The Chemoattractant Receptor-like Protein C5L2 Binds the C3a des-Arg77/Acylation-stimulating Protein*

David KalantDagger §, Stuart A. Cain§, Magdalena MaslowskaDagger §, Allan D SnidermanDagger , Katherine CianfloneDagger , and Peter N. Monk||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 Galpha 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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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.

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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). Galpha 16 was cloned from human monocyte mRNA and authenticated by sequencing. Human Galpha 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.

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 -80 °C.

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 beta -hexosaminidase from RBL intracellular granules (25) or as the stimulation of triglyceride synthesis in HSF and 3T3-L1 cells (17). For beta -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.

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.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
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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.


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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.


                              
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Table I
Summary of competition binding data for human chemoattractant receptors expressed in RBL cells

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).


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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).

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.


                              
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Table II
Competition binding data for human skin fibroblasts and C3aR-transfected HEK 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.


                              
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Table III
Summary of receptor activation data for human chemoattractant receptors expressed in RBL cells

Co-expression of C5L2 with Galpha 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 Galpha 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 Galpha 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 beta gamma subunits and ADP-ribosylated Galpha i. In C5L2+Galpha 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 Galpha 16 proteins. RBL cells were transfected with human CD88 (A), CD88+Galpha 16 (B), or C5L2+Galpha 16 (C) using monocistronic or bicistronic expression vectors. Functional association of CD88 with Galpha 16 was demonstrated using pertussis toxin treatment to inhibit endogenous Gi-like G proteins; CD88- (A) and CD88+Galpha 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 beta -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 beta -hexosaminidase. C, RBL cells transfected with C5L2+Galpha 16 were treated with 1 µM of the indicated complement fragments, and degranulation was measured as the secretion of beta -hexosaminidase expressed as a percentage of the total cellular content.

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).


                              
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Table IV
Stimulation of triglyceride synthesis in different cell lines

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


                              
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Table V
Assessment of triglyceride synthesis by complement fragments in 3T3-L1 preadipocytes

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).


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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.

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.


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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.

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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