a kind gift by O. Götze, Göttingen, Germany, the anti-FLAG M2 and M5
antibodies were obtained from Scientific Imaging Systems (Rochester,
NY), and the fluorescein isothiocyanate-labeled goat anti-mouse
antibody from Becton-Dickinson (San Jose, CA).
The following buffers and stock solutions were used: phosphate-buffered
saline (10 mM sodium phosphate, pH 7.4, 2.7 mM
KCl, adjusted with NaCl to a final conductivity of 15 millisiemens), HAG-CM (20 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.25% w/v bovine serum albumin, 0.5 mM glucose), and KRH (118 mM NaCl, 4.6 mM KCl, 25 mM NaHCO3, 1 mM KH2PO4, 11 mM glucose, 1.1 mM MgCl2).
Construction of Chimeras and N-terminal Deletion
Mutants--
The C5aR coding sequence with an additional FLAG epitope
sequence and an EcoRI site at the 5' end (21) plus a new
SalI site at position 708 was used for mutagenesis of the
C5a receptor sequence. Conservative mutations (which did not alter the
amino acid code) that created novel restriction sites (BsrGI
at position 519 = Tyr174 at the junction of TM4 and
EL2, and BssHII at position 597 = Ala201 at
the junction EL2 and TM5) were introduced using the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA). The same 5'-UTR plus FLAG
epitope sequence was added to the C3aR by subcloning after introduction
of a novel MfeI site at position +4 of the C3aR cDNA (9). Similarly, novel restriction sites (BsrGI at position 476 = Tyr160 at the junction of TM4 and EL2, and
NruI at position 1003 = Ala335 at the
junction of EL2 and TM5) were introduced into the C3aR coding sequence.
These changes to the C3aR did not alter the Kd for
C3a.3 The receptor N termini
(defined as codons 1-23 in the C3aR and codons 1-37 in the C5aR) were
exchanged by fusion PCR, as described previously (21). The other
chimeras were generated by subcloning via the BsrGI site and
the NruI/BssHII sites after suitable blunting of
the BssHII site according to standard techniques (22).
N-terminal deletions of the C3aR coding sequence (9, 16, and 22 residues) were generated by standard PCR using the cDNA clone
HNAFG09 (8) without further modifications. The final panel of 16 chimeras (including the wt receptors) plus three deletion mutants in
vector pcDNA3/Amp (CLONTECH) was characterized
by restriction analysis and DNA sequence analysis of all PCR-amplified
sequences. In all cases where no expression of the mutant receptor on
the cell surface could be detected, the complete coding sequence was determined.
Immunofluorescence and Flow Cytometry--
Human embryonic
kidney cells (HEK-293) were transfected using LipofectAMINE as
described previously (9). 3 days later, cells were harvested and
resuspended in phosphate-buffered saline + 0.5% w/v bovine serum
albumin at 1 × 107 cells/ml. 50 µl were incubated
with 50 µl of first step antibody (8H1: 10 µg/ml; W17/1: 5 µg/ml;
M2: 20 µg/ml; M5: 20 µg/ml) for 30 min at 0 °C, developed with
fluorescein isothiocyanate-conjugated goat anti-mouse antibody, and
examined flow-cytometrically on FACScan (Becton-Dickinson) using
mock-transfected cells as negative control. For immunofluorescence
studies of mutants that were not expressed on the cell surface,
LipofectAMINE-transfected HEK-293 cells were developed with the same
set of antibodies after prior fixation and permeabilization of the
cells in acetone, as described previously (21).
Binding Studies--
Binding assays were performed essentially
as described (9). Briefly, 1.5-3.0 × 105 transfected
cells were incubated with 20,000-40,000 cpm of
125I-labeled C3a and varying concentrations of unlabeled
ligand at room temperature in a total volume of 50 µl of HAG-CM.
After 30 min, unbound ligand was removed by vacuum filtration using the MultiScreen filtration system with a Durapore 0.45-µm membrane (Millipore, Bedford, MA) equilibrated with HAG-CM. The filter was
washed twice with 100 µl of HAG-CM and dried, and bound radioactivity was determined by Signal Transduction Assays--
HEK-293 (1.2 × 107) cells were plated into a T150 flask in Earl's
modified Eagle's medium supplemented with 10% fetal bovine serum.
