Role of the Second Extracellular Loop of Human C3a Receptor
in Agonist Binding and Receptor Function*
Ta-Hsiang
Chao,
Julia A.
Ember,
Meiying
Wang,
Yolanda
Bayon
,
Tony E.
Hugli, and
Richard D.
Ye§
From the Department of Immunology, The Scripps Research Institute,
La Jolla, California 92037
 |
ABSTRACT |
The C3a anaphylatoxin receptor (C3aR) is a G
protein-coupled receptor with an unusually large second extracellular
loop (e2 loop, ~172 amino acids). To determine the function of this
unique structure, chimeric and deletion mutants were prepared and
analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2
loop from the C5a receptor (C5aR) abolished binding of
125I-C3a and C3a-stimulated calcium mobilization.
However, as much as 65% of the e2 loop sequence (amino acids 198-308)
may be removed without affecting C3a binding or calcium responses. The
e2 loop sequences adjacent to the transmembrane domains contain
multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a
receptor mediated by intact C3a or by two C3a analog peptides. These
data suggest a two-site C3a-C3aR interaction model similar to that
established for C5a/C5aR. The anionic residues near the N and C termini
of the C3aR e2 loop constitute a non-effector secondary interaction
site with cationic residues in the C-terminal helical region of C3a,
whereas the C3a C-terminal sequence LGLAR engages the primary effector
site in C3aR.
 |
INTRODUCTION |
Human C3a is a 77-amino acid protein generated during
activation of the complement cascade (1). The anaphylatoxin C3a
together with C4a and C5a are involved in mediation of a variety of
inflammatory responses (2, 3). C3a is chemotactic for eosinophils and basophils, whereas C5a is chemotactic for eosinophils, basophils, and
neutrophils. It was recently reported that C3a activation causes
release of effectors from eosinophils, which in turn activate neutrophils (4). Whereas C5a is more potent than C3a in phagocyte chemotaxis and most other functions, C3a can be generated at an approximately 20-fold higher concentration in plasma (5-7). Recent studies suggest that C3a can activate human mast cells (8, 9) and
tonsilar lymphocytes (10). The underlying mechanisms for these newly
described functions of C3a remain incompletely understood.
Biochemical and pharmacological studies indicate that both C3a and C5a
bind and activate G protein-coupled receptors, which then transduce
signals through heterotrimeric G proteins (6, 11). To date, our
understanding of the anaphylatoxin receptors comes mostly from studies
of the C5a receptor. Molecular cloning of C5a receptors from various
species has revealed a primary structure containing seven putative
transmembrane domains similar to that of other members of the
rhodopsin-like G protein-coupled receptor superfamily (12-15). Members
of this superfamily include recently cloned receptors for many other
chemoattractants and chemokines. Studies using mutagenesis (16, 17) and
blocking antibodies (18, 19) indicate that anionic aspartate residues
located in the extracellular N-terminal region of the C5a receptor
(C5aR)1 constitute a
non-effector interaction site with C5a. High affinity binding and
function of the C5a protein requires an additional interaction of its
C-terminal region with an effector site in the C5a receptor (20). The
C-terminal region of C5a appears to interact with a binding pocket
formed by a cluster arrangement of multiple transmembrane helices (20,
21).
The human C3a receptor (C3aR) contain an exceptionally large
extracellular loop between the fourth and fifth transmembrane domains
(22-24). Subsequent cloning of C3aR from mouse (25, 26), rat (27), and
guinea pig (28) confirmed the presence of a large second extracellular
loop (e2 loop) in all four species. Extracellular portions of G
protein-coupled receptors are believed to provide the sites for
interaction with their specific agonists. The extracellular N-terminal
regions of G protein-coupled receptors for large glycoprotein hormones
are known to be essential for agonist binding (29). Recent studies have
also suggested a role for the extracellular loops of G protein-coupled
receptors for peptide agonist in both binding and ligand specificity
(30). Because the large e2 loop is a unique feature of C3aR, it may play a role in binding the cationic C3a molecule, especially in the
absence of anionic clusters in the N-terminal region of the C3aR. In
this study, we constructed chimeric and mutant C3a receptors by
replacement or progressive deletion of the e2 loop. Analysis of these
chimeric and mutant receptors by direct agonist binding and calcium
mobilization assays suggested that the large e2 loop is necessary for
high affinity C3a binding. Terminal ends on the e2 loop contain anionic
residues that may interact with residues in the cationic C3a protein.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Native C3a was purified from expired human plasma
(31). The peptide E7 (WWGKKYRASKLGLAR), the native C3a 18R peptide
(ITELRRQHARASHLGLAR), and the octapeptide (AAALGLAR) were synthesized
and characterized as described previously (32). Chemicals were
purchased from Sigma. Restriction enzymes were obtained from Life
Technologies, Inc.
