©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Probing the Message:Address Sites for Chemoattractant Binding to the C5a Receptor
MUTAGENESIS OF HYDROPHILIC AND PROLINE RESIDUES WITHIN THE TRANSMEMBRANE SEGMENTS (*)

(Received for publication, March 30, 1995; and in revised form, May 18, 1995)

Lee F. Kolakowski , Jr. (1) Bao Lu (1) Craig Gerard (1) (2) (3) (4) (5) Norma P. Gerard (1) (2) (3) (4) (5)(§)

From the  (1)Ina Sue Perlmutter Laboratory and (2)Department of Pediatrics, Children's Hospital, the (3)Departments of Medicine, Beth Israel and Brigham and Women's Hospitals, the (4)Center for Blood Research and the (5)Thorndike Laboratory of Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The C5a anaphylatoxin ligand-receptor interaction on polymorphonuclear granulocytes stimulates chemotaxis, degranulation, and the oxidative burst. The receptor is a member of the large G-protein-coupled family. The ligand is a cationic peptide of 72 amino acids derived from the C5 component of complement and has been shown to have a number of structural requirements for interaction with the receptor. In order to probe the potential interaction sites between ligand and receptor, we constructed a series of mutated receptor molecules, targeting cysteines, prolines, and additional amino acids of interest because of combinations of charge or hydrophobicity and putative location with respect to the membrane. Transfected mutant receptors were analyzed for cell surface expression, ligand binding, and ligand-activated phospholipase C activity. The receptors created can be placed generally in four distinct classes: those which bind and signal like the natural receptor; those which bind but fail to transduce signals; those which are expressed but neither bind nor transduce signal; and those which are not expressed at the cell surface.


INTRODUCTION

The human complement 5a (C5a) anaphylatoxin receptor is a member of the large family of G-protein-coupled receptors (GPCRs)()(1, 2) . A paradigm for receptor-ligand interactions among low molecular weight agonists such as retinal and catecholamines has the ligands binding to their receptors at a single site within the pocket formed by transmembrane segments(3, 4) . In contrast, peptidergic agonist-receptor systems such as those for C5a, interleukin-8, and pituitary glycoprotein hormones appear to display two regions of interaction: one receptor domain largely confers binding affinity, while a second site mediates ligand biological activity(5, 6) .

Classical studies of the C5a ligand demonstrate two regions of the protein that are required for high affinity binding and potent signaling via heterotrimeric G-proteins(2, 7, 8, 9, 10) . The conformationally critical disulfide bonds present in C5a knit a core domain essential for high affinity receptor binding. Chemical and exopeptidase cleavages demonstrate that the ligand COOH-terminal region encodes the receptor activating sequence, MQLGR(11, 12) . Further studies using site-directed mutagenesis have located distinct receptor contact points within the ligand NH-terminal region and the disulfide core(10) .

More recently, evidence for distinct binding and activation sites in the C5a receptor has been derived from mutagenesis and monoclonal antibody studies showing the importance of the receptor NH terminus for high affinity ligand binding(13, 14, 15) . Treatment with a spider venom metalloproteinase suggests that specific cleavage of the receptor blocks binding of intact C5a, but not COOH-terminal C5a agonist peptides(16) . Similarly to C5a, the cationic chemokines interleukin-8, and MGSA/GRO also recognize acidic NH-terminal receptor sequences, and selectivity in ligand binding for these highly homologous receptors can be transferred with molecules chimeric at the NH terminus(17, 18) .

Signal transduction by the C5a/C5a receptor pair in COS cells results in minimal activation of PI kinase and undetectable activation of PLC (1) . Previous studies indicate coupling of the C5a receptor to G in polymorphonuclear leukocytes to activate endogenous PLC2, by release of the subunits(45, 46) . As the PLC2 isoform is absent in COS cells, signaling through the C5a receptor can be assessed by taking advantage of the ability of G to substitute for the G subunit, which, following activation by ligand, stimulates the endogenous COS cell PLC(27) .

These findings suggest that C5a like other peptidergic ligands that utilize GPCRs conform to the message:address model of Schwyzer(19, 20) , where the ``address'' domain binds ``recognition'' sequences in the receptor and presents the ``message'' domain to the agonist site in the receptor.