Following a 16-h incubation at 37 °C, medium was removed and
replaced with serum-free medium, and cells were co-transfected with
cDNA encoding each chimera, truncation mutant, or wild type receptor and a cDNA encoding G
After 18-24 h the medium was aspirated off, and 100 µl of fresh
Earl's modified Eagle's medium containing 4 µM Fluo-3AM
(Molecular Probes, Eugene, OR; stock solution prepared at 2 mM in Me2SO containing 20% pluronic acid),
0.1% bovine serum albumin, and 2.5 µM probenecid (prepared with the addition of equal equivalents of NaOH) was added to
each well and incubated in a CO2 incubator for 1 h at 37 °C. Medium was aspirated and replaced with 100 µl of the same medium but without Fluo-3AM and incubated for 10 min at 37 °C. Cells
were washed three times with a Denley cell wash with KRH containing
0.1% bovine serum albumin, 2.5 µM probenecid, and 20 mM HEPES, pH 7.4 (buffer A), and after the last wash cells
were aspirated down to final volume of 100 µL. Ligand
concentration-response curves were prepared in 96-well polyproplyene
microplates at three times the final concentration in buffer A and
warmed to 37 °C.
Microplates containing the Fluo-3AM-loaded cells and the ligand plates
were placed in a fluorometric imaging plate reader where all 96 wells
are monitored simultaneously (24). At initiation of the reading
fluorescence is read every 1 s for 60 s and then every 3 s for the following 60 s. Agonist (50 µl) was added at 10 s, and the maximal fluorescent count above background after the
addition of agonist was used to define maximal activity for that
concentration of agonist. The fluorometric imaging plate reader
software normalizes fluorescent readings to give equivalent readings
for all wells at zero time.
The aim of this study was the identification of receptor
modules in the human C3aR and C5aR that determine binding affinity, specificity (i.e. discrimination from the noncognate
ligand), and signal transduction. For this purpose, chimeric C3a/C5a
receptors were constructed by systematically recombining four receptor
parts (the N terminus, TM1 to TM4, EL2, and TM5 to the C terminus) to generate all 16 possible receptor chimeras (including the wt
receptors). To normalize receptor expression and to facilitate
immunological detection of the receptors on the cell surface, the same
5'-UTR plus a sequence tag coding for an N-terminal FLAG epitope were introduced at the 5' end of each clone, as described previously for the
human C5aR (21). All receptor constructs were analyzed for (i) cell
surface expression by flow cytometry using a panel of monoclonal
antibodies directed against the N-terminal FLAG epitope, the C5aR N
terminus, and EL2 of the human C3aR, (ii) ligand binding of C3a and C5a
in competitive displacement studies, and (iii) functional response
(intracellular calcium mobilization) toward C3a, C5a, and a synthetic
C3a and C5a analogue peptide after cotransfection of G-16) and
analyzed for cell surface expression, binding of C3a and C5a, and
functional responsiveness (calcium mobilization) toward C3a, C5a, and a
C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any
reciprocal exchange. N-terminal substitution confirmed the two-binding
site model for the human C5aR, in which the receptor N terminus is
required for high affinity binding of the native ligand but not a C5a
analogue peptide. In contrast, the human C3a receptor did not require
the original N terminus for high affinity binding of and activation by
C3a, a result that was confirmed by N-terminal deletion mutants. This
indicates a completely different binding mode of the anaphylatoxins to
their corresponding receptors. The C5a analogue peptide, but not C5a,
was an agonist of the C3aR. Replacement of the C3aR N terminus by the
C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a
receptor, which bound and functionally responded to both ligands, C3a
and C5a.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helices and a C-terminal
"finger" (3-5). They act on their target cells by binding to and
activating highly ligand-specific membrane receptors belonging to the
family of seven transmembrane domain G-protein-coupled receptors. The
human C5a receptor (C5aR)1
was cloned in 1991 (6, 7), and in 1996 the corresponding receptor for
C3a was identified and cloned by our groups (8, 9). Both receptors are
closely related in sequence, especially in their transmembrane regions.
A highly characteristic and unique feature of the C3a receptor (C3aR)
is the unusually large second extracellular loop (EL2) of ~175
residues with as yet unknown function.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-counting (Cobra II Auto Gamma, Canberra Packard, Meriden, CT). Binding curves were analyzed using the software package
LIGAND (23).
