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W1993346410 abstract "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 β-arrestins (β-arr 1- and β-arr 2-EGFP) we show a persistent complex between C5aR and β-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 β-arr 1 and were internalized. In contrast, the phosphorylation-deficient receptors C5aR-A334,338 and C5aR-A332,334,338were not internalized even though observations by confocal microscopy indicated that β-arr 1-EGFP and/or β-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 β-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 β-arrestin 1 (β-arr 1-(319–418)-EGFP). Thus, the results suggest that the C5aR is internalized via a pathway dependent on β-arrestin, clathrin, and dynamin. 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 β-arrestins (β-arr 1- and β-arr 2-EGFP) we show a persistent complex between C5aR and β-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 β-arr 1 and were internalized. In contrast, the phosphorylation-deficient receptors C5aR-A334,338 and C5aR-A332,334,338were not internalized even though observations by confocal microscopy indicated that β-arr 1-EGFP and/or β-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 β-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 β-arrestin 1 (β-arr 1-(319–418)-EGFP). Thus, the results suggest that the C5aR is internalized via a pathway dependent on β-arrestin, clathrin, and dynamin. complement 5a C5a receptor blasticidine G-protein-coupled receptor green fluorescent protein enhanced GFP β-arrestin N-formyl peptide receptor The complement C5a1anaphylatoxin receptor (C5aR) is a member of the G-protein-coupled receptor family (GPCR) (1Boulay F. Mery L. Tardif M. Brouchon L. Vignais P. Biochemistry. 1991; 30: 2993-2999Crossref PubMed Scopus (192) Google Scholar, 2Gerard N.P. Gerard C. Nature. 1991; 349: 614-617Crossref PubMed Scopus (561) Google Scholar). It is abundantly expressed in myeloid cells and to a lower level in a variety of non-myeloid cells (3Zwirner J. Fayyazi A. Gotze O. Mol. Immunol. 1999; 36: 877-884Crossref PubMed Scopus (80) Google Scholar). 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 (4Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar). 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 β2-adrenergic receptor, is that, once activated and phosphorylated, the GPCRs form a stable complex with a family of adapters known as arrestins and β-arrestins (5McDonald P.H. Lefkowitz R.J. Cell. Signal. 2001; 13: 683-689Crossref PubMed Scopus (111) Google Scholar). There is growing evidence indicating that the formation of this complex is a general intermediate for endocytosis of most GPCRs. The β-arrestins interact with clathrin and the adapter protein complex AP-2 (6Goodman O.B. Krupnick J.G.J. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar, 7Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Ménard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (839) Google Scholar, 8Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar) and target the agonist-occupied receptors to pre-existing clathrin-coated pits for internalization (9Scott M.G. Benmerah A. Muntaner O. Marullo S. J. Biol. Chem. 2002; 277: 3552-3559Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Santini F. Gaidarov I. Keen J.H. J. Cell Biol. 2002; 156: 665-676Crossref PubMed Scopus (91) Google Scholar). Expression of a GTPase-defective dynamin mutant blocks the clathrin-dependent endocytic pathway in a dominant-negative manner (11Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar), and the agonist-mediated internalization of most GPCRs is inhibited (12Pierce K.L. Lefkowitz R.J. Nat. Rev. Neurosci. 2001; 2: 727-733Crossref PubMed Scopus (363) Google Scholar). 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 β-arrestins, can be internalized through a process independent of β-arrestin and dynamin (13Zhang J. Stephen S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 14Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 15Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry. 2001; 40: 3467-3475Crossref PubMed Scopus (47) Google Scholar, 16Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), whereas the 5-hydroxytryptamine 2A receptor is internalized via a pathway dependent on dynamin and independent of β-arrestin (17Bhatnagar A. Willins D.L. Gray J.A. Woods J. Benovic J.L. Roth B.L. J. Biol. Chem. 2001; 276: 8269-8277Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The internalization of the M2 muscarinic acetylcholine receptor appears to proceed through an atypical pathway that is independent of clathrin (18Roseberry A.G. Hosey M.M. J. Cell Sci. 2001; 114: 739-746PubMed Google Scholar).