Construction of Chimeric C3aR/C5aR and the e2 Loop Deletion
Mutants--
The N-terminal C3aR/C5aR chimeric receptor was
constructed by substituting amino acids 1-21 of the human C3aR with
amino acids 1-37 of the human C5aR. The e2 loop chimeric receptor was
generated by replacing amino acids 163-332 of C3aR with amino acids
175-200 of the human C5aR. Briefly, the human C3aR cDNA AZ3B (22)
and the human C5aR cDNA (12, 13) were used as templates for
amplification by PCR of overlapping cDNA fragments with specific
oligonucleotide primers. The fragments were then annealed and amplified
again with primers corresponding to the 5' and 3' end sequences of the resulting chimeric molecule (33). The full-length PCR products were
sequenced to confirm accuracy, digested with EcoRI, and
cloned into the same site in the pSFFV-neo expression vector (34). To
confirm surface expression of the C3aR/C5aR chimera, a hemagglutinin (HA) tag (YPYDVPDYA) was placed after the first methionine in the
C3aR/C5aR chimera for detection by the anti-HA monoclonal antibody
12CA5 (Boehringer Mannheim). By using the same PCR strategy, progressive deletions of the e2 loop of C3aR were accomplished, and the
resulting cDNAs were subcloned into the same expression vector.
Site-directed Mutagenesis--
Using the above PCR-based
methodology, synthetic oligonucleotides
Lys183-Lys186
(5'-tacaaatttggtctctccagctcattaaaatatccaaaaattttatggag-3'),
and Lys325-Lys326-Lys327
(5'-ttaggccaattcacaaaaaaaaaacaagtgccaacac-3'), where
mutated sequences are underlined, were used to construct the
corresponding D183K/D186K, D325K/D326K/D327K, and
D183K/D186K/D325K/D326K/D327K mutant C3a receptors. PCR products were
then subcloned into the pSFFV-neo expression vector. DNA sequencing was
performed to confirm accuracy.
Stable Expression in RBL-2H3 Cells--
Ten µg of DNA was
linearized with XbaI and transfected to RBL-2H3 (ATCC
CRL-2256) cells using LipofectAMINE reagent (Life Technologies, Inc.).
G418 was added to 300 µg/ml for selection and maintenance of stably
transfected cell lines in Dulbecco's modified Eagle's medium
supplemented with 10 mM Hepes, 100 mM nonessential amino acids, 2 mM L-glutamine, and
20% heat-inactivated fetal bovine serum (HyClone, Logan, UT).
Intracellular Ca2+ Measurement--
Transfected
RBL-2H3 cells (~5 × 106) were harvested using
trypsin-free cell dissociation buffer (Life Technologies, Inc.) and loaded with 5 µM Indo-1 AM (Molecular Probes, Eugene, OR)
by incubation at 37 °C for 30 min. Intracellular free calcium was
measured with ~2 × 105 cells in a 0.25-ml final
volume. Continuous fluorescent measurements of calcium-bound and free
Indo-1 were made using an SLM 8000 photon counting spectrofluorometer
(SLM-Aminco, Urbana, IL) detecting at 400 and 490 nm, respectively,
with an excitation wavelength of 340 nm. Intracellular free calcium
concentration ([Ca2+]i) was
calculated as 250 (F
Fmin)/(Fmax
F), where 250 is the Kd of Indo-1 for
calcium (in nM), F is the measured ratio of
emission at 400 and 490 nm (A/B), Fmax is the ratio A/B when Triton X-100 is added to the cells for release of the
entire intracellular calcium storage, and Fmin
is the ratio A/B when EGTA is added to cells to chelate the cytoplasmic
calcium (35).
C3a Binding Assays--
Iodination of C3a with
125I was performed using the IODO-BEAD iodination reagent
(Pierce). The average specific activity of 125I-labeled C3a
was 520 Ci/mmol. The saturation binding curve was generated using
increasing concentrations of labeled C3a with 100-fold excess
concentration of unlabeled C3a. For each duplicate measurement,
~5 × 105 cells resuspended in Earl's balanced salt
buffer (10 mM Hepes, 0.5% bovine serum albumin, pH 7.4)
were mixed with various concentrations of 125I-C3a and
unlabeled C3a in a final volume of 100 µl. The mixture was incubated
at room temperature for 60 min. Unbound 125I-C3a was
separated by spinning through a 100-µl phthalate oil cushion. Curve
fitting and statistical analysis were conducted as follows. The
saturation binding curve was determined by nonlinear regression
analysis using the GraphPad Prism program (GraphPad, San Diego, CA).
The program uses the least sum-of-squares method. Saturation binding
curves best fitted the one-site binding model (hyperbola,
Y = Bmax × X/Kd + X, where X
is the concentration of the ligand) with curve fitting values ranging
between 0.95 and 0.99.
Immunofluorescent Staining--
Transfected RBL-2H3 cells, grown
on 18-mm glass coverslips in six-well culture dishes, were washed with
1× phosphate-buffered saline and fixed in 1% paraformaldehyde for 3 min. After blocking with 2% goat serum in phosphate-buffered saline
for 20 min, the anti-HA tag, monoclonal antibody 12CA5, or the
anti-C5aR-(9-29) polyclonal antibody against the human C5aR N terminus
(18) was added to 2 µg/ml. The cells were incubated at room
temperature for 1 h, washed briefly with phosphate-buffered
saline, and incubated with rhodamine-conjugated secondary antibody at 7 µg/ml for an additional hour. Specimens were mounted and observed
under a Nikon epifluorescent microscope.