-16 (15 µg of each plasmid)
using LipofectAMINE Plus reagent (Life Technologies, Inc.) according to
the manufacturer's recommendation. Following a 24-h incubation at
37 °C, the cells were replated on poly-D-lysine coated
96-well black wall microplate (Becton-Dickinson) at 30,000 cells/well.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-16, which had
previously been shown to complement the signal transduction cascade of
the anaphylatoxin receptors in these cells (9, 25, 26). Flow cytometric
analysis revealed highly different surface expression levels among this panel of receptor chimeras, indicating determinants other than the
5'-UTR and N-terminal sequence that regulate cell surface expression.
Specifically, for 7 of 14 chimeras (Ch2 to Ch7 and Ch10; Table
I) reproducible and significant surface
expression could not be confirmed, which was compatible with and
explained the negative results obtained in the binding studies and
functional analyses for these mutants. This observation was unexpected
because in the case of C5aR/fMLP chimeric receptors, all of the
constructs were expressed on the cell surface, despite an even slightly
lower sequence homology between those two receptors (19). However, all
mutants with negative cell surface expression were expressed intracellularly as shown by immunofluorescence studies of
acetone-treated cells (data not shown), indicating a defect in correct
folding and/or transport of these mutants to the cell surface.
Combined results of cell surface expression, C3a/C5a bindings and
functional responsiveness towards C3a, C5a, and synthetic C3a/C5a
analogue peptides of all receptor mutants used in this
study
The Receptor N Termini Determine Ligand Affinity (C5aR) and Discrimination from the Noncognate Ligand (C3aR)-- Replacement of the C5aR N terminus by the C3aR N terminus (chimera Ch8, i.e. a C5aR with a C3aR N terminus) lead to an affinity loss in C5a binding of >2 orders of magnitude (i.e. above the sensitivity of this assay) without affecting the signal transduction properties of the C5a analogue synthetic peptide H-8315 (Table I), confirming previous data that showed the presence of a major ligand binding site within this part of the receptor, which contributes to ligand binding affinity (12-14). Conversely, in the reciprocal exchange mutant (Ch9, a C3aR with a C5aR N terminus) significant C5a binding could be detected (Table I and Fig. 1). The Kd = 33 ± 14 nM for C5a binding of this mutant was only about 1 order of magnitude higher than the Kd = 5.2 ± 2.5 nM of the wt C5aR. Because the C3aR does not bind C5a (Ch1; Table I), this provides direct evidence for a C5a interaction site within the C5aR N terminus. More important, this chimera responded functionally to C5a with an ED50 of ~60 nM (Table I and Fig. 2). Although C5a was about 2-3 orders of magnitude less potent on this receptor mutant than on the wt C5aR, this result clearly shows that the C3aR "core structure" (i.e. TM1 to C terminus) does accept C5a as an activating stimulus. This unexpected result was confirmed by the observation that the C5a analogue peptide H-8315 had similar activity on both anaphylatoxin receptors (Ch1 = C3aR and Ch16 = C5aR) as well as on the N-terminal exchange mutants (Ch8 and Ch9; Table I). This suggests that the N terminus of the C3aR partially determines ligand specificity by preventing the binding of the noncognate C5a. Once bound, C5a could activate the C3aR at physiologically relevant concentrations. As a consequence, previous investigations analyzing the functional activity of C5a analogue peptides on human granulocytes (27) have to be interpreted with caution because these cells harbor both anaphylatoxin receptors, both of which respond to this peptide. The C3a analogue peptide, however, appears to be more sequence-restricted because it did not activate the C5aR (see Ch8 and Ch16; Table I)
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EL2 Influences Correct Positioning of the Transmembrane Helix
Bundle--
Previous investigations had implicated Glu199
in the second extracellular loop of the human C5aR in ligand binding
(16). Indeed, replacement of EL2 from the C3aR in chimera Ch9 by the
C5aR homologue increased C5a binding affinity about 10-fold, thus
restoring wild type affinity of the resulting chimera Ch11 (Table I).
