COMMUNICATION
Chimeric Receptors of the Human C3a Receptor and C5a Receptor (CD88)*

Torsten CrassDagger §, Robert S. Ames§, Henry M. Sarauparallel , Mark A. Tornetta, James J. Foleyparallel , Jörg KöhlDagger , Andreas KlosDagger , and Wilfried BautschDagger **

From the Dagger  Institute of Medical Microbiology, Hannover Medical School, D-30623 Hannover, Germany and the Departments of  Molecular Biology and parallel  Pulmonary Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Galpha -16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any reciprocal exchange. N-terminal substitution confirmed the two-binding site model for the human C5aR, in which the receptor N terminus is required for high affinity binding of the native ligand but not a C5a analogue peptide. In contrast, the human C3a receptor did not require the original N terminus for high affinity binding of and activation by C3a, a result that was confirmed by N-terminal deletion mutants. This indicates a completely different binding mode of the anaphylatoxins to their corresponding receptors. The C5a analogue peptide, but not C5a, was an agonist of the C3aR. Replacement of the C3aR N terminus by the C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a receptor, which bound and functionally responded to both ligands, C3a and C5a.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Activation of the complement cascade leads to the generation of the 74-77-amino acid anaphylatoxins C3a and C5a. Acting as potent signaling molecules, C3a and C5a exhibit a broad spectrum of proinflammatory effects on different organs and cell types, which include smooth muscle contraction, increase in vascular permeability, chemotaxis, cell activation, and granule secretion. Despite a high overall similarity in their structure and functional effects, C3a and C5a exhibit qualitative and quantitative differences in their ability to elicit the above-mentioned reactions, suggesting distinct roles of each anaphylatoxin in the initiation and maintenance of inflammatory reactions. Increased serum levels of C3a and C5a have been reported in a number of disease states, like the adult respiratory distress syndrome, chronic polyarthritis, and psoriasis, suggesting an important role of the anaphylatoxins in the pathophysiology of these and other inflammatory diseases (for review see Refs. 1 and 2). A more detailed knowledge about the mode of action of the anaphylatoxins on their target cells might suggest novel and valuable therapeutic approaches for the treatment of many of these disease states.

C3a and C5a have similar solution structures consisting of a globular core composed of four tightly folded alpha -helices and a C-terminal "finger" (3-5). They act on their target cells by binding to and activating highly ligand-specific membrane receptors belonging to the family of seven transmembrane domain G-protein-coupled receptors. The human C5a receptor (C5aR)1 was cloned in 1991 (6, 7), and in 1996 the corresponding receptor for C3a was identified and cloned by our groups (8, 9). Both receptors are closely related in sequence, especially in their transmembrane regions. A highly characteristic and unique feature of the C3a receptor (C3aR) is the unusually large second extracellular loop (EL2) of ~175 residues with as yet unknown function.

Several mutagenesis experiments, including chimeras with the fMLP receptor, N-terminal deletion mutants, and site-directed mutagenesis of individual residues have revealed a two-point binding model for the C5aR (10-14). According to this model, a first binding site is located in the C5aR N terminus that binds core residues of the C5a ligand. This interaction provides about half of the overall binding energy and facilitates the interaction of the C terminus of the ligand with the second binding site, which encompasses several residues scattered among the extracellular loops and TM helices. Two of these, Glu199 in EL2 and Arg206 in TM5, have already been identified (15, 16). This model is confirmed by studies using synthetic peptides mimicking the C5a C terminus, which are able to bind to the C5aR and to induce signal transduction, although with much lower affinity and potency than the wild type ligand. As might be expected, the activity of such peptides is not affected by mutations in the C5aR N terminus, in contrast to what is observed with native C5a (12, 13).

The sites of interaction between C3a and the C3aR are not known. Like C5a, synthetic C3a analogue peptides of the C terminus are full receptor agonists, although most of these investigations have been performed in the heterologous guinea pig system. As the peptide length is extended toward the C3a N terminus, the activity of the peptides gradually increases. A sequence-optimized 21-mer "superagonist" peptide (17) has been identified that exhibits ~10% of the activity of wild type C3a versus the cloned human C3aR (18). These data would also indicate a two-point binding site in the C3aR, where the C terminus of the ligand is bound by the effector site, whereas the overall affinity is increased by additional interactions. However, no mutagenesis experiments of the C3aR have been reported yet.