Flow Cytometry--
Transfected RBL-2H3 cells were
harvested in trypsin-free cell dissociation buffer, blocked for 30 min
on ice with 1% goat serum in phosphate-buffered saline, and incubated
with a rabbit polyclonal antibody against C3aR for 1 h on ice. The
antibody was generated against the entire e2 loop, which was expressed in Escherichia coli and used as immunogen. Fluorescein
isothiocyanate-labeled goat anti-rabbit secondary antibody was then
added at 1:250 dilution. The cells were further incubated on ice for an
additional hour and assayed on a FACScan flow cytometer (Beckton
Dickinson, Mountain View, CA).
 |
RESULTS |
A prominent feature of the human C3aR is a large extracellular
loop between the fourth and fifth transmembrane domains (the e2 loop).
Subsequent cloning of C3aR from mouse, guinea pig, and rat confirmed
the presence of a large e2 loop in all four species (Fig.
1). A comparison between human C3aR and
C5aR indicated that C3aR, unlike C5aR, does not have a highly anionic
N-terminal segment, known to participate in the C5a-C5aR interaction.
Therefore, the unique structural features represented by the large e2
loop in c3aR suggests that it may play a significant role in
receptor-ligand interactions.

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 1.
Comparison of the e2 loop sequences of
C3a receptors from human (Hu), guinea pig
(Gp), rat (Rt), and
mouse (Mo). The sequences (top) are
aligned and identical amino acid residues are indicated by
asterisks. Two regions containing clusters of negatively
charged amino acids (Asp and Glu) are boxed. Transmembrane
and loop structures (bottom) are assigned based on
hydropathy analysis and comparisons with sequences of other G
protein-coupled receptors. Filled circles (residues 162-183
and 309-332, corresponding to the sequences set in bold)
represent sequences of the e2 loop essential for C3aR function as
determined by deletion mutagenesis (see text). A disulfide bond between
residues 95 and 172 is indicated (-S-S-). The sequences were
obtained from the GenBank with accession numbers U28488,
Z73157, and U62027 (human C3aR), U77460 and U97537 (mouse C3aR),
U86379 (rat C3aR), and U86378 (guinea pig C3aR).
|
|
To examine this possibility, we utilized stable transfectants of
RBL-2H3 cells that express the human C3aR for measuring agonist binding
and calcium mobilization. Cell surface expression of the recombinant
C3aR was confirmed by immunofluorescence staining with a rabbit
antibody against the e2 loop of the C3aR. A typical periplasmic
membrane staining pattern was observed in the transfected cells (Fig.
2B) but not in untransfected
RBL-2H3 (not shown). Direct binding of the transfected cells with
125I-labeled C3a revealed a high binding affinity
(Kd = 3.85 ± 0.15 nM; Fig.
2C and Table I). C3a
stimulation efficiently mobilized calcium with an EC50 of
2.52 ± 0.10 nM (Fig. 2D and Table
II). These results indicated that the
human C3aR was functionally expressed in the RBL-2H3 cells.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Exogenous expression and functional analysis
of the human C3aR in RBL-2H3 cells. A, schematic
representation of the C3aR with the extracellular (e) and
intracellular (i) loops marked and the transmembrane domains
(TM) numbered. B, cell surface expression of the
C3aR was detected by immunofluorescent staining using an anti-C3aR
antibody and a rhodamine-conjugated second antibody as described under
"Experimental Procedures." C, binding of
125I-C3a by the C3aR in transfected cells. Data shown are
the means of specific binding collected from three separate
measurements, each performed in duplicate. Inset, Scatchard
plot of the specific binding data. D, a representative
result of calcium mobilization in the transfected cells in response to
C3a. Relative fluorescent intensity was measured in cells loaded with
Indo-1 AM. The experiment was conducted as described under
"Experimental Procedures." Arrows mark the time of
addition of the indicated ligand and reagents.
|
|
To investigate the role of the N-terminal segment and the e2 loop of
C3aR in agonist binding, chimeric receptors were prepared by
interchanging portions of the human C3aR and C5aR as illustrated in
Fig. 3. Replacement of the N-terminal
segment of C3aR (21 amino acids) with the equivalent segment from C5aR
(37 amino acids) resulted in a very small effect to the binding
affinity for C3a (Fig. 3A, top panel, and Table I). This
structural change did not confer C5a binding capability on the chimera
either (data not shown). Similarly, N-terminal tagging with HA had
little influence on C3a binding (Fig. 3, A and B,
middle panels, and Table I). Taken together, these results
suggest that the C3aR differs from C5aR in its utilization of the
N-terminal segment for agonist binding.

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 3.
Construction and expression of C3aR/C5aR
chimeric receptors. A, schematic representation of the
C3aR constructs with or without HA tagging. The C3aR/C5aR N-terminal
chimera (top panel) was made by substituting the N-terminal
sequence of C3aR (amino acids 1-21) with the corresponding sequence
from C5aR (amino acids 1-37). The middle panel depicts an
N-terminal HA-tagged (YPYDVPDYA) wild-type C3aR. A C3aR/C5aR e2 loop
chimera (bottom panel) was prepared by replacing the entire
e2 loop of C3aR (amino acids 162-332) with its corresponding loop from
C5aR (amino acids 175-200). A similar construct was made by adding the
HA tag to the N terminus, and this construct was used for
immunofluorescent staining. B, confirmation of cell surface
expression of these receptors by immunofluorescent staining. Shown in
each panel are representative cells transfected with the
corresponding constructs on the left side. An anti-C5aR
polyclonal antibody was used for detecting expression of the C3aR/C5aR
N-terminal chimera. The anti-HA monoclonal antibody 12CA5 was used for
the other two receptors. Rhodamine-conjugated secondary antibodies were
used for detection of fluorescence.
|
|
The role of the e2 loop in agonist binding was next examined in a
chimera of C3aR in which the entire 172-residue e2 loop was replaced
with the corresponding 25-residue loop from the human C5aR (Fig.