Previous studies demonstrate that human C5a can bind and activate the murine C5a receptor with potency almost identical to the human receptor, suggesting that elements in common for both species provide the agonist site. Comparison of the murine C5a receptor to the human molecule demonstrates that the transmembrane segments are well conserved, while extracellular sequences are quite divergent, thus providing a dramatic ``mutagenesis'' result(1, 21) . Since data to date suggest that the address site for C5a is largely extracellular, we undertook site-directed mutagenesis of hydrophilic and charged residues within the transmembrane segments to investigate potential message sequences.


EXPERIMENTAL PROCEDURES

Materials

Human C5a anaphylatoxin was expressed in Escherichia coli(22) or obtained from Sigma. COS cells (CRL 1651) and 293 cells (CRL 1573) were obtained from The American Type Culture Collection, Bethesda, MD. I-Human C5a and H-inositol were from DuPont NEN. DEAE-dextran (average molecular weight 500,000) and chloroquine were from Sigma. Anti-FLAG M2 monoclonal antibody was obtained from IBI (New Haven, CT). Fluorescein isothiocyanate goat anti-mouse IgG was from Becton Dickinson (Lincoln Park, NJ), biotinylated anti-mouse IgG, horseradish peroxidase-labeled avidin-biotin conjugates from Vector Labs (Burlingame, CA), and mouse anti-horseradish peroxidase IgG1 was obtained from Zymed (South San Francisco, CA).

C5aR Mutagenesis

The human C5aR cDNA was modified to express an amino-terminal epitope by adding the FLAG epitope sequence (MDYKDDDDKEF) and converting the initiating methionine (ATG) to leucine (CTG)(23) . As described previously, this sequence has no significant effect on binding or expression(23) . The EcoRI/XbaI cassette encoding the FLAG-wild-type C5aR cDNA was subcloned into the mutagenesis vector pSELECT-1 (Promega Corp., Madison, WI). Oligonucleotide-directed mutagenesis was carried out using the Altered Sites mutagenesis protocols (Promega Corp.). All mutations were verified by DNA sequencing (Sequenase, U.S. Biotechnology Inc., Cleveland, OH)(24) . Mutant cDNAs were subcloned into the mammalian expression vector, pCDM8, for functional studies (25) .

Cell Cultures and Transfection

COS cells were maintained and transfected using DEAE-dextran as described previously(1) . Cells were subcultured at 8 10/10-cm plate 16-24 h before transfection. At transfection, media was replaced with DMEM containing 2% fetal bovine serum or Nu-serum (Collaborative Research, Bedford, MA), 1 mM chloroquine, 400 µg/ml DEAE-dextran, and 1 µg/ml plasmid DNA. Cells were incubated at 37 °C for 3.5-4 h, transfection medium aspirated and replaced with 10% MeSO in PBS for 2 min at room temperature, and finally replaced with culture medium. All functional studies were carried out 60-72 h later.

Human embryonic kidney 293 cells were maintained as described for COS cells and transfected with cationic liposomes as described previously(23) .

Binding Studies

Binding studies were conducted on intact 293 cells or on crude membrane preparations from COS cells. Membranes were prepared as follows: 60-75 h after transfection cells were washed twice with PBS, scraped into TEN (40 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl), and ruptured by one cycle of freeze-thaw in liquid N. Membranes were pelleted by centrifugation (4 °C, 36,000 g for 15 min), resuspended in TEN, and protein concentration determined by Coomassie Blue staining compared with BSA standards. 10 intact 293 cells or 100-200 µg of COS cell membrane protein were used in binding experiments; in Hank's balanced salt solution, 25 mM HEPES, 0.1% BSA, incubating with 0.1 nMI-C5a and varying amounts of unlabeled C5a on ice for 45 min. Unbound ligand was separated by filtration on glass fiber filters (Whatman GF/F) that were soaked overnight in 0.1% polyethyleneimine. Filters were washed three times with ice-cold binding buffer. Bound ligand was quantitated by liquid scintillation counting. Each binding experiment was conducted three times in triplicate. All binding data presented in Table 1were determined using membranes from transfected COS cells. Binding affinities (K) and sites/cell (B) were determined using the Ligand program (26) . The relative counts/minute bound at 0.1 nMI-C5a for each mutant compared to wild-type is also presented.



Cell Surface Expression

Initial expression studies were performed using FLAG-C5a receptor plasmids transfected in 293 cells. Three days after transfection, cells were suspended by trituration in PBS. Approximately 10 cells were incubated in PBS containing 3% BSA and 10 µg/ml anti-FLAG M2 antibody or isotype matched anti-horseradish peroxidase for 1 h on ice. Following staining with fluorescein isothiocyanate anti-mouse IgG, cells were fixed in 2% paraformaldehyde and analyzed by flow cytometry.