Receptor chimeras between the C5aR and the fMLP receptor have been generated and found to be expressed on the cell surface allowing for the delineation of sites involved in fMLP binding (19). Because the human C3aR also has high homology to the C5aR, we used an analogous approach to identify sequence modules in the anaphylatoxin receptors that are involved in ligand binding. Using a panel of 19 receptor mutants, we show that the binding behavior of C3a to the C3aR differs from the C5a-C5aR interaction, culminating in the generation of a true hybrid C3a/C5a receptor.

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Materials-- Human C3a was obtained from Advanced Research Technologies (San Diego, CA), human C5a was from Sigma, 125I-labeled C3a and C5a were from NEN Life Science Products, and the synthetic peptides H-1264 and H-8135 were from Bachem (King of Prussia, PA). The monoclonal mouse IgG1 anti-C3aR antibody 8H1 was generated in our laboratory,2 the monoclonal mouse IgG1 anti-C5aR antibody W17/1 (20) was a kind gift by O. Götze, Göttingen, Germany, the anti-FLAG M2 and M5 antibodies were obtained from Scientific Imaging Systems (Rochester, NY), and the fluorescein isothiocyanate-labeled goat anti-mouse antibody from Becton-Dickinson (San Jose, CA).

The following buffers and stock solutions were used: phosphate-buffered saline (10 mM sodium phosphate, pH 7.4, 2.7 mM KCl, adjusted with NaCl to a final conductivity of 15 millisiemens), HAG-CM (20 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.25% w/v bovine serum albumin, 0.5 mM glucose), and KRH (118 mM NaCl, 4.6 mM KCl, 25 mM NaHCO3, 1 mM KH2PO4, 11 mM glucose, 1.1 mM MgCl2).

Construction of Chimeras and N-terminal Deletion Mutants-- The C5aR coding sequence with an additional FLAG epitope sequence and an EcoRI site at the 5' end (21) plus a new SalI site at position 708 was used for mutagenesis of the C5a receptor sequence. Conservative mutations (which did not alter the amino acid code) that created novel restriction sites (BsrGI at position 519 = Tyr174 at the junction of TM4 and EL2, and BssHII at position 597 = Ala201 at the junction EL2 and TM5) were introduced using the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA). The same 5'-UTR plus FLAG epitope sequence was added to the C3aR by subcloning after introduction of a novel MfeI site at position +4 of the C3aR cDNA (9). Similarly, novel restriction sites (BsrGI at position 476 = Tyr160 at the junction of TM4 and EL2, and NruI at position 1003 = Ala335 at the junction of EL2 and TM5) were introduced into the C3aR coding sequence. These changes to the C3aR did not alter the Kd for C3a.3 The receptor N termini (defined as codons 1-23 in the C3aR and codons 1-37 in the C5aR) were exchanged by fusion PCR, as described previously (21). The other chimeras were generated by subcloning via the BsrGI site and the NruI/BssHII sites after suitable blunting of the BssHII site according to standard techniques (22). N-terminal deletions of the C3aR coding sequence (9, 16, and 22 residues) were generated by standard PCR using the cDNA clone HNAFG09 (8) without further modifications. The final panel of 16 chimeras (including the wt receptors) plus three deletion mutants in vector pcDNA3/Amp (CLONTECH) was characterized by restriction analysis and DNA sequence analysis of all PCR-amplified sequences. In all cases where no expression of the mutant receptor on the cell surface could be detected, the complete coding sequence was determined.

Immunofluorescence and Flow Cytometry-- Human embryonic kidney cells (HEK-293) were transfected using LipofectAMINE as described previously (9). 3 days later, cells were harvested and resuspended in phosphate-buffered saline + 0.5% w/v bovine serum albumin at 1 × 107 cells/ml. 50 µl were incubated with 50 µl of first step antibody (8H1: 10 µg/ml; W17/1: 5 µg/ml; M2: 20 µg/ml; M5: 20 µg/ml) for 30 min at 0 °C, developed with fluorescein isothiocyanate-conjugated goat anti-mouse antibody, and examined flow-cytometrically on FACScan (Becton-Dickinson) using mock-transfected cells as negative control. For immunofluorescence studies of mutants that were not expressed on the cell surface, LipofectAMINE-transfected HEK-293 cells were developed with the same set of antibodies after prior fixation and permeabilization of the cells in acetone, as described previously (21).

