©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Major Phosphorylation Sites in Human C5a Anaphylatoxin Receptor in Vivo(*)

(Received for publication, March 30, 1995)

Eric Giannini Laurence Brouchon François Boulay (§)

From theCommissariat à l'Energie Atomique, Laboratoire de Biochimie (CNRS/URA 1130), Département de Biologie Moléculaire et Structurale Centre d'Etudes Nucléaires, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interaction of human C5a anaphylatoxin with cell surface receptors mediates cell activation and receptor desensitization. Treatment of differentiated HL60 cells or transiently transfected COS-7 cells with C5a or phorbol 12-myristate 13-acetate (PMA) results in rapid hyperphosphorylation of the C5aR. In an attempt to gain more insight into the function of phosphorylation in the desensitization of C5aR, we have initiated experiments to identify phosphoacceptor sites at the amino acid level after stimulation of cells with either C5a or PMA. In this report we show that C5aR is phosphorylated exclusively on serine residues in both differentiated HL60 and transfected COS-7 cells irrespective of the stimulus used. Peptide mapping after cyanogen bromide cleavage of phosphorylated C5aR indicates that despite the presence of a protein kinase C consensus motif the third cytoplasmic loop is not phosphorylated when cells are challenged with either C5a or PMA. Thus, whether the cells are stimulated with C5a or PMA, the phosphorylation sites appear to be restricted to serine residues in the carboxyl tail. Phosphoamino acid analysis of a series of mutants in which an individual serine residue was replaced by a threonine residue indicates that the C5aR undergoes C5a-dependent phosphorylation to the maximal stoichiometry of 6 mol of PO(4)/mol of receptor at Ser, Ser, Ser, Ser, Ser, and Ser. Simultaneous substitution of serine residues by alanine at positions 332, 334, and 338 affected neither the binding of C5a nor the cell surface expression of the mutant, but resulted in a dramatic reduction (more than 80%) of both C5a- and PMA-mediated phosphorylation as compared to the wild type receptor. This result suggests that phosphorylation on the segment extending from Ser to Ser is required for the subsequent phosphorylation of the carboxyl-terminal tail of C5aR.


INTRODUCTION

Leukocytes respond to activating signals generated by chemotactic factors, such as N-formyl peptides, C5a, (^1)interleukin-8, or platelet-activating factor (PAF), with a complex array of biochemical events that culminate with the production of superoxide and the release of lysosomal enzymes(1) . The biological responses are initiated by binding of chemotactic factors to receptors that are all members of the G protein-coupled receptor family(2, 3, 4, 5, 6, 7) . Despite the persistent presence of chemotactic factors, the cellular responses are transient and cells become rapidly refractory to further stimulation with the same agonist(8, 9) . This phenomenon, termed homologous desensitization, is common to many hormonal and neurotransmitter signaling systems(10, 11) . Receptor phosphorylation appears to be a key mechanism by which many G protein-coupled receptors are regulated (12, 13, 14) .

It has been shown recently that the C5aR is phosphorylated rapidly upon binding of C5a, even in presence of staurosporine, a potent inhibitor of protein kinase C(15, 16) . Moreover, disruption of the signaling pathway by ADP-ribosylation of G(i) proteins with pertussis toxin does not reduce phosphorylation mediated by saturating doses of agonist(49) . It is therefore postulated that a specific G protein-coupled receptor kinase(s) (GRK) is involved in the process. Phosphorylation of C5aR is correlated with an attenuation of agonist-mediated GTPase activity in membranes prepared from agonist-treated cells and may explain the homologous desensitization of C5a mediated-responses(16) . In addition, C5aR but not the N-formyl peptide receptor (FPR), is phosphorylated in absence of agonist in cells stimulated with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C(15) . However, although the difference in phosphorylation between C5aR and FPR correlates with the attenuation of C5a-dependent GTPase activity in PMA-treated cells(16) , it is unclear whether protein kinase C has a functional role in the heterologous desensitization of C5aR in leukocytes stimulated with other chemoattractants. In differentiated HL60 cells, although N-formyl-Met-Leu-Phe is thought to activate protein kinase C it is unable to induce phosphorylation of unoccupied C5aR(15) , whereas in transfected RBL-2H3 cells thrombin and antigen have the capacity to induce phosphorylation of unoccupied C5aR(16) .

By analogy with the visual and beta-adrenergic systems(10, 17) , one can speculate that chemoattractant receptor phosphorylation impairs coupling with the G proteins. On the other hand, the rapid disappearance of agonist-occupied chemoattractant receptors from the cell surface and the apparent segregation of occupied receptors from the G protein partner (18, 19, 20, 21) suggest that the rapid loss of membrane receptors might represent an additional mechanism whereby signal transduction is rapidly attenuated in leukocytes. A definitive functional link between phosphorylation of the C5aR and uncoupling from the G protein is yet to be established. It is currently unknown whether a complete inhibition of the signal transduction requires a molecular interaction between the phosphorylated C5aR and arrestin-like molecules (22) , and whether there is some relationship between the phosphorylation of serine and/or threonine residues and internalization/sequestration of occupied C5aR.

