From the Mike Rosenbloom Laboratory for
Cardiovascular Research, Division of Medicine, McGill University Health
Centre, Montreal, Quebec H3A 1A1, Canada and the ¶ Department of
Neurology, University of Sheffield Medical School,
Sheffield, S10 2RX, United Kingdom
Received for publication, June 20, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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The orphan receptor C5L2 has recently been
described as a high affinity binding protein for complement fragments
C5a and C3a that, unlike the previously described C5a receptor (CD88),
couples only weakly to Gi-like G proteins (Cain,
S. A., and Monk, P. N. (2002) J. Biol. Chem.
277, 7165-7169). Here we demonstrate that C5L2 binds the metabolites
of C4a and C3a, C4a des-Arg77, and C3a
des-Arg77 (also known as the acylation-stimulating protein
or ASP) at a site distinct from the C5a binding site. The binding of
these metabolites to C5L2 does not stimulate the degranulation of
transfected rat basophilic leukemia cells either through endogenous rat
G proteins or when co-transfected with human G C5a and C3a have wide ranging effects in humans. Although
initially described as leukocyte chemoattractants and anaphylatoxins, it is now clear that C5a and C3a are involved in microbial host defense, immune regulation (1), and protection against toxic insult
(2-5). C5a and C3a are also reported to have psychopharmacological effects on feeding and drinking behavior (6, 7). Both complement fragments are rapidly desarginated by serum carboxypeptidase, which
modulates their function. Although C5a des-Arg74 retains
most of the activity of intact C5a, albeit with a generally lower
affinity for the C5a receptor (CD88),
1 C3a des-Arg77
activity is profoundly reduced relative to C3a with respect to immunologic function. No binding of the C3a des-Arg77 form
to the previously cloned and characterized C3a receptor (C3aR) is
observed in transfected RBL cells or mouse macrophage/monocytes (8)
and, unlike C3a, C3a des-Arg77 does not stimulate
eosinophil chemotaxis (9), prostanoid production by guinea pig
peritoneal macrophages and rat Kupffer cells (10), or human
monocyte-like U937 cell degranulation (11). However, the following
responses to C3a des-Arg77 have been reported. (i) The
cytotoxicity of NK cells is inhibited by both C3a and C3a
des-Arg77 (12). (ii) Cytokine production by human
monocyte/macrophages and PBMC is enhanced by these ligands but
inhibited in human tonsil-derived B cells (13, 14). (iii) Histamine
release from rat peritoneal mast cells is stimulated (15). In addition,
C3a des-Arg77 has well documented acylation-stimulating
properties and increases triacylglycerol synthesis in human adipocytes,
preadipocytes, and human skin fibroblasts (HSF), where this function as
an acylation-stimulating protein (ASP) was initially characterized
(16). This triglyceride-stimulating activity is also shared by C3a
(17). One hypothesis explaining this pattern of responses is that cells
may express two kinds of receptor; one, probably C3aR, binds only C3a,
and another, as yet unidentified receptor, binds both C3a and C3a
des-Arg77.
We have recently characterized a novel chemoattractant-binding protein,
C5L2, that has high affinity for C5a, C5a des-Arg74, and
C3a (18). Here we report that C5L2 also binds C3a
des-Arg77/ASP and is expressed in three C3a
des-Arg77/ASP-responsive cell types.
Cell Lines and Culture Conditions--
HSFs were obtained as
described previously (19). RBL-2H3, HEK 293, HSF, and 3T3-L1 cells were
routinely cultured in Dulbecco's modified Eagle's medium/F12 plus
10% (v/v) fetal calf serum at 37 °C, 5%
CO2. The media was supplemented with 400 mg/liter G-418 for
RBL-2H3 and 500 mg/liter G-418 for HEK 293 stably transfected cells.
Stable Transfection of RBL and HEK 293 Cells--
C5L2, C3aR and
CD88-transfected RBL-2H3 and HEK 293 cells were produced as
described (18). G Transient Transfection of HEK 293 Cells--
HEK 293 cells were
seeded into 6-well plates at 1 × 106 cells/well the
day before transfection. C5L2 in vector pEE6hCMV.neo (Celltech) or C3aR
in vector pcDNA1/AMP (Invitrogen) at 2 µg of DNA/well was
transfected with LipofectAMINE 2000 (5 µl/well) (Invitrogen) according to the manufacturer's protocol. Cells were assayed for binding/uptake 3 days post-transfection.
