title: Dynamics of protein kinase C-mediated phosphorylation of the complement C5a receptor on serine 334.
abstract: Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t((1/2)) approximately 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-beta and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nM) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t((1/2)) < 5 s) binding of beta-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.
authors.affiliation: Department of Cellular and Molecular Immunology, Georg-August-University, 37073 Göttingen, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Pollok-Kopp
authors.affiliation: Department of Cellular and Molecular Immunology, Georg-August-University, 37073 Göttingen, Germany.
authors.forename: Friederike
authors.initials: F
authors.lastname: Hüttenrauch
authors.affiliation: Department of Cellular and Molecular Immunology, Georg-August-University, 37073 Göttingen, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Rethorn
authors.affiliation: Department of Cellular and Molecular Immunology, Georg-August-University, 37073 Göttingen, Germany. Electronic address: mopperm@gwdg.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Oppermann
mesh_terms: D045163:Antibodies, Phospho-Specific / Q000494:pharmacology; D019390:Arrestins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D015936:Complement C5a / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007155:Immunologic Factors / Q000378:metabolism / Q000494:pharmacology; D008387:Marine Toxins; D008565:Membrane Proteins / Q000819:agonists / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000819:agonists / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D020298:U937 Cells; D000071557:beta-Arrestins
chemical_list: D045163:Antibodies, Phospho-Specific; D019390:Arrestins; C488033:C5AR1 protein, human; D007155:Immunologic Factors; D008387:Marine Toxins; D008565:Membrane Proteins; D010080:Oxazoles; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; D012694:Serine; C059041:calyculin A; D015936:Complement C5a; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D010749:Phosphoprotein Phosphatases