Prior to a clear demonstration of the role played by the phosphorylation of C5aR in uncoupling from G protein(s) and internalization/sequestration of occupied C5aR, a precise localization of phosphorylated sites is essential. Based on the hydropathy plot and the predicted topology of C5aR, several putative PKC phosphorylation sites can be identified in the cytoplasmic domain. The third intracellular loop between TMD5 and TMD6 contains the motif LRTWSRRA that conforms to the consensus sequence (R/K)XX(S/T)X(R/K)X for potential phosphorylation by protein kinase C. In addition, the predicted COOH-terminal tail contains 11 serine and threonine residues with several less favorable phosphorylation motifs for PKC.

In this report, we identify phosphorylation sites of C5aR in vivo. We show that the putative PKC phosphorylation motif in the third intracellular loop is not modified even when phosphorylation is mediated by PMA; both PMA-induced and C5a-induced phosphorylation of the C5aR map in the COOH-terminal cytoplasmic tail and are restricted to serine residues. In presence of agonist C5aR is phosphorylated with the maximal stoichiometry of 6 mol of PO(4)/mol of receptor. We also examine the effect of the simultaneous replacement of Ser, Ser, and Ser by alanine residues and determine that these residues are the main phosphorylation sites in both PMA- and C5a-induced phosphorylation.


EXPERIMENTAL PROCEDURES

Materials

Cell culture media and fetal calf serum were purchased from Boehringer Mannheim. Carrier-free [P]orthophosphoric acid and NaI were from Amersham Corp. Sepharose 4B-protein A was from Pharmacia Biotech Inc. Human recombinant C5a (hrC5a), PMA, N^6-O-2`-dibutyryl-adenosine 3`,5`-cyclic monophosphate (Bt(2)cAMP), and bovine serum albumin were from Sigma. Cyanogen bromide (CNBr) was from Merck.

Cells and Cell Culture

Promyelocytic HL60 cells and COS-7 cells were from the American Type Culture Collection (Rockville, MD). HL60 cells in suspension and COS-7 monolayers were grown in RPMI 1640 and Dulbecco's modified Eagle's medium, respectively. Culture media contained 10% heat-inactivated fetal calf serum, 100 µML-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. All cells were cultured at 37 °C in 5% CO(2). Differentiation of HL60 cells was induced by 1 mM Bt(2)cAMP for 3 days(23) . COS-7 cells were transfected by electroporation with 10 µg of plasmid DNA/2 10^6 cells as described previously (24) .

Oligonucleotide-directed Mutagenesis and Expression of C5aR

cDNA encoding human C5aR was excised from pCDM8-C5aR with HindIII and BamHI and subcloned into pSELECT-1 vector to generate a single-stranded DNA. The Promega pSELECT-1 mutagenesis protocol was followed to carry out mutagenesis reactions, using homemade oligonucleotides. Mutations were verified by direct sequencing with the dideoxynucleotide chain termination method with primer extension (25) using the Sequenase kit from U. S. Biochemical Corp. Methionine residue 120 was substituted for a leucine residue in order to disrupt a cyanogen bromide cleavage site. Serine residues in the carboxyl-terminal tail of C5aR were individually substituted with a threonine residue. The mutated cDNAs were excised from pSELECT-1 with HindIII and BamHI and subcloned into pCDM8-C5aR after digestion with the HindIII and BstEII. Coding sequences of all mutated cDNAs were sequenced after reinsertion into CDM8. The CDM8 vector carrying wild type C5aR or mutated C5aR was used to transfect COS-7 cells by electroporation.

In Vivo Phosphorylation and Immunoprecipitation

Differentiated HL60 cells were cultured in suspension at a density not exceeding 2 10^6 cells/ml. Transfected COS-7 cells were seeded into 100-mm dishes. Three days after differentiation or transfection, the cells were washed twice with phosphate-free buffer and metabolically labeled with [P]orthophosphoric acid (0.3-0.5 mCi/ml) as described previously(15) . Phosphorylation of C5aR was initiated with 50 nM C5a or 1 µM PMA. After 15 min of incubation at 37 °C, the cells were lysed in 500 µl of ice-cold lysis buffer consisting of 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, pH 7.5, supplemented with 5 µg/ml each leupeptin and soybean trypsin inhibitor, 100 ng/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, and pepstatin, 10 mM NaF, 1 mMp-nitrophenyl phosphate, and 10 mM sodium phosphate. Insoluble material was removed by centrifugation at 4 °C for 3 min at 15,000 g. Immunoprecipitation assays were performed by incubating cell lysates with 5 µl of affinity-purified anti-C5aR immunoglobulins (rabbit polyclonal antibody generated against the last 11 amino acid residues of C5aR) that were preadsorbed on 10 mg of protein A-Sepharose CL-4B. The immunoprecipitates were extensively washed as described previously (15) and analyzed by SDS-PAGE under reducing conditions (26) and autoradiography.

