©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutation of Glutamate 199 of the Human C5a Receptor Defines a Binding Site for Ligand Distinct from the Receptor N Terminus (*)

Peter N. Monk (§) , Michael D. Barker , Lynda J. Partridge , James E. Pease

From the (1)Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biochemistry, University of Sheffield, Sheffield S10 2UH, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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)()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 this report we show that another glutamate residue, Glu, 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 GlnR by at least 200-fold, suggesting that this lysine residue, probably analogous to Lys of hC5a, may interact with Glu.


EXPERIMENTAL PROCEDURES

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) .

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) .

Measurement of 5-Hydroxy-[H]tryptamine Release

Secretion was measured as the release of [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 CaLevels

RBL cells loaded with the fluorescent Ca 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.


RESULTS

Expression of Wild Type and Mutant Receptors for Human C5a in RBL Cells

Cells transfected with wild type (WTR) or Glu Gln substitution (GlnR) 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 GlnR-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 GlnR-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 GlnR 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 GlnR cells (Fig. 2). The difference between the response of GlnR 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 I-hC5a by hC5a and hC5a des-Arg

The lack of potency of 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-Argon Intracellular CaLevels in Transfected RBL Cells

The activation of RBL cells may also be monitored by the measurement of intracellular Ca 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 GlnR 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, GlnR cells required 10 nM hC5a for a maximal response (Fig. 4, a and b). The response of GlnR 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 GlnR 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 CaLevels in Transfected RBL Cells

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 Ca levels in RBL cells. As expected from the secretion assays, the maximal stimulation of increases in Ca levels in GlnR-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 GlnR cells showed no detectable response. However, the response of GlnR 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, GlnR 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.


DISCUSSION

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 of the human C5a receptor forms part of this additional site.

Glu 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 GlnR 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 GlnR 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 GlnR, 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.

To locate the site of the interaction between Glu 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 GlnR, 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 GlnR. 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; GlnR 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.

The role of this C-terminal Arg was further analyzed by the use of hC5a des-Arg, 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 GlnR-bearing cells to both hC5a and hC5a des-Arg would be identical; however, hC5a des-Arg is much less active than hC5a on GlnR cells, stimulating little release even at 700 nM. The retention of some stimulatory activity of hC5a des-Arg when used with GlnR-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 GlnR 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) .

The relative role of Arg 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 GlnR 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) .

In conclusion, we have shown that the mutation of Glu 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.


FOOTNOTES

*
This work was supported by awards from the Arthritis and Rheumatism Council and the British Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Krebs Institute for Biomolecular Research, Dept. of Molecular Biology and Biotechnology, University of Sheffield, P. O. Box 594, Sheffield S10 2UH, United Kingdom. Fax: 44-114-2768297.

The abbreviations used are: hC5aR, human complement fragment 5a receptor; Fluo3-am, acetoxymethyl ester of Fluo3; Cha, cyclohexylalanine; 5-HT, 5-hydroxytryptamine; RBL, rat basophilic leukemia; FACS, fluorescence-activated cell sorting; WTR, wild type hC5aR; GlnR, substitution of Gln for Glu of hC5aR.


ACKNOWLEDGEMENTS

We thank Paul E. Brown and Arthur J. G. Moir for oligonucleotide and peptide synthesis and John Lawry (Yorkshire Cancer Research Campaign) for FACS.


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