3A, bottom panel). Cell surface expression of the stable transfectants was confirmed by immunofluorescent staining with an
anti-HA tag antibody (Fig. 3B, bottom panel). The
transfected cells, however, displayed no specific binding for C3a, nor
did the cells respond to C3a with calcium mobilization (Table I). Thus,
replacement of the C3aR e2 loop with the e2 loop from human C5aR led to
an apparent loss of C3aR functions.
The human C3aR shares approximately 40% overall sequence homology with
human C5aR. Published data indicate that these two receptors
specifically respond to their respective agonists on both peripheral
blood leukocytes and transfected mammalian cells. To examine whether
exchanging the e2 loops between these two receptors produced altered
binding specificity, we evaluated binding and calcium mobilization
using human C5a as the agonist. No binding or calcium response to C5a
was detected in cells expressing the C5a e2 loop replacement in C3aR
mutant (data not shown). These results suggested that replacement of
the entire e2 loop in C3aR fails to change binding specificity of this
receptor from C3a to C5a.
Results from the above experiments indicated that the large e2 loop in
C3aR was required for the binding of C3a. However, although the large
e2 loop was present in C3a receptors from the four species cloned to
date, a comparison of the e2 loop sequences from human, mouse, guinea
pig, and rat revealed less sequence homology than exists in other
regions of the receptor. For example, the human and mouse C3a receptors
share 65% identity overall, but the e2 loop regions have only a 45%
sequence identity (25, 26). This finding raised the question of whether
only a limited portion of the e2 loop is important for C3a binding. To
address this issue, mutants created by sequential deletion of portions of the e2 loop sequence from the human C3aR were designed (Fig. 4). Six deletion mutants were created by
systematic removal of N-terminal segments beginning at residue 308 near
the C terminus of the e2 loop (Fig. 4A). Analysis of these
modified receptors in transfected RBL-2H3 cells showed that four of the
six mutant receptors were almost fully functional in agonist binding
and calcium mobilization assays, compared with the wild-type C3aR (Table II). Deletion of sequences N-terminal to residue 197 resulted in
a receptor with much reduced binding affinity and calcium mobilization capability as reflected by marked increases in the
Kd and EC50 values for
183-308
(Table II). Removal of the additional nine residues between 174 and 183 led to a receptor (
174-308) essentially devoid of all functions.
Deletion of this stretch of the C3aR sequence (amino acids 174-182)
likely disrupted the global conformation of C3aR because of its
proximity to a conserved cysteine residue at position 172. This
deletion also resulted in poor expression of the C3aR mutant in
transfected cells as determined by immunofluorescent microscopy using
an anti-HA tag (data not shown). Thus, deletion mutagenesis suggested
that the N terminus of the e2 loop contains a segment between residues 182-197 that is necessary for high affinity C3a binding.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 4.
Schematic representation of the N-terminal
and C-terminal deletions of the e2 loop. The e2 loop sequence is
shown at the top. A, the N-terminal e2 loop
deletion mutants were constructed by progressive deletion of the e2
loop from residue 308 to 173. B, the C-terminal e2 loop
deletion mutants were constructed by progressive deletion from residue
212 to 332. Both groups of deletion mutants were constructed by
alteration of the C3aR cDNA and subsequent expression in RBL-2H3
cells, as described under "Experimental Procedures."
|
|
Four additional mutant receptors were constructed by progressive
deletion of residues 212-332 near the C terminus of the e2 loop in
C3aR (Fig. 4B). When analyzed in transfected RBL-2H3 cells, all but one mutant receptor (i.e.
212-308) displayed
altered binding and calcium mobilization capabilities (Table II). There was a gradual decrease in C3a binding because residues 309-331 were
deleted from this region of the e2 loop. These deletions also reduced
the calcium response (Table II). These results suggested that deletions
at the C terminus of the e2 loop of C3aR also altered C3a binding.