Binding of I-Labeled C5a to Transfected COS-7 Cells

For receptor binding studies, human recombinant C5a (hrC5a) was labeled with I using chloramine T. In brief, 2 mCi of NaI were mixed with 1 nmol of hrC5a in 25 µl of phosphate buffer (200 mM, pH 7.5) at room temperature. Iodination was initiated with 2 µg of chloramine T and stopped after 1 min by the addition of 40 µg of Na(2)S(2)O(5). I-Labeled C5a was separated from free iodine by gel filtration on P6-DG from Bio-Rad (0.5 cm 20 cm) equilibrated in phosphate-buffered saline supplemented with 1% bovine serum albumin. All binding studies were carried out at 4 °C with subconfluent COS-7 cell monolayers 3 days after transfection as described previously(4) . Data were analyzed by computer fitting using the iterative nonlinear regression program Ligand(27) .

Indirect Immunofluorescence Microscopy

Transfected COS-7 cells were fixed, permeabilized, and treated for immunostaining as described previously(24) . Coverslips were mounted in Aquamount and viewed with a Zeiss IM epifluorescence microscope.

Cyanogen Bromide Cleavage and Phosphopeptide Analysis

For cyanogen bromide cleavage, P-labeled C5aR was eluted from protein A-Sepharose CL-4B with 98% formic acid. The eluted receptor was subjected to cyanogen bromide cleavage in 70% formic acid (50 mg of CNBr/ml) for 16 h at room temperature in the dark. Cleavage was terminated by freeze-drying. The CNBr peptides were solubilized in Laemmli buffer and were separated by multiphasic buffer system (Tris/BisTris/Bicine) for SDS-PAGE(28) . In brief, the gel consisted of 7.5% (w/v) acrylamide stacking, 10% separating, and 20% resolving gel supplemented with 0.2% linear polyacrylamide (M(r) > 5,000,000) to avoid cracking during the drying step. The gels were not fixed but dried immediately after electrophoresis and exposed to Fuji RX film at -80 °C. Relative molecular masses were determined after Coomassie Blue staining of molecular weight markers ranging from 2500 to 17,000 (BDH Chemicals).

Phosphoamino Acid Analysis

In situP-labeled C5aR was eluted from the Sepharose beads as described above. The eluate was freeze-dried in a SpeedVac, partially hydrolyzed in 200 µl of 6 M HCl at 110 °C for 90 min in a sealed glass tube, diluted with 1 ml of water, and dried. The dry residue was resolubilized in 10 µl of buffer at pH 1.9 and spotted on thin-layer cellulose plates (10 10 cm; Schleicher & Schüll) with 1 nmol each of phosphoserine, phosphothreonine, and phosphotyrosine as markers for identification of the phosphoamino acid positions by ninhydrin staining. Phosphoamino acids were separated by electrophoresis at pH 1.9 (Buffer 1: formic acid/acetic acid/H(2)O; 22/78/900 (v/v/v)) for 30 min at 1000 V, followed by a second electrophoresis at pH 3.5 (Buffer 2: pyridine/acetic acid/H(2)O; 5/50/945 (v/v/v)) for 20 min at 1000 V in the orthogonal direction(29) . After ninhydrin staining the thin layer plates were exposed either to x-ray film at -80 °C or to PhosphorImager screen.


RESULTS

Identification of in Vivo Phosphoamino Acids in C5aR

To determine the nature of the phosphoacceptor sites of C5aR in the C5a- and PMA-mediated phosphorylation state, we immunopurified the receptor from cells that were metabolically labeled with [P]orthophosphate. Labeled C5aR proteins were immunoprecipitated from cell lysates with an affinity-purified antibody directed against the last 11 residues of the carboxyl tail of C5aR (15) . For each immunoprecipitation, an aliquot was electrophoresed on SDS-polyacrylamide gels and visualized by autoradiography in order to check the degree of purity of the immunopurified receptor. We examined P-labeled C5aR isolated from differentiated HL60 cells (dHL60) or transiently transfected COS-7 cells that were stimulated with either 50 nM C5a or with 1 µM PMA. As shown in Fig.1A, the P-labeled C5aR migrated as two bands with relative molecular masses of 43-48 kDa and 120-130 kDa. Additional studies have provided evidence that the 120-130-kDa species is related to C5aR. When a glycosylation-defective mutant (the consensus glycosylation motif NYT being changed into QYT) was expressed in COS-7 cells, the immunopurified P-labeled C5aR migrated in SDS-PAGE as two bands of 39-40 kDa and 80-90 kDa (data not shown). Thus, the upper band is likely to represent a dimeric form of the C5aR. Although one cannot exclude the possibility that dimers are already formed in the plasma membrane, we favor the hypothesis that dimerization rather occurs during the immunoprecipitation step or when the immune complex is dissociated in the Laemmli buffer.