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 (19Giannini E. Brouchon L. Boulay F. J. Biol. Chem. 1995; 270: 19166-19172Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Mutants with combined amino acid replacements exhibit different capacities to incorporate phosphate on the remaining serine residues (20Naik N. Giannini E. Brouchon L. Boulay F. J. Cell Sci. 1997; 110: 2381-2390Crossref PubMed Google Scholar, 21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The phosphorylation of the first membrane proximal serine residue is dispensable for receptor desensitization, and agonist sequestration is apparently independent of desensitization (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).In the present study, we used confocal microscopy and the green fluorescent protein conjugate of β-arrestin 1, β-arrestin 2, and the dominant negative mutant β-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, β-arrestin 1 and β-arrestin 2 were found to have differential abilities to associate with the activated receptor. In contrast to β-arrestin 1, β-arrestin 2 redistributed to the plasma membrane regardless of whether C5aR was phosphorylated provided it was activated.DISCUSSIONFor 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 β-arrestin scaffolding proteins. In the present work, by using green fluorescent protein-tagged β-arrestin 1 and 2, we find that C5a stimulated a striking and rapid redistribution of β-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 β-arrestins as evidenced by the co-localization of C5aR and β-arrestins on the same endosomal compartments. Similar agonist-mediated translocation and association to endosomes have been previously described with a variety of GPCRs (6Goodman O.B. Krupnick J.G.J. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar, 23Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). The co-trafficking of β-arrestins and C5aR to perinuclear vesicles was consistently best observed with β-arrestin 2, suggesting that this isoform has a higher affinity for the activated wild-type receptor. The persistent association of β-arrestins with intracellular vesicular compartments that contain internalized GPCRs is not a general rule. The stability of β-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 β-arrestin 2 on endosomal compartments, “class A” receptors including the β2-adrenergic, dopamine D1A, and endothelin type A receptors rapidly dissociate from β-arrestins, which remain confined to the plasma membrane (14Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 30Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 34Schmidlin F. Dery O. Bunnett N.W. Grady E.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3324-3329Crossref PubMed Scopus (56) Google Scholar). 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 β-arrestins to agonist-occupied C5aR and from the observation that co-expression of β-arrestin 1 increases by 1.4-fold the rate of C5aR internalization, 2L. Braun and F. Boulay, unpublished results. it is likely that β-arrestins play an important role in C5aR internalization. In the present study, we provide strong evidence that β-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 β-arrestin 1. Second, the co-expression of C5aR with either the dominant negative mutant K44E of dynamin or β-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 (35Barlic J. Khandaker M.H. Mahon E. Andrews J. DeVries M.E. Mitchell G.B. Rahimpour R. Tan C.M. Ferguson S.S. Kelvin D.J. J. Biol. Chem. 1999; 274: 16287-16294Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), the C5aR appears to be internalized via clathrin-coated pits in a dynamin-dependent manner. The role of β-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 β-arrestin and dynamin (13Zhang J. Stephen S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 36Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 37Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (144) Google Scholar). 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 β-arrestin 1.2 Our conclusions strikingly contrast with recent studies suggesting that C5aR and FPR are internalized through a β-arrestin-, clathrin-, and dynamin-independent pathway (15Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry. 2001; 40: 3467-3475Crossref PubMed Scopus (47) Google Scholar, 16Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These divergent results might possibly be due to differences in the methods used to assess receptor internalization. In the study by Bennett et al. (16Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Here, we show by confocal microscopy that the phosphorylation of either two-serine pair is sufficient to allow the co-trafficking of β-arrestins with the receptor to intracellular vesicles. Whether β-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 (38Bennett T.A. Foutz T.D. Gurevich V.V. Sklar L.A. Prossnitz E.R. J. Biol. Chem. 2001; 276: 49195-49203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).Of particular interest is the observation by confocal microscopy that β-arrestins are recruited to the plasma membrane by phosphorylation-deficient mutant. The agonist-dependent recruitment of β-arr 2-EGFP but not β-arr 1-EGFP by C5aR-A332,334,338 provides additional support to the notion that β-arrestin 2 is the preferred isoform for targeting wild-type C5aR to clathrin-coated pits. Although the affinity of β-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 (20Naik N. Giannini E. Brouchon L. Boulay F. J. Cell Sci. 1997; 110: 2381-2390Crossref PubMed Google Scholar, 21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).In addition to their role as clathrin adapters, β-arrestins function as scaffolding proteins for several signaling pathways. Although β-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) (26Luttrell L.M. Ferguson S.S.G. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1250) Google Scholar), β-arrestin 2 forms a complex with and target c-Jun N-terminal kinase 3 (JNK3) to specific subcellular compartments and/or specific substrates (39McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar). Recently, β-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 (40Fong A.M. Premont R.T. Richardson R.M., Yu, Y.R. Lefkowitz R.J. Patel D.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7478-7483Crossref PubMed Scopus (260) Google Scholar). Our data indicating that β-arrestins can be recruited to the plasma membrane in the absence of receptor phosphorylation suggest that β-arrestin-mediated signaling is still possible in the absence of receptor internalization. With other chemoattractant receptors, the agonist-dependent recruitment of β-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 (41Arai H. Monteclaro F.S. Tsou C.L. Franci C. Charo I.F. J. Biol. Chem. 1997; 272: 25037-25042Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 42Richardson R.M. Ali H. Pridgen B.C. Haribabu B. Snyderman R. J. Biol. Chem. 1998; 273: 10690-10695Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 43Kraft K. Olbrich H. Majoul I. Mack M. Proudfoot A. Oppermann M. J. Biol. Chem. 2001; 276: 34408-34418Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar).In conclusion, we have demonstrated that C5a induces a marked redistribution of β-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 β-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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), suggesting a weaker interaction with β-arrestins and, thereby, a slightly prolonged interaction with the G protein. The complement C5a1anaphylatoxin receptor (C5aR) is a member of the G-protein-coupled receptor family (GPCR) (1Boulay F. Mery L. Tardif M. Brouchon L. Vignais P. Biochemistry. 1991; 30: 2993-2999Crossref PubMed Scopus (192) Google Scholar, 2Gerard N.P. Gerard C. Nature. 1991; 349: 614-617Crossref PubMed Scopus (561) Google Scholar). It is abundantly expressed in myeloid cells and to a lower level in a variety of non-myeloid cells (3Zwirner J. Fayyazi A. Gotze O. Mol. Immunol. 1999; 36: 877-884Crossref PubMed Scopus (80) Google Scholar). 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 (4Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar). 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 β2-adrenergic receptor, is that, once activated and phosphorylated, the GPCRs form a stable complex with a family of adapters known as arrestins and β-arrestins (5McDonald P.H. Lefkowitz R.J. Cell. Signal. 2001; 13: 683-689Crossref PubMed Scopus (111) Google Scholar). There is growing evidence indicating that the formation of this complex is a general intermediate for endocytosis of most GPCRs. The β-arrestins interact with clathrin and the adapter protein complex AP-2 (6Goodman O.B. Krupnick J.G.J. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar, 7Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Ménard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (839) Google Scholar, 8Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar) and target the agonist-occupied receptors to pre-existing clathrin-coated pits for internalization (9Scott M.G. Benmerah A. Muntaner O. Marullo S. J. Biol. Chem. 2002; 277: 3552-3559Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Santini F. Gaidarov I. Keen J.H. J. Cell Biol. 2002; 156: 665-676Crossref PubMed Scopus (91) Google Scholar). Expression of a GTPase-defective dynamin mutant blocks the clathrin-dependent endocytic pathway in a dominant-negative manner (11Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar), and the agonist-mediated internalization of most GPCRs is inhibited (12Pierce K.L. Lefkowitz R.J. Nat. Rev. Neurosci. 2001; 2: 727-733Crossref PubMed Scopus (363) Google Scholar). 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 β-arrestins, can be internalized through a process independent of β-arrestin and dynamin (13Zhang J. Stephen S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 14Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 15Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry. 2001; 40: 3467-3475Crossref PubMed Scopus (47) Google Scholar, 16Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), whereas the 5-hydroxytryptamine 2A receptor is internalized via a pathway dependent on dynamin and independent of β-arrestin (17Bhatnagar A. Willins D.L. Gray J.A. Woods J. Benovic J.L. Roth B.L. J. Biol. Chem. 2001; 276: 8269-8277Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The internalization of the M2 muscarinic acetylcholine receptor appears to proceed through an atypical pathway that is independent of clathrin (18Roseberry A.G. Hosey M.M. J. Cell Sci. 2001; 114: 739-746PubMed Google Scholar). 