COS cells were plated on fibronectin-coated glass slide dishes (Nunc, Naperville, IL) and transfected as described above. Cells were washed with PBS, and incubated in PBS containing 3% BSA for 10 min at room temperature to block nonspecific sites. Monoclonal anti-C5a receptor antibody S5/1 (13) (generously provided by Prof. Otto Gotze, Department of Immunology, University of Gottingen, Germany) was added at 10 µg/ml and incubated 30-60 min at room temperature. Cells were washed three times with PBS and subsequently stained with biotinylated goat anti-mouse IgG, followed by avidin-biotin complex, hydrogen peroxide and diaminobenzidine, and examined by light microscopy (Olympus BH-2 microscope). Controls for nonspecific staining included cells transfected with vector alone, and the staining pattern compared to that obtained with cells fixed in 2% paraformaldehyde prior to any antibody incubation, to confirm cell membrane expression.

Signal Transduction by Transfected Receptors

Ligand-stimulated activation of phosphatidyl inositol-specific phospholipase C was assessed as described previously (27) . Cells were co-transfected with C5a receptor or mutant and a plasmid encoding G, 48 h later they were washed with inositol-free DMEM (Life Technologies, Inc.), and incubated another 24 h in this medium containing 10 µCi/ml [H]inositol and 10% fetal bovine serum. Labeling medium was removed, and cell layers were incubated in triplicate in inositol-free DMEM, 10 mM LiCl, and 0-150 nM C5a for 30 min at 37 °C. Reactions were terminated by addition of an equal volume of 10% HClO containing 4 mg/ml phytic acid followed by incubation at 0 or -20 °C for at least 30 min. Inositol phosphates were purified by chromatography on Dowex-1 (formate; Bio-Rad) (28) and quantitated by liquid scintillation counting. C5a-induced PLC activation was compared with controls in the absence of ligand and untransfected cells. COS cells transfected with vector alone or with unrelated receptor cDNAs were not activated by C5a. All experiments were carried out in triplicate; values are expressed as the mean ± S.E. percent stimulation above base line and at least two independent experiments were conducted for each mutant receptor.


RESULTS

Mutations of Acidic and Cysteine Residues

As the carboxyl terminus of the C5a (arginine 74) is crucial for the full potency of signaling activity of the protein (9) our strategy utilized for this first phase of mutagenesis was to mutate residues that are negatively charged or can provide a partial negative charge to neutralize the guanidinium group. The mutants were made of the aspartic acid and cysteine residues within the transmembrane segments (Fig. 1). As shown in Table 1, the binding, signaling, and expression pattern of each mutant was investigated by transient expression in COS cells. Analysis of binding and cell surface expression were performed with transfection of the receptor cDNA alone, while signal transduction for phospholipase C activation was evaluated in cells co-transfected with G. Previous investigations demonstrated no change in binding or expression in the presence or absence of this G subunit(27) .


Figure 1: Schematic diagram of the human C5a receptor. The location of each mutation generated in the human C5a receptor (1) is shown as a filled circle, except GlnStop which is shown as a filled square. Cys and Cys form a disulfide bond (indicated by stippled line). A single consensus asparagine-linked glycosylation site is indicated. The seven transmembrane segments are shown boxed and bordered approximately by the dashed horizontal lines. The boundaries of each of the predicted transmembrane domains conform to the three-dimensional model proposed for rhodopsin(31) .



Within the transmembrane segments, the C5a receptor contains only two acidic residues (Asp and Asp). These amino acids, as well as Asn, were each mutated conservatively (Table 1). While the AspAsn mutant did not express efficiently on the cell surface, AspAsn and AsnGln mutants were expressed on the cell surface and bound ligand with slightly lower K than wild-type. These latter two mutations had some effect on the ability to express the receptors at the membrane, as the B for each is lower than wild-type. The AspAsn mutant is completely defective at stimulating PLC activity, while the AsnGln mutant is only 35% as effective as wild-type.

Among the cysteine residues mutated, three are required for binding of C5a (Cys, Cys, and Cys). Cys and Cys are positioned at the homologous positions to the amino acids in rhodopsin and the 2-adrenergic receptor that form an intramolecular disulfide bond(29, 30) . The mutations on the cysteines in the first and second extracellular loops are not expressed on the cell surface, as indicated by flow cytometry (Table 1). The binding deficit for these two mutations is expected.

