From the
C5a, a potent chemoattractant for monocytes, neutrophils, and
other leukocytes, binds to a cell surface receptor of the
seven-transmembrane superfamily. Here we report the effects of
substituting Gln for Glu
Complement fragment C5a is produced following activation of the
complement cascade and is a potent chemoattractant and secretagogue for
leukocytes. In vivo it is an important mediator of
inflammation and is thought to play a role in the pathogenesis of
several inflammatory disorders, including adult respiratory distress
syndrome and rheumatoid arthritis, and to exacerbate tissue damage in
arteriosclerosis and myocardial infarction(1) .
All the
effects of C5a appear to be mediated by a single class of G
protein-coupled receptors expressed on the cell surface. The sequence
of the human C5a receptor (hC5aR)
In this
report we show that another glutamate residue, Glu
A rabbit antiserum, raised against a peptide
analogue of the entire N-terminal sequence of the C5a receptor, was
used to sort the highest 5% of transfected cells on a Becton-Dickinson
Vantage FACS. RBL cells expressing the wild type but not the mutant
hC5aR were then cloned by limiting dilution(16) .
There is clear evidence that hC5a interacts with its receptor
through two or more separate sites. The N terminus of the receptor
contains a number of aspartate residues that appear to interact with
basic residues of hC5a(9, 11) . Peptides derived from
the C terminus of hC5a have been shown to bind to the receptor at an
additional site, hitherto uncharacterized(5, 11) . In
this report we present evidence to show that Glu
Glu
To
locate the site of the interaction between Glu
The role of this C-terminal Arg was further analyzed by the use of
hC5a des-Arg
The relative role of
Arg
In
conclusion, we have shown that the mutation of Glu
We thank Paul E. Brown and Arthur J. G. Moir for
oligonucleotide and peptide synthesis and John Lawry (Yorkshire Cancer
Research Campaign) for FACS.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of the human C5a receptor
(hC5aR) expressed in a model cell system for chemoattractant receptor
signaling, the rat basophilic leukemia cell line RBL-2H3. Both the
binding affinity for hC5a and the EC
for subsequent
cellular signals are reduced 5-10-fold by this substitution. A
peptide mimic of the C terminus of C5a also binds to, and activates,
hC5aR. The response to this peptide is reduced in cells bearing mutated
hC5aR, indicating that the mutation affects interactions with the C
terminus of hC5a. The C-terminal peptide contains only two basic
residues, a Lys and an Arg (assumed to be analogous to Lys
and Arg
of hC5a), which could act as counter-ions
for Glu
of the receptor. If the counter-ion on hC5a was
Arg
, then it would be expected that intact hC5a and hC5a
des-Arg
would have identical affinities and potencies when
interacting with mutant hC5aR. It was found, however, that the binding
affinity and potency (for receptor signaling events) of hC5a
des-Arg
was always lower than for intact hC5a.
Furthermore, the equivalent C-terminal peptide to hC5a des-Arg
(i.e. lacking the C-terminal Arg) could partially
activate the wild type but not the mutant receptor, whereas the
converse peptide, containing Arg but containing Met instead of Lys, had
equal potencies for both wild type and mutant receptors. Taken together
these data indicate that Glu
of hC5aR is not involved in
an interaction with Arg
of hC5a, but may interact with
Lys
of hC5a. Mutation of Glu
defines a
second ligand binding site on hC5aR, distinct from the previously
characterized site on the receptor N terminus. Unlike the N-terminal
binding site, this second site is associated not just with the
interaction with hC5a, but also with receptor activation.
(
)suggests that
the receptor has seven transmembrane domains linked by alternating
intra- and extracellular loops(2, 3) . Residues in the
extracellular N terminus and outer loops 2 and 3 are believed to be
involved in the binding of ligand, whereas regions of the intracellular
C terminus and inner loops are probably involved in coupling to (and
the activation of) G
proteins(4, 5, 6, 7) . The precise sites
of interaction between hC5a and hC5aR are still unknown, but it has
been suggested that three discontiguous regions of hC5a are necessary
for high affinity binding(8) . At the C terminus of hC5a,
mutation of Lys
, Leu
, or Arg
substantially inhibits binding while substitution of Gly for
Arg
in the disulfide-linked core of hC5a and mutation of
some N-terminal residues also affects binding affinity. More recently,
studies with antibodies against the hC5a receptor (9, 10) and receptor mutations (4, 11) have highlighted the role of the receptor N
terminus, containing a number of aspartate residues, in ligand binding.
