Phosphorylation of Key Serine Residues Is Required for Internalization of the Complement 5a (C5a) Anaphylatoxin Receptor via a beta -Arrestin, Dynamin, and Clathrin-dependent Pathway*

Laurence BraunDagger, Thierry Christophe§, and François Boulay

From the Département de Réponse et Dynamique Cellulaires/Biochimie et Biophysique des Systèmes Intégrés, (UMR 5092, Commissariat à l'Energie Atomique (CEA)/CNRS/Université Joseph Fourier), CEA/Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France

Received for publication, October 3, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human complement 5a (C5a) anaphylatoxin receptor (CD88) is a G protein-coupled receptor involved in innate host defense and inflammation. Upon agonist binding, C5a receptor (C5aR) undergoes rapid phosphorylation on the six serine residues present in the C-terminal region followed by desensitization and internalization. Using confocal immunofluorescence microscopy and green fluorescent protein-tagged beta -arrestins (beta -arr 1- and beta -arr 2-EGFP) we show a persistent complex between C5aR and beta -arrestins to endosomal compartments. Serine residues in the C5aR C terminus were identified that control the intracellular trafficking of the C5aR-arrestin complex in response to C5a. Two phosphorylation mutants C5aR-A314,317,327,332 and C5aR-A314,317,332,334, which are phosphorylated only on Ser334/Ser338 and Ser327/Ser338, respectively, recruited beta -arr 1 and were internalized. In contrast, the phosphorylation-deficient receptors C5aR-A334,338 and C5aR-A332,334,338 were not internalized even though observations by confocal microscopy indicated that beta -arr 1-EGFP and/or beta -arr 2-EGFP could be recruited to the plasma membrane. Altogether the results indicate that C5aR activation is able to promote a loose association with beta -arrestins, but phosphorylation of either Ser334/Ser338 or Ser327/Ser338 is necessary and sufficient for the formation of a persistent complex. In addition, it was observed that C5aR endocytosis was inhibited by the expression of the dominant negative mutants of dynamin (K44E) and beta -arrestin 1 (beta -arr 1-(319-418)-EGFP). Thus, the results suggest that the C5aR is internalized via a pathway dependent on beta -arrestin, clathrin, and dynamin.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The complement C5a1 anaphylatoxin receptor (C5aR) is a member of the G-protein-coupled receptor family (GPCR) (1, 2). It is abundantly expressed in myeloid cells and to a lower level in a variety of non-myeloid cells (3). In neutrophils, the activation of chemoattractant receptors including the C5aR triggers a complex array of cellular functions that results in directed cell migration and release of large amounts of proteolytic enzymes and reactive oxygen species (4). These responses are highly regulated since, despite the persistent presence of chemoattractants, the intracellular signaling events are transient.

As for many other GPCRs, this attenuated responsiveness is thought to result from receptor desensitization through their phosphorylation and rapid internalization. The current concept for GPCR desensitization, largely extrapolated from studies with rhodopsin and the beta 2-adrenergic receptor, is that, once activated and phosphorylated, the GPCRs form a stable complex with a family of adapters known as arrestins and beta -arrestins (5). There is growing evidence indicating that the formation of this complex is a general intermediate for endocytosis of most GPCRs. The beta -arrestins interact with clathrin and the adapter protein complex AP-2 (6-8) and target the agonist-occupied receptors to pre-existing clathrin-coated pits for internalization (9, 10). Expression of a GTPase-defective dynamin mutant blocks the clathrin-dependent endocytic pathway in a dominant-negative manner (11), and the agonist-mediated internalization of most GPCRs is inhibited (12). However, some GPCRs appear to depart from this standard model and are capable of utilizing alternative pathways. For instance, the angiotensin II 1A and N-formyl peptide receptors, although able to interact with beta -arrestins, can be internalized through a process independent of beta -arrestin and dynamin (13-16), whereas the 5-hydroxytryptamine 2A receptor is internalized via a pathway dependent on dynamin and independent of beta -arrestin (17). The internalization of the M2 muscarinic acetylcholine receptor appears to proceed through an atypical pathway that is independent of clathrin (18).

It has been previously established that the activated C5aR is phosphorylated on the 6 serine residues located in the C-terminal tail at positions 314, 317, 327, 332, 334, and 338 (19). Mutants with combined amino acid replacements exhibit different capacities to incorporate phosphate on the remaining serine residues (20, 21). Combined mutations at positions 332 and 334, at positions 334 and 338, or at all 3 positions yield phosphorylation-deficient mutants, whereas mutants for which the serine pairs Ser327/Ser338 or Ser334/Ser338 are conserved retain the capacity to be phosphorylated (21). This phosphorylation step plays a key role in the attenuation of the cellular response since phosphorylation-deficient mutants trigger sustained intracellular signaling events that result in a significant increase in C5a-mediated superoxide production by neutrophil-like differentiated HL-60 cells (21). The phosphorylation of the first membrane proximal serine residue is dispensable for receptor desensitization, and agonist sequestration is apparently independent of desensitization (21).