Cys is predicted to lie deep within TMS-5 and oriented toward TMS-4. To determine if mutants were properly expressed on the cell surface, indirect immunohistochemical staining of transfected COS cells was performed using the C5a receptor-specific monoclonal antibody S5/1(13) . Fig. 2shows typical results with wild-type receptor (Fig. 2A) and the CysSer (Fig. 2B) where both are expressed at high levels on the cell membrane of unpermeabilized cells. These results indicate that Cys is required only for the binding of C5a. Random mutagenesis at this site may reveal the spectrum of residues capable of substituting for cysteine.


Figure 2: Characterization of expression of C5a receptor mutants. Indirect immunoperoxidase staining of unpermeabilized transfected COS cells using a monoclonal anti-C5a receptor antibody was performed on all mutants which showed abnormal binding (see Tables I-III). Three patterns of staining were observed in all mutants and examples of each are shown. Panels A and B show the normal strong expression of C5a receptors on the cell surface (A, wild-type C5aR; B, CysSer). Panel C shows an example of mutations with aberrant staining (mutant ProAla). Panel D shows the expression pattern of mutant receptors with very low levels of staining (mutant ProAla). Panel E shows the staining of untransfected COS cells. Panels A-D are shown at 2 the magnification of panel E.



Mutation of the remaining transmembrane cysteines had differing effects on function of the receptor. CysSer in TMS-2 resulted in a receptor with normal binding affinity but only 30% of signal transduction in response to 150 nM C5a (Table 1). CysSer in TMS-4 resulted in a receptor that binds ligand almost as well as natural C5aR but produces no detectable PLC activation. Cys is positioned at approximately the same depth as Cys and is predicted to be oriented toward TMS-5(31) . The CysSer mutation binds ligand with wild-type affinity and 43% of wild-type activation of PLC. In contrast, mutation of both Cys and Cys yielded a functionally normal receptor for binding and signal transduction. The B for each of these mutants is close to that of wild-type, except for CysSer, which shows 4-fold greater number of sites.

Mutation of Transmembrane Proline Residues

The second strategy utilized in our mutagenesis experiments was to probe the role of the structural rigidity of the receptor in ligand binding by mutating transmembrane proline residues. The C5a receptor contains 16 proline residues, 7 of which are within or directly adjacent to transmembrane segments. Of these, 4 (prolines 170, 214, 257, 297) are well conserved among other members of the GPCR family and have been mutated in the muscarinic M3 receptor(32) . The pattern of functional importance of these ProAla mutations in the C5a receptor differs substantially from that of the M3 muscarinic receptor (Table 2). Among the 7 proline TMS residues investigated, three mutants (ProAla, ProAla, and ProAla) bind ligand at 0.1 nMI-C5a comparable to wild-type receptor, despite the observation that the B determined from Scatchard analysis for each of these mutants is substantially less than wild-type. The other four mutants (ProAla, ProAla, ProAla, and ProAla) are unable to bind ligand at wild-type levels. The expression patterns for these mutations were determined as described for the cysteine mutations above. Only one of the binding-impaired mutants expressed robustly (ProAla); the others were either expressed at very low levels or in aberrant patterns of staining in cells ( Table 2and Fig. 2C). The ability of mutants that bind ligand to stimulate PLC was determined. With the exception of ProAla, all other mutations were only partially effective (ProAla, ProAla, and ProAla) for generating intracellular signals.



Mutation of Third Extracellular Loop Proline Residues

Data from other receptors suggest that TMS6 and 7 may be a separate functional domain(33, 34) . A mutation was generated converting the LeuPro, yielding a non-functional receptor that demonstrates a normal immunostaining pattern (Table 2). This prompted us to produce the neighboring mutation ProLeu, generating a protein that binds minimally compared to untransfected cells and does not signal (Table 2).