Because deletion of a large portion of the e2 loop (from residues
198-308, 111 residues or 65% of the e2 loop) did not significantly affect C3a binding, we concluded that only the N and C termini of the
e2 loop contained elements necessary for C3a-C3aR interaction. Charged
residue side chains play important functions in receptor-ligand interaction as reported in other studies of G protein-coupled receptors
including C5aR (21). An examination of the e2 loop sequence identified
several negatively charged residues in the same N- and C-terminal
regions that were shown to affect function when deleted (Fig. 1,
boxed areas). This includes an aspartic acid triplet located
a few residues from the boundary of the fifth transmembrane domain
(Fig. 1, second boxed area). To test the function of these
residues, the triplet was replaced with three lysyl residues that carry
opposite charges on the side chain (Fig. 5). Functional analysis of this mutant
receptor (D325K/D326K/D327K) revealed a 6-fold decrease in C3a binding
affinity (Kd increased from 3.85 to 22.12 nM) and a 14-fold drop in calcium mobilization capability
(EC50 values changed from 2.52 to 34.56 nM),
suggesting that these negatively charged residues on C3aR contribute to
C3a binding (Table III). The effect of
D325K/D326K/D327K changes on C3a binding results from either important
alterations in charge-charge interactions within C3aR or between C3a
and its receptor. Because the N and C termini of the e2 loop should be proximal to one another at the membrane surface, we also examined the
function of anionic residues located near the amino end of the e2 loop.
Aspartic acids at positions 183 and 186 of the e2 loop have been
conserved in C3aR from four different species (Fig. 1), and these
residues are also located in a segment that affects C3a binding when
deleted (Table II). Replacement of these two residues by cationic
lysines resulted in a receptor (D183K/D186K) having a nearly 4-fold
decrease in binding affinity and a 40-fold decrease in calcium response
to C3a (Table III). The resultant receptor was also expressed at a
lower density on the cell surface, which may be partially responsible
for the reduced calcium mobilization. These results suggest a critical
role for aspartic acid residues at the N terminus of the e2 loop, in
either C3a binding or maintenance of overall receptor structure.


View larger version (67K):
[in this window]
[in a new window]
|
Fig. 5.
Construction and analysis of C3aR with Asp
Lys mutations. A, schematic representation of the
three Asp Lys mutant C3a receptors. The Asp and Lys residues are
marked in their relative positions within the e2 loop. The e2 loop
sequence is shown on the top, with the candidate Asp
residues in bold. B, flow cytometric analysis of
RBL-2H3 cells stably transfected with the
D183K/D186K/D325K/D326K/D327K mutant C3aR (dashed
line), wild-type C3aR (dotted line), and the expression
vector only (solid line). The cells were incubated with a
rabbit polyclonal antibody directed to the e2 loop and stained with a
fluorescein isothiocyanate-labeled goat anti-rabbit IgG secondary
antibody, as described under "Experimental Procedures."
C, calcium mobilization in RBL-2H3 cells transfected
with the D183K/D186K/D325K/D326K/D327K mutant C3aR
(right panels) and the wild-type C3aR (left
panels) (on next page). Representative traces are shown based on
three independent measurements. The intact C3a (100 nM,
top row), E7 peptide (200 nM, second
row from top), 18R peptide (200 nM,
third row from top), and the octapeptide (10 µM, bottom row) were added at the times
indicated. Relative fluorescence intensity is shown.
|
|
Finally, the combined effect of
Asp183-Asp186 and
Asp325-Asp326-Asp327 replacement by
lysines in a mutant C3aR (D183K/D186K/D325K/D326K/D327K) was measured.
Whereas the resultant receptor was expressed on the cell surface, as
determined by flow cytometry with an anti-C3aR antibody (Fig.
5B) and evidenced by low level calcium mobilization in
response to C3a (Fig. 5C), the binding affinity for C3a was elevated to micromolar range (Table III). These results suggested interaction between some or all of the negatively charged residues in
the e2 loop of C3aR and positively charged residues on the C3a
molecule. To examine this possibility, short C3a analog peptides, that
have previously been shown to activate C3aR (32), were used in the
calcium mobilization assays to evaluate wild-type and the
D183K/D186K/D325K/D326K/D327K mutant C3a receptors. The first two
analog peptides employed here are E7 (WWGKKYRASKLGLAR) and C3a-derived
18R (ITELRRQHARASHLGLAR), both having positively charged residues
N-terminal to the functionally essential sequence LGLAR. Like native
C3a, these two peptides were much less effective in stimulating calcium
mobilization from cells expressing the D183K/D186K/D325K/D326K/D327K
mutant receptor than from cells expressing the wild-type C3aR (Fig.
5C, compare right panels with left
panels). A semiquantitative analysis of the extent of calcium mobilization indicated that the intact C3a was 35-40% active on the
D183K/D186K/D325K/D326K/D327K mutant receptor as compared with the
wild-type C3aR, whereas the two peptides retained only ~2% activity
on the mutant receptor. This difference may be a result of additional
receptor contact sites in native C3a that are missing from the much
shorter analog peptides.
Having established that substitution of five aspartic acid
residues in the e2 loop by lysyl residues reduced the activity of both
C3a and two C3a analog peptides containing cationic residues, we next
examined whether peptides without the cationic resides were affected by
the Asp to Lys replacement. The octapeptide AAALGLAR can activate C3aR
when used at micromolar concentrations (Fig. 5C,
bottom row). Approximately 65-70% of this activity was
retained when the D183K/D186K/D325K/D326K/D327K mutant C3aR was
evaluated by the calcium mobilization assay. These results indicated
that a C3a effector peptide lacking additional cationic residues was minimally impacted by the Asp to Lys replacement in C3aR. These results
support our hypothesis that charge-charge interactions between C3aR and
C3a involve one or more of the aspartic acid side chains at positions
183, 186, 325, 326, and 327 of the e2 loop in C3aR.
 |
DISCUSSION |
Little was known about the function of the large extracellular
loop of the C3aR, a unique feature of this G protein-coupled receptor.