It has been shown that the receptor N terminus is required for high
affinity binding of hC5a but is not essential for receptor activation,
providing evidence that at least two receptor sites are involved in the
interaction with ligand(4, 10) . Peptides that mimic the
C-terminal region of hC5a retain full ability to bind to mutated hC5aR
in which the first 22 residues of the receptor N terminus have been
deleted; hC5a itself binds to this mutant with nearly a 1000-fold lower
affinity compared to wild type receptor(11) . These additional
interaction sites, distinct from the receptor N terminus, have been
proposed to be Asp
, as a counter ion for Arg
of hC5a, and Glu
/Glu
, possible
counter-ions for Arg
(12) . We have already shown
that although Asp
is not required for high affinity
binding of hC5a, it is necessary for correct transduction of the ligand
binding signal(13, 14) . The
Glu
/Glu
residues of hC5aR do not appear to
be involved in the interaction with ligand(13) .
, in
the second extracellular loop of hC5aR, is involved in the binding of
ligand and activation of the receptor. Mutation of this residue to Gln
reduces the affinity for hC5a of the receptor 10-fold and also
increases the EC
for the stimulation of RBL cell secretion
by both hC5a and hC5a des-Arg
. The responses to peptide
mimics of either the C5a C terminus (Peptide 1:
Tyr-Phe-Lys-Ala-Cha-Cha-Leu-D-Phe-Arg; Ref. 5) or the C5a
des-Arg
C terminus (Peptide 3:
Tyr-Phe-Lys-Ala-Cha-Cha-Leu-D-Phe) are similarly affected,
suggesting that Glu
interacts with a residue present in
Peptides 1 and 3. The substitution of Met for Lys in Peptide 1 (Peptide
2: Tyr-Phe-Met-Ala-Cha-Cha-Leu-D-Phe-Arg) reduces the activity
of the peptide on wild type but not Gln
R by at least
200-fold, suggesting that this lysine residue, probably analogous to
Lys
of hC5a, may interact with Glu
.
Materials
Recombinant human C5a, phorbol
myristate acetate, and calcium ionophore A23187 were obtained from
Sigma (Poole, Dorset, United Kingdom (UK)). Purified human C5a
des-Arg was a generous gift from T. J. Williams and P. J.
Jose (National Heart and Lung Institute, London). Radioiodinated human
C5a was purchased from NEN Ltd. (Stevenage, UK). Fluo3-am and Pluronic
F-127 were supplied by Molecular Probes (Cambridge, UK).
[
H]5-HT was from Amersham International (Bucks.,
UK). The vector pEE6hCMV.neo was supplied by Celltech Ltd. (Slough,
UK). Peptides were synthesized on a Milligen 9050 peptide synthesizer,
using using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl)
amino acid derivatives supplied by Novabiochem (Nottingham, UK) and
Milligen (Watford, UK), and analyzed by mass spectrometry.
Tissue Culture
RBL-2H3 cells were routinely
cultured in Dulbecco's modified Eagle's medium + 10% (v/v) fetal calf serum, which was supplemented with 400
mg/liter G-418 (Life Technologies, Inc.) for transfected cells, at 37
°C, 5% CO.
Transfection and DNA Manipulation
RBL-2H3 cells
were transfected by electroporation, as described
previously(15, 16) , using the pEE6hCMV.neo vector
containing either a wild type or a mutant hC5aR cDNA insert in which
the glutamate residue at position 199 was changed to an glutamine
(Gln) by the overlap extension method as described
previously(6) .
Measurement of
5-Hydroxy-[
Secretion was measured as the release of
[H]tryptamine
Release
H]5-HT from intracellular granules, as described
previously(14) . Release was calculated as a percentage of total
cell associated radioactivity (measured following cell lysis in 0.5%
(v/v) Triton X-100 in balanced salt solution).