Production of Anaphylatoxins--
Expression and purification of
the recombinant His6-tagged C5a, C5a des-Arg74,
and C3a were performed under denaturing conditions as described (21).
Recombinant C4a, C4a des-Arg77, and C3a
des-Arg77 were expressed and purified under non-denaturing
conditions by sonication in the presence of BugBuster Protein
Extraction Reagent (Novagen) using manufacturer's conditions. Plasma
C3a des-Arg77/ASP and plasma C3a were purified as described
previously (17).
Fluorescent Labeling of C3a des-Arg77/ASP
and C3a--
C3a des-Arg77/ASP and C3a were labeled with
FLUOS (Roche Molecular Biochemicals) at a molar ratio of 1:10 (ligand
to FLUOS) for 2 h according to the manufacturer's
recommendations. Labeled ligand was separated from free FLUOS on a
Sephadex G25 M column and stored in aliquots at Radiolabeled Ligand Competition Receptor Binding
Assays--
Competition binding assays were performed using 50 pM 125I-C5a or 125I-C3a
(PerkinElmer Life Sciences) on adherent C3aR-, CD88-, or C5L2-transfected RBL cells in 96-well microtiter plates (55,000 cells/well) at 4 °C as described previously (22). Competition assays
for HSF, 3T3-L1, U937, and HEK 293 were performed using 1 nM 125I-C3a or 125I-C3a
des-Arg77/ASP on adherent cells in 96-well microtiter
plates. Competition curves were generated by preincubating adherent
cells with increasing concentrations of unlabeled complement fragments.
The IC50, standard error values and linear regression
analyses were obtained by using GraphPad Prism 2.0 or Sigma Plot.
Production of Antiserum against C5L2--
Antiserum was raised
in rabbits using the extracellular N-terminal sequence of human C5L2
(MGNDSVSYEYGDYSDLSDRPVDC) coupled to keyhole limpet hemocyanin, as
described previously (23). The serum recognized RBL cells transfected
with human C5L2 (but not untransfected control cells) at dilutions as
low as 1/10,000, and binding to C5L2 was totally inhibited by
preincubation of serum with 100 µg/ml immunizing peptide.
Fluorescence-activated Cell Scanning for Ligand
Binding/Uptake Assays--
Cells were incubated with
the indicated concentrations of FLUOS-labeled C3a
des-Arg77/ASP or C3a for 30 min at 37 °C in binding
buffer (24) and washed three times with cold binding buffer. Cells were
then detached with 0.25% trypsin/0.02% EDTA in phosphate-buffered
saline (PBS), fixed with 1% paraformaldehyde, washed with 0.3% PBS,
and assayed by FACS. For anti-human C5L2 binding, cells were released
from the culture dishes with non-enzymatic cell dissociation solution (Sigma), pelleted (600 × g, 5 min), resuspended with
anti-C5L2 antiserum (1:2000 in 3% bovine serum albumin in PBS),
and incubated at 4 °C for 60 min. Again, cells were pelleted, washed
twice with PBS, and resuspended in fluorescein isothiocyanate-labeled
anti-rabbit IgG, (Sigma) at (1:1000 dilution in 3% bovine serum
albumin in PBS) and incubated at 4 °C for 60 min. Finally, cells
were pelleted, washed twice, and resuspended in 0.3% paraformaldehyde
in PBS for FACS analysis.
Cellular Activation Assays--
Cellular activation was measured
as the release of Analysis of Receptor Expression by RT-PCR--
Total RNA was
isolated by Trizol extraction from freshly isolated samples of the
tissues and cells. For RT-PCR, cDNA was produced from 3 µg of RNA
by reverse transcriptase, and 4% of the reaction was amplified by PCR
with 1.5 mM MgCl2 and 0.01 mM
tetramethyl ammonium chloride under the following protocol: 1 min at
94 °C; 1 min at 60 °C; and 2 min at 72 °C for 35 cycles.