Figure 1: Immunoprecipitation and phosphoamino acid analysis of P-labeled C5aR. A, phosphorylated C5aR was immunoprecipitated with affinity-purified IgG from P-labeled dHL-60 cells or transfected COS-7 cells after stimulation with either 50 nM C5a or 1 µM PMA. Immunoprecipitates were analyzed by SDS-PAGE under reducing conditions, and autoradiography. Immunoprecipitation of C5aR was completely blocked by addition of competing C5aR COOH-terminal peptide. All immunoprecipitates used in this study presented a similar pattern. B, P-labeled C5aR was eluted from Sepharose beads, hydrolyzed, and electrophoresed on cellulose TLC plates, as described under ``Experimental Procedures.'' Positions of phosphoserine (P-S), phosphothreonine (P-T), and phosphotyrosine (P-Y) were assessed by ninhydrin staining of the standards that were coelectrophoresed with the radioactive phosphoamino acids. Radioactive spots that migrate below phosphotyrosine are phosphopeptides arising during partial acid hydrolysis. Cellulose TLC plates were then overexposed to Fuji x-ray films at -80 °C. The figure is representative of at least three independent experiments.



To identify the nature of the phosphorylated residues, we subjected the P-labeled C5aR, eluted from the protein A-Sepharose beads, to limited acid hydrolysis. The phosphoamino acids were resolved on thin layer cellulose plates by two-dimensional electrophoresis and visualized by autoradiography (Fig.1B). From these experiments (n = 3), we observed that the P-labeled C5aR proteins contained exclusively phosphoserine residues whether they were purified from dHL-60 or from transfected COS-7 cells stimulated with either C5a or PMA. This result was quite unexpected in view of the elevated number of threonine residues present in the putative third intracellular loop and the cytoplasmic tail, which have been proved to be the main targets for kinases in numerous receptors.

Mapping of the Domains of C5aR Containing Phosphoserine Residues

Two potential regions of C5aR, i.e. the putative third intracellular hydrophilic loop that contains a potential PKC consensus motif (LRTWSRRA) and the COOH-terminal region that contains numerous serine residues, are likely to be phosphorylated upon agonist binding or after stimulation of cells with PMA. To define which of these two regions were preferentially modified when cells were stimulated with C5a or with PMA, we cleaved the immunopurified phosphorylated receptors at methionine residues with cyanogen bromide. The scarcity and the peculiar distribution of methionine residues in the primary amino acid sequence of C5aR allowed the generation of a limited number of well resolved CNBr fragments with molecular masses ranging from 8.8 to 16 kDa (Fig.2, upper part). The 13-kDa fragment represents the NH(2)-terminal part of the receptor, comprising the first two transmembrane domains (TMD) and the first intracellular and extracellular loops. The 16-kDa fragment encompasses residues 121 to methionine 264 and includes the putative second intracellular loop, between TMD3 and TMD4, and the third cytoplasmic loop with a consensus PKC-phosphorylation motif XRXXSRX, between TMD5 and TMD6. The 8.8-kDa fragment contains the serine/threonine-rich cytoplasmic tail with PKC phosphorylation sites in a less favorable context.


Figure 2: Mapping of the phosphorylated regions of C5aR after stimulation with either C5a or PMA. A, positions of methionine residues and putative phosphorylation sites in the primary sequence of the C5aR are indicated. The blackrectangles represent the putative TMDs. The serine/threonine-rich cytoplasmic tail and the putative PKC motif in the third intracellular loop between TMD5 and TMD6 have been expanded. The theoretical masses of the peptides generated by CNBr cleavage are indicated for the wild type and the mutant receptor Met Leu (M L). B, phosphopeptide analysis after CNBr cleavage of P-labeled C5aR. P-Labeled C5aR was immunoprecipitated with affinity-purified IgG from COS-7 cells that were transfected either with the wild type C5aR (Wt) or with the Met Leu mutant form (M L). Cells were stimulated with either 50 nM C5a (left panel) or with 1 µM PMA (right panel). The phosphorylated receptor was eluted from the Sepharose beads, treated with CNBr for 16 h, and the CNBr-peptides were separated by SDS-PAGE under reducing conditions using the multiphasic buffer system described by Wiltfang et al.(28) .



As illustrated in Fig.2(lower part), cleavage of wild type C5aR immunoprecipitated from C5a-stimulated COS-7 cells yielded a single phosphorylated fragment that migrated with an apparent molecular mass of 8-9 kDa on SDS-polyacrylamide gels. Uncleaved C5aR migrated in this electrophoretic system at the interface between the 10% separating gel and the 20% resolving gel. The lack of phosphorylated species in the range of 16 kDa suggests that the third intracellular loop is not phosphorylated after C5a stimulation. Surprisingly, the same pattern of phosphorylation was observed and a 16-kDa phosphorylated species was not detected after cleavage of wild type receptor immunopurified from PMA-treated cells. To rule out the possibility that the 8-9-kDa phosphorylated band is in fact the 16-kDa peptide with an aberrant electrophoretic behavior, we generated a mutant receptor in which the methionine residue at position 120 was replaced by leucine. With this mutant receptor, only two CNBr fragments with molecular masses of 8.8 and 29 kDa were expected (see Fig.2, upper part). As with the wild type receptor, the mutant receptor yielded a phosphorylated cyanogen bromide fragment in the range of 8-9 kDa whether the phosphorylation was mediated by C5a or by PMA. Lack of phosphorylated 16-kDa species could have resulted from an adsorption of this peptide on the glassware during freeze-drying. To rule out this possibility, experiments were performed in which the cleavage of C5aR was carried out in the gel slice. After washing, the gel slice was further loaded on a gel and phosphorylated peptides were resolved by SDS-PAGE. The same pattern of phosphorylated bands was observed (not shown). Thus, this strongly suggests that the third intracellular loop is not phosphorylated, despite the presence of an amino acid sequence that conforms to a PKC phosphorylation motif.