In the present study, we used confocal microscopy and the green fluorescent protein conjugate of beta -arrestin 1, beta -arrestin 2, and the dominant negative mutant beta -arrestin 1-(319-418) to examine the cellular trafficking of a series of C5aR mutants in response to C5a. Our results demonstrate that a minimal level of phosphorylation is required for internalization through a dynamin-, arrestin-, and clathrin-dependent internalization pathway. Moreover, beta -arrestin 1 and beta -arrestin 2 were found to have differential abilities to associate with the activated receptor. In contrast to beta -arrestin 1, beta -arrestin 2 redistributed to the plasma membrane regardless of whether C5aR was phosphorylated provided it was activated.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Reagents-- Bovine serum albumin fraction V, human recombinant C5a, 1,4-diazabicyclo(2.2.2)octane (DABCO), aprotinin, leupeptin, and pepstatin were purchased from Sigma. Goat anti-rabbit Alexa 568-conjugate antibody was from Molecular Probes, Inc. (Eugene, OR). Tissue culture media were from Invitrogen. Dithiobis(succinimidyl propionate) was from Pierce. Dithiothreitol cycloheximide, okadaic acid, and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride were from Roche Molecular Biochemicals. Puromycin and polyclonal anti-GFP antibodies were from Clontech. Blasticidine was from Invitrogen. The rabbit polyclonal antibodies directed against the last 10 amino acid residues of C5aR has been previously described (22). The dominant negative dynamin mutant (hemagglutinin-dynamin K44E) (11) were supplied by Dr. S. L. Schmid (Department of Cell Biology, Scripps Research Institute, La Jolla, CA).

beta -Arrestin-EGFP Fusion Proteins and C5aR Phosphorylation Mutants-- To construct a beta -arr 1-EGFP fusion, a HindIII-XbaI fragment encoding human beta -arrestin 1 (beta -arr 1) was isolated from plasmid pBJI-neo (supplied by Dr. DeBlasi Mario Negri, Instituto di Richerche Farmacologiche, Santa Maria Imbaro, Milan, Italy) and subcloned into pcDNA3.1 (Invitrogen). A HindIII-KpnI fragment encoding residues 1-410 of beta -arrestin 1 and a KpnI-SalI synthetic fragment encoding the last eight residues of beta -arrestin 1 were then ligated into pEGFP-N3 (Clontech). The beta -arr 1-(319-418)-EGFP fusion was prepared as follows; a 300-bp fragment encoding the last 100 residues of beta -arrestin 1 was amplified by PCR from plasmid beta -arr 1-EGFP with the sense primer 5'-CCCAAGCTTACCATGGTTTCCTACAAAGTGAAAGTG-3' and the reverse primer 5'-GTCGCCGTCCAGCTCGACCAG-3'. After cleavage with HindIII and BamHI, the PCR fragment was cloned into pEGFP-N3. The nucleotide sequence of the amplified fragment was verified by sequencing. To construct a beta -arr 2-EGFP fusion, a SacI-PstI fragment and a PstI-BamHI fragment encoding rat beta -arr 2 were isolated from plasmid beta -arr 2-GFP pS65-T (23) and cloned into pEGFP-N3.

Cell Culture and Transfection-- The stable expression of wild-type C5aR and mutant receptors C5aR-A314,317,332,334 and C5aR-A332,334,338 in the insulin-secreting cell line RINm5F has been previously described (20). RINm5F cells that stably expressed the mutant receptors C5aR-A314,317,327,332 and C5aR-A334,338 were obtained by transfecting cells with 1 µg of pPUR plasmid (Clontech) and 20 µg of CDM8 mutant receptors as described previously (24). DNA-mediated gene transfer into RINm5F cells was performed by electroporation. Cells were cultured in RPMI 1640 medium/GlutaMax I supplemented with 1 µg/ml puromycin and 10% heat-inactivated fetal calf serum. Resistant clones were assayed for receptor expression by testing their ability to bind 125I-labeled C5a as described previously (25). RINm5F cells expressing either wild-type or mutant C5aR were further transfected with 1 µg of pEF-Bsd plasmid (Invitrogen) and 20 µg of beta -arr-EGFP plasmids (beta -arr 1-, beta -arr 2-, and beta -arr 1-(319-418)-EGFP). Selection of beta -arr-EGFP-expressing clones was performed by culturing cells in the presence of 10 µg/ml blasticidine. Resistant clones were screened for beta -arr-EGFP expression by epifluorescence with an inverted microscope.

For transient transfection, HEK-293 cells were plated onto 6-well plates. Twenty-four hours after plating, cells were co-transfected with C5aR and either a control vector, beta -arr 1-(319-418)-EGFP, or hemagglutinin-dynamin K44E using a standard calcium phosphate co-precipitation protocol. After 60-72 h, cells were assayed for their ability to internalize C5a labeled with radioactive iodine as previously described (25).

Subcellular Fractionation-- Cells incubated with or without 50 nM C5a were washed with ice-cold phosphate-buffered saline and scraped into 0.25 M sucrose, 10 mM Tris, pH 7.4, 1 mM EDTA, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and disrupted by Dounce homogenization. Nuclei and unbroken cells were removed by centrifugation at 1000 × g for 10 min at 4 °C. A crude plasma membrane fraction was prepared by centrifugation of the supernatant at 3000 × g for 15 min. Then centrifugation of the resulting supernatant at 300,000 × g for 30 min at 4 °C gave rise to a supernatant containing cytosol.