Primers for human C5L2 were 5'-CCTGGTGGTCTACGGTTCAG-3' (sense) and
5'-GGGCAGGATTTGTGTCTGTT-3' (antisense). Primers for murine C5L2
(Ensembl gene identification number ENSMUSG00000041388) were
5'-ATGGCCGACTTGCTTTGT-3' (sense) and 5'-CCTTGGTCACCGCACTTTC-3'
(antisense). As control, glyceraldehyde 3-phosphate dehydrogenase (GAP)
was used as described previously for human GAP with the human primers
5'-GGTGAAGGTCGGAGTCAACGGATTTGG-3' (sense) and
5'-GGCCATGAGGTCCACCACCCTGTT-3' (antisense) (product size 978 bp) and
the mouse primers 5'-CAGTTATTACCTAGTGGGG-3' (sense) and
5'-CCAGTTGAGGTCTTTCCAACG-3' (antisense) (product size 756 bp). Reaction
products were separated on a 7.5% polyacrylamide gel and detected by
silver staining (Bio-Rad), and a 100-bp DNA ladder (New England
Biolabs) was used as standard.
C5L2 Is a Promiscuous Complement Fragment-binding Protein--
We
have shown previously that C5L2 has binding sites for C5a, C5a
des-Arg74, C4a, and C3a (18). Here we show that the
des-Arg77 forms of C4a and C3a are also ligands for this
receptor when expressed in the RBL-2H3 cell line (Fig.
1, A and B, and
Table I) and can compete strongly with
125I-C3a for C5L2 binding (Fig. 1A). In
contrast, C4a des-Arg77 and C3a des-Arg77/ASP
cannot compete effectively with 125I-C5a for C5L2 or CD88
binding (Fig. 1B, and Table I). Although C3aR and C5L2 bind
C3a with similar affinities, C3aR has no detectable affinity for C3a
des-Arg77/ASP (Table I). Similarly, although C4a can
compete with 125I-C3a for binding to both C3aR and C5L2,
suggesting a similar affinity for both receptors, C4a
des-Arg77 is >50-fold more effective at competing with
125I-C3a binding at C5L2 than at C3aR (Table I). The data
suggest either that C5L2 has two conformations with different ligand
binding profiles or that the receptor has two binding sites. As we have shown previously that the Bmax values for
125I-C3a and 125I-C5a binding to
C5L2-transfected RBL cells are identical (18), the most likely
explanation is that a single form of C5L2 has separate binding sites.
We propose that one site binds 125I-C3a and C3a
des-Arg77/ASP, at which all of the complement fragments
except C5a des-Arg74 can compete with similar affinities,
and that the second high affinity site, which preferentially binds
125I-C5a, can only be competed by C5a des-Arg74
and, to a lesser extent, C4a.
C3a des-Arg77/ASP Binds Directly to C5L2 but
Not to C3aR or CD88--
Because recombinant C3a
des-Arg77/ASP can clearly compete with 125I-C3a
(but not C5a) for binding to C5L2, we then directly measured the
affinity of C3a des-Arg77/ASP for C5L2 using protein
purified from human plasma as C3a des-Arg77/ASP and
tested for acylation-stimulating bioactivity. Plasma-purified human C3a
des-Arg77/ASP and C3a were both labeled with FLUOS.
Increasing concentrations of C3a des-Arg77/ASP were
incubated with HEK 293 cells transiently transfected with C5L2, and
binding and uptake were assessed by flow cytometry (Fig.
2A). FLUOS-C3a
des-Arg77/ASP clearly binds to C5L2 with half-maximal
fluorescence intensity at ~3 nM, whereas mock-transfected
cells (Fig. 2A, inset) show no binding of C3a
des-Arg77/ASP, even at a high concentration of 10 nM. For comparison purposes, the binding of FLUOS-C3a to
HEK 293 cells transiently transfected with C3aR is shown (Fig.
2B) with half-maximal binding of FLUOS-C3a at 2.5 nM. In separate experiments, FLUOS-C3a
des-Arg77/ASP binding to C3aR transfected cells was found
to be not significantly different from basal (basal fluorescence = 100%; FLUOS-C3a des-Arg77/ASP = 103% ±8%,
mean ± S.E., n = 3), and neither FLUOS-C3a
des-Arg77/ASP nor FLUOS-C3a showed binding to cells
transiently transfected with CD88, the C5a receptor (Fig.
2C).