Measurement of Changes in Intracellular
Ca
RBL cells loaded with the
fluorescent CaLevels
indicator Fluo3 were used as described
previously (14). Increases in fluorescence were detected on an
Orthocyte cytometer, acquiring 2000 cells in 5 s.
Measurement of C5a Binding
Cells were harvested
with non-enzymatic cell dissociation solution and binding assays
performed as described previously (15) using I-C5a.
Expression of Wild Type and Mutant Receptors for Human
C5a in RBL Cells
Cells transfected with wild type (WTR) or
Glu
Gln substitution (Gln
R) hC5aR
were selected by FACS using a polyclonal antiserum raised against an
N-terminal peptide analogue of hC5aR(15) . Cells expressing high
levels of receptor were used in subsequent experiments. Binding studies
performed using radioiodinated C5a indicated that WTR and
Gln
R-bearing cells expressed high levels of receptor
(40,000 and 120,000, respectively), but that the affinity of the mutant
receptor for hC5a was lowered 10-fold relative to WTR receptor (4.70
± 1.0 nM and 48.6 ± 26.3 nM,
respectively).
Stimulation of the Secretory Response of Transfected RBL
Cells by hC5a and C-terminal Peptides
The lowered affinity of
GlnR suggests that an interaction occurs between hC5a and
Glu
of the wild type hC5aR. This was investigated further
using assays of cellular activation initially assessed by measuring the
release of [
H]5-HT from intracellular granules.
C5a and three peptide analogues of the C terminus of hC5a (Peptides 1,
2, and 3) were used to stimulate WTR- and Gln
R-bearing
cells. WTR cells responded to nanomolar concentrations of hC5a
(EC
for secretion
10-20 nM) and
micromolar concentrations of Peptide 1 (EC
1
µM) (Fig. 1a), whereas Gln
R
cells required higher levels of both hC5a and Peptide 1 (EC
50-60 nM and 20 µM,
respectively) (Fig. 1b). Peptide 2 failed to stimulate
significant levels of secretion from either of the cell lines tested (Fig. 1, a and b), although concentrations of
peptide above
100 µM could not be used, due to
limited solubility in aqueous conditions. Peptide 3 stimulated
detectable 5-HT release only in WTR cells, but could not be used at
concentrations >50 µM, again due to limited solubility (Fig. 1, a and b).
Figure 1:
The
secretory response of RBL cells transfected with wild type and mutant
receptors for human hC5a. Transfected RBL cells were labeled with
[H]5-HT and stimulated with hC5a or Peptide 1, 2,
or 3. RBL cells were transfected with either wild type hC5a receptors (a) or a substitution mutant receptor Glu
Gln (b). The results are the means ± S.E. of three
separate experiments performed in duplicate.
Stimulation of the Secretory Response of Transfected RBL
Cells by hC5a and hC5a des-Arg
hC5a des-Arg has previously been shown to be much less potent than hC5a in
neutrophil activation assays(15) . If Arg
interacted with Glu
, then hC5a and hC5a
des-Arg
would have identical potencies on cells
transfected with the mutant receptor. Cells bearing WTR showed an
EC
for hC5a des-Arg
of 40-50
nM, compared to an EC
of >700 nM for
Gln
R cells (Fig. 2). The difference between the
response of Gln
R cells to hC5a des-Arg
and
hC5a suggests that Glu
of hC5aR does not interact with
Arg
of hC5a.
Figure 2:
The secretory response of transfected RBL
cells to hC5a des-Arg. RBL cells transfected with either
wild type hC5aR (filledsymbols) or Gln
hC5aR (opensymbols) and labeled with
[
H]5-HT were stimulated with the stated
concentrations of either hC5a (
,
) or hC5a des-Arg
(
,
). Results shown are means ± S.E. from
three experiments performed in duplicate.