Indirect Immunofluorescence Staining and Fluorescence Microscopy-- For all microscopic analyses, cells were seeded on polylysine-coated coverslips 48 h before the experiments. Protein synthesis was blocked by incubating cells in RPMI 1640 supplemented with 1% serum albumin fraction and 100 µg/ml cycloheximide for 3 h before stimulation with C5a. Cells were treated in the same medium with 50 nM C5a at 37 °C for various time periods ranging from 0 to 30 min. Cells not treated with the ligand were used as a control. Cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline supplemented with 1 mM MgCl2 and 1 mM CaCl2 for 30 min on ice, quenched with 50 mM NH4Cl in RPMI 1640 for 15 min at 4 °C, permeabilized with 0.1% Nonidet P-40 for 10 min at room temperature, and incubated with blocking buffer (2% serum albumin fraction in RPMI 1640). For indirect immunofluorescence staining of C5aR, fixed and permeabilized cells were incubated with affinity-purified rabbit polyclonal antibody to the C5aR C terminus for 1 h at room temperature. Cells were then washed 3 times and incubated with red-fluorescent Alexa 568-conjugate goat anti-rabbit antibody for 30 min at room temperature. After washing, cells were embedded in 1,4-diazabicyclo(2.2.2)octane (DABCO). For detailed microscopic analysis, we used a Leica TCS-SP2 confocal-scanning microscope. EGFP was excited at 488 nm with an argon laser, and its fluorescence was collected between 497 and 532 nm. Alexa 568 was excited at 543 nm, and fluorescence emission was collected between 557 and 667 nm.

Internalization Assay-- C5aR sequestration was performed as previously described (21). Briefly, HEK-293 cells were seeded in polyornithine-coated 6-well plates 48 h before co-transfection with plasmids containing the C5aR cDNA and either the DynK44E cDNA, beta -arr 1-(319-418)-EGFP cDNA, or the empty vector alone. Surface-expressed receptors were saturated with 125I-labeled C5a (100 nM) in Dulbecco's modified Eagle's medium, 1% serum albumin fraction, 20 mM Hepes, pH 7.5, for 60 min, at 4 °C. Control cells were incubated with an excess of unlabeled C5a to determine nonspecific binding. Internalization was initiated by diluting cells in 5 volumes of Dulbecco's modified Eagle's medium at 37 °C. At the indicated times, cells were washed and treated for 10 min with ice-cold buffer containing 0.15 M NaCl and 0.2 M acetic acid at pH 2.5 to remove 125I-labeled C5a bound to cell surface receptors, thus giving a measure of C5a internalization/sequestration. The kinetics of C5a internalization are expressed as the percentage of specifically bound 125I-labeled C5a that is internalized as follows: (cpm resistant to acid wash after warming minus cpm resistant to acid wash before warming)/(cpm specifically bound at 4 °C under saturating conditions).

Detection of C5aR and EGFP Conjugates by Immunoblotting-- Cell monolayers were lysed in 2-fold Laemmli sample buffer supplemented with 10 mM dithiothreitol and briefly sonicated with a microtip. Proteins were then separated by SDS-PAGE and transferred to a 0.2-µm Protran nitrocellulose filter for immunoblotting. Immunodetection of C5aR and EGFP conjugates was performed by affinity-purified rabbit anti-C5aR IgGs (1/200) and a rabbit polyclonal anti-GFP antiserum (1/400) followed by a 1-h incubation period time with 125I-labeled protein A. Immune complexes on membranes were visualized by autoradiography.

Immunoprecipitation and Western Blotting-- Immunoprecipitation experiments were performed by using stably transfected RINm5F cells in 100-mm dishes. Cells were stimulated as described in the figure legends and solubilized in 1 ml of immunoprecipitation buffer containing 150 mM NaCl, 10 mM Tris, pH 7.5, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 10 mM NaF, 10 mM NaPi, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 0.1 µM okadaic acid, 1 mM sodium orthovanadate, and 1 µg/ml aprotinin, leupeptin, and pepstatin. To detect C5aR-associated proteins, stimulated cells were subjected to covalent protein cross-linking by using the permeable and cleavable cross-linker dithiobis(succinimidyl propionate), as previously described (26). Immunoprecipitation of C5aR was performed with affinity-purified polyclonal anti-C5aR, as previously described (19). Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. Protein immunoblotting for full-length and truncated beta -arrestin was performed by using rabbit polyclonal anti-GFP antiserum. Immune complexes on nitrocellulose membrane were visualized by 125I-labeled protein A and autoradiography.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Cell Lines Expressing C5aR and beta -Arrestins-- To examine whether the C5aR interacts with the beta -arrestins and to what extent the phosphorylation of the C-terminal region of C5aR influences this interaction, we established RINm5F cell lines that expressed either wild-type or mutant C5aRs (see Fig. 1A) together with either beta -arr 1-EGFP, beta -arr 2-EGFP, or a dominant negative form of beta -arrestin 1 referred to as 1beta -arr 1-(319-418)-EGFP (27). RINm5F cells are adherent cells of rat insulinoma origin that have been previously shown to rapidly and efficiently internalize the C5aR in response to C5a binding (20). The expression of each beta -arrestin variant was assessed by Western blotting of whole cell lysates using anti-GFP polyclonal antibodies. As illustrated in Fig. 1B, single protein species of around 75, 40, and 70 kDa were immunodetected that corresponded to beta -arr 1-EGFP, beta -arr 1-(319-418)-EGFP, and beta -arr 2-EGFP, respectively.