C3a des-Arg77/ASP binding was further examined in cells
that are responsive to the acylation-stimulating properties of C3a
des-Arg77/ASP and compared with that in HEK cells
transfected with C3aR and CD88. 125I-C3a
des-Arg77/ASP does not bind to C3aR-transfected HEK cells
and does not compete with 125I-C3a (Table
II), as found previously (26). Similarly,
Bt2-cAMP-differentiated U937 macrophages (which are
reported to express the C3a receptor and respond to C3a) demonstrated
no specific C3a des-Arg77/ASP binding (data not shown). The
result was also negative for undifferentiated U937 cells (data not
shown). Also, C3a des-Arg77/ASP does not bind to HEK 293 cells transfected with CD88 (binding of 125I-C3a
des-Arg77/ASP, mock transfection 100% ± 4%,
n = 6; irrelevant receptor transfection, 102% ± 11%,
n = 6; CD88 transfection, 110% ± 22%, n = 6). Similar results were obtained for
125I-C3a binding to CD88 (irrelevant receptor transfection,
100% ± 6%, n = 6; CD88 transfection, 99% ± 17, n = 6). By contrast, human skin fibroblasts, which
respond to C3a des-Arg77/ASP by increasing triglyceride
synthesis (27), bind both 125I-C3a
des-Arg77/ASP and 125I-C3a with high affinity
(Table II). As observed in C5L2-transfected RBL cells, unlabeled C3a
des-Arg77/ASP is slightly less effective at competing for
125I-C3a binding than unlabeled C3a in both HSF- and
C5L2-transfected RBL cells (Tables II and I, respectively), whereas C3a
was an effective competitor for 125I-C3a
des-Arg77/ASP binding (Table II). Thus, C5L2 has binding
characteristics that overlap with both CD88 and C3aR but also has the
unique ability to bind C3a des-Arg77/ASP, which parallels
the binding characteristics of HSF cells.
C3a des-Arg77 Binding to C5L2 Does Not Stimulate
Degranulation in C5L2-Transfected RBL Cells--
We have shown
previously that C5a, C5a des-Arg74, C4a, and C3a binding to
C5L2 does not stimulate either an increase in intracellular Ca2+ or the degranulation of transfected RBL cells due to
weak coupling to endogenous Gi-like G proteins (18). We
also examined the effects of C3a des-Arg77/ASP and C4a
des-Arg77 and found that these ligands did not stimulate
degranulation in transfected RBL cells at concentrations of up to 10 µM (data not shown). In addition, there was no effect of
these two ligands on either CD88 or C3aR activation of degranulation
(Table III) although the expected
responses to C5a, C5a des-Arg74, and C3a, respectively, are
robust. Neither recombinant nor plasma-purified C3a
des-Arg77/ASP (nor any other ligand) is able to activate
endogenous G proteins in C5L2-transfected RBL cells.
Co-expression of C5L2 with G C3a des-Arg77/ASP Stimulates Triglyceride
Synthesis in Human Skin Fibroblasts but Not in Cells Expressing C3a
Receptor--
In HSF, both C3a des-Arg77/ASP and C3a can
stimulate triglyceride synthesis (TGS) at levels comparable with
insulin, a hormone well known to influence cellular triglyceride levels
(Table IV). C3a des-Arg77/ASP
appears to act via stimulation of the protein kinase C pathway (31),
and stimulation of this pathway by the phorbol ester PMA also results
in increased TGS (Table IV). Bioactivity of C3a is not dependent on
conversion of C3a to the des-arginated form, C3a
des-Arg77/ASP, because the presence of the carboxypeptidase
inhibitor (Plummer's inhibitor) has no effect on C3a bioactivity
(Table IV). Increased TGS is not simply a response to C3a binding,
however, as C3aR-transfected HEK cells and
Bt2-cAMP-differentiated U937 monocytic cells (which express
the C3aR and bind C3a) do not respond with an increase in TGS to either
C3a or C3a des-Arg77/ASP (Table IV). However, these cell
types may lack all or part of the machinery to mount an increase in
TGS, as there is no significant response to treatment with PMA or
insulin (Table IV).
Both C3a and C3a des-Arg77/ASP bind to the C5L2 receptor
expressed in RBL cells and HSF with comparable affinity, suggesting that C5L2 may be the C3a des-Arg77/ASP receptor on HSF. As
C5L2 has already been shown to bind several complement fragments, we
examined the acylation-stimulating properties of other C5L2 ligands in
cells that respond to C3a des-Arg77/ASP. Even at higher
concentrations than those usually used, there was no stimulation of
triglyceride synthesis in 3T3-L1 preadipocytes (Table
V) or in HSF (data not shown) with C5a,
C5a des-Arg74, C4a, or C4a des-Arg77, despite a
clear response to C3a des-Arg77/ASP in both cell types.