Unexpectedly, however, this replacement also strongly affected the
signal transduction properties toward C5a and the C5a analogue peptide,
which were virtually inactive on this mutant (Table I). This result,
which shows a critical involvement of the second extracellular loop in
signal transduction, may be explained by an indirect effect of this
loop on the correct positioning of the transmembrane helix bundle, thus
affecting signal transduction. Similarly, replacement of EL2 in the
C5aR by the C3aR counterpart (Ch16 Ch14; Table I) critically
affects both ligand binding (C3a as well as C5a) and signal
transduction despite the fact that EL2 of the C3aR does not by itself
prevent C5a binding and signal transduction, as already shown above
(Ch9; Table I). Again, this result is best explained by an indirect
effect of EL2 of the C3aR on the correct positioning of the TM helix
bundle. Thus, helix bundles and EL2 have to be regarded as a functional
unit in which the size of EL2 is of critical importance. Neither
elongation of the C5aR loop (compare Ch16
Ch14; Table I) nor
shortening of EL2 of the C3aR (Ch9
Ch11; Table I) are compatible
with correct functioning of the resulting receptor mutants, an
important caveat for any deletional mutagenesis experiments in the C3aR
loop. Furthermore, it is tempting to speculate that C3aR antagonists
may be found that bind within EL2 of the C3aR, thus functionally
"tethering" the helix bundle with consequences similar to those
observed by exchange of the C3aR loop by its C5aR counterpart (compare
Ch8
Ch10; Table I). Our data do not exclude the hypothesis that residues within EL2 of the C3aR are involved in ligand binding, although this idea appears less likely in view of the low sequence homology in EL2 of the C3aR sequences of different species (28). Similarly, all chimeras with "mixed" C3aR/C5aR helix bundles did not show any signal transduction ability (like Ch12 and Ch14; Table I),
suggesting that the transmembrane receptor modules also are not
completely compatible with each other despite the fact that within the
coding region these domains exhibit the highest sequence homology.
The C3aR N Terminus Is Not Required for Ligand Binding--
Apart
from the wt C3aR, only Ch9, a C3aR with a C5aR N terminus, showed
significant C3a binding (Fig. 1) and signal transduction (Fig. 2) both
with C3a and the C3a-like peptide H-1264. Because the same mutant Ch9
also bound C5a (Table I and Fig. 1), it is thus revealed as a true
hybrid C3a/C5a receptor. This finding suggests that the C3aR N terminus
determines receptor specificity, as explained above, but does not
participate in C3a binding or else that the C5aR N terminus complements
putative N-terminal residues involved in C3a binding despite the fact
that the sequence homology between C3aR and C5aR is rather low in this
receptor part. To exclude this latter possibility, however, N-terminal truncation mutants of the C3aR were generated (09,
16, and
22). The
09 and
16 mutants showed only slightly decreased
binding affinities and almost unaltered signal transduction properties, whereas the
22 deletion mutant was not properly expressed on the
cell surface (Table I). These results confirm that the N terminus of
the C3aR does only contribute to a minor degree or not at all to C3a
binding, in sharp contrast to what is known from the C5aR (11-14). The
ligand binding site(s) for C3a must reside in the other receptor
modules of the C3aR, although it is not possible to further delineate
them using this set of mutants due to the indirect effects on receptor
expression. Alternatively, mutagenesis of candidate residues is
required and will be greatly facilitated by the low sequence homology
(<50%) of the C3aR cloned from mouse, humans, and guinea pigs
(28).
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ACKNOWLEDGEMENT |
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We cordially thank D. Bitter-Suermann (Hannover, Germany) for continuous strong support.
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FOOTNOTES |
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* This work was supported in part by Deutsche Forschungsgemeinschaft Grant 1425/3-1 and Sonderforschungsbeveich Grant 244 "Chronische Entzündung" (to W. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed: Inst. of Medical Microbiology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany. Tel.: 49-511-532-4342; Fax: 49-511-532-4366; E-mail: Bautsch.Wilfried{at}MH-Hannover.de.
2 B. Sohns, J. Westermann, R. Frank, M. Grove, J. Köhl, A. Klos, and W. Bautsch, manuscript in preparation.
3 T. Crass, R. S. Ames, H. M. Sarau, M. A. Tornetta, J. J. Foley, J. Köhl, A. Klos, and W. Bautsch, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: C5aR, C5a receptor; C3aR, C3a receptor; Ch, receptor chimera; EL, extracellular loop; TM, transmembrane region; UTR, untranslated region; fMLP, formylmethionylleucylphenylalanine; PCR, polymerase chain reaction; wt, wild type.
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