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Fig. 1.   Construction of cell lines co-expressing wild-type or mutant receptors and beta -arrestins. A, primary amino acid sequence of the C-terminal region of C5aR. It has been previously established that the C5aR can be phosphorylated on serine residues at positions 314, 317, 327, 332, 334, and 338 (19). Alanine substitution mutants used in this study are shown. B, analysis of total protein extracts from RINm5F cells co-expressing C5aR and beta -arr 1-EGFP, beta -arr 1-(319-418)-EGFP, or beta -arr 2-EGFP. Immunoblotting was performed with a GFP-specific polyclonal antibody. C, detection of wild-type (wt) and mutant C5aR by immunoblotting with anti-C5aR rabbit serum. RINm5F cells were directly lysed in Laemmli sample buffer under reducing conditions at 37 °C before (-) and after (+) 10 min of stimulation with C5a. Based on previous studies (20), the retarded bands correspond to the phosphorylated form of C5aR.

Mutant receptors have been previously described and analyzed for their ability to be phosphorylated after C5a binding when expressed in COS-7 or RINm5F cells (20, 21). With respect to the ability/inability to be phosphorylated, wild-type C5aR, C5aR-A314,317,332,334, and C5aR-A332,334,338 behave similarly that they are expressed in COS-7 or RINm5F cells. Phosphorylated receptors, i.e. wild-type C5aR and C5aR-A314,317,332,334, have a reduced electrophoretic mobility on polyacrylamide gel in the presence of sodium dodecyl sulfate, whereas the phosphorylation-deficient mutant C5aR-A332,334,338 lacks this electrophoretic shift (20, 28). This hallmark of phosphorylation was used in the present study to analyze two additional mutants, A314,317,332,334 and C5aR-A334,338, when expressed in RINm5F cells (Fig. 1C). The absence of phosphorylation of the latter was further confirmed by receptor immunoprecipitation after metabolic labeling with radioactive orthophosphate (not shown).

Intracellular Trafficking of beta -Arrestins upon Activation of C5aR-- The cellular localization of beta -arrestin 1 in cells co-expressing C5aR and beta -arr 1-EGFP was first examined by subcellular fractionation of cells that were stimulated or not with C5a. Consistent with previous observations indicating that beta -arrestin 1 resides in the cytosol of unstimulated cells (29), beta -arr 1-EGFP was mainly associated with the cytosolic fraction of RINm5F cells in the absence of C5a stimulation (Fig. 2A). When cells transfected with both wild-type C5aR and beta -arr 1-EGFP were stimulated with C5a, a significant amount of beta -arr 1-EGFP was recruited to the plasma membrane (Fig. 2A). Under the same conditions of stimulation there was no translocation of beta -arr 1-(319-418)-EGFP (Fig. 2B). The translocation of beta -arr 1-EGFP to the plasma membrane of cells expressing the phosphorylation-deficient mutant C5aR-A332,334,338 receptor was not significantly detectable after C5a stimulation (Fig. 2C). Thus, activated C5aR recruits beta -arrestins to the plasma membrane. The redistribution of beta -arrestin 1-EGFP appears to be linked to the ability of C5aR to be phosphorylated and is unlikely to result from C5a-mediated signaling since the phosphorylation-deficient A332,334,338 mutant, which supports sustained signaling (21), fails to induce a significant translocation of beta -arrestin 1-EGFP to the plasma membrane.


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Fig. 2.   Translocation of beta -arrestin 1 to the membrane after agonist stimulation. RINm5F cells stably co-expressing wild-type C5aR (wt) and beta -arr 1-EGFP (A) or beta -arr 1-(319-418)-EGFP (B) or co-expressing the phosphorylation-deficient mutant C5aR-A332,334,338 and beta -arr 1-EGFP (C) were treated without (-) or with (+) 50 nM C5a for 5 min at 37 °C. After harvesting, cells were subjected to subcellular fractionation as described under "Experimental Procedures." Membrane fractions were dissolved in lysis buffer and subjected to SDS-PAGE and Western blot analysis using a GFP-specific polyclonal antibody. PM, plasma membrane.

To further examine the intracellular trafficking of C5aR and beta -arrestins at the cell level and to explore whether beta -arrestin 1 and beta -arrestin 2 were targeted to sites containing the C5aR, we used confocal microscopy to detect beta -arrestin-EGFP chimera and immuno-decorated C5aR in fixed and permeabilized cells. In resting cells, the C5aR was mainly recovered in the plasma membrane, as indicated by a thin red fluorescent staining at the cell periphery (Fig. 3, A and B, top panels). Both beta -arr 1-EGFP and beta -arr 2-EGFP were evenly distributed throughout the cytoplasm of C5aR-expressing cells. Consistent with previous reports (30), beta -arr 1-EGFP was also recovered in nucleus, whereas beta -arr 2-EGFP was completely excluded from this region. Merging of the red and green signals indicated a complete absence of co-localization of C5aR and fluorescent beta -arrestins. The addition of C5a rapidly led to the movement of both EGFP-tagged beta -arrestins from the cytosol to the plasma membrane. By 2 min, red and green fluorescence signals were still co-localized at the cell periphery. At further time points, distinct punctate foci of beta -arrestin-EGFP began to appear at sites below the plasma membrane (not shown), and by 10 min, the green fluorescence of beta -arrestin-EGFP chimera was seen as bright spots that localized in the perinuclear region. These fluorescent spots had the same size, shape, and location as those containing C5aR, as indicated by superimposition of confocal images where a yellow color denotes co-localization. As illustrated in Fig. 3, A and B (lower panels), this pattern was maintained over the longest time points monitored (30 min). Thus, C5a induces translocation of both beta -arrestins from the cytosol to the plasma membrane, where they colocalize with the C5aR, followed by the endocytosis of the C5aR and the association of beta -arrestins to C5aR-containing vesicles.


