Human Complement 5a (C5a) Anaphylatoxin Receptor (CD88) Phosphorylation Sites and Their Specific Role in Receptor Phosphorylation and Attenuation of G Protein-mediated Responses

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166–19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser334 was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser332 and Ser334 or Ser334 and Ser338, was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser334 and Ser338 is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8–2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms. Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166–19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser334 was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser332 and Ser334 or Ser334 and Ser338, was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser334 and Ser338 is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8–2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms. human complement 5a C5a receptor G protein-coupled receptor protein kinase C N-formyl-Met-Leu-Phe-Lys-OH N 6,O-2′-dibutyryl adenosine 3′,5′ cyclic monophosphate extracellular signal-regulated kinase mitogen-activated protein phorbol 12-myristate 13-acetate The capacity of phagocytic leukocytes to clear the host from invading microorganisms is dependent on their ability to migrate to sites of inflammation and to release large amounts of proteolytic enzymes and reactive oxygen species. Phagocytes do this in response to a variety of structurally diverse chemoattractants, including bacterialN-formylated peptides, the complement fragment C5a,1 leukotriene B4, platelet-activating factor, and interleukin 8 (reviewed in Ref. 1.Ye R. Boulay F. Adv. Pharmacol. 1997; 39: 221-289Crossref PubMed Scopus (105) Google Scholar). Chemoattractants elicit intracellular signaling through specific receptors that are coupled to the pertussis toxin-sensitive heterotrimeric Gi2 protein. This latter activates in turn second messenger-generating enzymes, including phospholipases C, D, and A2 (reviewed in Ref. 2.Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar), as well as kinases, such as phosphatidylinositol 3-kinase (3.Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar) and sphingosine kinase (4.Alemany R. Meyer zu Heringdorf D. van Koppen C.J. Jakobs K.H. J. Biol. Chem. 1999; 274: 3994-3999Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Ultimately, the NADPH-oxidase is activated and releases superoxide anions. This is a crucial bactericidal mechanism, but it is also believed to be a major cause of inflammatory disorders when inappropriately activated.The activation of the NADPH-oxidase complex responsible for the production of superoxide requires the coordinated action of multiple signaling pathways. The NADPH-oxidase becomes functional through a multistep assembly of several components at the plasma membrane, namely the flavocytochrome b 558, the small G proteins (Rac and Rap), and cytosolic factors (p47 phox, p67 phox, and p40 phox) (reviewed in Ref. 5.Babior B.M. Curr. Opin. Hematol. 1995; 2: 55-60Crossref PubMed Scopus (122) Google Scholar). The ability of chemoattractants to induce the production of superoxide varies from one chemoattractant to another. The activation of the NADPH-oxidase complex is correlated with the rapid increase in Ca2+ and diacylglycerol, which subsequently activate protein kinase C (PKC), and the phosphorylation of the cytosolic factor p47 phox (6.el Benna J. Faust L.P. Babior B.M. J. Biol. Chem. 1994; 269: 23431-23436Abstract Full Text PDF PubMed Google Scholar, 7.el Benna J. Faust L.P. Johnson J.L. Babior B.M. J. Biol. Chem. 1996; 271: 6374-6378Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The involvement of PKC in triggering the activation of NADPH oxidase is supported by the ability of phorbol 12-myristate 13-acetate (PMA) to induce a sustained superoxide production and the phosphorylation and translocation of p47 phox to the plasma membrane (8.Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google Scholar).Despite the persistent presence of chemoattractants, the intracellular signaling events are transient, with a time course in the minute range. This attenuated responsiveness is thought to result from the desensitization of receptors through their phosphorylation and rapid sequestration (reviewed in Refs. 9.Dohlman H.G. Thorner J. Caron M. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar and 10.Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). The current concept for desensitization of G protein-coupled receptors (GPCRs), largely extrapolated from numerous studies with rhodopsin and the β2-adrenergic receptor, is that arrestin proteins bind to the phosphorylated receptors and sterically prevent its interaction with the G protein (11.Wilden U. Hall S.W. Kühn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (568) Google Scholar, 12.Dolph P.J. Ranganathan R. Colley N.J. Hardy R.W. Socolich M. Zuker C.S. Science. 1993; 260: 1910-1916Crossref PubMed Scopus (263) Google Scholar, 13.Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (897) Google Scholar). In addition, β-arrestin acts as a clathrin adapter that targets the agonist-occupied β2-adrenergic receptor to the endocytic pathway via clathrin coated pits (14.Goodman 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, 15.Ferguson 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). Expression of a GTPase defective dynamin mutant (K44A) (16.Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar) specifically inhibits the clathrin-dependent endocytic pathway and the agonist-mediated internalization of GPCRs is inhibited. However, mounting evidence indicates that not all GPCRs are internalized through this pathway. For instance, internalization of the angiotensin II 1A and m2 muscarinic acetylcholine receptors in HEK293 cells is independent of β-arrestin and is not inhibited when the clathrin-coated vesicle pathway is blocked by overexpression of a dominant negative form of dynamin (17.Zhang J. Ferguson S.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, 18.Pals-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, 19.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).Although receptor desensitization is thought to be a key regulatory mechanism controlling the inflammatory potential of phagocytic cells, the impact of an altered chemoattractant receptor desensitization on intracellular signaling events and the release of reactive oxygen species has never been investigated in leukocytes. The C5a receptor (C5aR) is particularly well suited to examine this issue because previous studies have shown the rapid agonist-mediated phosphorylation of this receptor on serine residues in differentiated HL-60 cells and after heterologous expression (20.Tardif M. Mery L. Brouchon L. Boulay F. J. Immunol. 1993; 150: 3534-3545PubMed Google Scholar, 21.Ali H. Richardson R.M. Tomhave E.D. Didsbury J.R. Snyderman R. J. Biol. Chem. 1993; 268: 24247-24254Abstract Full Text PDF PubMed Google Scholar, 22.Giannini E. Brouchon L. Boulay F. J. Biol. Chem. 1995; 270: 19166-19172Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In transfected COS-7 cells, the six serine residues of the carboxyl-terminal domain, at positions 314, 317, 327, 332, 334, and 338, were identified as the major phosphorylation sites, but it is not known whether a stoichiometry of 6 phosphate groups per receptor is required for receptor desensitization (22.Giannini E. Brouchon L. Boulay F. J. Biol. Chem. 1995; 270: 19166-19172Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).In this paper, we show that phosphorylation of either of the two serine pairs (Ser332/Ser334 and Ser334/Ser338) is a prerequisite for full receptor phosphorylation. This strongly supports the notion that C5aR is sequentially phosphorylated upon agonist binding. However, phosphorylation of the first four serine residues was found to be dispensable for receptor desensitization. In stably transfected HL-60 cells, receptor sequestration was found to be independent of receptor desensitization. In addition, the results show for the first time that a nonphosphorylatable receptor is able to support sustained intracellular signaling events that result in a significant increase in the production of superoxide by neutrophil-like differentiated HL-60 cells. This indicates that cell adaptation to a persistent stimulus is to some extent dependent on receptor phosphorylation. The capacity of phagocytic leukocytes to clear the host from invading microorganisms is dependent on their ability to migrate to sites of inflammation and to release large amounts of proteolytic enzymes and reactive oxygen species. Phagocytes do this in response to a variety of structurally diverse chemoattractants, including bacterialN-formylated peptides, the complement fragment C5a,1 leukotriene B4, platelet-activating factor, and interleukin 8 (reviewed in Ref. 1.Ye R. Boulay F. Adv. Pharmacol. 1997; 39: 221-289Crossref PubMed Scopus (105) Google Scholar). Chemoattractants elicit intracellular signaling through specific receptors that are coupled to the pertussis toxin-sensitive heterotrimeric Gi2 protein. This latter activates in turn second messenger-generating enzymes, including phospholipases C, D, and A2 (reviewed in Ref. 2.Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar), as well as kinases, such as phosphatidylinositol 3-kinase (3.Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar) and sphingosine kinase (4.Alemany R. Meyer zu Heringdorf D. van Koppen C.J. Jakobs K.H. J. Biol. Chem. 1999; 274: 3994-3999Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Ultimately, the NADPH-oxidase is activated and releases superoxide anions. This is a crucial bactericidal mechanism, but it is also believed to be a major cause of inflammatory disorders when inappropriately activated. The activation of the NADPH-oxidase complex responsible for the production of superoxide requires the coordinated action of multiple signaling pathways. The NADPH-oxidase becomes functional through a multistep assembly of several components at the plasma membrane, namely the flavocytochrome b 558, the small G proteins (Rac and Rap), and cytosolic factors (p47 phox, p67 phox, and p40 phox) (reviewed in Ref. 5.Babior B.M. Curr. Opin. Hematol. 