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 (19Giannini E. Brouchon L. Boulay F. J. Biol. Chem. 1995; 270: 19166-19172Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Mutants with combined amino acid replacements exhibit different capacities to incorporate phosphate on the remaining serine residues (20Naik N. Giannini E. Brouchon L. Boulay F. J. Cell Sci. 1997; 110: 2381-2390Crossref PubMed Google Scholar, 21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The phosphorylation of the first membrane proximal serine residue is dispensable for receptor desensitization, and agonist sequestration is apparently independent of desensitization (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In the present study, we used confocal microscopy and the green fluorescent protein conjugate of β-arrestin 1, β-arrestin 2, and the dominant negative mutant β-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, β-arrestin 1 and β-arrestin 2 were found to have differential abilities to associate with the activated receptor. In contrast to β-arrestin 1, β-arrestin 2 redistributed to the plasma membrane regardless of whether C5aR was phosphorylated provided it was activated. DISCUSSIONFor 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 β-arrestin scaffolding proteins. In the present work, by using green fluorescent protein-tagged β-arrestin 1 and 2, we find that C5a stimulated a striking and rapid redistribution of β-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 β-arrestins as evidenced by the co-localization of C5aR and β-arrestins on the same endosomal compartments. Similar agonist-mediated translocation and association to endosomes have been previously described with a variety of GPCRs (6Goodman O.B. Krupnick J.G.J. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar, 23Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). The co-trafficking of β-arrestins and C5aR to perinuclear vesicles was consistently best observed with β-arrestin 2, suggesting that this isoform has a higher affinity for the activated wild-type receptor. The persistent association of β-arrestins with intracellular vesicular compartments that contain internalized GPCRs is not a general rule. The stability of β-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 β-arrestin 2 on endosomal compartments, “class A” receptors including the β2-adrenergic, dopamine D1A, and endothelin type A receptors rapidly dissociate from β-arrestins, which remain confined to the plasma membrane (14Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 30Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 34Schmidlin F. Dery O. Bunnett N.W. Grady E.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3324-3329Crossref PubMed Scopus (56) Google Scholar). 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 β-arrestins to agonist-occupied C5aR and from the observation that co-expression of β-arrestin 1 increases by 1.4-fold the rate of C5aR internalization, 2L. Braun and F. Boulay, unpublished results. it is likely that β-arrestins play an important role in C5aR internalization. In the present study, we provide strong evidence that β-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 β-arrestin 1. Second, the co-expression of C5aR with either the dominant negative mutant K44E of dynamin or β-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 (35Barlic J. Khandaker M.H. Mahon E. Andrews J. DeVries M.E. Mitchell G.B. Rahimpour R. Tan C.M. Ferguson S.S. Kelvin D.J. J. Biol. Chem. 1999; 274: 16287-16294Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), the C5aR appears to be internalized via clathrin-coated pits in a dynamin-dependent manner. The role of β-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 β-arrestin and dynamin (13Zhang J. Stephen S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 36Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 37Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (144) Google Scholar). 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 β-arrestin 1.2 Our conclusions strikingly contrast with recent studies suggesting that C5aR and FPR are internalized through a β-arrestin-, clathrin-, and dynamin-independent pathway (15Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry. 2001; 40: 3467-3475Crossref PubMed Scopus (47) Google Scholar, 16Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These divergent results might possibly be due to differences in the methods used to assess receptor internalization. In the study by Bennett et al. (16Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Here, we show by confocal microscopy that the phosphorylation of either two-serine pair is sufficient to allow the co-trafficking of β-arrestins with the receptor to intracellular vesicles. Whether β-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 (38Bennett T.A. Foutz T.D. Gurevich V.V. Sklar L.A. Prossnitz E.R. J. Biol. Chem. 2001; 276: 49195-49203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).Of particular interest is the observation by confocal microscopy that β-arrestins are recruited to the plasma membrane by phosphorylation-deficient mutant. The agonist-dependent recruitment of β-arr 2-EGFP but not β-arr 1-EGFP by C5aR-A332,334,338 provides additional support to the notion that β-arrestin 2 is the preferred isoform for targeting wild-type C5aR to clathrin-coated pits. Although the affinity of β-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 (20Naik N. Giannini E. Brouchon L. Boulay F. J. Cell Sci. 1997; 110: 2381-2390Crossref PubMed Google Scholar, 21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).In addition to their role as clathrin adapters, β-arrestins function as scaffolding proteins for several signaling pathways. Although β-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) (26Luttrell L.M. Ferguson S.S.G. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1250) Google Scholar), β-arrestin 2 forms a complex with and target c-Jun N-terminal kinase 3 (JNK3) to specific subcellular compartments and/or specific substrates (39McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar). Recently, β-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 (40Fong A.M. Premont R.T. Richardson R.M., Yu, Y.R. Lefkowitz R.J. Patel D.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7478-7483Crossref PubMed Scopus (260) Google Scholar). Our data indicating that β-arrestins can be recruited to the plasma membrane in the absence of receptor phosphorylation suggest that β-arrestin-mediated signaling is still possible in the absence of receptor internalization. With other chemoattractant receptors, the agonist-dependent recruitment of β-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 (41Arai H. Monteclaro F.S. Tsou C.L. Franci C. Charo I.F. J. Biol. Chem. 1997; 272: 25037-25042Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 42Richardson R.M. Ali H. Pridgen B.C. Haribabu B. Snyderman R. J. Biol. Chem. 1998; 273: 10690-10695Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 43Kraft K. Olbrich H. Majoul I. Mack M. Proudfoot A. Oppermann M. J. Biol. Chem. 2001; 276: 34408-34418Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar).In conclusion, we have demonstrated that C5a induces a marked redistribution of β-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 β-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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), suggesting a weaker interaction with β-arrestins and, thereby, a slightly prolonged interaction with the G protein. 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 β-arrestin scaffolding proteins. In the present work, by using green fluorescent protein-tagged β-arrestin 1 and 2, we find that C5a stimulated a striking and rapid redistribution of β-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 β-arrestins as evidenced by the co-localization of C5aR and β-arrestins on the same endosomal compartments. Similar agonist-mediated translocation and association to endosomes have been previously described with a variety of GPCRs (6Goodman O.B. Krupnick J.G.J. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar, 23Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). The co-trafficking of β-arrestins and C5aR to perinuclear vesicles was consistently best observed with β-arrestin 2, suggesting that this isoform has a higher affinity for the activated wild-type receptor. The persistent association of β-arrestins with intracellular vesicular compartments that contain internalized GPCRs is not a general rule. The stability of β-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 β-arrestin 2 on endosomal compartments, “class A” receptors including the β2-adrenergic, dopamine D1A, and endothelin type A receptors rapidly dissociate from β-arrestins, which remain confined to the plasma membrane (14Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 30Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 34Schmidlin F. Dery O. Bunnett N.W. Grady E.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3324-3329Crossref PubMed Scopus (56) Google Scholar). 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 β-arrestins to agonist-occupied C5aR and from the observation that co-expression of β-arrestin 1 increases by 1.4-fold the rate of C5aR internalization, 2L. Braun and F. Boulay, unpublished results. it is likely that β-arrestins play an important role in C5aR internalization. In the present study, we provide strong evidence that β-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 β-arrestin 1. Second, the co-expression of C5aR with either the dominant negative mutant K44E of dynamin or β-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 (35Barlic J. Khandaker M.H. Mahon E. Andrews J. DeVries M.E. Mitchell G.B. Rahimpour R. Tan C.M. Ferguson S.S. Kelvin D.J. J. Biol. Chem. 1999; 274: 16287-16294Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), the C5aR appears to be internalized via clathrin-coated pits in a dynamin-dependent manner. The role of β-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 β-arrestin and dynamin (13Zhang J. Stephen S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 36Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 37Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (144) Google Scholar). 