Displacement of
The lack of potency of hC5a des-ArgI-hC5a by hC5a and hC5a
des-Arg
on Glu
cells could be due to the loss of the
ability of mutant receptors to bind this ligand. This was investigated
by displacement studies using radioiodinated hC5a. The relative
abilities of hC5a des-Arg
and hC5a to displace labeled
hC5a appeared identical on cells bearing wild type and mutant receptors (Fig. 3). In both cases, hC5a des-Arg
was
approximately 100-fold less potent at displacement than hC5a which
strongly suggests that Arg
of hC5a does not interact with
Glu
of hC5aR and that the primary interaction of hC5a
des-Arg
with the receptor is not affected by the
substitution of Gln for Glu
.
Figure 3:
Displacement of I-hC5a by
hC5a and hC5a des-Arg
. RBL cells bearing either the wild
type hC5aR (filledsymbols) or Gln
mutant receptors (opensymbols) were incubated
with a fixed concentration of
I-hC5a and the stated
concentrations of unlabeled hC5a (
,
) or hC5a des-Arg
(
,
) and the specific binding determined as
described under ``Experimental Procedures.'' The results are
the means of a representative experiment performed in
duplicate.
Effects of hC5a and hC5a des-Arg
The activation of RBL cells may also be monitored by
the measurement of intracellular Caon
Intracellular Ca
Levels in Transfected RBL
Cells
levels
([Ca
]
) with the long
wavelength dye, Fluo3-am. We have shown previously that this response
requires 100-fold lower concentrations of ligand than the secretion of
5-HT(7, 14) . Stimulation with hC5a caused a transient
increase in Fluo3 fluorescence in WTR cells indicative of an increase
in [Ca
]
. This increase
was maximal at 30 s after hC5a addition (result not shown). The dose
response of this peak increase in fluorescence in WTR and
Gln
R cells showed a reduction in the sensitivity of the
mutant receptor to hC5a (Fig. 4, a and b),
whereas WTR cells respond maximally at
1 nM hC5a,
Gln
R cells required
10 nM hC5a for a maximal
response (Fig. 4, a and b). The response of
Gln
R cells to hC5a des-Arg
showed a similar
shift relative to WTR-bearing cells; WTR cells showed no additional
increase in response when the concentration of hC5a des-Arg
was
10 nM, but Gln
R cells required
250 nM for a maximal response (Fig. 4, a and b).
Figure 4:
Changes in intracellular Ca levels in transfected RBL cells stimulated by hC5a
des-Arg
. RBL cells transfected with either wild type
receptor (a) or Gln
mutant receptors (b) were labeled with Fluo3-am and stimulated with the stated
concentrations of hC5a or hC5a des-Arg
. Increases in
Fluo3-associated fluorescence were measured by flow cytometry, 30 s
after stimulation. Results are the means ± S.E. of three
separate experiments performed in duplicate. ND, not
determined.
Effects of C-terminal Peptides on Intracellular
Ca
The lack of solubility of Peptides 2 and 3 was such
that little or no secretion of 5-HT was measurable. This problem was
overcome by the use of peptides to stimulate changes in intracellular
CaLevels in Transfected RBL
Cells
levels in RBL cells. As expected from the
secretion assays, the maximal stimulation of increases in
Ca
levels in Gln
R-bearing cells with
Peptide 1 required increased concentrations relative to WTR (1
µM and
100 µM, respectively) (Fig. 5, a and b). Similarly, Peptide 3
stimulated only a small increase in intracellular Ca
at high peptide concentrations in WTR cells, whereas
Gln
R cells showed no detectable response. However, the
response of Gln
R and WTR cells to Peptide 2 did not fit
this pattern. The WTR cells required 100 µM Peptide 2 for
a near-maximal response (Fig. 5a), compared to 1
µM Peptide 1. In contrast, Gln
R cells had a
similar dose response to both Peptide 1 and Peptide 2, which was near
maximal levels at 100 µM. The mutant and wild type
receptor-bearing cells responded similarly to peptide 2 (Fig. 5b), suggesting that the substitution of Met for
the Lys residue in Peptide 2 removes a site of interaction between the
peptide and hC5aR.