In this study we examined the role of the e2 loop of C3aR in agonist
binding and receptor activation. Construction and analysis of chimeric
receptors between C3aR and C5aR led to the conclusion that the e2 loop
but not the N-terminal region of the C3aR is indispensable for C3a
binding. Because sequences of the e2 loop are the least conserved
across species, progressive deletion was adopted to localize the
specific regions of the loop important for C3a binding. It was
surprising to find that nearly two-thirds of the loop sequence could be
removed without affecting C3a binding. This finding parallels a recent
study reporting the presence of an isoform of the guinea pig C3aR,
which lacks 35 amino acids (residues 254-288) from the large
extracellular loop and maintains activity (36). The observation that
this shorter C3aR isoform also binds C3a with high affinity was in
agreement with our own result, further indicating that part of the C3aR loop structure is not essential for agonist binding. Only when portions
of the e2 loop sequences near the transmembrane domains were deleted
did the resulting receptors display reduced binding affinity for C3a.
Data from our subsequent studies suggested that anionic residues at the
terminal ends of the e2 loop likely form contact sites for C3a. Our
findings differ from a recent report claiming that residues 185-193 of
the e2 loop represent an immunodominant domain but not a ligand-binding
region because antibodies interacting with this region failed to block
C3a-induced calcium mobilization (37). In contrast, our data showed
that when sequences within this region (residues 174-197) were deleted
or when the aspartate residues were replaced by lysyl residues, both
calcium mobilization and agonist binding decreased. Consequently, we
conclude that both the N- and C-terminal ends of the e2 loop are
involved in agonist binding.
Previous studies of the C5a receptor resulted in the identification of
charged amino acids in the second extracellular loop, the fifth
transmembrane domain, and the N-terminal regions that serve as contact
sites for C5a binding (17, 21, 38). These results support a two-site
binding model, which was proposed earlier based on both biochemical and
functional assays (39). The model predicts the presence of a
non-effector or internal recognition site and an effector or activation
site within the C5a protein, which interact with corresponding binding
sites on C5aR. With the cloning of the C5aR cDNA, it has been shown
that negatively charged residues in the e2 loop, as well as in the
N-terminal region of C5aR, interact with positively charged residues in
C5a at positions 12, 37, and 40 and in the effector region near the C
terminus. Interactions between C5aR and the non-effector site residues
12, 37, and 40 in C5a likely are followed by insertion of the
C-terminal effector domain of C5a into a binding pocket formed by the
C5aR transmembrane domains, thereby leading to receptor-mediated cellular activation (20, 21). These findings provide important clues
for our understanding of C3a-C3aR interactions. There are similarities
between the modes of action for C3a and C5a, e.g. each
having an effector site at the C terminus and cationic residues in
other parts of the molecules that participate in binding. However, there are major structural differences as well between C3aR and C5aR.
The human C3aR has a relatively short (21 amino acids) N-terminal region containing only one aspartate residue, which does not align with
aspartate residues at the N-terminal end of C5aR. In our studies,
replacement of the C3aR N-terminal region with the N-terminal region of
C5aR resulted in a chimeric C3aR that displays wild-type levels of
binding affinity for C3a. This finding suggested that the N-terminal
region of the human C3aR does not play as important a role in agonist
recognition as was the case for C5aR. Likewise, C3aR and C5aR interact
only with their respective agonists and retain high specificity even in
chimeric form. Replacement of either the N-terminal region or the e2
loop of the C3aR with the equivalent structure from the human C5a did
not confer C5a binding capability. These results differ from a previous
report indicating a promiscuous response for anaphylatoxins in
Xenopus oocytes (40).
Our combined results support a model that depicts a non-effector C3a
binding/recognition site in the extracellular loop of the receptor, as
proposed in a recent review article (7). This recognition site contains
negatively charged aspartate residues in the N and C termini of the e2
loop. These anionic residues may interact with positively charged amino
acid side chains near the C terminus of the C3a molecule, thus
facilitating agonist binding (Fig. 6).
Our results indicate that the aspartate residues in both terminal
segments of the e2 loop play a concerted role in C3a binding.
Significant C3a binding was maintained when only one of the two
aspartic residue clusters in the e2 loop was replaced by lysyl
residues, but not when both were replaced. However, complete replacement of the five anionic residues
(D183K/D186K/D325K/D326K/D327K mutant) caused a very significant
reduction in C3a binding. Results obtained from calcium mobilization
studies using synthetic C3a analog peptides also supported the presence
of charge-charge interactions between the wild-type C3aR and these
peptide agonists.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
A model proposed for the interaction of C3a
with its receptor. The receptor is schematically shown as a
cylinder divided into seven sections corresponding to the seven
transmembrane domains. The two negatively charged regions of the
receptor's e2 loop are marked by signs and highlighted in the
shaded area. Positively charged residues in the C3a molecule
near the C-terminal effector region are indicated by + signs. The model
depicts possible charge-charge interactions between C3a and its
receptor. This schematic has been adapted from Ember and Hugli
(7).
|
|
Similar to the intact C3a molecule, the peptides E7 and 18R contain the
effector sequence LGLAR at their C-terminal end and two to three
cationic residues near the N terminus. In comparison with C3a, the
peptides were much less effective in mobilizing intracellular calcium
in the transfected cells expressing the D183K/D186K/D325K/D326K/D327K
mutant receptor than in the wild-type C3aR. It is possible that there
are multiple, cooperating recognition sites for C3a-C3aR interaction.