Treatment of HSF or 3T3-L1 preadipocytes with other peptides of
similar charge and size (lysozyme, cytochrome C) also has no effect on
triglyceride synthesis or binding of C3a
des-Arg77/ASP.2
These results suggest that the triglyceride synthesis stimulation is
both peptide- and receptor-specific, with both C3a
des-Arg77/ASP and C3a as the appropriate ligands
interacting with the receptor C5L2. All ligands that stimulate C5L2,
C3aR, or CD88 to increase TGS or degranulation also act as competitors
for either 125I-C3a or 125I-C5a binding. The
converse is not true; some C5L2 ligands (e.g. C5a) bind C5L2
but fail to activate the receptor (as assessed by TGS). C4a also binds
to both C3aR and C5L2 receptors but activates neither, whereas C3a
binds to and activates both receptors, but induces different responses
(degranulation versus TGS). Activation requires binding to
the appropriate receptor, but ligand binding per se does not
necessarily cause activation. This may be explicable in terms of the
physical separation of binding and activation sites on chemoattractant
receptors such as CD88 (25, 32). The two binding sites tentatively
identified on C5L2 may also have different roles, one involved solely
in ligand binding and one involved in both binding and activation of
TGS. Thus, C5a, which binds to the first site on C5L2, may be able to
sterically hinder the binding of ligands that interact primarily with
the second site (C3a and C3a des-Arg77/ASP) without
activation of receptor. The ability of C5a to influence binding to the
second site is presumably dependent on the C-terminal Arg residue, as
C5a des-Arg74 cannot compete for 125I-C3a
binding to C5L2.
C5L2 mRNA and Cell Surface Protein Are Expressed in Adipose
Tissue, Skin Fibroblasts, and 3T3-L1 Preadipocytes--
Although C3a
des-Arg77/ASP is regarded as biologically inactive in most
myeloid systems, the acylation-stimulating properties of this
complement fragment are well documented in adipocytes and related cells
(33). We therefore investigated the expression of C5L2 in human adipose
tissue, HSF, and 3T3-L1 preadipocytes, because fibroblasts,
preadipocytes, and adipocytes are all known to respond directly to C3a
and C3a des-Arg77/ASP by an increase in triglyceride
synthesis (Table IV) and glucose transport (17). We performed RT-PCR
using species-specific sets of primers to detect expression in human
adipocytes, HSF, and mouse 3T3-L1 preadipocyte mRNA. Both primer
sets (human and murine) produced a band as seen on polyacrylamide
electrophoresis gels at sizes similar to those expected for a C5L2
transcript (Fig. 4). As the DNA markers
are standardized for agarose gels and not polyacrylamide gels, the
human adipose tissue PCR product was extracted from an agarose gel and
sequenced. We confirmed the authenticity of the transcript as that of
C5L2. By contrast, RT-PCR of RNA from the human monocytic cell line
U937 and non-transfected HEK 293 cells did not result in any PCR
product using C5L2 primers despite equal levels of
glyceraldehyde-3-phosphate dehydrogenase (Fig. 4).
These results were further confirmed using an antiserum specific to the
N-terminal region of human C5L2. FACS analysis clearly demonstrates
that HSFs (Fig. 5A) express
endogenous C5L2 on their cell surface, although the fluorescent
intensity was lower than that of HEK 293 cells overexpressing stably
transfected C5L2 (Fig. 5B). In contrast, untransfected HEK
293 cells did not bind the anti-serum (Fig. 5C). As the
antiserum does not appear to recognize murine C5L2, cells transfected
with mouse C5L2 were negative (data not shown), and we were unable to
test for the expression of C5L2 on the surface of the murine 3T3-L1
cells.