Intracellular Loop Mutations

The third strategy utilized to determine elements of functional importance in the C5a receptor involved mutagenesis of intracellular loops. A recurrent theme in receptor function is the interaction of heterotrimeric G-proteins with intracellular loop residues, specifically in the third intracellular loop and the proximal COOH-terminal tail. Seven point mutations were generated and expressed (Table 3). Binding studies with each of these mutants show that three have no effect on binding (TrpPhe, GlyAla, SerAla) but have modest to severe effects on levels of stimulation of PLC. The four remaining mutants showed diminished levels of ligand binding at 0.1 nMI-C5a, but rather dramatic effects on PLC stimulation, indicating a perturbation in the interaction with G. A single point mutation in the first intracellular loop (ArgGlu), that disrupts both cell surface expression and signal transduction, is unusual as most reported mutations of this loop have little or no effect on expression or G-protein coupling(35) . The B for this construct is very low, but other mutants with similar B values (e.g. ProAla) have normal signaling capacity.




DISCUSSION

G-protein-coupled receptors appear to have evolved a common strategy for the recognition of large peptide ligands. Ligand binding to elements in the NH-terminal and extracellular loop regions, constituting a high affinity binding site, leads to the presentation of ligand sequences which activate receptors. The identification of the latter sites has been approached in the present work by site-directed mutagenesis of transmembrane sequences. As the activating sequence of C5a is highly polar, we limited our mutagenesis to residues with polar or structural characteristics.

Role of Hydrophilic and Cysteine Residues in the C5a Receptor

Results from other GPCRs have demonstrated that charged and other polar side chains are crucial for maintaining receptor conformation and ligand binding. In the 2-adrenergic receptor, the conserved aspartic acid residue in TMS-3 is required to provide a counter ion to the catecholamine charge. In rhodopsin retinal is linked via a Schiff base to Lys, and the resulting charge is thought to be balanced by Glu ((36) and reviewed in (37) ). Some mutations at these sites in rhodopsin are active in the dark. Thus, charged residues within the TMS can be involved in direct interaction with the ligand and/or play a role in the equilibrium between active and inactive forms.

In mutagenesis experiments of other receptors, the aspartic acid residues in the canonical tripeptide ``DRY'' and the TMS-2 motif ``LAVAD'' have been implicated in G-protein interaction and conformational changes during activation(31) . The C5a receptor also encodes an asparagine residue (Asn) at the position where the amino group of catecholamine drugs binds to Asp of 2-adrenergic receptors(38) . Conservative mutations at these sites demonstrate that Asp is essential for the proper expression and trafficking of the C5a receptor. Asp and Asn bind ligand nearly as well as wild-type, but both are defective at stimulation of PLC via G (Table 1). This indicates that these residues are crucial components for the generation of the active form of the receptor. Because of differences in PLC activation and because both provide wild-type-like binding, they may be different than similar sites in the 2-adrenergic receptor.

The results from mutagenesis of all transmembrane cysteine residues show that three are required for binding (Cys, Cys, and Cys) (Table 1). It has been previously determined for other GPCRs, such as rhodopsin, that cysteines in extracellular loops one and two are linked in a disulfide bond which stabilizes the structure(39, 40) . The data from CysSer and CysSer mutants are consistent with these residues participating in a similar disulfide bond in the C5a receptor. The CysSer mutant does not bind or signal and is expressed well at the surface of COS cells (Fig. 2B). Predictions of the structure of rhodopsin suggest that a cysteine residue, present at the homologous position (Cys), is oriented within the transmembrane pocket toward TMS-4(31) . In bovine rhodopsin Cys can be replaced by serine with little or no effect on function(29) . It is unclear whether the carboxyl-terminal arginine of C5a directly contacts this cysteine residue, as it is predicted to lie deep within the plane of the transmembrane segments. Alternatively, Cys may be important for structural changes associated with agonist binding and activation.

The CysSer mutant binds C5a almost as well as the wild-type receptor, but when co-transfected with G, fails to activate PLC. This cysteine is predicted to be almost at the same depth within the membrane as Cys and is oriented toward Cys in structural models of GPCRs.

The remaining cysteine mutants all bind ligand with wild-type characteristics and activate PLC. Two (CysSer and CysSer) have diminished ability to stimulate PLC.