The localization of one of these sites in the e2 loop of the human C3aR
should facilitate the identification of other agonist recognition and
receptor activation sites within the C3aR.
 |
ACKNOWLEDGEMENT |
We thank Dr. Philippe Pfeifer for providing
purified human C3a.
 |
FOOTNOTES |
*
This work was supported by United States Public Health
Service Grants AI41670 (to T. E. H.) and AI40176 and AI33503 (to
R. D. Y.) and by a grant-in-aid from the American Heart Association (to R. D. Y.) and was done during the tenure of an Established Investigatorship (to R. D. Y.) from the American Heart Association. This is publication 11727-IMM from The Scripps Research Institute.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.
On leave of absence from Instituto de Biología y
Genética Molecular, Facultad de Medicina, Calle Ramón y
Cajal, Valladolid 47005, Spain. Current address: Sidney Kimmel Cancer
Center, 10835 Altman Row, San Diego, CA 92121.
§
To whom correspondence should be addressed: Dept. of Pharmacology
(MC868), University of Illinois, 835 S. Wolcott Ave., Chicago, IL
60612. Tel.: 312-996-5087; Fax: 312-996-7857; E-mail:
yer{at}uic.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
C5aR, C5a receptor;
C3aR, C3a receptor;
e2 loop, the second extracellular loop;
PCR, polymerase chain reaction;
HA, hemagglutinin.
 |
REFERENCES |
-
Hugli, T. E.
(1989)
Curr. Top. Microbiol. Immunol.
153,
181-208
-
Cochrane, C. G.,
and Muller-Eberhard, H. J.
(1968)
J. Exp. Med.
127,
368-371
-
Goldstein, I. M.
(1988)
in
Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I., Goldstein, I. M., and Snyderman, R., eds), pp. 55-74, Raven Press, New York
-
Daffern, P. J.,
Pfeifer, P. H.,
Ember, J. A.,
and Hugli, T. E.
(1995)
J. Exp. Med.
181,
2119-2127[Abstract]
-
Chenoweth, D. E.,
Erickson, B. W.,
and Hugli, T. E.
(1979)
Biochem. Biophys. Res. Commun.
68,
227-231
-
Gerard, C.,
and Gerard, N. P.
(1994)
Annu. Rev. Immunol.
12,
775-808[CrossRef][Medline]
[Order article via Infotrieve]
-
Ember, J. A.,
and Hugli, T. E.
(1997)
Immunopharmacology
38,
3-15[CrossRef][Medline]
[Order article via Infotrieve]
-
Nilsson, G.,
Johnell, M.,
Hammer, C. H.,
Tiffany, H. L.,
Nilsson, K.,
Metcalfe, D. D.,
Siegbahn, A.,
and Murphy, P. M.
(1996)
J. Immunol.
157,
1693-1698[Abstract]
-
Hartmann, K.,
Henz, B. M.,
Kruger-Krasagakes, S.,
Kohl, J.,
Burger, R.,
Guhl, S.,
Haase, I.,
Lippert, U.,
and Zuberbier, T.
(1997)
Blood
89,
2863-2870[Abstract/Free Full Text]
-
Fischer, W. H.,
and Hugli, T. E.
(1997)
J. Immunol.
159,
4279-4286[Abstract]
-
Siciliano, S. J.,
Rollins, T. E.,
and Springer, M. S.
(1990)
J. Biol. Chem.
265,
19568-19574[Abstract/Free Full Text]
-
Gerard, N. P.,
and Gerard, C.
(1991)
Nature
349,
614-617[CrossRef][Medline]
[Order article via Infotrieve]
-
Boulay, F.,
Mery, L.,
Tardif, M.,
Brouchon, L.,
and Vignais, P.
(1991)
Biochemistry
30,
2993-2999[Medline]
[Order article via Infotrieve]
-
Gerard, C.,
Bao, L.,
Orozco, O.,
Pearson, M.,
Kunz, D.,
and Gerard, N. P.
(1992)
J. Immunol.
149,
2600-2606[Abstract/Free Full Text]
-
Perret, J. J.,
Raspe, E.,
Vassart, G.,
and Parmentier, M.
(1992)
Biochem. J.
288,
911-917[Medline]
[Order article via Infotrieve]
-
Mery, L.,
and Boulay, F.
(1993)
Eur. J. Haematol.
51,
282-287[Medline]
[Order article via Infotrieve]
-
DeMartino, J. A.,
Van-Riper, G.,
Siciliano, S. J.,
Molineaux, C. J.,
Konteatis, Z. D.,
Rosen, H.,
and Springer, M. S.
(1994)
J. Cell. Biochem.
269,
14446-14450
-
Morgan, E. L.,
Ember, J. A.,
Sanderson, S. D.,
Scholz, W.,
Bucher, R.,
Ye, R. D.,
and Hugli, T. E.