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Fig. 3.   Agonist-stimulated colocalization of C5aR with beta -arrestin-EGFP. Representative laser-scanning micrographs showing the distribution of wild-type C5aR (left panels) in RINm5F cells expressing either beta -arr 1-EGFP (A, middle panels) or beta -arr 2-EGFP (B, middle panels) after treatment for various periods of time with 50 nM of C5a. After stimulation, cells were fixed, permeabilized, and stained as described under "Experimental Procedures." Colocalization of C5aR and beta -arrestin-EGFP is shown in the overlay images (right panels). Confocal images are representative of more than four independent experiments. Bar, 8 µm.

Binding of beta -Arrestins to Wild-type and Mutant C5aRs-- The ability of beta -arrestin-EGFP to interact with the C5a-occupied receptors was further assessed. Cells expressing C5aR and either beta -arr 1-EGFP or beta -arr 2-EGFP were treated with C5a for 5 min and subjected to reversible cross-linking with dithiobis(succinimidyl propionate). C5aR was immunoprecipitated with an antibody directed against its C terminus. After SDS-PAGE, beta -arrestin-EGFP that was associated with immunoprecipitated receptors was detected by immunoblotting. The treatment of cells with C5a strikingly increased the interaction of C5aR with either beta -arrestin 1 or beta -arrestin 2, as shown in Fig. 4, A and B (lanes 1 and 2), by the apparition of an additional band migrating below two contaminating species that were detected whether C5aR was expressed or not (not shown). No interaction could be detected either between wild-type C5aR and beta -arr 1-(319-418)-EGFP (Fig. 4A, lanes 3 and 4) or between the phosphorylation-deficient mutant C5aR-A332,334,338 and the two beta -arrestins (Fig. 4, A, lanes 5 and 6, and B, lanes 3 and 4, respectively). Altogether the results indicate that there is a stable physical association between beta -arrestins and C5aR provided that C5aR is activated and phosphorylated.


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Fig. 4.   Cross-linking of beta -arrestin to C5a-stimulated C5aR. RINm5F cells that stably co-expressed either wild-type C5aR (wt) or A332,334,338 mutant receptor and either beta -arr 1-EGFP, beta -arr 1-(319-418)-EGFP (A), or beta -arr 2-EGFP (B) were incubated without (-) or with (+) 50 nM C5a for 15 min at 37 °C. beta -Arrestin-receptor complexes were stabilized by covalent cross-linking with dithiobis(succinimidyl propionate). After cross-linking, wild-type C5aR or C5aR-A332,334,338 mutant was immunoprecipitated with an affinity-purified rabbit polyclonal antibody directed to the C-terminal end of C5aR. The presence of beta -arrestin in the immunoprecipitates was further detected by Western blot with anti-GFP antibodies and is indicated by an arrow. Results are representative of three independent experiments.

Effect of beta -arr 1-(319-418)-EGFP Expression on C5aR Internalization-- To determine whether clathrin is required for agonist-induced endocytosis of C5aR, we analyzed C5aR internalization in RINm5F-C5aR cells stably transfected with beta -arr 1-(319-418)-EGFP. This fragment, which constitutively binds to clathrin but is unable to interact with phosphorylated GPCRs, acts as a dominant negative mutant that inhibits agonist-stimulated endocytosis of GPCRs via the classical clathrin-dependent internalization pathway (31). Our observation that the stimulation of cells with C5a did not induce translocation of beta -arr 1-(319-418)-EGFP from the cytosol to the plasma membrane fraction (Fig. 2B) nor its association with the activated receptor (Fig. 4A) was further confirmed by confocal microscopy as shown in Fig. 5. The dominant negative mutant beta -arr 1-(319-418)-EGFP was distributed throughout the cell in superficial and perinuclear vesicles. Although a punctate distribution of green fluorescence was observable at the cell periphery, there was no clear overlap with C5aR regardless of whether cells were stimulated with C5a.


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Fig. 5.   Effect of dominant negative beta -arr 1-(319-418)-EGFP on C5aR endocytosis. RINm5F cells stably expressing wild-type C5aR together with beta -arr 1-(319-418)-EGFP were treated with 50 nM C5a for 0, 5, 10, and 30 min at 37 °C. After stimulation, cells were fixed, permeabilized, and stained as described under "Experimental Procedures." Confocal microscopic images in the right panels are formed by superimposition of images from the two other panels in the same row. Bar, 8 µm.

Because beta -arr 1-(319-418)-EGFP constitutively interacts with clathrin but not with GPCR (31), the expression of this truncated mutant is expected to inhibit the agonist-induced internalization of C5aR if a clathrin-dependent pathway is the major endocytic pathway involved in C5aR-internalization. In control RINm5F-C5aR cells, i.e. not transfected with beta -arrestin-EGFP, C5aR is clearly recovered in vesicles that accumulate in the perinuclear region upon C5a addition (20). In cells co-expressing beta -arr 1-(319-418)-EGFP, C5aR largely remained at the plasma membrane of the majority of cells, even 30 min after C5a application (Fig. 5). These results indicate that the expression of beta -arr 1-(319-418)-EGFP strongly inhibits C5aR endocytosis and provide strong evidence that C5aR is mainly internalized via the classical clathrin-dependent pathway.