Binding Studies-- Binding assays were performed essentially as described (9). Briefly, 1.5-3.0 × 105 transfected cells were incubated with 20,000-40,000 cpm of 125I-labeled C3a and varying concentrations of unlabeled ligand at room temperature in a total volume of 50 µl of HAG-CM. After 30 min, unbound ligand was removed by vacuum filtration using the MultiScreen filtration system with a Durapore 0.45-µm membrane (Millipore, Bedford, MA) equilibrated with HAG-CM. The filter was washed twice with 100 µl of HAG-CM and dried, and bound radioactivity was determined by gamma -counting (Cobra II Auto Gamma, Canberra Packard, Meriden, CT). Binding curves were analyzed using the software package LIGAND (23).

Signal Transduction Assays-- HEK-293 (1.2 × 107) cells were plated into a T150 flask in Earl's modified Eagle's medium supplemented with 10% fetal bovine serum. Following a 16-h incubation at 37 °C, medium was removed and replaced with serum-free medium, and cells were co-transfected with cDNA encoding each chimera, truncation mutant, or wild type receptor and a cDNA encoding Galpha -16 (15 µg of each plasmid) using LipofectAMINE Plus reagent (Life Technologies, Inc.) according to the manufacturer's recommendation. Following a 24-h incubation at 37 °C, the cells were replated on poly-D-lysine coated 96-well black wall microplate (Becton-Dickinson) at 30,000 cells/well.

After 18-24 h the medium was aspirated off, and 100 µl of fresh Earl's modified Eagle's medium containing 4 µM Fluo-3AM (Molecular Probes, Eugene, OR; stock solution prepared at 2 mM in Me2SO containing 20% pluronic acid), 0.1% bovine serum albumin, and 2.5 µM probenecid (prepared with the addition of equal equivalents of NaOH) was added to each well and incubated in a CO2 incubator for 1 h at 37 °C. Medium was aspirated and replaced with 100 µl of the same medium but without Fluo-3AM and incubated for 10 min at 37 °C. Cells were washed three times with a Denley cell wash with KRH containing 0.1% bovine serum albumin, 2.5 µM probenecid, and 20 mM HEPES, pH 7.4 (buffer A), and after the last wash cells were aspirated down to final volume of 100 µL. Ligand concentration-response curves were prepared in 96-well polyproplyene microplates at three times the final concentration in buffer A and warmed to 37 °C.

Microplates containing the Fluo-3AM-loaded cells and the ligand plates were placed in a fluorometric imaging plate reader where all 96 wells are monitored simultaneously (24). At initiation of the reading fluorescence is read every 1 s for 60 s and then every 3 s for the following 60 s. Agonist (50 µl) was added at 10 s, and the maximal fluorescent count above background after the addition of agonist was used to define maximal activity for that concentration of agonist. The fluorometric imaging plate reader software normalizes fluorescent readings to give equivalent readings for all wells at zero time.

    RESULTS AND DISCUSSION
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The aim of this study was the identification of receptor modules in the human C3aR and C5aR that determine binding affinity, specificity (i.e. discrimination from the noncognate ligand), and signal transduction. For this purpose, chimeric C3a/C5a receptors were constructed by systematically recombining four receptor parts (the N terminus, TM1 to TM4, EL2, and TM5 to the C terminus) to generate all 16 possible receptor chimeras (including the wt receptors). To normalize receptor expression and to facilitate immunological detection of the receptors on the cell surface, the same 5'-UTR plus a sequence tag coding for an N-terminal FLAG epitope were introduced at the 5' end of each clone, as described previously for the human C5aR (21). All receptor constructs were analyzed for (i) cell surface expression by flow cytometry using a panel of monoclonal antibodies directed against the N-terminal FLAG epitope, the C5aR N terminus, and EL2 of the human C3aR, (ii) ligand binding of C3a and C5a in competitive displacement studies, and (iii) functional response (intracellular calcium mobilization) toward C3a, C5a, and a synthetic C3a and C5a analogue peptide after cotransfection of Galpha -16, which had previously been shown to complement the signal transduction cascade of the anaphylatoxin receptors in these cells (9, 25, 26). Flow cytometric analysis revealed highly different surface expression levels among this panel of receptor chimeras, indicating determinants other than the 5'-UTR and N-terminal sequence that regulate cell surface expression. Specifically, for 7 of 14 chimeras (Ch2 to Ch7 and Ch10; Table I) reproducible and significant surface expression could not be confirmed, which was compatible with and explained the negative results obtained in the binding studies and functional analyses for these mutants. This observation was unexpected because in the case of C5aR/fMLP chimeric receptors, all of the constructs were expressed on the cell surface, despite an even slightly lower sequence homology between those two receptors (19). However, all mutants with negative cell surface expression were expressed intracellularly as shown by immunofluorescence studies of acetone-treated cells (data not shown), indicating a defect in correct folding and/or transport of these mutants to the cell surface.