Together the results indicate that the carboxyl-terminal tail of unoccupied C5aR is the exclusive target for a PMA-stimulated kinase, presumably PKC, and that the same domain is also phosphorylated by a staurosporine-insensitive kinase(15, 16, 49) , presumably a member of the GRK family, which specifically recognizes the agonist-occupied state of C5aR.

Identification of Phosphorylated Serine Residues in Agonist-dependent Phosphorylation

As a rule, it is difficult to characterize the phosphorylation sites in the primary sequence of heptahelical receptors, primarily because of problems in sequencing phosphorylated Ser/Thr-rich peptides and difficulties in purifying sufficient amount of phosphorylated material to isolate the COOH-terminal domain after proteolytic cleavage. The observation that phosphorylation occurs exclusively on serine residues in the carboxyl tail of C5aR rendered the identification of phosphorylated serine residues possible without turning to a strategy involving peptide sequencing or mass spectrometry. We reasoned that if a serine residue is phosphorylated, its replacement by threonine might generate a phosphothreonine detectable by phosphoamino acid analysis, inasmuch as the kinase has no strict requirement for a serine residue at this particular position. Six mutants in which serine at position 314, 317, 327, 332, 334, or 338 was replaced by threonine, were transiently expressed in COS-7 cells. On the basis of the level of phosphate incorporation, all the mutant receptors were found to be transported to cell surface with an efficiency similar to that of the wild type receptor (not shown). After stimulation with C5a, the phosphorylated mutant receptors were immunopurified and subjected to phosphoamino acid analysis. As illustrated in Fig.3, a phosphothreonine was detectable whatever the position of the Ser/Thr substitution, suggesting that all serine residues could be phosphorylated upon C5a binding. The results indicate that C5aR undergoes C5a-dependent phosphorylation to the maximal stoichiometry of 6 mol of PO(4)/mol of receptor at the following positions: Ser, Ser, Ser, Ser, Ser, and Ser.


Figure 3: Phosphoamino acid analysis of P-labeled C5aR mutants. Individual serine residues in the carboxyl tail of the C5aR were replaced with a threonine residue, and each mutant was expressed in COS-7 cells. Three days post-transfection, the cells were metabolically labeled with P-labeled orthophosphoric acid and stimulated with 50 nM C5a. After immunoprecipitation, P-labeled C5aR mutants were processed for phosphoamino acid analysis, as described under ``Experimental Procedures.'' Cellulose TLC plates were then exposed to Fuji x-ray films at -80 °C or analyzed with a PhosphorImager. For each mutant, the autoradiograph is representative of at least two independent experiments. P-S, phosphoserine; P-T, phosphothreonine; P-Y, phosphotyrosine.



Identification of Phosphorylated Sites in PMA-dependent Phosphorylation

Surprisingly, although the cyanogen bromide experiment strongly indicates that PMA-mediated phosphorylation exclusively occurs in the COOH-terminal region (see Fig.2B), we could not clearly observe phosphothreonine when the same strategy was applied to mutant receptors phosphorylated after PMA stimulation. Only the mutant Ser Thr yielded a very weak phosphothreonine signal, suggesting that Ser is a phosphoacceptor site in the presence of PMA (the amount of radioactive material under the phosphothreonine spot represented less than 5% of that present at the level of the phosphoserine). Since after PMA stimulation the level of radioactivity incorporated in immunopurified C5aR represents about 20-30% of that found after agonist-dependent phosphorylation, no more than two residues are expected to be phosphorylated in the presence of PMA, if one assumes that phosphorylation is restricted to two particular serine residues. In fact, if two serine residues were predominantly phosphorylated by a PMA-activated kinase, one should have obtained a ratio of phosphoserine/phosphothreonine close to the unit in at least two of the Ser Thr mutants. The lack of a clear detection of phosphothreonine with all mutants may result from two mechanisms that are not mutually exclusive. i) The PMA-activated kinase may have a strict requirement for serine residues, and ii) phosphorylation may occur randomly on the COOH-terminal tail of C5aR with a maximal stoichiometry of 2 mol of PO(4)/mol of C5aR. A random phosphorylation would subsequently reduce the level of phosphate incorporation at a particular position, thereby making the detection of the phosphothreonine difficult.