To have a more precise measurement of the effect of beta -arrestin on the internalization process, we assayed the capacity of RINm5F-C5aR cells expressing or not beta -arr 1-(319-418)-EGFP to internalize radiolabeled C5a (see "Experimental Procedures"). Paradoxically, although the expression of beta -arr 1-(319-418)-EGFP has a marked inhibitory effect on the cellular trafficking of C5aR, no dramatic change in the amount of 125I-labeled C5a that was sequestered (i.e. resistant to acid treatment) could be observed compared with control cells (data not shown). Thus, it appears that even though it is not routed to deep endosomal compartments, C5aR is still able to sequester radiolabeled C5a in beta -arr 1-(319-418)-EGFP-expressing RINm5F cells. In these cells, agonist sequestration may be due to a relative abundance of endogenous receptor-interacting proteins that stabilize the agonist-receptor complex in a high affinity state insensitive to mild acid treatment.

Effect of Dominant Negative K44E Dynamin and beta -arr 1-(319-418)-EGFP on C5aR Internalization in HEK-293 Cells-- We further examined the internalization of 125I-labeled C5a in HEK-293 cells when the C5aR was transiently overexpressed in the absence or the presence of beta -arr 1-(319-418)-EGFP. As shown in Fig. 6, when C5aR was expressed alone, ~45-50% of the radiolabeled C5a bound at 4 °C was resistant to acid treatment after warming at 37 °C, whereas little (less than 10%) radiolabeled C5a was internalized or sequestered when beta -arr 1-(319-418)-EGFP was co-expressed with C5aR. This result is consistent with the confocal microscopy experiments and suggests a critical role of clathrin in C5aR internalization.


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Fig. 6.   Sequestration of C5aR is inhibited by expression of beta -arr 1-(319-418) and DynK44E. HEK-293 cells transiently co-transfected with C5aR and either a control vector (empty vector), beta -arr 1-(319-418)-EGFP, or dominant negative dynamin (DynK44E) were allowed to bind 125I-labeled C5a at 4 °C, and the capacity of cells to internalize surface bound ligand was assessed as described under "Experimental Procedures." After different periods of incubation at 37 °C, internalization was stopped by transferring cells into a chilled low pH buffer to remove bound ligand that had not been internalized. Results are presented as the mean percentage ± S.E. (n = 3) of saturably bound 125I-labeled C5a at 4 °C that is internalized in the different transfection conditions.

Number of reports have shown that agonist stimulation of GPCRs promotes the formation of receptor-containing vesicles, which are pinched off from the plasma membrane and translocated into endocytic compartments. The pinching or sealing off of the vesicles from the plasma membrane is dependent upon dynamin, a GTPase-containing molecule (11, 32). Because beta -arrestins are thought to act as scaffolding proteins in coupling GPCRs to clathrin-coated vesicles (6, 23, 33), we investigated the role of dynamin in C5aR internalization by transiently co-expressing in HEK-293 cells both C5aR and dynamin K44E, a mutant previously shown to inhibit the endocytosis via clathrin-coated pits in a dominant negative manner (11). As illustrated in Fig. 6, expression of dynamin K44E markedly impaired the ability of C5aR to internalize radiolabeled C5a. Thus, altogether the results suggest that internalization of C5aR, like most GPCRs, is an arrestin-, clathrin-, and dynamin-dependent process.

Identification of Residues within the C5aR C Terminal Region That Allow Interaction with beta -Arrestin-- Previous studies show that C5aR is phosphorylated in a hierarchical manner on the six serine residues located in the C-terminal cytoplasmic portion (19, 21). By substituting alanine for serine (see Fig. 1B), we have previously shown that the internalization of the non-phosphorylable mutant C5aR-A332,334,338 is severely impaired compared with wild-type receptor, whereas the mutant C5aR-A314,317,332,334 that is phosphorylated on serine 327 and/or serine 338 showed a rate of internalization in between that of wild-type and C5aR-A332,334,338 receptor (20). To pinpoint serine residues that stabilize receptor/beta -arrestin interaction, we further analyzed RINm5F cells that stably expressed two additional C5aR mutants, C5aR-A314,317,327,332 and C5aR-A334,338, that are phosphorylated and non-phosphorylated, respectively.

Confocal microscopy was used to assess the intracellular distribution of the aforementioned mutant receptors and beta -arr 1-EGFP at several time points after the addition of C5a. As shown in Fig. 7, A and B, substitution of alanine for serine either at positions 314, 317, 327, and 332 or at positions 314, 317, 332, and 334 affected neither the C5a-mediated internalization of these two mutant receptors nor their ability to recruit beta -arr 1-EGFP. The complex was found to traffic into the same endosomal vesicles, as indicated by merging confocal images. In marked contrast, mutations either at positions 334 and 338 or at positions 332, 334, and 338 completely abolished the movement of these two mutant receptors from the cell periphery to the vesicular compartment in the perinuclear region (Fig. 8, A and B). Interestingly, upon C5a application, beta -arr 1-EGFP clearly translocated to the plasma membrane of cells expressing the A334,338 mutant receptor, whereas very little, if any, translocation occurred with cells expressing the A332,334,338 mutant. In the latter case, beta -arr 1-EGFP remained restricted to the cytoplasm even after a prolonged exposure to C5a (Fig. 8B).