Figure 5:
Changes in intracellular Ca levels in transfected RBL cells stimulated by hC5a C-terminal
peptides. RBL cells were transfected with either wild type receptor (a) or Gln
mutant receptors (b) were
labeled with Fluo3-am and stimulated with the stated concentrations of
Peptide 1, 2, or 3. Increases in Fluo3-associated fluorescence were
measured by flow cytometry, 30 s after stimulation. Results are the
means ± S.E. of three separate experiments performed in
duplicate. ND, not determined.
The peptides had no effect on untransfected cells
or RBL cells transfected with an irrelevant receptor (the type A
receptor for human IL-8) at concentrations of 50-100 µM (results not shown), indicating that the peptide stimulation of
responses in RBL cells is specific to hC5aR.
of the
human C5a receptor forms part of this additional site.
is a residue conserved in C5a receptors from the four species so
far characterized (human, murine, bovine, and canine; Ref. 18), which
suggests that it plays an important role in receptor activity. The
substitution of the uncharged Gln residue for Glu
lowers
the apparent affinity of the receptor for hC5a by approximately a
factor of 10 without affecting the expression of the receptor at the
cell surface. When expressed in the rat cell line RBL-2H3, ligation of
hC5aR causes a pertussis toxin-sensitive discharge of granule contents
and increases in intracellular Ca
and
F-actin(7, 11, 14, 17) . Previous
studies have shown that the binding affinity of hC5aR expressed in
either Chinese hamster ovary or RBL cells can be affected by the number
of receptors at the cell surface; an RBL cell line containing 166,000
receptors/cell (compared to 120,000 in Gln
R cells) had a K
for hC5a of 20
nM(14, 15) . Decreases in binding affinity may
be due to an excess of receptor over G protein, producing a
subpopulation of receptors in a low affinity state which are inactive
for signal transduction. We have therefore used the secretion of
[
H]5-HT and increases in intracellular
Ca
as a measure of the interaction between ligand and
receptor that is not affected by receptors not coupled to G proteins.
In secretion assays RBL cells containing Gln
R clearly
showed a dose response to hC5a that reflected the lowered binding
affinity relative to WTR. However, this change in the ability of hC5a
to activate mutant receptors is not due simply to a decrease in binding
affinity. In studies using Gln
R, hC5a des-Arg
effectively displaced labeled hC5a at 100-fold excess, whereas in
secretion assays hC5a des-Arg
was a poor stimulus relative
to hC5a, even at >100-fold higher concentrations. This suggest that
the mutation of Glu
does not affect the primary binding
site for ligand but does disrupt a second site that is responsible for
cellular activation. These results also indicate that Arg
of hC5a does not interact with Glu
of hC5aR.
and hC5a,
we used peptides modeled on the structure of the C terminus of hC5a.
Peptide 1 has been shown to activate hC5aR even when the N terminus of
the receptor has been deleted(5, 11) . Our results show
that Peptide 1 can still activate Gln
R, but with a
20-fold lowering of receptor activity relative to wild type
receptor. This suggests that the site of interaction between
Glu
and hC5a is retained on Peptide 1. Only two
positively charged residues that might act as counter-ions for
Glu
are present in Peptide 1: the C-terminal Arg
(analogous to Arg
of hC5a) and the Lys (presumably an
analog of Lys
of hC5a, a residue conserved in C5a from all
species so far investigated; Ref. 19). Additional peptides, in which
either the Lys was changed to Met (Peptide 2) or the C-terminal Arg was
omitted (Peptide 3), were synthesized and assayed for the ability to
stimulate secretion from transfected RBL cells. However, Peptide 2 was
insoluble in aqueous solutions above 100 µM, and
concentrations below this failed to cause secretion of 5-HT. Peptide 3
was soluble at concentrations
50 µM and could
stimulate 5-HT release in WTR cells only. We have shown previously that
increases in intracellular Ca
are stimulated at hC5a
concentrations 100-fold lower than secretion(14) , and so this
assay of cellular activation was used to study the effects of these
peptides. Cells with WTR required 10- and 100-fold lower concentrations
of hC5a and Peptide 1, respectively, to stimulate maximal increases in
intracellular Ca
compared to Gln
R. This
is similar to the difference in concentrations required for the
stimulation of secretion. However, Peptide 2 did not appear to
discriminate between wild type and mutant receptors; similar dose
responses were measured in cells with both receptor types. This result
strongly suggests that the Lys residue changed to Met in Peptide 2
interacts with Glu
in wild type receptors. Receptors
containing Gln
cannot interact with this Lys and so have
a lowered affinity (as shown by the Ca
response) for
Peptide 1, quite close in fact to that observed for Peptide 2. That
Peptide 2 retains any activity at all may be due in part to the
C-terminal Arg residue. Peptide 3, which lacks this Arg residue,
stimulated increases in intracellular Ca
only in WTR
cells; Gln
R cells did not respond. This suggests that the
residue interacting with Glu
of hC5aR is unlikely to be
the C-terminal Arg and that, in the absence of an interaction with the
peptide Lys residue, this Arg is essential for receptor activation.
