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 ϳ 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-␤ 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 ␤-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.

PMX-53 as a Dual CD88 Antagonist and an Agonist for Mas-Related Gene 2 (MrgX2) in Human Mast Cells

“…CD88 undergoes phosphorylation in response to agonist stimulation resulting in their desensitization (Christophe et al, 2000;Braun et al, 2003). Furthermore, phosphorylation-deficient mutants of CD88 are resistant to desensitization (Pollok-Kopp et al, 2007). Solinski et al (2010) showed that MrgX1 is one of the few GPCRs that does not associate with ␤-arrestin and is resistant to agonist-induced receptor internalization.…”

International Union of Basic and Clinical Pharmacology. LXXXVII. Complement Peptide C5a, C4a, and C3a Receptors

“…However, internalization of the receptor, one mechanism for desensitization, was found to be dependent only on amino acids 335-350 in a series of truncated C5a 1 receptor mutants, beyond the majority of the phosphorylation sites . Similarly, another study found that although Ser334, Ser327, Ser332, and Ser338 could be phosphorylated by PK-Cb, these sites were functionally redundant for internalization and desensitization and even that, at high concentrations of C5a, b-arrestin binding to the C terminus could actually inhibit phosphorylation (Pollok-Kopp et al, 2007). Thus, the role of phosphorylation in the control of receptor function remains unclear.…”

Surface masking shapes the traffic of the neuropeptide Y Y2 receptor

“…These sites could be primed for internalization by phosphorylation, as indicated for C5a receptor [36], vasopressin and oxytocin receptors [3, 15] and opioid receptors [18]. Physical association with protein kinases and arrestins could in turn help mobilization of masked receptors.…”