                              
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Table I
Combined results of cell surface expression, C3a/C5a bindings and functional responsiveness towards C3a, C5a, and synthetic C3a/C5a analogue peptides of all receptor mutants used in this study

The Receptor N Termini Determine Ligand Affinity (C5aR) and Discrimination from the Noncognate Ligand (C3aR)-- Replacement of the C5aR N terminus by the C3aR N terminus (chimera Ch8, i.e. a C5aR with a C3aR N terminus) lead to an affinity loss in C5a binding of >2 orders of magnitude (i.e. above the sensitivity of this assay) without affecting the signal transduction properties of the C5a analogue synthetic peptide H-8315 (Table I), confirming previous data that showed the presence of a major ligand binding site within this part of the receptor, which contributes to ligand binding affinity (12-14). Conversely, in the reciprocal exchange mutant (Ch9, a C3aR with a C5aR N terminus) significant C5a binding could be detected (Table I and Fig. 1). The Kd = 33 ± 14 nM for C5a binding of this mutant was only about 1 order of magnitude higher than the Kd = 5.2 ± 2.5 nM of the wt C5aR. Because the C3aR does not bind C5a (Ch1; Table I), this provides direct evidence for a C5a interaction site within the C5aR N terminus. More important, this chimera responded functionally to C5a with an ED50 of ~60 nM (Table I and Fig. 2). Although C5a was about 2-3 orders of magnitude less potent on this receptor mutant than on the wt C5aR, this result clearly shows that the C3aR "core structure" (i.e. TM1 to C terminus) does accept C5a as an activating stimulus. This unexpected result was confirmed by the observation that the C5a analogue peptide H-8315 had similar activity on both anaphylatoxin receptors (Ch1 = C3aR and Ch16 = C5aR) as well as on the N-terminal exchange mutants (Ch8 and Ch9; Table I). This suggests that the N terminus of the C3aR partially determines ligand specificity by preventing the binding of the noncognate C5a. Once bound, C5a could activate the C3aR at physiologically relevant concentrations. As a consequence, previous investigations analyzing the functional activity of C5a analogue peptides on human granulocytes (27) have to be interpreted with caution because these cells harbor both anaphylatoxin receptors, both of which respond to this peptide. The C3a analogue peptide, however, appears to be more sequence-restricted because it did not activate the C5aR (see Ch8 and Ch16; Table I)


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Fig. 1.   Competitive binding curves of Ch1 (C3aR, using 125I-labeled C3a as tracer; circles), Ch9 (using 125I-labeled C3a as tracer; triangles), and Ch16 (C5aR, using 125I-labeled C5a as tracer; squares) transiently transfected into HEK-293 cells and competitively displaced by increasing concentrations of either C3a (filled symbols) or C5a (open symbols).


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Fig. 2.   Functional response (intracellular calcium mobilization) of Ch1, Ch9, and Ch16 toward C3a (circles), C5a (squares), and the synthetic peptides H-8135 (C5a analogue; triangles) and H-1264 (C3a analogue; diamonds).