To further characterize the region of the carboxyl terminus of C5aR that is phosphorylated in the presence of PMA, we constructed a mutant in which Ser, Ser, and Ser were replaced with alanine residues, leaving only Ser, Ser, and Ser as potential phosphorylation sites. This mutant proved to be correctly transported to the surface of transfected COS-7 cells, as evidenced by an intense surface immunostaining of permeabilized cells (data not shown). The level of surface expression of the mutant receptor was further assessed by binding of I-labeled C5a to the transfected COS-7 cell monolayers. The binding capacity and the affinity of mutant and wild type receptors were very similar (Fig.4). The dissociation constant (K) was about 10 nM for wild type and mutant receptors, and the maximal binding values (B(max)) were comparable (about 10^6 sites/cell for the wild type and 8.7 10^5 sites/cell for the mutant). The number of binding sites per cell was consistently slightly lower in cells transfected with the mutant cDNA, but the reduction never exceeded more than 15% (n = 3). However, when phosphorylation of the mutant was assayed in COS-7 cells, we observed a dramatic reduction in the level of phosphorylation although the mutant receptor behaved as a wild type receptor with respect to surface expression and binding of C5a. It exhibited no detectable basal phosphorylation and only a very weak phosphorylation was observed when cells were challenged with either C5a or PMA, as compared with the wild type receptor (Fig.5). Taking into account the lower level of expression of the mutant receptor, we estimated that the incorporation of phosphate represented less than 15% of that found in the wild type receptor (n = 2), although three additional phosphorylation sites (at positions 314, 317, and 327) were present in the COOH-terminal cytoplasmic tail. These data provide strong supporting evidence that the mutated serine residues are primary phosphoacceptor sites in the basal phosphorylation and when cells are stimulated with either PMA or C5a. In addition, they suggest that the mechanism of phosphorylation proceeds in a hierarchical manner. The lack of phosphorylation at Ser, Ser, and/or Ser may prevent the kinase(s) to efficiently proceed further.


Figure 4: Surface expression of Ser Ala C5aR mutant. Identical amounts of COS-7 cells (2 10^6 cells) were transfected with the vector alone (mock-transfected), 10 µg of wild type (WT) or mutant cDNA (A), and 3 days post-transfection, cell monolayers were assayed for their ability to bind I-labeled C5a in the presence or absence of 250 nM unlabeled C5a as described previously(4) . Binding parameters were calculated by computer fitting using the iterative nonlinear regression program Ligand(27) . The figure is representative of three experiments.




Figure 5: C5a- and PMA-induced phosphorylation of wild type C5aR and Ser Ala C5aR mutant. Equal amounts of COS-7 cells (2 10^6 cells) were transfected with 10 µg of wild type or mutant (A) cDNA. The ability of C5a or PMA to induce phosphorylation of the wild type and the mutant receptors was assayed after prelabeling cells with [P]orthophosphoric acid. Three days post-transfection, cells were stimulated with either the buffer alone, 50 nM C5a, or 1 µM PMA for 15 min at 37 °C, lysed, and then treated for receptor immunoprecipitation as described under ``Experimental Procedures''. Immunoprecipitates were analyzed by 10% SDS-PAGE under reducing conditions followed by autoradiography or PhosphorImager analysis for quantification. Results are representative of two independent experiments.




DISCUSSION

To our knowledge this study represents the first attempt to localize phosphorylation sites in C5aR in vivo. It demonstrates that the Ser/Thr-rich carboxyl terminus of C5aR is the main target for kinases in both C5a- and PMA-dependent phosphorylation. Surprisingly, only phosphoserine residues were found to constitute the phosphoacceptor sites, despite the existence of five threonine residues within the cytoplasmic tail. A similar pattern of phosphorylation has been described recently for another receptor, the luteinizing hormone/CG receptor, in which the human chorionic gonadoptropin- and PMA-stimulated phosphorylation occurs exclusively on serine residues of the COOH-terminal cytoplasmic tail(30) . Such a situation is nevertheless not a rule among G protein-coupled receptors. In the case of rhodopsin, the photolyzed receptor is sequentially phosphorylated at multiple serine and threonine residues in the COOH-terminal region(31) . (^2)More recently, it has been shown with a fusion protein containing the last 47 amino acids of FPR that the carboxyl tail of FPR is sequentially phosphorylated by GRK2 on multiple serine and threonine residues(32) . However, it remains to be established in this latter case that the pattern of phosphorylation and the kinase selectivity are identical in vivo.