In summary, we have shown that adipocytes, HSF, and 3T3-L1
preadipocytes, cell types that have been shown to bind both C3a and C3a
des-Arg77/ASP and to respond to these ligands with
increased triglyceride synthesis, also express C5L2. C5L2 binds both
ligands with high affinity, suggesting that it may be a functional C3a
des-Arg77/ASP and C3a receptor when expressed in
appropriate cell types. In contrast, C5a and C5a des-Arg74,
which bind preferentially to a different site on C5L2, do not stimulate
triglyceride synthesis. The role of C5L2 in cellular responses to
complement fragments is clearly complex and remains to be elucidated.
16. C3a
des-Arg77/ASP and C3a can potently stimulate triglyceride
synthesis in human skin fibroblasts and 3T3-L1 preadipocytes. Here we
show that both cell types and human adipose tissue express C5L2
mRNA and that the human fibroblasts express C5L2 protein at the
cell surface. This is the first demonstration of the expression of C5L2
in cells that bind and respond to C3a des-Arg77/ASP and
C3a. Thus C5L2, a promiscuous complement fragment-binding protein with
a high affinity site that binds C3a des-Arg77/ASP, may
mediate the acylation-stimulating properties of this peptide.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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ABSTRACT
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REFERENCES
16 was cloned from human monocyte
mRNA and authenticated by sequencing. Human G
16 and
either C5L2 or CD88 were ligated into the bicistronic expression vector
pIRES (Clontech). Stable transfection of RBL-2H3
cells with pIRES constructs was achieved by electroporation (20). Cells
underwent three rounds of fluorescence-activated cell sorting (FACS)
using anti-CD88 antibody (clone S5/1; Serotec) or anti-hemagglutinin peptide antibody (Roche Molecular Biochemicals, clone 12CA5) for C5L2-expressing cells, selecting the top 5% of receptor-positive cells
in each round. HEK 293 cells were transfected (see below) and then
sorted with two rounds of FACS using FLUOS-C3a
des-Arg77/ASP binding, selecting the top 50% of the
population of positive cells each time.
80 °C.
-hexosaminidase from RBL intracellular granules
(25) or as the stimulation of triglyceride synthesis in HSF and 3T3-L1
cells (17). For
-hexosaminidase assays, EC50 and
standard error values were obtained by iterative curve fitting using
GraphPad Prism 2.0. For triglyceride synthesis, cells were incubated
with 100 µM [3H]oleate complexed to albumin
(molar ratio 5:1) for 4 h. Triglyceride synthesis was calculated
as [3H]oleate incorporation into triglyceride.
RESULTS AND DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
C3a des-Arg77/ASP and C4a
des-Arg77 bind to RBL cells expressing C5L2. RBL cells
stably transfected with C5L2 were incubated with the stated
concentrations of complement fragments for 10 min prior to the addition
of 50 pM 125I-C3a (A) or 50 pM 125I-C5a (B). Results are the
means of n (n shown in Table I) separate
experiments performed in triplicate ± S.E.
Summary of competition binding data for human chemoattractant receptors
expressed in RBL cells
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Fig. 2.
C5L2 shows saturable binding/uptake of C3a
des-Arg77/ASP. HEK 293 cells were transiently
transfected with C5L2 (A), C3aR (B),
or CD88 (C), and 3 days later cells were incubated for 30 min with the indicated concentrations of FLUOS-labeled C3a
des-Arg77/ASP or C3a, respectively. Binding/uptake was
assessed by FACS, and the percentage of cells above a fluorescence
intensity of 8 was determined. The fluorescence histograms for mock
versus receptor transfected cells at the highest ligand
concentration are shown in the insets (panels
A and B).
Competition binding data for human skin fibroblasts and
C3aR-transfected HEK cells
Summary of receptor activation data for human chemoattractant receptors
expressed in RBL cells
16 Does Not Enable a
Degranulatory Response--
The C5a receptor CD88 can couple
effectively to the pertussis toxin (PT)-sensitive G proteins
Gi2 and Gi3 (28) and also to the
toxin-insensitive Gq-family member, G16 (29,
30). We reasoned that the moderate response of ligand coupling to C5L2 could be due to the absence of human G16 from RBL cells,
which we tested by co-transfecting cells with human G
16
and either CD88 or C5L2. The bicistronic vector pIRES was used to
increase the likelihood that equal amounts of receptor and G protein
would be expressed in transfected cells. With transfection of CD88
alone (Fig. 3A), increasing
concentrations of PT inhibit the degranulation response. In
co-transfected cells (Fig. 3B), CD88 clearly couples strongly to G
16, and the degranulation response to C5a
is resistant to doses of PT that could substantially inhibit
degranulation in cells transfected with CD88 alone. At a higher dose of
PT (10 ng/ml), a small inhibition of degranulation is observed,
presumably due to stabilization of interactions between free
subunits and ADP-ribosylated G
i. In
C5L2+G
16 co-transfected cells, treatment with high
concentrations (1 µM) of intact or des-Arg complement
fragments still does not stimulate degranulation (Fig. 3C).