The Role of Proline Residues in the C5a Receptor

Structural studies of bacteriorhodopsin show that proline residues within the transmembrane segments introduce kinks in the helices, as the amine hydrogen is not available for helical stabilization(41) . The data presented in Table 2show that mutation of helical proline residues yields a number of changes in receptor function. Among the prolines mutated, several are required for the proper trafficking to the cell surface as demonstrated by binding and immunohistochemistry experiments. Three fail to express well at the cell surface (ProAla, ProAla, and ProAla), and the expression of ProAla appears to cause dramatic changes in the morphology of COS cells (Fig. 2C). Pro, unlike Pro and Pro, is not widely conserved in other GPCRs but is present in other chemotactic receptors (formylmethionylleucylphenylalanine and interleukin-8 receptors). The effects of these mutations are likely the result of misfolding of the receptors in the endoplasmic reticulum and regulation of this process by chaperonins. A general defect in the processing of opsin in Drosophila (ninaA) results from mutations in a cis-trans-prolyl isomerase, indicating that proline residues in the TMS play a specific role in the ability of these receptors to form proper structures(42) . Mutations of proline residues corresponding to Pro, Pro, Pro, and Pro in the C5a receptor have been made in the rat M3 muscarinic receptor (Pro, Pro, Pro, and Pro in the rat M3 muscarinic receptor, respectively)(32) . The rat ProAla mutations at positions 242, 505, and 540 all affect the B for N-methylscopolamine binding, and the affinity of the Pro mutant is two orders of magnitude higher than wild-type. As shown in Table 2, the ProAla and ProAla mutations in the C5a receptor behave in a similar fashion to those in the muscarinic receptor, but ProAla provides 76% of the wild-type binding and is diminished in its activation of PLC. ProAla is expressed on the cell surface and binds ligand weakly, analogous to ProAla in the muscarinic receptor.

Additional mutations (LeuPro and ProLeu) were made in the extracellular loop connecting TMS6-7 (Table 2). Both of these mutants are expressed on the cell surface. ProAla binds ligand (36% of wild-type), and both fail to activate PLC. Studies of rhodopsin and the 2-adrenergic receptor indicate that TMS6-7 are tightly associated in the membrane and are able to function in ``split'' receptor experiments caused by cleavage of rhodopsin with trypsin (43) or by co-expression of partial receptors(34) . Our results indicate that the structure of the extracellular loop is critical for adopting the necessary structure for activating G-proteins.

Other Mutations

Mutations in the intracellular loops of the receptor were constructed and characterized (Table 3). These mutations are unlikely to affect the direct interaction of C5a with the receptor, but may shed light on the contact points of G-proteins with the C5a receptor. In vivo the C5a receptor has been shown to interact directly with G(44, 45) , but due to the lack of expression of G-responsive PLC isotypes within COS cells, we utilize co-transfections with G in order to study the receptor's ability to interact with G-protein(27) . Among 7 intracellular loop residues mutated, three (TrpPhe, GlyAla, SerAla) have no effect on maximal binding of C5a relative to the wild-type receptor, indicating that these sites are not essential for precoupling of G-protein to the receptor. The stimulation of PLC in each of these is diminished relative to wild-type. Two additional point mutants (ThrAla and ThrArg) are somewhat diminished in their ability to bind ligand, and the ability of these receptors to stimulate PLC is completely abrogated. This indicates this region of the third intracellular loop is important for efficient coupling to G-proteins. Truncation of the C5a receptor at Gly diminishes binding to 30% of wild-type, and as expected abolishes the ability to activate PLC (Table 3).

Using site-directed mutagenesis, and subsequent binding and signaling studies of the mutant C5a receptors, we have identified amino acids that are involved in the activity of the message site. These amino acids located in the transmembrane segments are Asp, Asn, Cys, and Cys. Identification of the specific role of each of these amino acids is beyond the scope of this work, but the nature of these functional groups (acidic, thiol, or amide) suggests that hydrogen bonding or ionic interactions within the 7TMS bundle are crucial for providing ligand binding and G-protein activation. Proline mutations and intracellular loop mutations of the receptor have distinct behavior, for when they are defective in function, the number of binding sites is also decreased, suggesting a processing defect.

The message:address model suggests that a unique site in the receptor provides the message sequence. These studies do not distinguish direct effects on the message sequence from effects on other aspects of the change in state from inactive to activated receptor. As agonists and antagonists are developed for the C5a receptor, these mutants will provide additional insights to C5a receptor function and aid in the identification of the second message site.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL36162 and HL71910 and by Pfizer Central Research, Groton, CT. 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: Ina Sue Perlmutter Laboratory, Children's Hospital, 320 Longwood Ave., Boston, MA 02115. Tel.: 617-355-6174; Fax: 617-730-0422.

The abbreviations used are: GPCR, G-protein-coupled receptor; C5aR, C5a anaphylatoxin receptor; BSA, bovine serum albumin; PBS, phosphate-buffered saline; MeSO, dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; PLC, phospholipase C; TMS, transmembrane segment.


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