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Fig. 7.   Trafficking of beta -arr 1-EGFP with C5aR into endocytic vesicles is mediated by specific serine residues in the receptor C-terminal tail. RINm5F cells were stably co-transfected either with beta -arr 1-EGFP and either C5aR-A314,317,327,332 (A) or C5aR-A314,317,332,334 (B). Cells were treated with 50 nM of C5a for 0, 2, 5, 10, and 30 min at 37 °C. Cells were fixed, permeabilized, and stained as described under "Experimental Procedures." Colocalization of C5aR and beta -arrestin-EGFP is shown in the overlay images. Confocal images are representative of at least three independent experiments. Bar, 8 µm.


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Fig. 8.   Differential translocation of beta -arr 1-EGFP and beta -arr 2-EGFP upon phosphorylation-deficient mutant C5aR activation. RINm5F cells that stably co-expressed either beta -arr 1-EGFP plus A334,338 C5aR (A), A332,334,338 C5aR (B), or beta -arr 2-EGFP plus C5aR-A332,334,338 C5aR (C) were treated with 50 nM of C5a for 0, 10, and 30 min at 37 °C. Cells were fixed, permeabilized, and stained as described under "Experimental Procedures." Colocalization of C5aR and beta -arrestin-EGFP is shown in the overlay images. Confocal images are representative of at least three independent experiments. Bar, 8 µm.

Surprisingly, although cross-linking and immunoprecipitation experiments did not demonstrate a C5a-mediated association of the A332,334,338 mutant with any beta -arrestin isoform (Fig. 4, A and B), we did observe a change in the intracellular distribution pattern of beta -arr 2-EGFP in response to C5a (Fig. 8C). By 2 min, an increase in the level of green fluorescence was observable at the cell periphery that also contained the mutant receptor (not shown). This pattern of fluorescence remained stable with time. By 30 min, we could only observe the presence of few receptor- and beta -arrestin 2-containing vesicles in a minority of cells, indicating a slow rate of internalization of this phosphorylation-deficient mutant. Thus, in the absence of phosphorylation, the binding of C5a brings about a conformational change that is apparently sufficient to mediate a loose interaction with beta -arrestin 2 but not with beta -arrestin 1. Phosphorylation of only two serine residues either at positions 327/338 or at positions 334/338 is sufficient to trigger a firm association with beta -arrestins and the subsequent endocytosis of C5aR.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For most GPCRs, the primary effect of agonist binding is to bring about a conformational change that triggers the coupling to heterotrimeric G proteins and promotes the phosphorylation of the receptor and its interaction with the beta -arrestin scaffolding proteins. In the present work, by using green fluorescent protein-tagged beta -arrestin 1 and 2, we find that C5a stimulated a striking and rapid redistribution of beta -arrestin 1 and 2 from the cytosol to the plasma membrane followed by the formation of a long-lasting complex between wild-type C5aR and both beta -arrestins as evidenced by the co-localization of C5aR and beta -arrestins on the same endosomal compartments. Similar agonist-mediated translocation and association to endosomes have been previously described with a variety of GPCRs (6, 23). The co-trafficking of beta -arrestins and C5aR to perinuclear vesicles was consistently best observed with beta -arrestin 2, suggesting that this isoform has a higher affinity for the activated wild-type receptor. The persistent association of beta -arrestins with intracellular vesicular compartments that contain internalized GPCRs is not a general rule. The stability of beta -arrestin-receptor interactions seems to differ from receptor to receptor. For instance, although "class B" receptors, such as the angiotensin II type 1A and the neurokinin 1 receptors, colocalize with beta -arrestin 2 on endosomal compartments, "class A" receptors including the beta 2-adrenergic, dopamine D1A, and endothelin type A receptors rapidly dissociate from beta -arrestins, which remain confined to the plasma membrane (14, 30, 34). In this respect, C5aR behaves as a "class B" receptor. The significance of this prolonged colocalization is not known, but it is likely to regulate the dephosphorylation and recycling of C5aR.

Judging from the persistent association of beta -arrestins to agonist-occupied C5aR and from the observation that co-expression of beta -arrestin 1 increases by 1.4-fold the rate of C5aR internalization,2 it is likely that beta -arrestins play an important role in C5aR internalization. In the present study, we provide strong evidence that beta -arrestins target the activated C5aR to clathrin-coated pits in a dynamin-dependent manner. First, confocal microscopy experiments with RINm5F cells clearly show that activated C5aR remains at the periphery of cells expressing the clathrin-interacting domain of beta -arrestin 1. Second, the co-expression of C5aR with either the dominant negative mutant K44E of dynamin or beta -arr 1-(319-418)-EGFP results in a severe reduction of internalization/sequestration of radiolabeled C5a in HEK-293 cells. Thus, similarly to the interleukin 8 receptor (35), the C5aR appears to be internalized via clathrin-coated pits in a dynamin-dependent manner. The role of beta -arrestins and dynamin in the endocytosis of GPCRs is not a general rule since it has been reported that the internalization of several GPCRs, including the M2 muscarinic and angiotensin II type A receptors, is independent of beta -arrestin and dynamin (13, 36, 37). As C5aR, the leukocyte chemoattractant receptors N-formyl peptide receptor (FPR) and formyl peptide receptor-like 1 (FPRL1) appear to behave as "class B" receptors since they also form a persistent complex with beta -arrestin 1.2 Our conclusions strikingly contrast with recent studies suggesting that C5aR and FPR are internalized through a beta -arrestin-, clathrin-, and dynamin-independent pathway (15, 16). These divergent results might possibly be due to differences in the methods used to assess receptor internalization. In the study by Bennett et al. (16), C5aR internalization was detected by flow cytometry with an antibody directed to the receptor N-terminal domain, whereas our conclusions are based on confocal microscopy and the uptake of radiolabeled C5a. Our results do not, however, exclude the possibility that, under certain experimental conditions, the internalization of C5aR could proceed through an alternative pathway.