, which has been shown to be considerably less
active than hC5a in cellular assays(20) . As expected, the
EC
of the secretory response of cells bearing WTR to hC5a
des-Arg
was much higher than that seen with hC5a. If
Glu
interacted with Arg
of hC5a, the
response of Gln
R-bearing cells to both hC5a and hC5a
des-Arg
would be identical; however, hC5a des-Arg
is much less active than hC5a on Gln
R cells,
stimulating little release even at 700 nM. The retention of
some stimulatory activity of hC5a des-Arg
when used with
Gln
R-bearing cells (i.e. when potential
interactions with both Lys
and Arg
have been
lost) suggests that additional interaction sites are present on hC5a
which are partly responsible for receptor activation. The use of
Peptide 3, which cannot stimulate a Ca
response in
Gln
R cells, indicates that these additional sites may
occur outside of the C-terminal region of hC5a. Additional interaction
sites on hC5a, possibly binding to the receptor N terminus could act by
increasing the probability of C-terminal regions correctly interacting
with the receptor or by causing receptor activation independently of
the hC5a C terminus. However, a number of antibodies have been raised
against the hC5a binding site on the N-terminal domain of hC5aR which,
although they can partially inhibit both binding and receptor
activation by hC5a, have no stimulatory activity
themselves(9, 10) . In addition, a mutant form of hC5a
lacking five C-terminal residues (C5a-(1-69)) has been shown to
be capable of binding to hC5aR but is devoid of activity (21). This
suggests that binding to this N-terminal site does not cause receptor
activation and that the first hypothesis is more likely to be correct.
The use of C5a-(1-69) also indicates that the presence of
Lys
is not sufficient to activate hC5aR in the absence of
the C-terminal residues. The same authors also found that the
C-terminal pentapeptide of hC5a (MQLGR) alone is also inactive and does
not bind to receptors, perhaps because the absence of Lys
prevents peptide activity(20) .
compared to Lys
in receptor activation
can be seen by the use of hC5a and hC5a des-Arg
in
Ca
response assays. The difference in the EC
values between WTR and Gln
R cells when hC5a is used
is approximately 10-fold, but when hC5a des-Arg
is used
this difference increases to nearly 100-fold. Thus the loss of the
interaction with Lys
in the absence of Arg
causes an additional 10-fold loss of the capacity of ligand to
activate the receptor. However, the loss of interactions with both of
these residues in the C terminus of hC5a des-Arg
does not
completely inhibit receptor activation. It is unclear from our data if
residues 69-73 in the C terminus of hC5a (DMQLG) can activate
hC5aR, but other workers have shown that modification of the
hydrophobicity or chirality of this sequence can significantly alter
the activity of C-terminal peptides(19, 22) .
of the
human C5a receptor to Gln lowers the ligand binding affinity
approximately 10-fold. This decrease in affinity appears to be due to
the loss of an interaction between Glu
and Lys
of hC5a, a residue that has already been shown to be important in
receptor binding(7) . Mutations of this and other residues of
hC5a and the human C5a receptor are currently being investigated.
R, substitution of Gln for
Glu
of hC5aR.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.