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 β-arrestin 1.2 Our conclusions strikingly contrast with recent studies suggesting that C5aR and FPR are internalized through a β-arrestin-, clathrin-, and dynamin-independent pathway (15Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry. 2001; 40: 3467-3475Crossref PubMed Scopus (47) Google Scholar, 16Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These divergent results might possibly be due to differences in the methods used to assess receptor internalization. In the study by Bennett et al. (16Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Here, we show by confocal microscopy that the phosphorylation of either two-serine pair is sufficient to allow the co-trafficking of β-arrestins with the receptor to intracellular vesicles. Whether β-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 (38Bennett T.A. Foutz T.D. Gurevich V.V. Sklar L.A. Prossnitz E.R. J. Biol. Chem. 2001; 276: 49195-49203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Of particular interest is the observation by confocal microscopy that β-arrestins are recruited to the plasma membrane by phosphorylation-deficient mutant. The agonist-dependent recruitment of β-arr 2-EGFP but not β-arr 1-EGFP by C5aR-A332,334,338 provides additional support to the notion that β-arrestin 2 is the preferred isoform for targeting wild-type C5aR to clathrin-coated pits. Although the affinity of β-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 (20Naik N. Giannini E. Brouchon L. Boulay F. J. Cell Sci. 1997; 110: 2381-2390Crossref PubMed Google Scholar, 21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In addition to their role as clathrin adapters, β-arrestins function as scaffolding proteins for several signaling pathways. Although β-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) (26Luttrell L.M. Ferguson S.S.G. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1250) Google Scholar), β-arrestin 2 forms a complex with and target c-Jun N-terminal kinase 3 (JNK3) to specific subcellular compartments and/or specific substrates (39McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar). Recently, β-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 (40Fong A.M. Premont R.T. Richardson R.M., Yu, Y.R. Lefkowitz R.J. Patel D.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7478-7483Crossref PubMed Scopus (260) Google Scholar). Our data indicating that β-arrestins can be recruited to the plasma membrane in the absence of receptor phosphorylation suggest that β-arrestin-mediated signaling is still possible in the absence of receptor internalization. With other chemoattractant receptors, the agonist-dependent recruitment of β-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 (41Arai H. Monteclaro F.S. Tsou C.L. Franci C. Charo I.F. J. Biol. Chem. 1997; 272: 25037-25042Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 42Richardson R.M. Ali H. Pridgen B.C. Haribabu B. Snyderman R. J. Biol. Chem. 1998; 273: 10690-10695Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 43Kraft K. Olbrich H. Majoul I. Mack M. Proudfoot A. Oppermann M. J. Biol. Chem. 2001; 276: 34408-34418Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In conclusion, we have demonstrated that C5a induces a marked redistribution of β-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 β-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 (21Christophe T. Rabiet M.-J. Tardif M. Milcent M.-D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), suggesting a weaker interaction with β-arrestins and, thereby, a slightly prolonged interaction with the G protein." @default.
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W1993346410 title "Phosphorylation of Key Serine Residues Is Required for Internalization of the Complement 5a (C5a) Anaphylatoxin Receptor via a β-Arrestin, Dynamin, and Clathrin-dependent Pathway" @default.
W1993346410 cites W1533171633 @default.
W1993346410 cites W1546290898 @default.
W1993346410 cites W1947826208 @default.
W1993346410 cites W1967965496 @default.
W1993346410 cites W1971800733 @default.
W1993346410 cites W1975386197 @default.
W1993346410 cites W1975593747 @default.
W1993346410 cites W1980205661 @default.
W1993346410 cites W1988518158 @default.
W1993346410 cites W2001647007 @default.
W1993346410 cites W2003819446 @default.
W1993346410 cites W2003832177 @default.
W1993346410 cites W2006272522 @default.
W1993346410 cites W2011643835 @default.
W1993346410 cites W2012571494 @default.
W1993346410 cites W2022669416 @default.
W1993346410 cites W2024875771 @default.
W1993346410 cites W2025472785 @default.
W1993346410 cites W2033680805 @default.
W1993346410 cites W2037204799 @default.
W1993346410 cites W2041654178 @default.
W1993346410 cites W2045106976 @default.
W1993346410 cites W2047703003 @default.
W1993346410 cites W2049132647 @default.
W1993346410 cites W2057511211 @default.
W1993346410 cites W2059327659 @default.
W1993346410 cites W2065674919 @default.
W1993346410 cites W2066071036 @default.
W1993346410 cites W2067698573 @default.
W1993346410 cites W2080815758 @default.
W1993346410 cites W2087773298 @default.
W1993346410 cites W2091073917 @default.
W1993346410 cites W2092532048 @default.
W1993346410 cites W2103481821 @default.
W1993346410 cites W2135319033 @default.
W1993346410 cites W2141030777 @default.
W1993346410 cites W2151312020 @default.
W1993346410 cites W2151526347 @default.
W1993346410 cites W2152568000 @default.
W1993346410 cites W2169295770 @default.
W1993346410 cites W329126969 @default.
W1993346410 cites W93284168 @default.
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