(1993)
J. Immunol.
151,
377-388[Abstract/Free Full Text]
-
Opperman, M.,
Raedt, U.,
Hebell, T.,
Schmidt, B.,
Zimmermann, B.,
and Gotze, O.
(1993)
J. Immunol.
151,
3785-3794[Abstract/Free Full Text]
-
DeMartino, J. A.,
Konteatis, Z. D.,
Siciliano, S. J.,
Van-Riper, G.,
Underwood, D. J.,
Fischer, P. A.,
and Springer, M. S.
(1995)
J. Biol. Chem.
270,
15966-15969[Abstract/Free Full Text]
-
Siciliano, S. J.,
Rollins, T. E.,
DeMartino, J.,
Konteatis, Z.,
Malkowitz, L.,
Van-Riper, G.,
Bondy, S.,
Rosen, H.,
and Springer, M. S.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
1214-1218[Abstract]
-
Roglic, A.,
Prossnitz, E. R.,
Cavanagh, S. L.,
Pan, Z.,
Zou, A.,
and Ye, R. D.
(1996)
Biochim. Biophys. Acta
1305,
39-43[Medline]
[Order article via Infotrieve]
-
Crass, T.,
Raffetseder, U.,
Martin, U.,
Grove, M.,
Klos, A.,
Kohl, J.,
and Bautsch, W.
(1996)
Eur. J. Immunol.
26,
1944-1950[Medline]
[Order article via Infotrieve]
-
Ames, R. S.,
Li, Y.,
Sarau, H. M.,
Nuthulaganti, P.,
Foley, J. J.,
Ellis, C.,
Zeng, Z.,
Su, K.,
Jurewicz, A. J.,
Hertzberg, R. P.,
Bergsma, D. J.,
and Kumar, C.
(1996)
J. Biol. Chem.
271,
20231-20234[Abstract/Free Full Text]
-
Tornetta, M. A.,
Foley, J. J.,
Sarau, H. M.,
and Ames, R. S.
(1997)
J. Immunol.
158,
5277-5282[Abstract]
-
Hsu, M. H.,
Ember, J. A.,
Wang, M.,
Prossnitz, E. R.,
Hugli, T. E.,
and Ye, R. D.
(1997)
Immunogenetics
47,
64-72[CrossRef][Medline]
[Order article via Infotrieve]
-
Fukuoka, Y.,
Ember, J. A.,
and Hugli, T. E.
(1998)
Biochem. Biophys. Res. Commun.
242,
663-668[CrossRef][Medline]
[Order article via Infotrieve]
-
Ember, J. A.,
Jagels, M. A.,
and Hugli, T. E.
(1998)
in
The Human Complement System in Health and Disease (Volanalsis, J. E., and Frank, M. M., eds), pp. 241-284, Marcel Dekker, New York
-
Phang, T.,
Kundu, G.,
Hong, S.,
Ji, I.,
and Ji, T. H.
(1998)
J. Biol. Chem.
273,
13841-13847[Abstract/Free Full Text]
-
Samson, M.,
LaRosa, G.,
Libert, F.,
Paindavoine, P.,
Detheux, M.,
Vassart, G.,
and Parmentier, M.
(1997)
J. Biol. Chem.
272,
24934-24941[Abstract/Free Full Text]
-
Hugli, T. E.,
Gerard, C.,
Kawahara, M.,
Scheetz, M. E.,
Barton, R.,
Briggs, S.,
Koppel, G.,
and Russell, S.
(1981)
Mol. Cell. Biochem.
41,
59-66[Medline]
[Order article via Infotrieve]
-
Ember, J. A.,
Johansen, N. L.,
and Hugli, T. E.
(1991)
Biochemistry
30,
3603-3612[Medline]
[Order article via Infotrieve]
-
Higuchi, R.,
Krummel, B.,
and Saiki, R. K.
(1988)
Nucleic Acids Res.
16,
7351-7367[Abstract]
-
Fuhlbrigge, R. C.,
Fine, S. M.,
Unanue, E. R.,
and Chaplin, D. D.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
5649-5653[Abstract]
-
Grynkiewicz, G.,
Poenie, M.,
and Tsien, R. Y.
(1985)
J. Biol. Chem.
260,
3440-3450[Abstract]
-
Fukuoka, Y.,
Ember, J. A.,
and Hugli, T. E.
(1998)
J. Immunol.
161,
2977-2984[Abstract/Free Full Text]
-
Hawlisch, H.,
Frank, R.,
Hennecke, M.,
Baensch, M.,
Sohns, B.,
Arseniev, L.,
Bautsch, W.,
Kola, A.,
Klos, A.,
and Kohl, J.
(1998)
J. Immunol.
160,
2947-2958[Abstract/Free Full Text]
-
Monk, P. N.,
Barker, M. D.,
Partridge, L. J.,
and Pease, J. E.
(1995)
J. Biol. Chem.
270,
16625-16629[Abstract/Free Full Text]
-
Chenoweth, D. E.,
and Hugli, T. E.
(1980)
Mol. Immunol.
17,
151-161[CrossRef][Medline]
[Order article via Infotrieve]
-
Ames, R. S.,
Nuthulaganti, P.,
and Kumar, C.
(1996)
FEBS Lett.
395,
157-159[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.