One could argue that the exclusive detection of phosphoserine is inherent to our methodology that used a rabbit polyclonal antibody directed against the COOH-terminal peptide (DTMAQKTQAV) to immunopurify C5aR. Indeed, phosphorylation of threonine residues at positions 342 and 347 may impair the ability of the antibody to precipitate C5aR and thereby lead to the exclusive immunoprecipitation of a population of receptors that are not phosphorylated on these two residues. However, this possibility is unlikely because identical amounts of [S]methionine-pulse-labeled C5aR were immunoprecipitated from COS-7 cells whether cells were treated or not treated with C5a (data not shown). This indicates that none of the threonine residues present in the antigenic epitope are phosphorylated or that their phosphorylation does not impair the formation of an immune complex (if so, phosphothreonine should have been detected). Thus, the phosphoacceptor sites are likely to be confined to serine residues of the COOH-terminal cytoplasmic tail for agonist- and PMA-dependent phosphorylation. Interestingly, four of the phosphoacceptor sites of C5aR, i.e. Ser, Ser, Ser, and Ser, are conserved between human(3, 4) , mouse(33) , and dog C5aR(34) , whereas Ser is replaced by an alanine residue in the dog receptor and Ser is replaced by a threonine residue in the mouse receptor. High degree of interspecies conservation of hydroxy amino acids at the former positions provides additional support for a key role of these residues in the regulation of C5aR.

The observation that the third cytoplasmic loop is not phosphorylated with either C5a or PMA, despite the presence of a putative PKC consensus motif, is not surprising if one considers that the relatively short third cytoplasmic loop of chemoattractant receptors is probably not a crucial region for coupling to the G proteins. Mutagenesis approaches(35) , competition strategies with fusion protein (36) or synthetic peptides representing intracellular regions of FPR (37) were recently used to probe the sites of interaction with the G protein(s). In all the cases, it was concluded that the third cytoplasmic loop of FPR, and presumably that of other members of the chemoattractant receptor subfamily, was not critical for receptor/G protein coupling, whereas the second intracellular loop and carboxyl-terminal tail of FPR appeared to serve as the major contact sites with the G(i) protein(s). Therefore, it is not unexpected to find the incorporation of phosphate in the COOH terminus of C5aR. However, the region encompassing residues 332-338, which contains the main phosphorylation sites, is apparently not critical for binding and G protein coupling as suggested by a previous report demonstrating that deletion of the COOH-terminal region distal from position 327 does not affect signal transduction in Xenopus oocytes(38) . In contrast, the third intracellular loops of other members of the G protein-coupled receptor superfamily, such as adrenergic and muscarinic receptors, are much longer and include a sequence rich with serine/threonine residues. In these receptors, the third loop has been implicated as being of major importance in signal transduction and G protein selectivity(39, 40) . Moreover, the phosphorylation sites of the human mAChR m2 subtype expressed in Sf9 cells were assigned to serine and threonine residues in the central part of the third intracellular loop(41) , and the agonist phosphorylation of alpha(2)-adrenergic receptor is dramatically reduced if part of the third cytoplasmic loop is deleted (42) . In the latter case, four consecutive serine residues in the third intracellular loop have been described as the sites for GRK2-mediated phosphorylation and desensitization(43) .

The present study provides convincing evidence that the phosphoacceptor sites in the carboxyl tail of the C5aR expressed in COS-7 cells, and it is tempting to speculate that the phosphorylation of C5aR follows the same rules in myeloid cells. Several reasons lead us to believe that the results reflect, at least in part, the positions of the phosphorylation sites in receptors constitutively expressed in dHL60 cells. 1) Phosphoamino acid analysis of C5aR immunopurified from myeloid cells clearly indicates that serine residues are the main phosphorylated sites, and 2) cyanogen bromide cleavage of C5aR immunopurified from C5a- or PMA-treated dHL-60 cells yielded the same pattern of phosphopeptides as that of C5aR purified from COS-7 cells (data not shown). In dHL60 cells, as in COS-7, both PMA-mediated phosphorylation of unoccupied C5aR and C5a-mediated phosphorylation are restricted to serine residues of the carboxyl-terminal tail. However, we are unable to demonstrate whether in neutrophils or neutrophil-like cells the occupied C5aR is phosphorylated on the same serine residues, because presently we know very little about the kinase(s) that phosphorylates C5aR in myeloid cells. Although protein kinase C is likely to be involved in the basal phosphorylation of non-liganded receptor, protein kinase C is definitely not the chief enzyme involved in the phosphorylation of agonist-occupied C5aR. Indeed, C5a-dependent phosphorylation is mainly resistant to the action of PKC inhibitors, such as staurosporine(15, 16) , and is not abolished after disruption of the signal transduction pathway by pertussis toxin(49) . Therefore, it is postulated that the main kinase(s) involved in the phosphorylation of C5aR is a member of the G protein-coupled receptor kinase family.