In a previous study, we have shown that the phosphorylation of C5aR occurs through a hierarchical process on serine residues located in the C-terminal domain (21). The two most distal serine residues (i.e. Ser334 and Ser338) serve as primary phosphorylation sites. This step is strictly required for the phosphorylation of the other residues. Partial phosphorylation on Ser327/Ser338 as well as Ser334/Ser338 is sufficient to confer a wild-type phenotype of desensitization (21). Here, we show by confocal microscopy that the phosphorylation of either two-serine pair is sufficient to allow the co-trafficking of beta -arrestins with the receptor to intracellular vesicles. Whether beta -arrestin binding is required to inhibit the association of C5aR with the G protein is presently not known. In the case of the FPR it has been shown by an elegant in vitro reconstitution assay that a partial phosphorylation is sufficient to inhibit FPR-G protein interactions independently of arrestin binding (38).

Of particular interest is the observation by confocal microscopy that beta -arrestins are recruited to the plasma membrane by phosphorylation-deficient mutant. The agonist-dependent recruitment of beta -arr 2-EGFP but not beta -arr 1-EGFP by C5aR-A332,334,338 provides additional support to the notion that beta -arrestin 2 is the preferred isoform for targeting wild-type C5aR to clathrin-coated pits. Although the affinity of beta -arrestins for these mutant receptors is most likely low, it may be sufficient to impose structural constraints that result in the sequestration of C5a in its binding site. This could explain previous results where radiolabeled C5a has been found to be significantly sequestered by phosphorylation-deficient mutant receptors (20, 21).

In addition to their role as clathrin adapters, beta -arrestins function as scaffolding proteins for several signaling pathways. Although beta -arrestin 1 has been shown to recruit and activate c-Src kinase to the plasma membrane, thereby allowing the activation of extracellular signal-regulated kinases (Erk1 and Erk2) (26), beta -arrestin 2 forms a complex with and target c-Jun N-terminal kinase 3 (JNK3) to specific subcellular compartments and/or specific substrates (39). Recently, beta -arrestin 2 has been shown to be essential, presumably through its binding to phosphorylated receptors, for the directed migration of mouse lymphocyte in a gradient of stromal-derived factor 1 (40). Our data indicating that beta -arrestins can be recruited to the plasma membrane in the absence of receptor phosphorylation suggest that beta -arrestin-mediated signaling is still possible in the absence of receptor internalization. With other chemoattractant receptors, the agonist-dependent recruitment of beta -arrestin 2 by phosphorylation-deficient mutant receptors may also occur to variable levels, depending on the receptor and the sets of residues that are mutated. This could explain why chemotaxis and the activation of MAP kinase mediated by phosphorylation-deficient mutants of chemoattractant receptors are variably affected (41-43).

In conclusion, we have demonstrated that C5a induces a marked redistribution of beta -arrestins to the plasma membrane, where they participate in clathrin-mediated endocytosis of C5aR. Moreover, we further establish that phosphorylation of two serine pairs, namely Ser327/Ser338 or Ser332/Ser338, is sufficient to stabilize the interaction between beta -arrestins and C5aR. In these serine pairs, the phosphorylation of Ser338 is likely to play a key role since its replacement by an alanine yields a mutant with a better ability to transduce signal (21), suggesting a weaker interaction with beta -arrestins and, thereby, a slightly prolonged interaction with the G protein.

    FOOTNOTES

* This study was supported by grants from the Commissariat à l'Energie Atomique, CNRS, and the University Joseph Fourier.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a postdoctoral fellowship from the Association pour la Recherche sur le Cancer. Present address: Plasticité et Expression des Génomes Microbiens (FRE 2383, CNRS/Université Joseph Fourier), 460 rue de la Piscine, 38041 Grenoble Cedex 9, France.

§ Present address: Euroscreen s.a. 47, Rue Adrienne Bolland, 6041 Gosselies Belgium.

To whom correspondence should be addressed. Tel.: 33-438-78-31-38; Fax: 33-438-78-51-85; E-mail: fboulay@cea.fr.

Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M210120200

2 L. Braun and F. Boulay, unpublished results.

    ABBREVIATIONS

The abbreviations used are: C5a, complement 5a; C5aR, C5a receptor; Bsd, blasticidine; GPCR, G-protein-coupled receptor; GFP, green fluorescent protein; EGFP, enhanced GFP; beta -arr, beta -arrestin; FPR, N-formyl peptide receptor.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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