EL2 Influences Correct Positioning of the Transmembrane Helix Bundle-- Previous investigations had implicated Glu199 in the second extracellular loop of the human C5aR in ligand binding (16). Indeed, replacement of EL2 from the C3aR in chimera Ch9 by the C5aR homologue increased C5a binding affinity about 10-fold, thus restoring wild type affinity of the resulting chimera Ch11 (Table I). Unexpectedly, however, this replacement also strongly affected the signal transduction properties toward C5a and the C5a analogue peptide, which were virtually inactive on this mutant (Table I). This result, which shows a critical involvement of the second extracellular loop in signal transduction, may be explained by an indirect effect of this loop on the correct positioning of the transmembrane helix bundle, thus affecting signal transduction. Similarly, replacement of EL2 in the C5aR by the C3aR counterpart (Ch16 right-arrow Ch14; Table I) critically affects both ligand binding (C3a as well as C5a) and signal transduction despite the fact that EL2 of the C3aR does not by itself prevent C5a binding and signal transduction, as already shown above (Ch9; Table I). Again, this result is best explained by an indirect effect of EL2 of the C3aR on the correct positioning of the TM helix bundle. Thus, helix bundles and EL2 have to be regarded as a functional unit in which the size of EL2 is of critical importance. Neither elongation of the C5aR loop (compare Ch16 right-arrow Ch14; Table I) nor shortening of EL2 of the C3aR (Ch9 right-arrow Ch11; Table I) are compatible with correct functioning of the resulting receptor mutants, an important caveat for any deletional mutagenesis experiments in the C3aR loop. Furthermore, it is tempting to speculate that C3aR antagonists may be found that bind within EL2 of the C3aR, thus functionally "tethering" the helix bundle with consequences similar to those observed by exchange of the C3aR loop by its C5aR counterpart (compare Ch8 right-arrow Ch10; Table I). Our data do not exclude the hypothesis that residues within EL2 of the C3aR are involved in ligand binding, although this idea appears less likely in view of the low sequence homology in EL2 of the C3aR sequences of different species (28). Similarly, all chimeras with "mixed" C3aR/C5aR helix bundles did not show any signal transduction ability (like Ch12 and Ch14; Table I), suggesting that the transmembrane receptor modules also are not completely compatible with each other despite the fact that within the coding region these domains exhibit the highest sequence homology.

The C3aR N Terminus Is Not Required for Ligand Binding-- Apart from the wt C3aR, only Ch9, a C3aR with a C5aR N terminus, showed significant C3a binding (Fig. 1) and signal transduction (Fig. 2) both with C3a and the C3a-like peptide H-1264. Because the same mutant Ch9 also bound C5a (Table I and Fig. 1), it is thus revealed as a true hybrid C3a/C5a receptor. This finding suggests that the C3aR N terminus determines receptor specificity, as explained above, but does not participate in C3a binding or else that the C5aR N terminus complements putative N-terminal residues involved in C3a binding despite the fact that the sequence homology between C3aR and C5aR is rather low in this receptor part. To exclude this latter possibility, however, N-terminal truncation mutants of the C3aR were generated (Delta 09, Delta 16, and Delta 22). The Delta 09 and Delta 16 mutants showed only slightly decreased binding affinities and almost unaltered signal transduction properties, whereas the Delta 22 deletion mutant was not properly expressed on the cell surface (Table I). These results confirm that the N terminus of the C3aR does only contribute to a minor degree or not at all to C3a binding, in sharp contrast to what is known from the C5aR (11-14). The ligand binding site(s) for C3a must reside in the other receptor modules of the C3aR, although it is not possible to further delineate them using this set of mutants due to the indirect effects on receptor expression. Alternatively, mutagenesis of candidate residues is required and will be greatly facilitated by the low sequence homology (<50%) of the C3aR cloned from mouse, humans, and guinea pigs (28).

    ACKNOWLEDGEMENT

We cordially thank D. Bitter-Suermann (Hannover, Germany) for continuous strong support.

    FOOTNOTES

* This work was supported in part by Deutsche Forschungsgemeinschaft Grant 1425/3-1 and Sonderforschungsbeveich Grant 244 "Chronische Entzündung" (to W. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: Inst. of Medical Microbiology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany. Tel.: 49-511-532-4342; Fax: 49-511-532-4366; E-mail: Bautsch.Wilfried{at}MH-Hannover.de.

2 B. Sohns, J. Westermann, R. Frank, M. Grove, J. Köhl, A. Klos, and W. Bautsch, manuscript in preparation.

3 T. Crass, R. S. Ames, H. M. Sarau, M. A. Tornetta, J. J. Foley, J. Köhl, A. Klos, and W. Bautsch, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: C5aR, C5a receptor; C3aR, C3a receptor; Ch, receptor chimera; EL, extracellular loop; TM, transmembrane region; UTR, untranslated region; fMLP, formylmethionylleucylphenylalanine; PCR, polymerase chain reaction; wt, wild type.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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