The observation that C5aR is equally well phosphorylated in dHL60 cells, C0S-7 cells, or rat insulinoma cells (RINm5F) suggests that the kinase(s) involved in the process is either widely distributed or that occupied C5aR is efficiently phosphorylated by more than one member of the GRK family. Messenger RNAs for both GRK2 and GRK6 are abundantly expressed in myeloid cells, and both kinases might be involved in the regulation of the chemoattractant receptors(44, 45) . GRK2 binds to, and phosphorylates, the carboxyl terminus of FPR (32) and PAF receptor in vitro(46) . However, presently there is very little evidence that GRK2 phosphorylates the carboxyl terminus of FPR and C5aR in vivo. The observation that PAF induces the translocation of GRK2 from the cytosol to the plasma membrane of neutrophils provides indirect evidence that PAF receptors interact with and is probably regulated by GRK2 in neutrophils(44) . In contrast, the lack of induction of GRK2 translocation by C5a suggests that the C5aR does not interact with this kinase. However, this conclusion is based on a single report and needs further confirmation. It has been suggested previously on the basis of phosphorylation studies with synthetic peptides that GRK2 is an acidotropic kinase, which prefers acidic amino acids on the NH(2)-terminal side of serine or threonine residues(47) . In this context, it is worth noting that in C5aR the sequences EESVV and RES could be potential phosphorylation sites for GRK2. Interestingly, the amino acid alignment of the carboxyl-terminal sequence of C5aR with that of FPR reveals that Ser and Ser in C5aR correspond to Ser and Thr in FPR, which are phosphorylated by GRK2 in vitro. In both receptors, these residues are preceded by glutamic or a aspartic acid residues (see Fig.6). Other serine residues that are in a neutral (PSLL) or an electropositive environment (RKSL, KSF, and TRST) are probably not the best substrates for GRK2. Localization of phosphorylation sites in other G protein-coupled receptors has not been delineated precisely, except in the case of rhodopsin, and presently it is difficult to extract a consensus phosphorylation sequence for the different GRKs. However, if one compares the carboxyl-terminal sequences of C5aR, FPR, interleukin-8 receptor, PAF receptor, and rhodopsin, it appears that the main phosphoacceptor sites of rhodopsin and C5aR in vivo, as well as those of FPR in vitro, are clustered in a region that presents striking similarities from one receptor to the next (Fig.6).


Figure 6: Amino acid alignment of the COOH-terminal domains present in C5aR, FPR, rhodopsin, PAF receptor, and interleukin-8 receptor. Alignment was performed with DNAstar software using the J. Hein method. A consensus sequence was extracted when a residue was present in the same position in at least two sequences. Hydroxy amino acids for which phosphorylation has been demonstrated are boxed. Numbers refer to the positions of amino acids in C5aR.



On the basis of the lack of phosphorylation of the mutant Ser Ala, we propose that C5aR is multiply phosphorylated in a sequential manner. Ser, Ser, and/or Ser are the primary phosphoacceptor sites whose phosphorylation is essential to allow the kinase to proceed further on other serine residues. A sequential phosphorylation has been demonstrated previously in the case of rhodopsin in which the light-dependent phosphorylation occurs first on serine 338 and subsequently on serine 343 and threonine 336(31) . In the case of C5aR, an immediate question is whether the basal phosphorylation plays any role in the phosphorylation process. It is tempting to speculate that the basal phosphorylation of C5aR on one of the key serine residues (332, 334, or 338) is important to ``prime'' the agonist-occupied receptor as a good substrate for a specific kinase, which will sequentially modify other serine residues. Such a mechanism has been proposed for the phosphorylation of glycogen synthase by glycogen synthase kinase-3 and casein kinase II. In this system, a primary phosphorylation of glycogen synthase by casein kinase II, is required to transform the protein into a substrate for glycogen synthase kinase-3, which sequentially modifies 4 serine residues(48) . It will be interesting to test whether the replacement of serine residues at positions 332, 334, and 338 of C5aR with negatively charged residues could boost the C5a-mediated incorporation of phosphate.

In conclusion, further studies are required to establish how phosphorylation of the serine residues of the COOH-terminal tail of C5aR contributes to the shut-down of signal transduction. The observation that the mutant Ser Ala is not phosphorylated further is of major importance to examine the contribution of serine phosphorylation in homologous desensitization and to investigate whether the agonist-induced internalization of the C5aR is dependent on its phosphorylation status. Examination of additional combinations of mutations to address this issue is under way.


FOOTNOTES

*
This work was supported by grants from the Commissariat à l'Energie Atomique (CEA), the Centre National de la Recherche Scientifique (CNRS/URA 1130), and the Association pour la Recherche sur le Cancer (ARC). 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 and reprint requests should be addressed: DBMS/Laboratoire de Biochimie (CEA; CNRS/URA 1130), Centre d'Etudes Nucléaires, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. Tel.: 33-76-88-31-38; Fax: 33-76-88-51-85.

^1
The abbreviations used in this paper are: C5a, activation peptide from fifth component of complement; C5aR, C5a receptor; PMA, phorbol 12-myristate 13-acetate; GRK, G protein-coupled receptor kinase; FPR, N-formyl peptide receptor; PAGE, polyacrylamide gel electrophoresis; TMD, transmembrane domain; Bicine, N,N-bis(2-hydroxyethyl)glycine; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

^2
McDowell, J. H., Nawrocki, J. P., and Hargrave, P. A. (1993) Biochemistry32, 4968-4974.


ACKNOWLEDGEMENTS

We are grateful to Drs. Nishigandha Naik and Alexandra Fuchs for critically reading the manuscript. We thank Professor P. V. Vignais for constant support during this work.


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