1995; 2: 55-60Crossref PubMed Scopus (122) Google Scholar). The ability of chemoattractants to induce the production of superoxide varies from one chemoattractant to another. The activation of the NADPH-oxidase complex is correlated with the rapid increase in Ca2+ and diacylglycerol, which subsequently activate protein kinase C (PKC), and the phosphorylation of the cytosolic factor p47 phox (6.el Benna J. Faust L.P. Babior B.M. J. Biol. Chem. 1994; 269: 23431-23436Abstract Full Text PDF PubMed Google Scholar, 7.el Benna J. Faust L.P. Johnson J.L. Babior B.M. J. Biol. Chem. 1996; 271: 6374-6378Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The involvement of PKC in triggering the activation of NADPH oxidase is supported by the ability of phorbol 12-myristate 13-acetate (PMA) to induce a sustained superoxide production and the phosphorylation and translocation of p47 phox to the plasma membrane (8.Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google Scholar). Despite the persistent presence of chemoattractants, the intracellular signaling events are transient, with a time course in the minute range. This attenuated responsiveness is thought to result from the desensitization of receptors through their phosphorylation and rapid sequestration (reviewed in Refs. 9.Dohlman H.G. Thorner J. Caron M. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar and 10.Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). The current concept for desensitization of G protein-coupled receptors (GPCRs), largely extrapolated from numerous studies with rhodopsin and the β2-adrenergic receptor, is that arrestin proteins bind to the phosphorylated receptors and sterically prevent its interaction with the G protein (11.Wilden U. Hall S.W. Kühn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (568) Google Scholar, 12.Dolph P.J. Ranganathan R. Colley N.J. Hardy R.W. Socolich M. Zuker C.S. Science. 1993; 260: 1910-1916Crossref PubMed Scopus (263) Google Scholar, 13.Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (897) Google Scholar). In addition, β-arrestin acts as a clathrin adapter that targets the agonist-occupied β2-adrenergic receptor to the endocytic pathway via clathrin coated pits (14.Goodman 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, 15.Ferguson 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). Expression of a GTPase defective dynamin mutant (K44A) (16.Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar) specifically inhibits the clathrin-dependent endocytic pathway and the agonist-mediated internalization of GPCRs is inhibited. However, mounting evidence indicates that not all GPCRs are internalized through this pathway. For instance, internalization of the angiotensin II 1A and m2 muscarinic acetylcholine receptors in HEK293 cells is independent of β-arrestin and is not inhibited when the clathrin-coated vesicle pathway is blocked by overexpression of a dominant negative form of dynamin (17.Zhang J. Ferguson S.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, 18.Pals-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, 19.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Although receptor desensitization is thought to be a key regulatory mechanism controlling the inflammatory potential of phagocytic cells, the impact of an altered chemoattractant receptor desensitization on intracellular signaling events and the release of reactive oxygen species has never been investigated in leukocytes. The C5a receptor (C5aR) is particularly well suited to examine this issue because previous studies have shown the rapid agonist-mediated phosphorylation of this receptor on serine residues in differentiated HL-60 cells and after heterologous expression (20.Tardif M. Mery L. Brouchon L. Boulay F. J. Immunol. 1993; 150: 3534-3545PubMed Google Scholar, 21.Ali H. Richardson R.M. Tomhave E.D. Didsbury J.R. Snyderman R. J. Biol. Chem. 1993; 268: 24247-24254Abstract Full Text PDF PubMed Google Scholar, 22.Giannini E. Brouchon L. Boulay F. J. Biol. Chem. 1995; 270: 19166-19172Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In transfected COS-7 cells, the six serine residues of the carboxyl-terminal domain, at positions 314, 317, 327, 332, 334, and 338, were identified as the major phosphorylation sites, but it is not known whether a stoichiometry of 6 phosphate groups per receptor is required for receptor desensitization (22.Giannini E. Brouchon L. Boulay F. J. Biol. Chem. 1995; 270: 19166-19172Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In this paper, we show that phosphorylation of either of the two serine pairs (Ser332/Ser334 and Ser334/Ser338) is a prerequisite for full receptor phosphorylation. This strongly supports the notion that C5aR is sequentially phosphorylated upon agonist binding. However, phosphorylation of the first four serine residues was found to be dispensable for receptor desensitization. In stably transfected HL-60 cells, receptor sequestration was found to be independent of receptor desensitization. In addition, the results show for the first time that a nonphosphorylatable receptor is able to support sustained intracellular signaling events that result in a significant increase in the production of superoxide by neutrophil-like differentiated HL-60 cells. This indicates that cell adaptation to a persistent stimulus is to some extent dependent on receptor phosphorylation.