It appears unlikely that C5L2 couples to G proteins usually associated
with leukocyte chemoattractant receptors, although this does not
eliminate the possibility of coupling to other signaling pathways.
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Fig. 3.
CD88 but not C5L2 stimulates degranulation
coupled to G 16 proteins. RBL
cells were transfected with human CD88 (A),
CD88+G
16 (B), or C5L2+G
16
(C) using monocistronic or bicistronic expression vectors.
Functional association of CD88 with G
16 was demonstrated
using pertussis toxin treatment to inhibit endogenous
Gi-like G proteins; CD88- (A) and
CD88+G
16- (B) transfected RBL cells were
treated for 4 h with 0-10 ng/ml PT prior to the addition of C5a.
Degranulation was measured as secretion of
-hexosaminidase expressed
as a percentage of the maximal release in the presence of 1 µM C5a with no PT. Typical release under these conditions
was 80% of total cellular
-hexosaminidase. C, RBL cells
transfected with C5L2+G
16 were treated with 1 µM of the indicated complement fragments, and
degranulation was measured as the secretion of
-hexosaminidase
expressed as a percentage of the total cellular content.
Stimulation of triglyceride synthesis in different cell lines
Assessment of triglyceride synthesis by complement fragments in 3T3-L1
preadipocytes
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Fig. 4.
C5L2 is expressed in cells that show binding
and response to C3a des-Arg77/ASP and C3a. RT-PCRs of
human adipose tissue, human skin fibroblasts, and mouse 3T3-L1
preadipocytes with primers for C5L2 show bands of expected size (human,
798 bp; mouse, 739 bp) after polyacrylamide gel electrophoresis and
silver staining. Cell lines that are negative for C3a
des-Arg77/ASP binding and response (HEK 293, U937 monocytic
cells) show no band. For control, human (Lanes 1-4) and
murine (Lane 6) glyceraldehyde 3-phosphate dehydrogenase
(GAP) was used. Lane 1, human adipose tissue;
lane 2, human skin fibroblasts; lane 3, HEK 293;
lane 4, U937 monocytic cells; lane 5, 100-bp DNA
ladder with 1000 bp indicated; lane 6; 3T3-L1
preadipocytes.
View larger version (16K):
[in a new window]
Fig. 5.
Human fibroblasts demonstrate cell surface
expression of human C5L2. HSF cells (A), HEK 293 cells
stably transfected with C5L2 (B), and untransfected HEK 293 cells (C) were detached non-enzymatically and incubated at
4 °C with either rabbit anti-C5L2 (continuous
line) or rabbit non-immune serum (NI serum;
broken line) as control. After washing, the cells
were incubated with goat anti-rabbit IgG conjugated to fluorescein
isothiocyanate. After washing and fixing with paraformaldehyde,
cellular fluorescence was measured by FACS.
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FOOTNOTES |
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* This research was funded by Arthritis Research Campaign Project Grant M0648 (to P. N. M.) and by the Canadian Institute for Health Research (to K. C.).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: Dept. of
Neurology, E Floor, University of Sheffield Medical School, Beech Hill Rd., Sheffield, S10 2RX, United Kingdom. Tel.: 44-114-2261312; Fax:
44-114-2760095; E-mail: p.monk@shef.ac.uk.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M206169200
2 D. Kalant, M. Maslowska, A. D. Sniderman, and K. Cianflone, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CD88, human C5a receptor; C3aR, human C3a receptor; HSF, human skin fibroblasts; ASP, acylation-stimulating protein; RBL, rat basophilic leukaemia cell line; HEK 293, human embryonic kidney 293 cell; FACS, fluorescence-activated cell scanning; FLUOS, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; PBS, phosphate-buffered saline; RT, reverse transcription; PT, pertussis toxin; PMA, phorbol 12-myristate 13-acetate; TGS, triglyceride synthesis.
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